CN111757937A - 腺苷碱基编辑器的用途 - Google Patents

腺苷碱基编辑器的用途 Download PDF

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CN111757937A
CN111757937A CN201880081042.9A CN201880081042A CN111757937A CN 111757937 A CN111757937 A CN 111757937A CN 201880081042 A CN201880081042 A CN 201880081042A CN 111757937 A CN111757937 A CN 111757937A
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D.R.刘
N.高德利
M.S.帕克
G.纽比
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Harvard College
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Abstract

本公开提供了用于治疗血液疾病/病症如镰状细胞病、血色素沉着症、血友病和beta地中海贫血的方法和组合物。例如，本公开提供了靶向HBG1/2的启动子以生成增加胎儿血红蛋白表达的点突变的治疗性引导RNA。作为另一个实例，本公开提供了靶向HBB、因子VIII和HFE中的突变的治疗性引导RNA，以治疗镰状细胞病、beta地中海贫血、血友病和血色素沉着症。本公开还提供了融合蛋白，其包含Cas9(例如，Cas9切口酶)结构域和使DNA中的腺苷脱氨基的腺苷脱氨酶。在一些实施方案中，融合蛋白与核酸如引导RNA(gRNA)复合，其将融合蛋白靶向DNA序列(例如，HBG1或HBG2启动子序列，或HFE，GBB或F8基因序列)。此类复合物可以用于增加胎儿血红蛋白的表达或校正HFE中的点突变(例如，C282Y)。

Description

腺苷碱基编辑器的用途
发明背景
靶向编辑核酸序列，例如靶向切割或将特定修饰靶向引入基因组DNA中，是用于研究基因功能的非常有前景的方法，并且还具有为人类遗传性疾病提供新疗法的潜力。由于许多遗传性疾病原则上可以通过在基因组中的特定位置处实现特定的核苷酸变化来治疗(例如，与疾病相关的基因的特定密码子中的A至G或T至C的变化)，开发可编程的方法来实现此类精确的基因编辑既代表强大的新研究工具，又代表基于基因编辑的治疗学的潜在新方法。
发明概述
本公开提供了用于治疗血液疾病/病症如镰状细胞病、血色素沉着症、血友病和beta地中海贫血的方法和组合物。例如，本公开提供了靶向HBG1/2的启动子以生成增加胎儿血红蛋白表达的点突变的治疗性引导RNA。作为另一个实例，本公开提供了靶向HBB、因子VIII和HFE中的突变(例如致病性突变)的治疗性引导RNA，以治疗镰状细胞病、beta地中海贫血(例如Hb C和Hb E)、血友病和血色素沉着症。本文提供的引导RNA可以与碱基编辑器蛋白(例如，腺苷碱基编辑器)复合以在基因或基因启动子中生成点突变，其可以校正致病性突变，生成非致病性点突变或调控(例如增加)基因的表达。
本文提供了使用腺苷脱氨酶和核酸可编程DNA结合蛋白(例如Cas9)修饰多核苷酸(例如DNA)的组合物、试剂盒和方法。本公开的一些方面提供了核碱基编辑蛋白，其在DNA的背景下催化腺苷的水解脱氨基作用(形成肌苷，其如鸟嘌呤(G)一样碱基配对)。没有作用于DNA的已知天然存在的腺苷脱氨酶。相反，已知的腺苷脱氨酶作用于RNA(例如，tRNA或mRNA)。为了克服这个缺点，将第一脱氧腺苷脱氨酶演化成接受DNA底物并将脱氧腺苷(dA)脱氨基成脱氧肌苷。此类腺苷脱氨酶描述于2017年8月3日提交的国际申请号：PCT/US2017/045,381中；其全部内容在此通过引用并入。来自大肠杆菌的作用于tRNA的腺苷脱氨酶(adenosine deaminase acting on tRNA)(ADAT)(TadA，代表tRNA腺苷脱氨酶A(tRNAadenosine deaminase A))与dCas9或Cas9切口酶结构域共价融合，并且组装了在构建体的脱氨酶部分中含有突变的融合蛋白。除大肠杆菌TadA(ecTadA)外，其他天然存在的腺苷脱氨酶，如人ADAR(作用于RNA的腺苷脱氨酶(adenosine deaminase acting on RNA))、小鼠ADA(腺苷脱氨酶)和人ADAT2，也可以与dCas9或Cas9切口酶结构域融合以生成腺苷核碱基编辑器(ABE)融合蛋白构建体。这些融合蛋白的定向演化导致将A-T碱基对有效转化为G-C碱基对的可编程的腺苷碱基编辑器，其具有低脱靶修饰和低插入/缺失(随机插入或缺失)形成率，特别是与目前的Cas9核酸酶介导的HDR基因组编辑方法相比。本文公开的ABE既可以用于校正与疾病相关的点突变，也可以用于引入抑制疾病的点突变(例如，单核苷酸多态性)。
通过演化腺苷脱氨酶制备核碱基编辑蛋白的脱氨酶结构域中的突变。例如，能够使DNA中的腺苷脱氨基的ecTadA变体包括以下突变的一个或多个：SEQ ID NO:1的W23L、W23R、R26G、H36L、N37S、P48S、P48T、P48A、I49V、R51L、N72D、L84F、S97C、D108N、A106V、H123Y、G125A、A142N、S146C、D147Y、R152H、R152P、E155V、I156F、K157N和K161T。但是应该理解，可以在其他腺苷脱氨酶中进行同源突变以生成能够使DNA中的腺苷脱氨基的变体。图7示出了可以有用于本文公开的方法的ecTadA变体。
在本文提供的实例中，具有经演化的融合蛋白的一般结构(如ecTadA(D108X；X＝G，V或N)-XTEN-nCas9)的示例性核碱基编辑器在细胞如真核细胞(例如，Hek293T哺乳动物细胞)中催化A至G转换突变。在其他实例中，示例性核碱基编辑器含有两个ecTadA结构域和核酸可编程DNA结合蛋白(napDNAbp)。两个ecTadA结构域可以是相同的(例如，同二聚体)，也可以是两个不同的ecTadA结构域(例如，异二聚体(例如，野生型ecTadA和ecTadA(A106V/D108N)))。例如，核碱基编辑器可以具有一般结构ecTadA-ecTadA*-nCas9，其中ecTadA*代表包含SEQ ID NO:1的一个或多个突变的演化的ecTadA。本文提供的含有ecTadA变体的核碱基编辑器的另外的实例证明了哺乳动物细胞中核碱基编辑器的性能改进。
不希望受任何特定理论的束缚，本文所述的腺苷核苷碱基编辑器通过使用ecTadA变体使DNA中的A碱基脱氨基，经由肌苷形成引起A至G突变而起作用。肌苷优先与C形成氢键，从而在DNA复制期间导致A至G突变。当与Cas9(或另一核酸可编程的DNA结合蛋白)共价拴系时，腺苷脱氨酶(例如ecTadA)定位于感兴趣的基因，并催化ssDNA底物中的A至G突变。该编辑器可以用于靶向和回复(revert)疾病相关基因中的单核苷酸多态性(SNP)，所述疾病相关基因需要A至G回复。该编辑器还可以用于靶向和回复疾病相关基因中的单核苷酸多态性(SNP)，所述疾病相关基因需要通过将与T相对的A突变为G来进行T至C回复。然后可以例如通过碱基切除修复机制用C替换T，或者可以在随后的DNA复制轮次中改变T。因此，本文所述的腺苷碱基编辑器可以使A核碱基脱氨基，以给出与疾病或病症无关的核苷酸序列。在一些方面，本文所述的腺苷碱基编辑器可以用于使基因启动子中的腺苷(A)核碱基脱氨基。在一些实施方案中，脱氨基引起诱导基因的转录。诱导基因转录引起由该基因编码的蛋白质的表达(例如，基因产物)增加。与碱基编辑器结合的引导RNA(gRNA)包含与启动子中的靶核酸序列互补的引导序列。在一些实施方案中，靶核酸序列是HBG1和/或HBG2基因的启动子中的核酸序列。在一些实施方案中，启动子中的靶核酸序列包含核酸序列5′-CTTGGGGGCCCCTTCCCCACACTA-3′(SEQ ID NO:838)，5′-CTTGGGGGCCCCTTCCCCACACT-3′(SEQID NO:839)，5′-CTTGGGGGCCCCTTCCCCACAC-3′(SEQ ID NO:840)，5′-CTTGGGGGCCCCTTCCCCACA-3′(SEQ ID NO:841)，5′-CTTGGGGGCCCCTTCCCCAC-3′(SEQ ID NO:842)，5′-CTTGGGGGCCCCTTCCCCA-3′(SEQ ID NO:843)，5′-CTTGGGGGCCCCTTCCCC-3′(SEQ IDNO:844)或5′-CTTGGGGGCCCCTTCCC-3′(SEQ ID NO:845)。在一些实施方案中，启动子中的靶核酸序列包含核酸序列5′-CTTGGGGGCCCCTTCCCCAC-3′(SEQ ID NO:842)。在一些实施方案中，启动子中的靶核酸序列包含SEQ ID NO:838-845的任一个的核酸位置14(以粗体显示)处的T，并且与位置14处的T互补的A核碱基的脱氨基导致靶核酸序列中的T至C突变。在一些实施方案中，靶核酸进一步包含SEQ ID NO:838-845的任一个的5′末端处的5′-CCT-3′。在一些实施方案中，引导RNA包含与启动子中靶核酸序列100％互补的至少15、16、17、18、19、20、21、22、23、24或26个连续核酸。在一些实施方案中，gRNA的引导序列包含核酸序列5′-UCAUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:846)，5′-CAUGUGGGGAAGGGGCCCCCAAG-3′(SEQID NO:847)，5′-AUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:848)，5′-UGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:849)，5′-GUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:850)，5′-UGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:851)5′-GGGGAAGGGGCCCCCAAG-3′(SEQ IDNO:852)或5′-GGGAAGGGGCCCCCAAG-3′(SEQ ID NO:853)。在一些实施方案中，gRNA的引导序列包含核酸序列5′-GUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:850)。在一些实施方案中，gRNA的引导序列包含SEQ ID NO:254-280的任一个的核酸序列。在一些实施方案中，碱基编辑器包含融合蛋白，所述融合蛋白包含(i)核酸可编程DNA结合蛋白(napDNAbp)和(ii)腺苷脱氨酶，所述腺苷脱氨酶和与感兴趣的靶序列互补的引导RNA组合使用以使核碱基脱氨基。在一些实施方案中，核酸可编程DNA结合蛋白(napDNAbp)是Cas9结构域。在一些实施方案中，Cas9结构域是Cas9切口酶(nCas9)。腺苷脱氨酶可以是本文所述的任何腺苷脱氨酶。在一些实施方案中，腺苷脱氨酶是包含SEQ ID NO:1的一个或多个突变的大肠杆菌TadA(ecTadA)。在一些实施方案中，腺苷脱氨酶是包含SEQ ID NO:1的氨基酸序列的ecTadA。
因此，在一些方面，本文所述的碱基编辑器和引导RNA复合物可以用于治疗由HBG1和/或HBG2基因的启动子中的C至T突变引起的疾病或病症。在一些实施方案中，疾病或病症是血液的疾病或病症。在某些实施方案中，疾病或病症是贫血。在一些实施方案中，贫血是镰状细胞贫血。例如，HBG1和/或HBG2基因的启动子中的-198C至T突变引起γ-珠蛋白表达水平的降低，其可以促进镰状细胞病(SCD)和β地中海贫血的发展。启动子中腺苷核碱基的脱氨基导致启动子中的T-A碱基对突变为HBG1和/或HBG2基因的启动子中的C-G碱基对。腺苷核碱基的脱氨基促进了在遗传性胎儿血红蛋白持续存在症(HPFH)中促进的序列。在一些实施方案中，使启动子中的腺苷核碱基脱氨基引起HBG1基因的转录增加。在一些实施方案中，使启动子中的腺苷核碱基脱氨基引起HBG1蛋白的量增加。在一些实施方案中，使启动子中的腺苷核碱基脱氨基引起HBG2基因的转录增加。在一些实施方案中，使启动子中的腺苷核碱基脱氨基引起HBG2蛋白的量增加。在一些实施方案中，使启动子中的腺苷核碱基脱氨基引起HBG1和HBG2基因两者的转录增加。在一些实施方案中，使启动子中的腺苷核碱基脱氨基引起HBG1和HBG2蛋白两者的量的增加。
在又一方面，本文所述的腺苷碱基编辑器可以用于使基因中的腺苷(A)核碱基脱氨基以校正基因中的点突变。在一些实施方案中，基因可以包含密码子中的突变，与野生型密码子相比，所述突变导致由该突变体密码子编码的氨基酸的变化。与碱基编辑器结合的引导RNA(gRNA)包含与基因中的靶核酸序列互补的引导序列。在一些实施方案中，靶核酸序列是HFE基因中的核酸序列。在一些实施方案中，HFE基因包含C至T突变。通过本文所述的碱基编辑器和引导RNA复合物对与T互补的A核碱基进行脱氨基校正该C至T突变(例如，参见图7)。在一些实施方案中，启动子中的靶核酸序列包含核酸序列。在一些实施方案中，HFE基因编码包含Cys至Tyr突变(例如C282Y)的人血色素沉着症(HFE)蛋白质。通过本文所述的碱基编辑器和引导RNA复合物对与T互补的A核碱基进行脱氨基校正HFE蛋白中的Cys至Tyr突变，从而导致野生型蛋白质的表达。在一些实施方案中，HFE基因中的靶核酸序列包含核酸序列5′-GGGTGCTCCACCTGGTACGTATAT-3′(SEQ ID NO:854)，5′-GGGTGCTCCACCTGGTACGTATA-3′(SEQ ID NO:855)，5′-GGGTGCTCCACCTGGTACGTAT-3′(SEQ ID NO:856)，5′-GGGTGCTCCACCTGGTACGTA-3′(SEQ ID NO:857)，5′-GGGTGCTCCACCTGGTACGT-3′(SEQ ID NO:858)，5′-GGGTGCTCCACCTGGTACG-3′(SEQ ID NO:859)，5′-GGGTGCTCCACCTGGTAC-3′(SEQ IDNO:860)或5′-GGGTGCTCCACCTGGTA-3′(SEQ ID NO:861)。在一些实施方案中，靶核酸进一步包含SEQ ID NO:854-861的核酸序列的任一个的5′末端处的5′-CCT-3′。在一些实施方案中，HFE基因中的靶核酸序列包含核酸序列5′-GGGTGCTCCACCTGGTACGT-3′(SEQ ID NO:858)。在一些实施方案中，引导RNA包含与HFE基因中的靶核酸序列100％互补的至少15、16、17、18、19、20、21、22、23、24或26个连续核酸。在一些实施方案中，gRNA的引导序列包含核酸序列5′-AUAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:862)，5′-UAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:863)，5′-AUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:864)，5′-UACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:865)，5′-ACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:866)，5′-CGUACCAGGUGGAGCACCC-3′(SEQ ID NO:867)，5′-GUACCAGGUGGAGCACCC-3′(SEQ IDNO:868)或5′-UACCAGGUGGAGCACCC-3′(SEQ ID NO:869)。在一些实施方案中，gRNA的引导序列进一步包含SEQ ID NO:862-869的核酸序列的任一个的5′末端处的G。在一些实施方案中，gRNA的引导序列包含核酸序列5’-GACGUACCAGGUGGAGCACCC-3’(SEQ ID NO:870)。在一些实施方案中，碱基编辑器包含融合蛋白，所述融合蛋白包含(i)核酸可编程DNA结合蛋白(napDNAbp)和(ii)腺苷脱氨酶，所述腺苷脱氨酶和与感兴趣的靶序列互补的引导RNA组合使用以使核碱基脱氨基。在一些实施方案中，核酸可编程DNA结合蛋白(napDNAbp)是Cas9结构域。在一些实施方案中，Cas9结构域是Cas9切口酶(nCas9)。腺苷脱氨酶可以是本文所述的任何腺苷脱氨酶。在一些实施方案中，腺苷脱氨酶是包含SEQ ID NO:1的一个或多个突变的大肠杆菌TadA(ecTadA)。在一些实施方案中，腺苷脱氨酶是包含SEQ ID NO:1的氨基酸序列的ecTadA。
因此，在一些方面，本文所述的碱基编辑器和引导RNA复合物可以用于治疗由HFE基因中的C至T突变引起的疾病或病症。在一些实施方案中，病症是铁贮积病症(ironstorage disorder)。在一些实施方案中，铁贮积病症是遗传性血色素沉着症(HHC)。在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基导致HFE基因中的T-A碱基对突变为HFE基因中的C-G碱基对。在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基引起从HFE基因转录的HFE蛋白功能的增加。在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基导致校正与遗传性血色素沉着症(HHC)相关的序列。在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基改善铁贮积病症的一种或多种症状。
在一些实施方案中，本文提供的腺苷脱氨酶能够使核酸分子中的腺苷脱氨基。在一些实施方案中，使腺苷核碱基脱氨基的方法包括使细胞中的核酸分子与复合物在体外接触，所述复合物包含(i)腺苷碱基编辑器和(ii)引导RNA。在一些实施方案中，使腺苷核碱基脱氨基的方法包括使细胞中的核酸分子与复合物在体内接触，所述复合物包含(i)腺苷碱基编辑器和(ii)引导RNA。在一些实施方案中，使腺苷核碱基脱氨基的方法包括使细胞中的核酸分子与复合物接触，所述复合物包含(i)腺苷碱基编辑器和(ii)引导RNA，其中细胞在受试者中。在一些实施方案中，细胞是永生化的淋巴样干细胞(lymphoblastoid cell)(LCL)。本公开的其他方面提供了融合蛋白，其包含Cas9结构域和腺苷脱氨酶结构域，例如，能够使DNA中的腺苷脱氨基的工程化脱氨酶结构域。在一些实施方案中，融合蛋白包含核定位序列(NLS)、肌苷碱基切除修复抑制剂(例如，dISN)和/或一个或多个接头中的一种或多种。
在一些方面，本公开提供了能够使脱氧核糖核酸(DNA)底物中的腺苷脱氨基的腺苷脱氨酶。在一些实施方案中，腺苷脱氨酶来自细菌，例如大肠杆菌或金黄色葡萄球菌。在一些实施方案中，腺苷脱氨酶是TadA脱氨酶。在一些实施方案中，TadA脱氨酶是大肠杆菌TadA脱氨酶(ecTadA)。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的D108X突变，或另一种腺苷脱氨酶中相应的突变，其中X是除野生型蛋白质中发现的氨基酸之外的任何氨基酸。在一些实施方案中，X是G、N、V、A或Y。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的D108N突变，或另一种腺苷脱氨酶中相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的A106X突变，或另一种腺苷脱氨酶中相应的突变，其中X是除野生型蛋白质中发现的氨基酸之外的任何氨基酸。在一些实施方案中，X是V、G、I、L或A。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的A106V突变，或另一种腺苷脱氨酶中相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的A106X和D108X突变，或另一种腺苷脱氨酶中相应的突变，其中X是除野生型蛋白质中发现的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的A106V和D108N突变，或另一种腺苷脱氨酶中相应的突变。在一些实施方案中，腺苷脱氨酶包含A106X、D108X、D147X、E155X、L84X、H123X和I156X突变，或另一种腺苷脱氨酶中相应的突变，其中X是除野生型蛋白质中发现的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含A106V、D108N、D147Y、E155V、L84F、H123Y和I156F突变，或另一种腺苷脱氨酶中相应的突变。在一些实施方案中，腺苷脱氨酶包含A106X、D108X、D147X、E155X、L84X、H123X、I156X、H36X、R51X、S146X和K157X突变，或另一种腺苷脱氨酶中相应的突变，其中X是除野生型蛋白质中发现的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含A106V、D108N、D147Y、E155V、L84F、H123Y、I156F、H36L、R51L、S146C和K157N突变，或另一种腺苷脱氨酶中相应的突变。在一些实施方案中，腺苷脱氨酶包含A106X、D108X、D147X、E155V、L84X、H123X、I156X、H36X、R51X、S146X、K157X、W23X、P48X和R152X突变，或另一种腺苷脱氨酶中相应的突变，其中X是除野生型蛋白质中发现的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含A106V、D108N、D147Y、E155V、L84F、H123Y、I156F、H36L、R51L、S146C、K157N、W23R、P48A和R152P突变，或另一种腺苷脱氨酶中相应的突变。应当理解，本文提供的腺苷脱氨酶可以含有任何组合的本文提供的一种或多种突变。
在另一方面，本公开提供了具有扩大的靶序列相容性的腺苷核碱基编辑器(ABE)。通常，天然ecTadA使tRNAArg的反密码子环的序列UAC(例如靶序列)中腺嘌呤脱氨基。不希望受到任何特定理论的束缚，为了扩大包含一个或多个ecTadA结构域的ABE(如本文提供的任何腺苷脱氨酶)的效用，优化腺苷脱氨酶蛋白以识别前间隔区序列内的多种靶序列，而不影响腺苷核碱基编辑复合物的编辑效率。在一些实施方案中，靶序列是5’-NAN-3’，其中N是T、C、G或A。例如，靶序列显示在图3A中。在一些实施方案中，靶序列包含5’-TAC-3’。在一些实施方案中，靶序列包含5’-GAA-3’。
在一些实施方案中，碱基编辑器包含SEQ ID NO:707的氨基酸序列或由之组成。在一些实施方案中，碱基编辑器包含SEQ ID NO:708的氨基酸序列或由之组成。在一些实施方案中，碱基编辑器包含SEQ ID NO:709的氨基酸序列或由之组成。在一些实施方案中，碱基编辑器包含SEQ ID NO:710的氨基酸序列或由之组成。在一些实施方案中，碱基编辑器包含SEQ ID NO:711的氨基酸序列或由之组成。
在另一方面，本公开提供了包含包括融合蛋白的复合物的药物组合物，所述融合蛋白包含(i)核酸可编程DNA结合蛋白(napDNAbp)，(ii)腺苷脱氨酶，和(iii)将融合蛋白导向感兴趣的靶序列的引导RNA；以及任选地药学上可接受的赋形剂。腺苷脱氨酶可以是本文所述的任何腺苷脱氨酶结构域或其任何组合。在一些实施方案中，napDNAbp是Cas9结构域。在一些实施方案中，Cas9结构域是Cas9切口酶(nCas9)。在一些实施方案中，引导RNA包含将融合蛋白导向HBG1和/或HBG2基因的启动子的引导核酸序列。在一些实施方案中，引导核酸序列包含SEQ ID NO:846-853的核酸序列。在一些实施方案中，引导RNA包含将融合蛋白导向HFE基因的核酸序列。在一些实施方案中，引导核酸序列包含SEQ ID NO:862-870的核酸序列。在一些实施方案中，将药物组合物施用于有此需要的受试者(例如，治疗疾病或病症)。在一些实施方案中，受试者患有血液疾病。在一些实施方案中，血液疾病是贫血。在一些实施方案中，贫血是镰状细胞贫血。在一些实施方案中，受试者患有铁贮积病症。在一些实施方案中，铁贮积病症是遗传性血色素沉着症(HHC)。在又一方面，本公开提供了包含核酸构建体的试剂盒，所述核酸构建体包含(a)编码融合蛋白的核酸序列，所述融合蛋白包含(i)核酸可编程DNA结合蛋白(napDNAbp)和(ii)腺苷脱氨酶；(b)驱动(a)的序列表达的异源启动子；和(c)编码引导RNA主链的表达构建体，其中构建体包含定位成允许将与靶序列相同或互补的核酸序列克隆到引导RNA主链中的克隆位点。腺苷脱氨酶可以是本文所述的任何腺苷脱氨酶结构域或其任何组合。在一些实施方案中，napDNAbp是Cas9结构域。在一些实施方案中，Cas9结构域是Cas9切口酶(nCas9)。在一些实施方案中，引导RNA包含将融合蛋白导向HBG1和/或HBG2基因的启动子的核酸序列。在一些实施方案中，靶核酸序列包含SEQ IDNO:838-845的任一个的核酸序列。在一些实施方案中，引导RNA包含将融合蛋白导向HFE基因的核酸序列。在一些实施方案中，靶核酸序列包含SEQ ID NO:854-861的任一个的核酸序列。
以上概述旨在以非限制性方式说明本文公开的技术的一些实施方案、优点、特征和用途。根据详述、附图、实施例和权利要求，本文公开的技术的其他实施方案、优点、特征和用途将是显而易见的。
附图简要说明
图1A至1C显示由A·T至G·C碱基编辑器(ABE)进行的碱基编辑的范围和概述。图1A：校正ClinVar数据库中已知的致病性人类SNP所需的碱基对变化的相对分布。图1B：腺苷(A)的水解脱氨基形成肌苷(I)，其被聚合酶读作鸟苷(G)。图1C：ABE介导的A·T至G·C碱基编辑策略。ABE含有已知不在自然界存在的假定的脱氧腺苷脱氨酶和催化受损的Cas9。它们以引导RNA编程的方式结合感兴趣的双链DNA靶标，暴露“R环”复合物中单链DNA的小泡(bubble)。脱氧腺苷脱氨酶结构域将该单链泡内的脱氧腺苷催化形成脱氧肌苷。DNA修复或复制后，在靶位点处的原始A·T碱基对被G·C碱基对替换。
图2A至2C显示ABE的蛋白质演化和工程化。图2A：从RNA腺苷脱氨酶开始演化DNA脱氧腺苷脱氨酶的策略。大肠杆菌细胞的文库各自包含表达与催化死亡Cas9(dCas9)融合的突变体ecTadA(TadA*)基因的质粒和表达需要一个或多个A·T至G·C突变以恢复抗生素抗性的有缺陷的抗生素抗性基因的选择质粒。将来自在每一轮突变和选择中存活的TadA*变体的突变输入到哺乳动物ABE构造中，并在人细胞中对其可编程A·T至G·C碱基编辑活性进行测定。图2B：每一轮演化产生的经演化的ABE的亚组的基因型。有关此工作中表征的所有57种演化的ABE基因型的列表，参见图7。二聚化状态(单体、演化的TadA*结构域的同二聚体或野生型TadA–演化的TadA*的异二聚体)和接头长度(以氨基酸的数目)也被列出。基于从中鉴定出突变的演化的轮次对突变进行着色。图2C：与和tRNAArg2复合的金黄色葡萄球菌TadA(PDB 2B3J)(未显示)的结构比对的大肠杆菌TadA脱氨酶结构(PDB 1Z3A)的三个视图。tRNA的UAC反密码子环是野生型TadA的内源性底物。左侧显示TadA二聚体和tRNA反密码子环；中间显示底物腺苷上游的U的核糖的2’羟基附近的残基；右侧显示在ABE演化期间突变并围绕UAC底物的残基。对野生型残基进行着色，以使其对应于从每一轮演化出现的突变，并对应于图2B中的基因型表。
图3A至3C显示在人基因组DNA位点处早期和中期演化的ABE介导A·T至G·C碱基编辑。图3A：19个人基因组DNA测试位点(左)以及在人染色体上的相应位置(右)的表格。对于每个位点，经编辑的A的序列上下文(靶基序)以红色显示。PAM序列以蓝色显示。图3B：在六个人基因组DNA位点处，第1轮和第2轮ABE在HEK293T细胞中的A·T至G·C碱基编辑效率。图3C：在六个人基因组DNA位点处，第3轮、第4轮和第5轮ABE在HEK293T细胞中的A·T至G·C碱基编辑效率。显示ABE2.9编辑用于比较。值和误差条反映在不同日期进行的三个独立的生物学重复的平均值和s.d.。同二聚体表示融合的TadA*–TadA*–Cas9切口酶构造；异二聚体表示融合的wtTadA–TadA*–Cas9切口酶构造。序列从上到下对应于SEQ ID NO:91-109。
图4A和4B显示晚期演化的ABE以更高的活性和更宽的序列相容性介导基因组DNA编辑。图4A：在六个人基因组DNA位点处，第6轮和第7轮ABE在HEK293T细胞中的A·T至G·C碱基编辑效率。显示ABE5.3编辑用于比较。图4B：在扩大的一组人基因组位点处，第6轮和第7轮ABE在HEK293T细胞中的A·T至G·C碱基编辑效率。位点1-17共同包括靶标A侧翼的每个可能的NAN序列上下文。显示ABE5.3编辑用于比较。值和误差条反映在不同日期进行的三个独立的生物学重复的平均值和s.d.。图4A和4B中显示的所有ABE均是wtTadA–TadA*–Cas9切口酶的异二聚体构造。
图5A至5C显示晚期ABE的活性窗口和产物纯度。图5A：在两个人基因组DNA位点中的前间隔区位置1-9(其一起在这些位置的每一个处放置了一个A)处，晚期ABE在HEK293T细胞中的相对A·T至G·C碱基编辑效率。将值相对于每个ABE在两个位点的每一个处观察到的最大效率＝1进行标准化。图5B：在所有19个测试的人基因组DNA位点中的前间隔区位置1-18和20处，晚期ABE在HEK293T细胞中的相对A·T至G·C碱基编辑效率。将值相对于每个ABE在19个位点的每一个处观察到的最大效率＝1进行标准化。图5C：在用ABE7.10或ABE7.9和相应的sgRNA处理的HEK293T细胞中，或在未经处理的HEK293T细胞中，两个代表性人基因组DNA位点处的产物分布。右侧显示插入/缺失频率。对于图5A和5B，值和误差条反映在不同日期进行的三个独立的生物学重复的平均值和s.d.。序列从上到下对应于SEQ ID NO:110-111。
图6A至6C显示ABE7.10介导的碱基编辑和Cas9介导的HDR的比较，以及将ABE7.10应用于两种与疾病相关的SNP。图6A：用ABE7.10或用Cas9核酸酶和靶向五个人基因组DNA位点的ssDNA供体模板(遵循CORRECT HDR方法41)处理的HEK293T细胞中的A·T至G·C碱基编辑效率。图6B：在如图6A中所述处理的HEK293T细胞中插入/缺失形成的比较。图6C：ABE在安装抑制疾病的SNP或校正诱导疾病的SNP中的应用。上图：HEK293T细胞中HBG1和HBG2基因的启动子区中ABE7.10介导的-198TàC突变(在与测序数据表中所示的链互补的链上)。靶A在前间隔区的位置7处。下图：LCL细胞HFE基因中ABE7.10介导的C282Y突变的回复。该突变是遗传性血色素沉着症的常见原因。靶A在前间隔区的位置5处。
图7显示此工作中描述的所有ABE的基因型表。基于其中鉴定出突变的演化的轮次对突变进行着色。
图8A至8D显示另外的早期ABE变体的碱基编辑效率。图8A：在六个人基因组靶DNA位点处，与Cas9切口酶融合的各种野生型RNA腺嘌呤脱氨酶在HEK293T细胞中的A·T至G·C碱基编辑效率。值反映在不同日期进行的三个生物学重复的平均值和标准偏差。图8B：在六个人基因组靶DNA位点处，具有改变的融合定向和接头长度的ABE2编辑器在HEK293T细胞中的A·T至G·C碱基编辑效率。图8C：在六个人基因组靶DNA位点处，与催化失活的烷基腺苷糖基化酶(AAG)或内切核酸酶V(EndoV)融合(这两种蛋白结合DNA中的肌苷)的ABE2编辑器在HEK293T细胞中的A·T至G·C碱基编辑效率。图8D：在有或没有AAG的情况下，在位点1处HAP1细胞中ABE2.1的A·T至G·C碱基编辑效率。图8B和8C中的值和误差条反映在不同日期进行的三个独立的生物学重复的平均值和s.d.。
图9显示用ABE2.1和靶向六个人基因组位点的每一个的sgRNA处理的HEK293T细胞的高通量DNA测序分析。显示了一个代表性重复。显示来自未经处理的HEK293T细胞的数据用于比较。
图10A至10C显示另外的ABE2和ABE3变体的碱基编辑效率，以及在大肠杆菌中向TadA*–dCas9融合物添加A142N对抗生素选择存活的影响。图10A：在六个人基因组靶DNA位点处，具有不同的经工程化的二聚化状态的ABE2变体在HEK293T细胞中的A·T至G·C碱基编辑效率。绘制的数据是三个生物学重复的平均值。图10B：在六个人基因组靶DNA位点处，以其二聚化状态(TadA*–TadA*–Cas9切口酶的同二聚体，或野生型TadA–TadA*–Cas9切口酶的异二聚体)，以TadA–TadA接头的长度，以及以TadA–Cas9切口酶接头的长度而不同的ABE3.1变体在HEK293T细胞中的A·T至G·C碱基编辑效率。有关ABE基因型和构造，参见图7。图10C：大肠杆菌细胞在具有256μg/mL的壮观霉素的2xYT琼脂上的菌落形成单位，所述大肠杆菌细胞表达靶向壮观霉素抗性基因中I89T缺陷的sgRNA和缺乏或含有演化第4轮中鉴定的A142N突变的TadA*-dCas9编辑器。在靶位点处成功进行A·T至G·C碱基编辑恢复壮观霉素抗性。图10A和10B中的值和误差条反映在不同日期进行的三个独立的生物学重复的平均值和s.d.。
图11A和11B显示另外的ABE5变体的碱基编辑效率。图11A：在六个人基因组靶DNA位点处，具有从第5轮文库的壮观霉素选择分离的两对突变的两个ABE3.1变体在HEK293T细胞中的A·T至G·C碱基编辑效率。图11B：在六个人基因组靶DNA位点处，具有不同接头长度的ABE5变体在HEK293T细胞中的A·T至G·C碱基编辑效率。有关ABE基因型和构造，参见图7。值和误差条反映在不同日期进行的三个独立的生物学重复的平均值和s.d.。
图12A至12C显示在17个基因组位点处ABE7变体的碱基编辑效率。在17个人基因组靶DNA位点处，ABE7.1-7.5(图12A)和ABE7.6-7.10(图12B)在HEK293T细胞中的A·T至G·C碱基编辑效率。有关ABE基因型和构造，参见图7。图12C：在六个人基因组靶DNA位点处，ABE7.8-7.10在U2OS细胞中的A·T至G·C碱基编辑效率。与HEK293T细胞相比，在U2OS细胞中观察到的较低的编辑效率与两种细胞系之间的转染效率差异一致；在本研究使用的条件下，在U2OS细胞中观察到约40％至55％的典型转染效率，与之相比在HEK293T细胞中为65-80％。值和误差条反映在不同日期进行的三个独立的生物学重复的平均值和s.d.。
图13显示几轮演化和工程化增加了ABE的持续合成能力。计算出的在6至17个人基因组靶DNA位点处，从每轮演化和工程化出现的最具活性的ABE的邻近的靶A之间的平均标准化连锁不平衡(LD)。较高的LD值表明，如果相同DNA链(相同的测序读出)中邻近的A也被编辑，则ABE更可能编辑A。LD值标准化为0至1，以独立于编辑效率。值和误差条反映来自在不同日期进行的三个独立的生物学重复的标准化LD值的平均值和s.d.。
图14A和14B显示用五个晚期ABE变体和靶向在HBG1和HBG2的启动子中-198T的sgRNA处理的HEK293T细胞的高通量DNA测序分析。显示了在HBG1(图14A)和HBG2(图14B)启动子靶标处的DNA序列的一个代表性复制。ABE介导的碱基编辑在与测序数据表中所示的链互补的链上安装了-198T→C突变。显示来自未经处理的HEK293T细胞的数据用于比较。
图15显示使用碱基编辑器ABE 7.10和靶向HBG1/2的启动子区域的12个gRNA的碱基编辑数据。gRNA靶序列的身份是(1)SEQ ID NO:259、(2)SEQ ID NO:260、(3)SEQ ID NO:266、(4)SEQ ID NO:267、(5)SEQ ID NO:268、(6)SEQ ID NO:269、(7)SEQ ID NO:270、(8)SEQ ID NO:276、(9)SEQ ID NO:277、(10)SEQ ID NO:278、(11)SEQ ID NO:279和(12)SEQID NO:280。
定义
如本文和权利要求书中所用，除非上下文另外明确指出，否则单数形式“一种”、“一个”和“该/所述”包括单数和复数。因此，例如，提及“试剂”包括单一试剂和多个此类试剂。
术语“脱氨酶”或“脱氨酶结构域”是指催化脱氨基反应的蛋白质或酶。在一些实施方案中，脱氨酶是腺苷脱氨酶，其催化腺嘌呤或腺苷的水解脱氨基作用。在一些实施方案中，脱氨酶或脱氨酶结构域是腺苷脱氨酶，分别催化腺苷或脱氧腺苷水解脱氨基为肌苷或脱氧肌苷。在一些实施方案中，腺苷脱氨酶催化脱氧核糖核酸(DNA)中腺嘌呤或腺苷的水解脱氨基作用。本文提供的腺苷脱氨酶(例如，工程化的腺苷脱氨酶、演化的腺苷脱氨酶)可以来自任何生物体，例如细菌。在一些实施方案中，脱氨酶或脱氨酶结构域是来自生物体的天然存在的脱氨酶的变体。在一些实施方案中，脱氨酶或脱氨酶结构域在自然界中不存在。例如，在一些实施方案中，脱氨酶或脱氨酶结构域与天然存在的脱氨酶至少50％、至少55％、至少60％、至少65％、至少70％、至少75％至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同。在一些实施方案中，腺苷脱氨酶来自细菌，例如大肠杆菌(E.coli)、金黄色葡萄球菌(S.aureus)、鼠伤寒沙门氏菌(S.typhi)、腐败希瓦氏菌(S.putrefaciens)、流感嗜血杆菌(H.influenzae)或新月柄杆菌(C.crescentus)。在一些实施方案中，腺苷脱氨酶是TadA脱氨酶。在一些实施方案中，TadA脱氨酶是大肠杆菌TadA脱氨酶(ecTadA)。在一些实施方案中，TadA脱氨酶是截短的大肠杆菌TadA脱氨酶。例如，相对于全长ecTadA，截短的ecTadA可以缺少一个或多个N端氨基酸。在一些实施方案中，截短的ecTadA可以相对于全长ecTadA缺少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、6、17、18、19或20个N端氨基酸残基。在一些实施方案中，截短的ecTadA可以相对于全长ecTadA缺少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、6、17、18、19或20个C端氨基酸残基。在一些实施方案中，ecTadA脱氨酶不包含N端甲硫氨酸。
在一些实施方案中，TadA脱氨酶是N端截短的TadA。在某些实施方案中，腺苷脱氨酶包含氨基酸序列：
MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD(SEQ ID NO:1)。
在一些实施方案中，TadA脱氨酶是全长大肠杆菌TadA脱氨酶。例如，在某些实施方案中，腺苷脱氨酶包含氨基酸序列：
MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD(SEQ ID NO:84)
然而，应当理解，可用于本申请的另外的腺苷脱氨酶对于熟练技术人员而言是显而易见的，并且在本公开的范围内。例如，腺苷脱氨酶可以是ADAT的同源物。示例性ADAT同源物包括但不限于：
金黄色葡萄球菌(Staphylococcus aureus)TadA:
MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN(SEQ ID NO:8)
枯草芽孢杆菌(Bacillus subtilis)TadA:
MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE(SEQ ID NO:9)
鼠伤寒沙门氏菌(Salmonella typhimurium)(S.typhimurium)TadA:
MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV(SEQ ID NO:371)
腐败希瓦氏菌(Shewanella putrefaciens)(S.putrefaciens)TadA:
MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE(SEQ ID NO:372)
流感嗜血杆菌(Haemophilus influenzae)F3031(H.influenzae)TadA:
MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK(SEQ ID NO:373)
新月柄杆菌(Caulobacter crescentus)(C.crescentus)TadA:
MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI(SEQ ID NO:374)
硫还原地杆菌(Geobacter sulfurreducens)(G.sulfurreducens)TadA:
MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP(SEQ ID NO:375)
术语“碱基编辑器(BE)”或“核碱基编辑器(NBE)”是指包含能够对核酸序列(例如，DNA或RNA)内的碱基(例如，A、T、C、G或U)进行修饰的多肽的试剂。在一些实施方案中，碱基编辑器能够使核酸内的碱基脱氨基。在一些实施方案中，碱基编辑器能够使DNA分子内的碱基脱氨基。在一些实施方案中，碱基编辑器能够使DNA中的腺嘌呤(A)脱氨基。在一些实施方案中，碱基编辑器是融合蛋白，其包含与腺苷脱氨酶融合的核酸可编程DNA结合蛋白(napDNAbp)。在一些实施方案中，碱基编辑器是与腺苷脱氨酶融合的Cas9蛋白。在一些实施方案中，碱基编辑器是与腺苷脱氨酶融合的Cas9切口酶(nCas9)。在一些实施方案中，碱基编辑器是与腺苷脱氨酶融合的核酸酶无活性的Cas9(dCas9)。在一些实施方案中，碱基编辑器与碱基切除修复抑制剂(例如，UGI结构域或dISN结构域)融合。在一些实施方案中，融合蛋白包含与脱氨酶和碱基切除修复抑制剂(例如UGI或dISN结构域)融合的Cas9切口酶。在一些实施方案中，融合蛋白的dCas9结构域包含SEQ ID NO:52的D10A和H840A突变，或SEQID NO:108-357中的任一个中的相应的突变，其使Cas9蛋白的核酸酶活性失活。在一些实施方案中，融合蛋白包含D10A突变并且包含SEQ ID NO:52的残基840处的组氨酸，或SEQ IDNO:108-357中的任一个中的相应的突变，其使得Cas9能够仅切割核酸双链体的一条链。Cas9切口酶的实例显示在SEQ ID NO:35中。
如本文所用，术语“接头”是指连接两个分子或部分，例如融合蛋白的两个结构域，诸如例如核酸酶无活性的Cas9结构域和核酸编辑结构域(例如腺苷脱氨酶结构域)的键(例如共价键)、化学基团或分子。在一些实施方案中，接头接合RNA可编程核酸酶的gRNA结合结构域，包括Cas9核酸酶结构域和核酸编辑蛋白的催化结构域。在一些实施方案中，接头接合dCas9和核酸编辑蛋白。通常，接头位于两个基团、分子或其他部分之间或侧翼有两个基团、分子或其他部分，并且经由共价键与每一个连接，从而连接两者。在一些实施方案中，接头是一个氨基酸或多个氨基酸(例如肽或蛋白质)。在一些实施方案中，接头是有机分子、基团、聚合物或化学部分。在一些实施方案中，接头包含SEQ ID NO:10、37-40、384-386、685-688或800-801的任一个的氨基酸序列。在一些实施方案中，接头的长度为5-100个氨基酸，例如长度为5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35-40、40-45、45-50、50-60、60-70、70-80、80-90、90-100、100-150或150-200个氨基酸。也考虑了更长或更短的接头。在一些实施方案中，接头包含氨基酸序列SGSETPGTSESATPES(SEQ ID NO:10)，其也可以称为XTEN接头。在一些实施方案中，接头包含氨基酸序列SGGS(SEQ ID NO:37)。在一些实施方案中，接头包含氨基酸序列(SGGS)2-SGSETPGTSESATPES-(SGGS)2(SEQ ID NO:800)，其也可以称为(SGGS)2-XTEN-(SGGS)2。在一些实施方案中，接头包含(SGGS)n(SEQ ID NO:37)、(GGGS)n(SEQ ID NO:38)、(GGGGS)n(SEQID NO:39)、(G)n、(EAAAK)n(SEQ ID NO:40)、(GGS)n、SGSETPGTSESATPES(SEQ ID NO:10)、(SGGS)n-SGSETPGTSESATPES-(SGGS)n(SEQ ID NO:801)或(XP)n基序，或这些中任何的组合，其中n独立地是1和30之间的整数，并且其中X是任何氨基酸。在一些实施方案中，n是1、2、3、4、5、6、7、8、9、10、11、12、13、14或15。
如本文所用，术语“突变”是指序列(例如核酸或氨基酸序列)内的残基用另一个残基取代或序列内一个或多个残基的缺失或插入。本文通常通过鉴定初始残基，随后是序列内残基的位置和新取代的残基的身份来描述突变。用于产生本文提供的氨基酸取代(突变)的各种方法在本领域中是熟知的，并且由例如Green and Sambrook,Molecular Cloning:ALaboratory Manual(第4版,Cold Spring Harbor Laboratory Press,Cold SpringHarbor,N.Y.(2012))提供。
术语“核定位序列”或“NLS”是指促进蛋白质输入细胞核(例如通过核转运)的氨基酸序列。核定位序列是本领域已知的，并且对于熟练技术人员是显而易见的。例如，NLS序列描述于2001年11月23日提交的Plank等人的国际PCT申请PCT/EP2000/011690，2001年5月31日公布为WO/2001/038547，其内容通过引用并入本文，用于其对示例性的核定位序列的公开内容。在一些实施方案中，NLS包含氨基酸序列PKKKRKV(SEQ ID NO:4)、MDSLLMNRRKFLYQFKNVRWAKGRRETYLC(SEQ ID NO:5)、MKRTADGSEFEPKKKRKV(SEQ ID NO:342)或KRTADGSEFEPKKKRKV(SEQ ID NO:343)。
术语“核酸可编程DNA结合蛋白”或“napDNAbp”是指与核酸(例如DNA或RNA)，例如引导核酸结合的蛋白质，所述核酸将napDNAbp引导至特定核酸序列。例如，Cas9蛋白可以与引导RNA结合，所述引导RNA将Cas9蛋白引导至与引导RNA互补的特定DNA序列。在一些实施方案中，napDNAbp是2类微生物CRISPR-Cas效应物。在一些实施方案中，napDNAbp是Cas9结构域，例如核酸酶活性Cas9、Cas9切口酶(nCas9)或核酸酶无活性Cas9(dCas9)。核酸可编程DNA结合蛋白的实例包括但不限于Cas9(例如dCas9和nCas9)、CasX、CasY、Cpf1、C2c1、C2c2、C2C3和Argonaute。然而，应当理解，核酸可编程DNA结合蛋白还包括结合RNA的核酸可编程蛋白。例如，napDNAbp可以与将napDNAbp引导至RNA的核酸结合。其他核酸可编程DNA结合蛋白也在本公开的范围内，但它们可以未在本公开中具体列出。
术语“Cas9”或“Cas9结构域”是指包含Cas9蛋白或其片段的RNA引导的核酸酶(例如，包含Cas9的活性、无活性或部分活性的DNA切割结构域，和/或Cas9的gRNA结合结构域的蛋白质)。Cas9核酸酶有时也称为casn1核酸酶或CRISPR(聚簇规则间隔短回文重复)相关核酸酶。CRISPR是适应性免疫系统，其提供针对移动遗传元件(病毒、可转座元件和接合质粒)的保护。CRISPR簇含有间隔区，与先前的移动元件互补的序列，并靶向侵入核酸。CRISPR簇得以转录并加工成CRISPR RNA(crRNA)。在II型CRISPR系统中，对pre-crRNA的正确加工需要反式编码的小RNA(tracrRNA)、内源性核糖核酸酶3(rnc)和Cas9蛋白。tracrRNA充当用于pre-crRNA的核糖核酸酶3辅助加工的引导。随后，Cas9/crRNA/tracrRNA以内切核水解方式切割与间隔区互补的线性或环状dsDNA靶标。首先以内切核水解方式切割不与crRNA互补的靶链，然后以3'-5'外切核水解方式修剪(trim)。在自然界中，DNA结合和切割通常需要蛋白质和这两种RNA。然而，单一引导RNA(“sgRNA”或简称“gNRA”)可以经工程化以将crRNA和tracrRNA两者的方面并入单一RNA种类中。参见例如Jinek M.,Chylinski K.,Fonfara I.,Hauer M.,Doudna J.A.,Charpentier E.Science337:816-821(2012)，其全部内容通过引用并入本文。Cas9识别CRISPR重复序列中的短基序(PAM或前间隔区相邻基序)，以帮助区分自我与非自我。Cas9核酸酶序列和结构是本领域技术人员熟知的(参见例如“Completegenome sequence of an M1 strain of Streptococcus pyogenes.”Ferretti et al.,J.J.,McShan W.M.,Ajdic D.J.,Savic D.J.,Savic G.,Lyon K.,Primeaux C.,SezateS.,Suvorov A.N.,Kenton S.,Lai H.S.,Lin S.P.,Qian Y.,Jia H.G.,Najar F.Z.,RenQ.,Zhu H.,Song L.,White J.,Yuan X.,Clifton S.W.,Roe B.A.,McLaughlin R.E.,Proc.Natl.Acad.Sci.U.S.A.98:4658-4663(2001)；“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.”Deltcheva E.,Chylinski K.,SharmaC.M.,Gonzales K.,Chao Y.,Pirzada Z.A.,Eckert M.R.,Vogel J.,Charpentier E.,Nature 471:602-607(2011)；和“A programmable dual-RNA-guided DNA endonucleasein adaptive bacterial immunity.”Jinek M.,Chylinski K.,Fonfara I.,Hauer M.,Doudna J.A.,Charpentier E.Science337:816-821(2012)，其各自的全部内容通过引用并入本文)。已经在各种物种中描述了Cas9直系同源物，包括但不限于酿脓链球菌(S.pyogenes)和嗜热链球菌(S.thermophilus)。基于本公开，其他合适的Cas9核酸酶和序列对于本领域技术人员将是显而易见的，并且此类Cas9核酸酶和序列包括来自Chylinski,Rhun,and Charpentier,“The tracrRNA and Cas9 families of type II CRISPR-Casimmunity systems”(2013)RNA Biology 10:5,726-737中公开的生物体和基因座的Cas9序列；其全部内容通过引用并入本文。在一些实施方案中，Cas9核酸酶具有无活性的(例如失活的)DNA切割结构域，也就是说，Cas9是切口的酶。
核酸酶失活的Cas9蛋白可以互换地称为“dCas9”蛋白(代表核酸酶-“死亡的”Cas9)。用于生成具有无活性的DNA切割结构域的Cas9蛋白(或其片段)的方法是已知的(参见例如Jinek et al.,Science.337:816-821(2012)；Qi et al.,“Repurposing CRISPR asan RNA-Guided Platform for Sequence-Specific Control of Gene Expression”(2013)Cell.28；152(5):1173-83，其各自的全部内容通过引用并入本文)。例如，已知Cas9的DNA切割结构域包括两个亚结构域，即HNH核酸酶亚结构域和RuvC1亚结构域。HNH亚结构域切割与gRNA互补的链，而RuvC1亚结构域切割非互补链。这些亚结构域内的突变可以沉默Cas9的核酸酶活性。例如，突变D10A和H840A完全使酿脓链球菌Cas9的核酸酶活性失活(Jinek et al.,Science.337:816-821(2012)；Qi et al.,Cell.28；152(5):1173-83(2013))。在一些实施方案中，提供了包含Cas9的片段的蛋白质。例如，在一些实施方案中，蛋白质包含两个Cas9结构域的一个：(1)Cas9的gRNA结合结构域；或(2)Cas9的DNA切割结构域。在一些实施方案中，包含Cas9或其片段的蛋白质称为“Cas9变体”。Cas9变体与Cas9或其片段共享同源性。例如，Cas9变体与野生型Cas9至少约70％相同、至少约80％相同、至少约90％相同、至少约95％相同、至少约96％相同、至少约97％相同、至少约98％相同、至少约99％相同、至少约99.5％相同或至少约99.9％相同。在一些实施方案中，Cas9变体与野生型Cas9相比，可以具有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、21、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50个或更多个氨基酸变化。在一些实施方案中，Cas9变体包含Cas9的片段(例如，gRNA结合结构域或DNA切割结构域)，使得该片段与野生型Cas9的相应的片段至少约70％相同、至少约80％相同、至少约90％相同、至少约95％相同、至少约96％相同、至少约97％相同、至少约98％相同、至少约99％相同、至少约99.5％相同或至少约99.9％相同。在一些实施方案中，片段是相应的野生型Cas9的氨基酸长度的至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％相同、至少96％、至少97％、至少98％、至少99％或至少99.5％。
在一些实施方案中，片段的长度为至少100个氨基酸。在一些实施方案中，片段的长度为至少100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1050、1100、1150、1200、1250或1300个氨基酸。在一些实施方案中，野生型Cas9对应与来自酿脓链球菌(Streptococcus pyogenes)的Cas9(NCBI参考序列：NC_017053.1，SEQ ID NO:47(核苷酸)；SEQ ID NO:48(氨基酸))。
ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGGCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGCGCCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGGCCATAGTTTACATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACTGGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCAATAGACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGA(SEQ ID NO:47)
Figure BDA0002539874730000221
Figure BDA0002539874730000222
(单下划线：HNH结构域；双下划线：RuvC结构域)
在一些实施方案中，野生型Cas9对应于，或包含SEQ ID NO:49(核苷酸)和/或SEQID NO:50(氨基酸)：
ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAGGA(SEQ ID NO:49)
Figure BDA0002539874730000241
Figure BDA0002539874730000242
(单下划线：HNH结构域；双下划线：RuvC结构域)
在一些实施方案中，野生型Cas9对应与来自酿脓链球菌的Cas9(NCBI参考序列：NC_002737.2，SEQ ID NO:51(核苷酸)；和Uniport参考序列：Q99ZW2，SEQ ID NO:52(氨基酸)。
ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGA(SEQ IDNO:51)
Figure BDA0002539874730000261
Figure BDA0002539874730000271
SQLGGD(SEQ ID NO:52)(单下划线：HNH结构域；双下划线：RuvC结构域)
在一些实施方案中，Cas9是指来自以下的Cas9：溃疡棒杆菌(Corynebacteriumulcerans)(NCBI Ref:NC_015683.1、NC_017317.1)；白喉棒杆菌(Corynebacteriumdiphtheria)(NCBI Ref:NC_016782.1、NC_016786.1)；Spiroplasma syrphidicola(NCBIRef:NC_021284.1)；间型普雷沃氏菌(Prevotella intermedia)(NCBI Ref:NC_017861.1)；台湾螺原体(Spiroplasma taiwanense(NCBI Ref:NC_021846.1)；海豚链球菌(Streptococcus iniae)(NCBI Ref:NC_021314.1)；波罗的海贝尔氏菌(Belliellabaltica)(NCBI Ref:NC_018010.1)；Psychroflexus torquisI(NCBI Ref:NC_018721.1)；嗜热链球菌(Streptococcus thermophilus)(NCBI Ref:YP_820832.1)、无害李斯特氏菌(Listeria innocua)(NCBI Ref:NP_472073.1)、空肠弯曲杆菌(Campylobacter jejuni)(NCBI Ref:YP_002344900.1)；嗜热脂肪地芽孢杆菌(Geobacillus stearothermophilus)(NCBI Ref:NZ_CP008934.1)；或脑膜炎奈瑟氏球菌(Neisseria.meningitides)(NCBI Ref:YP_002342100.1)或者是指来自任何其他生物体的Cas9。
在一些实施方案中，dCas9对应于，或包含部分或全部的Cas9氨基酸序列，所述Cas9氨基酸序列具有一个或多个使Cas9核酸酶活性失活的突变。例如，在一些实施方案中，dCas9结构域包含SEQ ID NO:52的D10A和H840A突变，或另一种Cas9中的相应的突变。在一些实施方案中，dCas9包含SEQ ID NO:53的氨基酸序列
dCas9(D10A和H840A)：
Figure BDA0002539874730000272
Figure BDA0002539874730000281
Figure BDA0002539874730000282
(单下划线：HNH结构域；双下划线：RuvC结构域)。
在一些实施方案中，Cas9结构域包含D10A突变，而在SEQ ID NO:52中提供的氨基酸序列中的位置840处，或在SEQ ID NO:108-357中提供的任何氨基酸序列中的相应的位置处的残基仍然是组氨酸。不希望受任何特定理论的束缚，催化残基H840的存在维持Cas9的活性以切割含有与靶向的A相对的T的非编辑的(例如，非脱氨基的)链。H840的回复(例如，从dCas9的A840)不导致含有A的靶链的切割。此类Cas9变体能够基于gRNA定义的靶序列在特定位置处产生单链DNA断裂(切口)，导致非编辑的链的修复，最终导致非编辑的链上的T至C变化。该过程的示意图显示于图1C中。简而言之，并且不希望受任何特定理论的束缚，A-T碱基对的A可以通过腺苷脱氨酶(例如，使DNA中的腺苷脱氨基的经工程化的腺苷脱氨酶)脱氨基成肌苷(I)。对具有T的非编辑的链产生切口有助于经由错配修复机制去除T。UGI结构域或催化无活性的肌苷特异性核酸酶(dISN)可以抑制肌苷特异性核酸酶(例如，空间上)，从而防止肌苷(I)的去除。
在其他实施方案中，提供了具有除D10A和H840A之外的突变的dCas9变体，其例如导致核酸酶失活的Cas9(dCas9)。举例来说，此类突变包括D10和H840处的其他氨基酸取代，或Cas9的核酸酶结构域内的其他取代(例如，HNH核酸酶亚结构域和/或RuvC1亚结构域中的取代)。在一些实施方案中，提供了dCas9的变体或同源物(例如SEQ ID NO:53的变体)，其与SEQ ID NO:10至少约70％相同、至少约80％相同、至少约90％相同、至少约95％相同、至少约98％相同、至少约99％相同、至少约99.5％相同或至少约99.9％相同。在一些实施方案中，提供了dCas9的变体(例如SEQ ID NO:53的变体)，其具有比SEQ IDNO:53短或长约5个氨基酸、约10个氨基酸、约15个氨基酸、约20个氨基酸、约25个氨基酸、约30个氨基酸、约40个氨基酸、约50个氨基酸、约75个氨基酸、约100个氨基酸或更多的氨基酸序列。
在一些实施方案中，如本文提供的Cas9融合蛋白包含Cas9蛋白的全长氨基酸序列，例如本文提供的Cas9序列之一。然而，在其他实施方案中，如本文提供的融合蛋白不包含全长Cas9序列，而仅包含其片段。例如，在一些实施方案中，本文提供的Cas9融合蛋白包含Cas9片段，其中所述片段结合crRNA和tracrRNA或sgRNA，但不包含功能性核酸酶结构域，例如其中其仅包含截短形式的核酸酶结构域或根本没有核酸酶结构域。
本文提供了合适的Cas9结构域和Cas9片段的示例性氨基酸序列，并且对于本领域技术人员来说，Cas9结构域和片段的另外合适的序列将是显而易见的。
在一些实施方案中，Cas9是指来自以下的Cas9：溃疡棒杆菌(Corynebacteriumulcerans)(NCBI Ref:NC_015683.1、NC_017317.1)；白喉棒杆菌(Corynebacteriumdiphtheria)(NCBI Ref:NC_016782.1、NC_016786.1)；Spiroplasma syrphidicola(NCBIRef:NC_021284.1)；间型普雷沃氏菌(Prevotella intermedia)(NCBI Ref:NC_017861.1)；台湾螺原体(Spiroplasma taiwanense(NCBI Ref:NC_021846.1)；海豚链球菌(Streptococcus iniae)(NCBI Ref:NC_021314.1)；波罗的海贝尔氏菌(Belliellabaltica)(NCBI Ref:NC_018010.1)；Psychroflexus torquisI(NCBI Ref:NC_018721.1)；嗜热链球菌(Streptococcus thermophilus)(NCBI Ref:YP_820832.1)；无害李斯特氏菌(Listeria innocua)(NCBI Ref:NP_472073.1)；空肠弯曲杆菌(Campylobacter jejuni)(NCBI Ref:YP_002344900.1)；嗜热脂肪地芽孢杆菌(Geobacillus stearothermophilus)(NCBI Ref:NZ_CP008934.1)；或脑膜炎奈瑟氏球菌(Neisseria.meningitides)(NCBI Ref:YP_002342100.1)。
应当理解，另外的Cas9蛋白(例如，核酸酶死亡Cas9(dCas9)、Cas9切口酶(nCas9)或核酸酶活性Cas9)，包括其变体和同源物，都在本公开的范围内。示例性Cas9蛋白包括但不限于下文提供的那些。在一些实施方案中，Cas9蛋白是核酸酶死亡Cas9(dCas9)。在一些实施方案中，dCas9包含氨基酸序列(SEQ ID NO:34)。在一些实施方案中，Cas9蛋白是Cas9切口酶(nCas9)。在一些实施方案中，nCas9包含氨基酸序列(SEQ ID NO:35)。在一些实施方案中，Cas9蛋白是核酸酶活性Cas9。在一些实施方案中，核酸酶活性Cas9包含氨基酸序列(SEQ ID NO:36)。
示例性的催化无活性的Cas9(dCas9)：
DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(SEQ ID NO:34)
示例性的Cas9切口酶(nCas9)：
DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(SEQ ID NO:35)
示例性的催化活性Cas9：
DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(SEQ ID NO:36)。
在一些实施方案中，Cas9是指来自构成单细胞原核微生物的结构域和界的古生菌(arehaea)(例如纳古生菌(nanoarchaea))的Cas9。在一些实施方案中，Cas9是指CasX或CasY，其已经描述于例如Burstein et al.,“New CRISPR–Cas systems fromuncultivated microbes.”Cell Res.2017Feb 21.doi:10.1038/cr.2017.21，其全部内容通过引用并入本文。使用基因组分辨的宏基因组学，鉴定了许多CRISPR-Cas系统，包括在生命的古生菌结构域中首次报道的Cas9。这种趋异的Cas9蛋白在研究很少的纳古生菌中作为活性CRISPR-Cas系统的一部分发现。在细菌中，发现了两个以前未知的系统，CRISPR-CasX和CRISPR-CasY，它们是迄今发现的最紧凑的系统之一。在一些实施方案中，Cas9是指CasX或CasX的变体。在一些实施方案中，Cas9是指CasY或CasY的变体。应当理解，其他RNA引导的DNA结合蛋白可以用作核酸可编程DNA结合蛋白(napDNAbp)，并且在本公开的范围内。
在一些实施方案中，本文提供的任何融合蛋白的核酸可编程DNA结合蛋白(napDNAbp)可以是CasX或CasY蛋白。在一些实施方案中，napDNAbp是CasX蛋白。在一些实施方案中，napDNAbp是CasY蛋白。在一些实施方案中，napDNAbp包含与天然存在的CasX或CasY蛋白至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同的氨基酸序列。在一些实施方案中，napDNAbp是天然存在的CasX或CasY蛋白。在一些实施方案中，napDNAbp包含与SEQ ID NO:417-419中的任一个至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同的氨基酸序列。在一些实施方案中，napDNAbp包含SEQ ID NO:417-419中的任一个的氨基酸序列。应当理解，根据本公开也可以使用来自其他细菌物种的CasX和CasY。
CasX(uniprot.org/uniprot/F0NN87；uniprot.org/uniprot/F0NH53)
>tr|F0NN87|F0NN87_SULIH CRISPR-相关的Casx蛋白质OS＝冰岛硫化叶菌(Sulfolobus islandicus)(菌株HVE10/4)GN＝SiH_0402PE＝4SV＝1
MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG(SEQ ID NO:417)
>tr|F0NH53|F0NH53_SULIR CRISPR相关的蛋白质,Casx OS＝冰岛硫化叶菌(Sulfolobus islandicus)(菌株REY15A)GN＝SiRe_0771PE＝4SV＝1
MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG(SEQ ID NO:418)
CasY(ncbi.nlm.nih.gov/protein/APG80656.1)
>APG80656.1 CRISPR-相关的蛋白质CasY[未培养的Parcubacteria组细菌]
MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPREIVSAINDDYVGLYGLSNFDDLYNAEKRNEEKVYSVLDFWYDCVQYGAVFSYTAPGLLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIKFLNKKEISRANGSLDKLKKDIIDCFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQKKLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNRNRGEVLFNKLKEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELKKAMMDITDAWRGQEQEEELEKRLRILAALTIKLREPKFDNHWGGYRSDINGKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVVSSLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQEALIKERLEAEKKKKPKKRKKKSDAEDEKETIDFKELFPHLAKPLKLVPNFYGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKDFFIKRLQKIFSVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQSRSRKSAAIDKNRVRLPSTENIAKAGIALARELSVAGFDWKDLLKKEEHEEYIDLIELHKTALALLLAVTETQLDISALDFVENGTVKDFMKTRDGNLVLEGRFLEMFSQSIVFSELRGLAGLMSRKEFITRSAIQTMNGKQAELLYIPHEFQSAKITTPKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHYFGYELTRTGQGIDGGVAENALRLEKSPVKKREIKCKQYKTLGRGQNKIVLYVRSSYYQTQFLEWFLHRPKNVQTDVAVSGSFLIDEKKVKTRWNYDALTVALEPVSGSERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEITGDSAKILDQNFISDPQLKTLREEVKGLKLDQRRGTFAMPSTKIARIRESLVHSLRNRIHHLALKHKAKIVYELEVSRFEEGKQKIKKVYATLKKADVYSEIDADKNLQTTVWGKLAVASEISASYTSQFCGACKKLWRAEMQVDETITTQELIGTVRVIKGGTLIDAIKDFMRPPIFDENDTPFPKYRDFCDKHHISKKMRGNSCLFICPFCRANADADIQASQTIALLRYVKEEKKVEDYFERFRKLKNIKVLGQMKKI(SEQ ID NO:419)
如本文所用，术语“有效量”是指足以引起期望的生物学反应的生物活性剂的量。例如，在一些实施方案中，核碱基编辑器的有效量可以指足以诱导由核碱基编辑器突变的特异性结合的靶位点的突变的核碱基编辑器的量。在一些实施方案中，本文提供的融合蛋白，例如包含核酸可编程DNA结合蛋白和脱氨酶结构域(例如腺苷脱氨酶结构域)的融合蛋白的有效量可以指足以诱导融合蛋白特异性结合和编辑的靶位点的编辑的融合蛋白的量。如熟练技术人员将理解的，试剂，例如融合蛋白、核碱基编辑器、脱氨酶、杂合蛋白、蛋白质二聚体、蛋白质(或蛋白质二聚体)和多核苷酸的复合物，或多核苷酸的有效量可以随各种因素而变化，诸如例如随期望的生物学反应，例如随待编辑的特定等位基因、基因组或靶位点，随靶向的细胞或组织和使用的试剂而变化。
如本文所用，术语“核酸”和“核酸分子”是指包含核碱基和酸性部分(例如核苷、核苷酸或核苷酸的聚合物)的化合物。通常，聚合核酸，例如包含三个或更多个核苷酸的核酸分子是线性分子，其中相邻的核苷酸经由磷酸二酯连接彼此连接。在一些实施方案中，“核酸”是指个别的核酸残基(例如核苷酸和/或核苷)。在一些实施方案中，“核酸”是指包含三个或更多个个别核苷酸残基的寡核苷酸链。如本文所用，术语“寡核苷酸”和“多核苷酸”可以可互换地使用以指核苷酸的聚合物(例如，至少三个核苷酸的串)。在一些实施方案中，“核酸”涵盖RNA以及单链和/或双链DNA。核酸可以是天然存在的，例如在基因组、转录物、mRNA、tRNA、rRNA、siRNA、snRNA、质粒、粘粒、染色体、染色单体或其他天然存在的核酸分子的背景下。另一方面，核酸分子可以是非天然存在的分子，例如重组DNA或RNA、人工染色体、工程化的基因组或其片段，或合成DNA、RNA、DNA/RNA杂交体，或包括非天然存在的核苷酸或核苷。此外，术语“核酸”、“DNA”、“RNA”和/或类似术语包括核酸类似物，例如具有除磷酸二酯主链之外的类似物。核酸可以从天然来源纯化，使用重组表达系统产生并任选地纯化、化学合成等。在适当的情况下，例如在化学合成分子的情况下，核酸可以包含核苷类似物，例如具有化学修饰的碱基或糖、和主链修饰的类似物。除非另有说明，核酸序列以5'至3'方向呈现。在一些实施方案中，核酸是或包含天然核苷(例如腺苷、胸苷、鸟苷、胞苷、尿苷、脱氧腺苷、脱氧胸苷、脱氧鸟苷和脱氧胞苷)；核苷类似物(例如2-氨基腺苷、2-硫代胸苷、肌苷、吡咯并嘧啶、3-甲基腺苷、5-甲基胞苷、2-氨基腺苷、C5-溴尿苷、C5-氟尿苷、C5-碘尿苷、C5-丙炔基-尿苷、C5-丙炔基-胞苷、C5-甲基胞苷、2-氨基腺苷、7-脱氮腺苷、7-脱氮鸟苷、8-氧代腺苷、8-氧代鸟苷、O(6)-甲基鸟嘌呤和2-硫代胞苷)；化学修饰的碱基；生物修饰的碱基(例如甲基化碱基)；插入的碱基；修饰的糖(例如2'-氟核糖、核糖、2'-脱氧核糖、阿拉伯糖和己糖)；和/或修饰的磷酸基团(例如硫代磷酸酯和5'-N-亚磷酰胺连接)。
如本文所用，术语“启动子”是指核酸序列的控制区域，在该区域控制核酸序列的其余部分的起始和转录速率。启动子还可以含有调节蛋白和分子(如RNA聚合酶和其他转录因子)可以结合的子区域。
术语“蛋白质”、“肽”和“多肽”在本文中可互换使用，并且是指通过肽(酰胺)键连接在一起的氨基酸残基的聚合物。该术语是指具有任何大小、结构或功能的蛋白质、肽或多肽。通常，蛋白质、肽或多肽将是至少三个氨基酸长。蛋白质、肽或多肽可以指个别的蛋白质或蛋白质的集合。蛋白质、肽或多肽中的一个或多个氨基酸可以被修饰，例如通过添加化学实体如碳水化合物基团、羟基、磷酸基团、法呢基、异法呢基、脂肪酸基团，用于缀合、官能化或其他修饰的接头等。蛋白质、肽或多肽也可以是单个分子或者可以是多分子复合物。蛋白质、肽或多肽可以仅仅是天然存在的蛋白质或肽的片段。蛋白质、肽或多肽可以是天然存在的、重组的或合成的，或其任何组合。如本文所用的术语“融合蛋白”是指包含来自至少两种不同蛋白质的蛋白质结构域的杂合多肽。一种蛋白质可以位于融合蛋白的氨基端(N端)部分或羧基端(C端)蛋白质，从而分别形成“氨基端融合蛋白”或“羧基端融合蛋白”。蛋白质可以包含不同的结构域，例如核酸结合结构域(例如指导蛋白质与靶位点结合的Cas9的gRNA结合结构域)和核酸编辑蛋白的核酸切割结构域或催化结构域。在一些实施方案中，蛋白质包含蛋白质性部分，例如构成核酸结合结构域的氨基酸序列，和有机化合物，例如可以起核酸切割试剂作用的化合物。在一些实施方案中，蛋白质与核酸例如RNA复合或缔合。本文提供的任何蛋白质可以通过本领域已知的任何方法产生。例如，本文提供的蛋白质可以经由重组蛋白质表达和纯化产生，其特别适用于包含肽接头的融合蛋白。用于重组蛋白质表达和纯化的方法是熟知的，并且包括Green and Sambrook,Molecular Cloning:ALaboratory Manual(第4版,Cold Spring Harbor Laboratory Press,Cold SpringHarbor,N.Y.(2012))描述的那些，其全部内容通过引用并入本文。
术语“RNA可编程核酸酶”和“RNA引导的核酸酶”在本文中可互换使用，并且是指与一个或多个不是切割靶标的RNA形成复合物(例如，结合或缔合)的核酸酶。在一些实施方案中，当与RNA形成复合物时，RNA可编程核酸酶可以称为核酸酶:RNA复合物。通常，结合的RNA称为引导RNA(gRNA)。gRNA可以作为两个或更多个RNA的复合物或者作为单个RNA分子存在。作为单个RNA分子存在的gRNA可以称为单引导RNA(sgRNA)，尽管“gRNA”可互换使用以指作为单个分子或作为两个或更多个分子的复合物存在的引导RNA。通常，作为单一RNA种类存在的gRNA包含两个结构域：(1)与靶核酸共享同源性(例如，并指导Cas9复合物与靶物的结合)的结构域，称为引导序列；和(2)结合Cas9蛋白的结构域。在一些实施方案中，结构域(1)与HBG1和/或HBG2基因的启动子中的序列共享同源性。在一些实施方案中，结构域(1)与HBG1和/或HBG2基因的启动子中的序列共享同源性。在一些实施方案中，结构域(1)与序列5’-GTGGGGAAGGGGCCCCCAAGAGG-3’(SEQ ID NO:2)共享同源性。在一些实施方案中，结构域(1)与HFE基因的启动子中的序列共享同源性。在一些实施方案中，结构域(1)与序列5’-TATACGTACCAGGTGGAGCACCCAGG-3’(SEQ ID NO:3)共享同源性。在一些实施方案中，结构域(2)对应已知为tracrRNA的序列，并且包含茎-环结构。例如，在一些实施方案中，结构域(2)与Jinek et al.,Science 337:816-821(2012)中提供的tracrRNA相同或同源，其全部内容通过引用并入本文。gRNA的其他实例(例如包括结构域2的那些)可以在2013年9月6日提交的题为“Switchable Cas9 Nucleases and Uses Thereof”的美国临时专利申请U.S.S.N.61/874,682和2013年9月6号提交的题为“Delivery System For FunctionalNucleases”的美国临时专利申请U.S.S.N.61/874,746中找到，每篇的全部内容通过引用以其整体并入本文。在一些实施方案中，gRNA包含结构域(1)和(2)中的两个或更多个，并且可以称为“延伸的gRNA”。例如，延伸的gRNA将例如结合两个或更多个Cas9蛋白并在两个或更多个不同区域处结合靶核酸，如本文所述。gRNA包含互补靶位点的核苷酸序列，其介导核酸酶/RNA复合物与所述靶位点的结合，提供了核酸酶:RNA复合物的序列特异性。在一些实施方案中，RNA可编程核酸酶是(CRISPR相关系统)(CRISPR-associated system)Cas9内切核酸酶，例如来自酿脓链球菌的Cas9(Csn1)(参见例如“Complete genome sequence of anM1 strain of Streptococcus pyogenes.”Ferretti J.J.,McShan W.M.,Ajdic D.J.,Savic D.J.,Savic G.,Lyon K.,Primeaux C.,Sezate S.,Suvorov A.N.,Kenton S.,LaiH.S.,Lin S.P.,Qian Y.,Jia H.G.,Najar F.Z.,Ren Q.,Zhu H.,Song L.,White J.,YuanX.,Clifton S.W.,Roe B.A.,McLaughlin R.E.,Proc.Natl.Acad.Sci.U.S.A.98:4658-4663(2001)；“CRISPR RNA maturation by trans-encoded small RNA and host factorRNase III.”Deltcheva E.,Chylinski K.,Sharma C.M.,Gonzales K.,Chao Y.,PirzadaZ.A.,Eckert M.R.,Vogel J.,Charpentier E.,Nature471:602-607(2011)；和“Aprogrammable dual-RNA-guided DNA endonuclease in adaptive bacterialimmunity.”Jinek M.,Chylinski K.,Fonfara I.,Hauer M.,Doudna J.A.,CharpentierE.Science 337:816-821(2012)，每篇的全部内容通过引用并入本文。
因为RNA可编程核酸酶(例如Cas9)使用RNA:DNA杂交来靶向DNA切割位点，所以这些蛋白质原则上能够被靶向到由引导RNA规定的任何序列。使用RNA可编程核酸酶例如Cas9进行位点特异性切割(例如，以修饰基因组)的方法是本领域已知的(参见例如Cong,L.etal.,Multiplex genome engineering using CRISPR/Cas systems.Science 339,819-823(2013)；Mali,P.et al.,RNA-guided human genome engineering via Cas9.Science339,823-826(2013)；Hwang,W.Y.et al.,Efficient genome editing in zebrafishusing a CRISPR-Cas system.Nature biotechnology 31,227-229(2013)；Jinek,M.etal.,RNA-programmed genome editing in human cells.eLife 2,e00471(2013)；Dicarlo,J.E.et al.,Genome engineering in Saccharomyces cerevisiae usingCRISPR-Cas systems.Nucleic acids research(2013)；Jiang,W.et al.RNA-guidedediting of bacterial genomes using CRISPR-Cas systems.Nature biotechnology31,233-239(2013)；每篇的全部内容通过引用并入本文)。
如本文所用，术语“受试者”是指个体生物体，例如个体哺乳动物。在一些实施方案中，受试者是人。在一些实施方案中，受试者是非人哺乳动物。在一些实施方案中，受试者是非人灵长类动物。在一些实施方案中，受试者是啮齿动物。在一些实施方案中，受试者是绵羊、山羊、牛、猫或狗。在一些实施方案中，受试者是脊椎动物、两栖动物、爬行动物、鱼、昆虫、苍蝇或线虫。在一些实施方案中，受试者是研究动物。在一些实施方案中，受试者是经遗传工程化的，例如基因遗传化的非人受试者。受试者可以是任何一个性别和处于任何发展阶段的。
术语“靶位点”是指由脱氨酶或包含脱氨酶的融合蛋白(例如本文提供的dCas9-腺苷脱氨酶融合蛋白)脱氨基的核酸分子内的序列。
术语“治疗/处理”是指如本文所述旨在逆转、缓解疾病或病症或其一种或多种症状、延迟疾病或病症或其一种或多种症状的发作或抑制疾病或病症或其一种或多种症状进展的临床干预。如本文所用，术语“治疗/处理”是指如本文所述旨在逆转、缓解疾病或病症或其一种或多种症状、延迟疾病或病症或其一种或多种症状的发作或抑制疾病或病症或其一种或多种症状进展的临床干预。在一些实施方案中，可以在一种或多种症状已经得以形成之后和/或疾病已经得到诊断之后施用治疗。在其他实施方案中，可以在没有症状的情况下施用治疗，例如用于预防或延迟症状的发作或抑制疾病的发作或进展。例如，可以在症状发作之前(例如，鉴于症状的历史和/或鉴于遗传或其他易感性因素)施用治疗于易感个体。治疗也可以在症状消退后继续进行，例如以预防或延迟其复发。
如本文中在蛋白质或核酸的背景中使用，术语“重组”是指自然界中不存在，但是作为人工程化的产物的蛋白质或核酸。例如，在一些实施方案中，重组蛋白质或核酸分子包含氨基酸或核苷酸序列，其相比于任何天然存在的序列包含至少一个、至少两个、至少三个、至少四个、至少五个、至少六个或至少七个突变。
发明详述
本公开的一些方面涉及使核碱基腺嘌呤脱氨基的蛋白质。本公开提供了腺苷脱氨酶蛋白，其能够使脱氧核糖核酸(DNA)中的脱氧腺苷残基的腺嘌呤脱氨基(即去除胺基)。例如，本文提供的腺苷脱氨酶能使DNA的脱氧腺苷残基的腺嘌呤脱氨基。本公开的其他方面提供了融合蛋白，其包含腺苷脱氨酶(例如，如本文所述的使DNA中的脱氧腺苷脱氨基的腺苷脱氨酶)和能够结合特定核苷酸序列的结构域(例如，Cas9或Cpf1蛋白)。腺苷脱氨酶对腺苷的脱氨基作用可以导致点突变，该过程在本文中称为核酸编辑。例如，腺嘌呤可以转化为通常与胞嘧啶碱基配对的肌苷残基。此类融合蛋白尤其可用于核酸序列的靶向编辑。此类融合蛋白可以用于体外靶向编辑DNA，例如，用于产生突变体细胞或动物；用于引入靶向突变，例如，用于校正离体细胞中的遗传缺陷，例如，在自受试者获得的细胞中，随后将所述细胞重新引入相同或另一个受试者中；以及，用于在体内引入靶向突变，例如，校正遗传缺陷或者在受试者中引入疾病相关基因的失活突变。作为实例，可以使用本文提供的核碱基编辑器使可以通过产生A至G或T至C突变来治疗的疾病得到治疗。不希望受到任何特定理论的束缚，可以通过诱导血红蛋白如胎儿血红蛋白(其通常在成年人中沉默)的表达来治疗某些贫血，如镰状细胞贫血。作为一个实例，在驱动HBG1和HBG2基因表达的启动子中将-198T突变为C导致HBG1和HBG2表达增加。
另一个实例，由基因中的G至A突变引起的一类病症是铁贮积病症，其中HFE基因包含导致C282Y突变体HFE蛋白表达的G至A突变。因此，本文所述的腺苷碱基编辑器可以用于此类G至A突变的靶向编辑(例如靶向基因组编辑)。本发明提供了利用脱氨酶和核碱基编辑器的脱氨酶、融合蛋白、核酸、载体、细胞、组合物、方法、试剂盒、系统等。
在一些实施方案中，本文提供的核碱基编辑器可以通过将一个或多个蛋白质结构域融合在一起，从而产生融合蛋白而制备。在某些实施方案中，本文提供的融合蛋白包含一个或多个改善融合蛋白的碱基编辑活性(例如，效率、选择性和特异性)的特征。例如，本文提供的融合蛋白可以包含具有降低的核酸酶活性的Cas9结构域。在一些实施方案中，本文提供的融合蛋白可以具有不具有核酸酶活性的Cas9结构域(dCas9)，或切割双链DNA分子的一条链的Cas9结构域，称为Cas9切口酶(nCas9)。不希望受任何特定理论的束缚，催化残基(例如H840)的存在维持Cas9切割含有与靶向的A相对的T的非编辑(例如，非脱氨基)链的活性。Cas9的催化残基的突变(例如，D10至A10)阻止含有靶向的A残基的编辑链的切割。此类Cas9变体能够基于gRNA定义的靶序列在特定位置处产生单链DNA断裂(切口)，导致非编辑链的修复，最终导致非编辑链上的T至C的变化。
腺苷脱氨酶
本公开的一些方面提供腺苷脱氨酶。在一些实施方案中，本文提供的腺苷脱氨酶能够使腺嘌呤脱氨基。在一些实施方案中，本文提供的腺苷脱氨酶能够使DNA的脱氧腺苷残基中的腺嘌呤脱氨基。腺苷脱氨酶可以衍生自任何合适的生物体(例如，大肠杆菌)。在一些实施方案中，腺嘌呤脱氨酶是天然存在的腺苷脱氨酶，其包括对应于本文提供的任何突变(例如，ecTadA中的突变)的一个或多个突变。本领域技术人员将能够通过本领域熟知的方法鉴定任何同源蛋白质中和相应编码核酸中的相应的残基，例如通过序列比对和同源残基的测定。因此，本领域技术人员能够在任何天然存在的腺苷脱氨酶(例如，与ecTadA具有同源性)中产生对应于本文所述的任何突变(例如ecTadA中鉴定的任何突变)的突变。在一些实施方案中，腺苷脱氨酶来自原核生物。在一些实施方案中，腺苷脱氨酶来自细菌。在一些实施方案中，腺苷脱氨酶来自大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、鼠伤寒沙门氏菌(Salmonella typhi)、腐败希瓦氏菌(Shewanella putrefaciens)、流感嗜血杆菌(Haemophilus influenzae)、新月柄杆菌(Caulobacter crescentus)或枯草芽孢杆菌(Bacillus subtilis)。在一些实施方案中，腺苷脱氨酶来自大肠杆菌。
在一些实施方案中，腺苷脱氨酶包含与SEQ ID NO:1、64-84、420-437、672-684、802-805的任一个所示的任一个氨基酸序列或与本文提供的任何腺苷脱氨酶至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同的氨基酸序列。应当理解，本文提供的腺苷脱氨酶可以包括一个或多个突变(例如，本文提供的任何突变)。本公开提供具有一定百分比同一性加上本文所述的任何突变或其组合的任何脱氨酶结构域。在一些实施方案中，腺苷脱氨酶包含与SEQ ID NO:1、64-84、420-437、672-684、802-805中所示的任一个氨基酸序列或本文提供的任何腺苷脱氨酶相比，具有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、21、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50个或更多个突变的氨基酸序列。在一些实施方案中，腺苷脱氨酶包含与SEQ ID NO:1、64-84、420-437、672-684、802-805中所示的任一个氨基酸序列或本文提供的任何腺苷脱氨酶相比，具有至少5个、至少10个、至少15个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个、至少60个、至少70个、至少80个、至少90个、至少100个、至少110个、至少120个、至少130个、至少140个、至少150个、至少160个、至少170个相同的连续氨基酸残基。
在一些实施方案中，腺苷脱氨酶包含在ecTadA SEQ ID NO:1中的D108X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的D108G、D108N、D108V、D108A或D108Y突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的D108N突变，或另一种腺苷脱氨酶中的相应的突变。然而，应当理解，可以类似地比对另外的脱氨酶以鉴定可以突变的同源氨基酸残基，如本文提供。
在一些实施方案中，腺苷脱氨酶包含在ecTadA SEQ ID NO:1中的A106X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的A106V突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的E155X突变，或另一种腺苷脱氨酶中的相应的突变，其中X的存在表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的E155D、E155G或E155V突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的E155V突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的D147X突变，或另一种腺苷脱氨酶中的相应的突变，其中X的存在表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的D147Y突变，或另一种腺苷脱氨酶中的相应的突变。
应当理解，本文提供的任何突变(例如，基于SEQ ID NO:1的ecTadA氨基酸序列)可以引入到其他腺苷脱氨酶中，例如金黄色葡萄球菌TadA(saTadA)或其他腺苷脱氨酶(例如，细菌腺苷脱氨酶)。对本领域技术人员而言，如何鉴定与ecTadA中的突变残基同源的来自其他腺苷脱氨酶的氨基酸残基是显而易见的。因此，在ecTadA中鉴定的任何突变可以在具有同源氨基酸残基的其他腺苷脱氨酶中产生。还应当理解，本文提供的任何突变可以在ecTadA或另一种腺苷脱氨酶中单独或以任何组合产生。例如，腺苷脱氨酶可以含有ecTadASEQ ID NO:1中的D108N、A106V、E155V和/或D147Y突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中以下突变的组(突变的组由“；”分隔)，或另一种腺苷脱氨酶中的相应的突变：D108N和A106V；D108N和E155V；D108N和D147Y；A106V和E155V；A106V和D147Y；E155V和D147Y；D108N、A106V和E55V；D108N、A106V和D147Y；D108N、E55V和D147Y；A106V、E55V和D147Y；和D108N、A106V、E55V和D147Y。然而，应当理解，本文提供的相应的突变的任何组合可以在腺苷脱氨酶(例如，ecTadA)中产生。在一些实施方案中，腺苷脱氨酶包含图7中所示的一个或多个突变，其鉴定了在ecTadA中产生的个别突变和突变的组合。在一些实施方案中，腺苷脱氨酶包含本文提供的任何突变或突变的组合。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的L84X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的L84F突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的H123X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的H123Y突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的I156X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的I156F突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含选自下组的一个、两个、三个、四个、五个、六个或七个突变：SEQ ID NO:1中的L84X、A106X、D108X、H123X、D147X、E155X和I156X，或另一种腺苷脱氨酶中相应的一个突变或多个突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸的存在。
在一些实施方案中，腺苷脱氨酶包含选自下组的一个、两个、三个、四个、五个、六个或七个突变：SEQ ID NO:1中的L84F、A106V、D108N、H123Y、D147Y、E155V和I156F，或另一种腺苷脱氨酶中相应的一个突变或多个突变。在一些实施方案中，腺苷脱氨酶包含选自下组的一个、两个、三个、四个、五个或六个突变：SEQ ID NO:1中的S2A、I49F、A106V、D108N、D147Y和E155V，或另一种腺苷脱氨酶中相应的一个突变或多个突变。在一些实施方案中，腺苷脱氨酶包含选自下组的一个、两个、三个、四个或五个突变：SEQ ID NO:1中的H8Y、A106T、D108N、N127S和K160S，或另一种腺苷脱氨酶中相应的一个突变或多个突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的A142X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的A142N、A142D、A142G突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的A142N突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的H36X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的H36L突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的N37X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的N37T或N37S突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的N37S突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的P48X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的P48T、P48S、P48A或P48L突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的P48T突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的P48S突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的P48A突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的R51X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的R51H或R51L突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的R51L突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的S146X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示野生型腺苷脱氨酶中除相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的S146R或S146C突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的S146C突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的K157X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的K157N突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的W23X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的W23R或W23L突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的W23R突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ IDNO:1中的W23L突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的R152X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的R152P或R52H突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的R152P突变，或另一种腺苷脱氨酶中的相应的突变。在一些实施方案中，腺苷脱氨酶包含SEQID NO:1中的R152H突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的R26X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的R26G突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的I49X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的I49V突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的N72X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的N72D突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的S97X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的S97C突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的G125X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的G125A突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含ecTadA SEQ ID NO:1中的K161X突变，或另一种腺苷脱氨酶中的相应的突变，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的K161T突变，或另一种腺苷脱氨酶中的相应的突变。
在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的W23X、H36X、N37X、P48X、I49X、R51X、N72X、L84X、S97X、A106X、D108X、H123X、G125X、A142X、S146X、D147X、R152X、E155X、I156X、K157X和/或K161X突变的一个或多个，或另一种腺苷脱氨酶中的一个或多个相应的突变，其中X的存在表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的W23L、W23R、H36L、P48S、P48A、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、R152P、E155V、I156F和/或K157N突变的一个或多个，或另一种腺苷脱氨酶中的一个或多个相应的突变。在一些实施方案中，腺苷脱氨酶包含对应于SEQ ID NO:1的图7中提供的突变的一个或多个，或另一种腺苷脱氨酶中的一个或多个相应的突变。
在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的A106X和D108X的一个或两个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸的存在。在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的A106V和D108N的一个或两个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成。
在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的A106X、D108X、D147X和E155X的一个、两个、三个或四个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸的存在。在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的A106V、D108N、D147Y和E155V的一个、两个、三个或四个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的A106V、D108N、D147Y和E155V突变，或另一种腺苷脱氨酶中的相应的突变，或由之组成。
在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的L84X、A106X、D108X、H123X、D147X、E155X和I156X的一个、两个、三个、四个、五个、六个或七个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸的存在。在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的L84F、A106V、D108N、H123Y、D147Y、E155V和I156F的一个、两个、三个、四个、五个、六个或七个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的L84F、A106V、D108N、H123Y、D147Y、E155V和I156F突变，或另一种腺苷脱氨酶中的相应的突变，或由之组成。
在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的H36X、R51X、L84X、A106X、D108X、H123X、S146X、D147X、E155X、I156X和K157X的一个、两个、三个、四个、五个、六个、七个、八个、九个、十个或十一个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸的存在。在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的H36L、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、E155V、I156F和K157N的一个、两个、三个、四个、五个、六个、七个、八个、九个、十个或十一个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的H36L、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、E155V、I156F和K157N突变，或另一种腺苷脱氨酶中的相应的突变，或由之组成。
在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的H36X、P48X、R51X、L84X、A106X、D108X、H123X、S146X、D147X、E155X、I156X和K157X的一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个或十二个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸的存在。在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的H36L、P48S、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、E155V、I156F和K157N的一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个或十二个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的H36L、P48S、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、E155V、I156F和K157N突变，或另一种腺苷脱氨酶中的相应的突变，或由之组成。
在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的H36X、P48X、R51X、L84X、A106X、D108X、H123X、A142X、S146X、D147X、E155X、I156X和K157X的一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个、十二个或十三个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸的存在。在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的H36L、P48S、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、E155V、I156F和K157N的一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个、十二个或十三个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的H36L、P48S、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、E155V、I156F和K157N突变，或另一种腺苷脱氨酶中的相应的突变，或由之组成。
在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的W23X、H36X、P48X、R51X、L84X、A106X、D108X、H123X、A142X、S146X、D147X、E155X、I156X和K157X的一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个、十二个、十三个或十四个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸的存在。在一些实施方案中，腺苷脱氨酶包含选自SEQID NO:1中的W23L、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、E155V、I156F和K157N的一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个、十二个、十三个或十四个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的W23L、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、E155V、I156F和K157N突变，或另一种腺苷脱氨酶中的相应的突变，或由之组成。
在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的W23X、H36X、P48X、R51X、L84X、A106X、D108X、H123X、S146X、D147X、R152X、E155X、I156X和K157X的一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个、十二个、十三个或十四个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸的存在。在一些实施方案中，腺苷脱氨酶包含选自SEQID NO:1中的W23R、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、R152P、E155V、I156F和K157N的一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个、十二个、十三个或十四个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的W23R、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、R152P、E155V、I156F和K157N突变，或另一种腺苷脱氨酶中的相应的突变，或由之组成。
在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的W23X、H36X、P48X、R51X、L84X、A106X、D108X、H123X、A142X、S146X、D147X、R152X、E155X、I156X和K157X的一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个、十二个、十三个、十四个或十五个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成，其中X表示除野生型腺苷脱氨酶中相应的氨基酸之外的任何氨基酸的存在。在一些实施方案中，腺苷脱氨酶包含选自SEQ ID NO:1中的W23L、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、R152P、E155V、I156F和K157N的一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个、十二个、十三个、十四个或十五个突变，或另一种腺苷脱氨酶中的相应的一个或多个突变，或由之组成。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1中的W23L、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、R152P、E155V、I156F和K157N突变，或另一种腺苷脱氨酶中的相应的突变，或由之组成。
在一些实施方案中，腺苷脱氨酶包含对应于SEQ ID NO:1的图7中提供的突变的一个或多个，或另一种腺苷脱氨酶中的相应的突变的一个或多个。在一些实施方案中，腺苷脱氨酶包含图7中提供的SEQ ID NO:1的变体，或另一种腺苷脱氨酶中的相应的变体，或由之组成。
应当理解，腺苷脱氨酶(例如，第一或第二腺苷脱氨酶)可以包含图7中所示的任何腺苷脱氨酶(例如，ecTadA腺苷脱氨酶)中提供的一种或多种突变。在一些实施方案中，腺苷脱氨酶包含图7中所示的任何腺苷脱氨酶(例如，ecTadA腺苷脱氨酶)的突变的组合。例如，腺苷脱氨酶可以包含突变W23R、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、R152P、E155V、I156F和K157N(相对于SEQ ID NO:1)，其显示为图7中的ABE7.10。在一些实施方案中，腺苷脱氨酶可以包含突变H36L、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、E155V、I156F和K157N(相对于SEQ ID NO:1)。在一些实施方案中，腺苷脱氨酶包含以下相对于SEQ ID NO:1的任何突变的组合，其中组合的每个突变由“_”分开，并且突变的每个组合在括号之间：(A106V_D108N)、(R107C_D108N)、(H8Y_D108N_S127S_D147Y_Q154H)、(H8Y_R24W_D108N_N127S_D147Y_E155V)、(D108N_D147Y_E155V)、(H8Y_D108N_S127S)、(H8Y_D108N_N127S_D147Y_Q154H)、(A106V_D108N_D147Y_E155V)、(D108Q_D147Y_E155V)、(D108M_D147Y_E155V)、(D108L_D147Y_E155V)、(D108K_D147Y_E155V)、(D108I_D147Y_E155V)、(D108F_D147Y_E155V)、(A106V_D108N_D147Y)、(A106V_D108M_D147Y_E155V)、(E59A_A106V_D108N_D147Y_E155V)、(E59A cat dead_A106V_D108N_D147Y_E155V)、(L84F_A106V_D108N_H123Y_D147Y_E155V_I156Y)、(L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)、(D103A_D014N)、(G22P_D103A_D104N)、(G22P_D103A_D104N_S138A)、(D103A_D104N_S138A)、(R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F)、(E25G_R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F)、(E25D_R26G_L84F_A106V_R107K_D108N_H123Y_A142N_A143G_D147Y_E155V_I156F)、(R26Q_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F)、(E25M_R26G_L84F_A106V_R107P_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F)、(R26C_L84F_A106V_R107H_D108N_H123Y_A142N_D147Y_E155V_I156F)、(L84F_A106V_D108N_H123Y_A142N_A143L_D147Y_E155V_I156F)、(R26G_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F)、(E25A_R26G_L84F_A106V_R107N_D108N_H123Y_A142N_A143E_D147Y_E155V_I156F)、(R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F)、(A106V_D108N_A142N_D147Y_E155V)、(R26G_A106V_D108N_A142N_D147Y_E155V)、(E25D_R26G_A106V_R107K_D108N_A142N_A143G_D147Y_E155V)、(R26G_A106V_D108N_R107H_A142N_A143D_D147Y_E155V)、(E25D_R26G_A106V_D108N_A142N_D147Y_E155V)、(A106V_R107K_D108N_A142N_D147Y_E155V)、(A106V_D108N_A142N_A143G_D147Y_E155V)、(A106V_D108N_A142N_A143L_D147Y_E155V)、(H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)、(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)、(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)、(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)、(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N)、(N37T_P48T_M70L_L84F_A106V_D108N_H123Y_D147Y_I49V_E155V_I156F)、(N37S_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K161T)、(H36L_L84F_A106V_D108N_H123Y_D147Y_Q154H_E155V_I156F)、(N72S_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F)、(H36L_P48L_L84F_A106V_D108N_H123Y_E134G_D147Y_E155V_I156F)、(H36L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N)、(H36L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F)、(L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T)、(N37S_R51H_D77G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)、(R51L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N)、(D24G_Q71R_L84F_H96L_A106V_D108N_H123Y_D147Y_E155V_I156F_K160E)、(H36L_G67V_L84F_A106V_D108N_H123Y_S146T_D147Y_E155V_I156F)、(Q71L_L84F_A106V_D108N_H123Y_L137M_A143E_D147Y_E155V_I156F)、(E25G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L)、(L84F_A91T_F104I_A106V_D108N_H123Y_D147Y_E155V_I156F)、(N72D_L84F_A106V_D108N_H123Y_G125A_D147Y_E155V_I156F)、(P48S_L84F_S97C_A106V_D108N_H123Y_D147Y_E155V_I156F)、(W23G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)、(D24G_P48L_Q71R_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L)、(L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F)、(H36L_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)、(N37S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_K161T)、(L84F_A106V_D108N_D147Y_E155V_I156F)、(R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K161T)、(L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K161T)、(L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E_K161T)、(L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E)、(R74Q L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)、(R74A_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)、(L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)、(R74Q_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)、(L84F_R98Q_A106V_D108N_H123Y_D147Y_E155V_I156F)、(L84F_A106V_D108N_H123Y_R129Q_D147Y_E155V_I156F)、(P48S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F)、(P48S_A142N)、(P48T_I49V_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_L157N)、(P48T_I49V_A142N)、(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)、(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F_K157N)、(H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)、(H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)、(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)、(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)、(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F_K157N)、(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)、(W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)、(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T)、(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152H_E155V_I156F_K157N)、(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)、(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)、(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_E155V_I156F_K157N)、(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_R152P_E155V_I156F_K157N)、(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T)、(W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)、(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N)。
在一些实施方案中，腺苷脱氨酶包含与SEQ ID NO:1、64-84、420-437、672-684、802-805的任一个，或本文提供的任何腺苷脱氨酶至少60％、65％、70％、75％、80％、85％、90％、95、98％、99％或99.5％相同的氨基酸序列。在一些实施方案中，腺苷脱氨酶包含与SEQ ID NO:1、64-84、420-437、672-684、802-805中所示的任一个氨基酸序列，或本文提供的任何腺苷脱氨酶相比，具有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、21、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50个或更多个突变的氨基酸序列。在一些实施方案中，腺苷脱氨酶包含与SEQID NO:1、64-84、420-437、672-684、802-805中所示的任一个氨基酸序列，或本文提供的任何腺苷脱氨酶相比，具有至少5个、至少10个、至少15个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个、至少60个、至少70个、至少80个、至少90个、至少100个、至少110个、至少120个、至少130个、至少140个、至少150个、至少160个或至少166个相同的连续氨基酸残基的氨基酸序列。在一些实施方案中，腺苷脱氨酶包含SEQ ID NO:1、64-84、420-437、672-684、802-805的任一个，或本文提供的任何腺苷脱氨酶的氨基酸序列。在一些实施方案中，腺苷脱氨酶由SEQ ID NO:1、64-84、420-437、672-684、802-805的任一个，或本文提供的任何腺苷脱氨酶的氨基酸序列组成。下面提供的ecTadA序列来自ecTadA(SEQID NO:1)，不存在N端甲硫氨酸(M)。下面提供的saTadA序列来自saTadA(SEQ ID NO:8)，不存在N端甲硫氨酸(M)。为清楚起见，用于鉴定各种氨基酸突变的氨基酸编号方案衍生自针对大肠杆菌TadA的ecTadA(SEQ ID NO:1)和针对金黄色葡萄球菌TadA的saTadA(SEQ IDNO:8)。相对于SEQ ID NO:1(ecTadA)或SEQ ID NO:8(saTadA)的氨基酸突变用下划线表示。
ecTadA
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD(SEQ ID NO:64)
ecTadA(D108N)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD(SEQ ID NO:65)
ecTadA(D108G)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD(SEQ ID NO:66)
ecTadA(D108V)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARVAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD(SEQ ID NO:67)
ecTadA(H8Y、D108N和N127S)
SEVEFSYEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARNAKTGAAGSLMDVLHHPGMSHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD(SEQ ID NO:68)
ecTadA(H8Y、D108N、N127S和E155D)
SEVEFSYEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARNAKTGAAGSLMDVLHHPGMSHRVEITEGILADECAALLSDFFRMRRQDIKAQKKAQSSTD(SEQ ID NO:69)
ecTadA(H8Y、D108N、N127S和E155G)
SEVEFSYEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHA EIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARNAKTGAAG SLMDVLHHPGMSHRVEITEGILADECAALLSDFFRMRRQGIKAQKKAQSSTD(SEQ ID NO:70)
ecTadA(H8Y、D108N、N127S和E155V)
SEVEFSYEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARNAKTGAAGSLMDVLHHPGMSHRVEITEGILADECAALLSDFFRMRRQVIKAQKKAQSSTD(SEQ ID NO:71)
ecTadA(A106V、D108N、D147Y和E155V)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSYFFRMRRQVIKAQKKAQSSTD(SEQ ID NO:72)
ecTadA(L84F、A106V、D108N、H123Y、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:73)
ecTadA(S2A、I49F、A106V、D108N、D147Y、E155V)
AEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPFGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSYFFRMRRQVIKAQKKAQSSTD(SEQ ID NO:74)
ecTadA(H8Y、A106T、D108N、N127S、K160S)
SEVEFSYEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGTRNAKTGAAGSLMDVLHHPGMSHRVEITEGILADECAALLSDFFRMRRQEIKAQSKAQSSTD(SEQ ID NO:75)
ecTadA(R26G、L84F、A106V、R107H、D108N、H123Y、A142N、A143D、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDEGEVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVHNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNDLLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:76)
ecTadA(E25G、R26G、L84F、A106V、R107H、D108N、H123Y、A142N、A143D、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDGGEVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVHNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNDLLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:77)
ecTadA(E25D、R26G、L84F、A106V、R107K、D108N、H123Y、A142N、A143G、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDDGEVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVKNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNGLLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:78)
ecTadA(R26Q、L84F、A106V、D108N、H123Y、A142N、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDEQEVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:79)
ecTadA(E25M、R26G、L84F、A106V、R107P、D108N、H123Y、A142N、A143D、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDMGEVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVPNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNDLLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:80)
ecTadA(R26C、L84F、A106V、R107H、D108N、H123Y、A142N、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDECEVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVHNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:81)
ecTadA(L84F、A106V、D108N、H123Y、A142N、A143L、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNLLLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:82)
ecTadA(R26G、L84F、A106V、D108N、H123Y、A142N、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDEGEVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:83)
ecTadA(E25A、R26G、L84F、A106V、R107N、D108N、H123Y、A142N、A143E、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDAGEVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVNNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNELLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:420)
ecTadA(L84F、A106V、D108N、H123Y、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:421)
ecTadA(N37T、P48T、L84F、A106V、D108N、H123Y、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHTNRVIGEGWNRTIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:422)
ecTadA(N37S、L84F、A106V、D108N、H123Y、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHSNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:423)
ecTadA(H36L、L84F、A106V、D108N、H123Y、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:424)
ecTadA(L84F、A106V、D108N、H123Y、S146R、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLRYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:425)
ecTadA(H36L、P48L、L84F、A106V、D108N、H123Y、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRLIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:426)
ecTadA(H36L、L84F、A106V、D108N、H123Y、D147Y、E155V、K57N、I156F)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRMRRQVFNAQKKAQSSTD(SEQ ID NO:427)
ecTadA(H36L、L84F、A106V、D108N、H123Y、S146C、D147Y、E155V、I156F)SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:428)
ecTadA(L84F、A106V、D108N、H123Y、S146R、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLRYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:429)
ecTadA(N37S、R51H、L84F、A106V、D108N、H123Y、D147Y、E155V、I156F)SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHSNRVIGEGWNRPIGHHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:430)
ecTadA(R51L、L84F、A106V、D108N、H123Y、D147Y、E155V、I156F、K157N)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRMRRQVFNAQKKAQSSTD(SEQ ID NO:431)
ecTadA(R51H、L84F、A106V、D108N、H123Y、D147Y、E155V、I156F、K157N)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGHHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRMRRQVFNAQKKAQSSTD(SEQ ID NO:432)
ecTadA(P48S)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRSIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD(SEQ ID NO:672)
ecTadA(P48T)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRTIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD(SEQ ID NO:673)
ecTadA(P48A)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD(SEQ ID NO:674)
ecTadA(A142N)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECNALLSDFFRMRRQEIKAQKKAQSSTD(SEQ ID NO:675)
ecTadA(W23R)
SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD(SEQ ID NO:676)
ecTadA(W23L)
SEVEFSHEYWMRHALTLAKRALDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD(SEQ ID NO:677)
ecTadA(R152P)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMPRQEIKAQKKAQSSTD(SEQ ID NO:678)
ecTadA(R152H)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMHRQEIKAQKKAQSSTD(SEQ ID NO:679)
ecTadA(L84F、A106V、D108N、H123Y、D147Y、E155V、I156F)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRMRRQVFKAQKKAQSSTD(SEQ ID NO:680)
ecTadA(H36L、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、E155V、I156F、K157N)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRPIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMRRQVFNAQKKAQSSTD(SEQ ID NO:681)
ecTadA(H36L、P48S、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、E155V、I156F、K157N)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRSIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMRRQVFNAQKKAQSSTD(SEQ ID NO:682)
ecTadA(H36L、P48A、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、E155V、I156F、K157N)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHA EIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAG SLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMRRQVFNAQKKAQSSTD(SEQ ID NO:683)
ecTadA(W23L、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、R152P、E155V、I156F、K157N)
SEVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHA EIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAG SLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD(SEQ ID NO:684)
ecTadA(H36L、P48S、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、E155V、I156F、K157N)
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRSIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRMRRQVFNAQKKAQSSTD(SEQ ID NO:802)
ecTadA(W23L、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、E155V、I156F、K157N)
SEVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRMRRQVFNAQKKAQSSTD(SEQ ID NO:803)
ecTadA(W23L、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、R152P、E155V、I156F、K157N)
SEVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECNALLCYFFRMPRQVFNAQKKAQSSTD(SEQ ID NO:804)
ecTadA(W23R、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、R152P、E155V、I156F、K157N)
SEVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD(SEQ ID NO:805)
核碱基编辑器的Cas9结构域
在一些方面，核酸可编程DNA结合蛋白(napDNAbp)是Cas9结构域。本文提供了非限制性的示例性Cas9结构域。Cas9结构域可以是核酸酶活性Cas9结构域、核酸酶无活性Cas9结构域或Cas9切口酶。在一些实施方案中，Cas9结构域是核酸酶活性结构域。例如，Cas9结构域可以是切割双链核酸的两条链(例如，双链DNA分子的两条链)的Cas9结构域。在一些实施方案中，Cas9结构域包含如SEQ ID NO:108-357中所示的氨基酸序列的任一个。在一些实施方案中Cas9结构域包含与SEQ ID NO:108-357中所示的氨基酸序列的任一个至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同的氨基酸序列。在一些实施方案中，Cas9结构域包含与SEQ ID NO:108-357中所示的任一个氨基酸序列相比具有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、21、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50个或更多个突变的氨基酸序列。在一些实施方案中，Cas9结构域包含具有与SEQ ID NO:108-357中所示的任一个氨基酸序列相比具有至少10、至少15、至少20、至少30、至少40、至少50、至少60、至少70、至少80、至少90、至少100、至少150、至少200、至少250、至少300、至少350、至少400、至少500、至少600、至少700、至少800、至少900、至少1000、至少1100、或至少1200个相同的连续氨基酸残基的氨基酸序列。
在一些实施方案中，Cas9结构域是核酸酶无活性的Cas9结构域(dCas9)。例如，dCas9结构域可以结合双链核酸分子(例如，经由gRNA分子)而不切割双链核酸分子的任一条链。在一些实施方案中，核酸酶无活性dCas9结构域包含SEQ ID NO:52中所示的氨基酸序列的D10X突变和H840X突变，或SEQ ID NO:108-357中所提供的任何氨基酸序列中的相应的突变，其中X是任何氨基酸变化。在一些实施方案中，核酸酶无活性dCas9结构域包含SEQ IDNO:52中所示的氨基酸序列的D10A突变和H840A突变，或SEQ ID NO:108-357中提供的任何氨基酸序列中的相应的突变。作为一个实例，核酸酶无活性的Cas9结构域包含SEQ ID NO:54中所示的氨基酸序列(克隆载体pPlatTET-gRNA2，登录号BAV54124)。
MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(SEQ ID NO:54；参见例如，Qi et al.,“Repurposing CRISPR as an RNA-guidedplatform for sequence-specific control of gene expression.”Cell.2013；152(5):1173-83，其全部内容通过引用并入本文)。
基于本公开和本领域的知识，另外的合适的核酸酶无活性dCas9结构域对于本领域技术人员将是显而易见的，并且在本公开的范围内。此类另外的示例性合适的核酸酶无活性Cas9结构域包括但不限于D10A/H840A、D10A/D839A/H840A和D10A/D839A/H840A/N863A突变体结构域(参见例如Prashant et al.,CAS9 transcriptional activators fortarget specificity screening and paired nickases for cooperative genomeengineering.Nature Biotechnology.2013；31(9):833-838，其全部内容通过引用并入本文)。在一些实施方案中，dCas9结构域包含与本文提供的任一个dCas9结构域至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同的氨基酸序列。在一些实施方案中，Cas9结构域包含与SEQ ID NO:108-357中所示的任一个氨基酸序列相比，具有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、21、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50个或更多个突变的氨基酸序列。在一些实施方案中，Cas9结构域包含与SEQ ID NO:108-357中所示的任一个氨基酸序列相比，具有至少10个、至少15个、至少20个、至少30个、至少40个、至少50个、至少60个、至少70个、至少80个、至少90个、至少100个、至少150个、至少200个、至少250个、至少300个、至少350个、至少400个、至少500个、至少600个、至少700个、至少800个、至少900个、至少1000个、至少1100个、或至少1200个相同的连续氨基酸残基的氨基酸序列。
在一些实施方案中，Cas9结构域是Cas9切口酶。Cas9切口酶可以是能够仅切割双链核酸分子(例如双链DNA分子)的一条链的Cas9蛋白。在一些实施方案中，Cas9切口酶切割双链核酸分子的靶链，这意味着Cas9切口酶切割与结合到Cas9的gRNA(例如，sgRNA)碱基配对(互补)的链。在一些实施方案中，Cas9切口酶包含D10A突变并且具有SEQ ID NO:52的位置840处的组氨酸，或在SEQ ID NO:108-357中的任一个中的突变。作为一个实例，Cas9切口酶可以包含如SEQ ID NO:35中所示的氨基酸序列。在一些实施方案中，Cas9切口酶切割双链核酸分子的非靶标、非碱基编辑链，这意味着Cas9切口酶切割不与结合到Cas9的gRNA(例如，sgRNA)碱基配对的链。在一些实施方案中，Cas9切口酶包含H840A突变并且具有在SEQID NO:52的位置10处的天冬氨酸残基或在SEQ ID NO:108-357的任一个中的相应的突变。在一些实施方案中，Cas9切口酶包含与本文提供的任一个Cas9切口酶至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同的氨基酸序列。基于本公开和本领域的知识，另外的合适的Cas9切口酶对于本领域技术人员将是显而易见的，并且在本公开的范围内。
具有降低的PAM排他性(exclusivity)的Cas9结构域
本公开的一些方面提供了具有不同PAM特异性的Cas9结构域。通常，Cas9蛋白，例如来自酿脓链球菌的Cas9(spCas9)，需要规范的NGG PAM序列来结合特定的核酸区域，其中“NGG”中的“N”是腺嘌呤(A)、胸腺嘧啶(T)、鸟嘌呤(G)或胞嘧啶(C)，并且G是鸟嘌呤。这可以限制在基因组内编辑期望的碱基的能力。在一些实施方案中，本文提供的碱基编辑融合蛋白需要定位于精确的位置处，例如，其中靶碱基在4碱基区域(例如“脱氨基作用窗口”)内，其在PAM的上游的约15个碱基。参见Komor,A.C.,et al.,“Programmable editing of atarget base in genomic DNA without double-stranded DNA cleavage”Nature533,420-424(2016)，其全部内容在此通过引用并入。在一些实施方案中，脱氨基作用窗口在2、3、4、5、6、7、8、9或10碱基区域内。在一些实施方案中，脱氨基作用窗口在PAM上游的5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24或25个碱基。因此，在一些实施方案中，本文提供的任何融合蛋白可以含有能够结合不含规范的(例如，NGG)PAM序列的核苷酸序列的Cas9结构域。本领域已经描述了结合非规范PAM序列的Cas9结构域，并且其对于熟练技术人员而言是显而易见的。例如，结合非规范PAM序列的Cas9结构域已经描述于Kleinstiver,B.P.,et al.,“Engineered CRISPR-Cas9 nucleases with altered PAMspecificities”Nature 523,481-485(2015)；and Kleinstiver,B.P.,et al.,“Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 bymodifying PAM recognition”Nature Biotechnology 33,1293-1298(2015)；每篇的全部内容在此通过引用并入。
在一些实施方案中，Cas9结构域是来自金黄色葡萄球菌的Cas9结构域(SaCas9)。在一些实施方案中，SaCas9结构域是核酸酶活性的SaCas9、核酸酶无活性的SaCas9(SaCas9d)或SaCas9切口酶(SaCas9n)。在一些实施方案中，SaCas9包含氨基酸序列SEQ IDNO:55。在一些实施方案中，SaCas9包含SEQ ID NO:55的N579X突变或SEQ ID NO:108-357中提供的任何氨基酸序列中的相应的突变，其中X是除N之外的任何氨基酸。在一些实施方案中，SaCas9包含SEQ ID NO:55的N579A突变或SEQ ID NO:108-357中提供的任何氨基酸序列中的相应的突变。
在一些实施方案中，SaCas9结构域、SaCas9d结构域或SaCas9n结构域可以结合具有非规范PAM的核酸序列。在一些实施方案中，SaCas9结构域、SaCas9d结构域或SaCas9n结构域可以结合具有NNGRRT PAM序列的核酸序列，其中N＝A、T、C或G，并且R＝A或G。在一些实施方案中，SaCas9结构域包含SEQ ID NO:55的E781X、N967X和R1014X突变中的一个或多个，或SEQ ID NO:108-357中提供的任何氨基酸序列中的相应的突变，其中X是任何氨基酸。在一些实施方案中，SaCas9结构域包含SEQ ID NO:55中的E781K、N967K和R1014H突变，或者在SEQ ID NO:108-357中提供的任何氨基酸序列中的一个或多个相应的突变中的一个或多个。在一些实施方案中，SaCas9结构域包含SEQ ID NO:55的E781K、N967K或R1014H突变或在SEQ ID NO:108-357中提供的任何氨基酸序列中的相应的突变。
在一些实施方案中，本文提供的任何融合蛋白的Cas9结构域包含与SEQID NO:55-57中的任一个至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同的氨基酸序列。在一些实施方案中，本文提供的任何融合蛋白的Cas9结构域包含SEQ ID NO:55-57中任一个的氨基酸序列。在一些实施方案中，本文提供的任何融合蛋白的Cas9结构域由SEQ ID NO:55-57中任一个的氨基酸序列组成。
示例性的SaCas9序列
Figure BDA0002539874730000651
可以将SEQ ID NO:55的残基N579(其是加下划线且粗体的)突变(例如突变为A579)以产生SaCas9切口酶。
示例性的SaCas9n序列
Figure BDA0002539874730000652
Figure BDA0002539874730000661
SEQ ID NO:56的残基A579(其可以从SEQ ID NO:55的N579突变以产生SaCas9切口酶)是加下划线且粗体的。
示例性的SaKKH Cas9
Figure BDA0002539874730000662
SEQ ID NO:57的残基A579(其可以从SEQ ID NO:55的N579突变以产生SaCas9切口酶)是加下划线且粗体的。SEQ ID NO:57的残基K781、K967和H1014(其可以从SEQ ID NO:55的E781、N967和R1014突变以产生SaKKH Cas9)是加下划线且斜体的。
在一些实施方案中，Cas9结构域是来自酿脓链球菌的Cas9结构域(SpCas9)。在一些实施方案中，SpCas9结构域是核酸酶活性的SpCas9、核酸酶无活性的SpCas9(SpCas9d)或SpCas9切口酶(SpCas9n)。在一些实施方案中，SpCas9包含氨基酸序列SEQ ID NO:58。在一些实施方案中，SpCas9包含SEQ ID NO:58的D9X突变或在SEQ ID NO:108-357中提供的任何氨基酸序列中的相应的突变，其中X是除D之外的任何氨基酸。在一些实施方案中，SpCas9包含SEQ ID NO:58的D9A突变或在SEQ ID NO:108-357中提供的任何氨基酸序列中的相应的突变。在一些实施方案中，SpCas9结构域、SpCas9d结构域或SpCas9n结构域可以结合具有非规范PAM的核酸序列。在一些实施方案中，SpCas9结构域、SpCas9d结构域或SpCas9n结构域可以结合具有NGG、NGA或NGCG PAM序列的核酸序列。在一些实施方案中，SpCas9结构域包含SEQ ID NO:58的D1134X、R1334X和T1336X突变或SEQ ID NO:108-35中提供的任何氨基酸序列中的相应的突变中的一个或多个，其中X是任何氨基酸。在一些实施方案中，SpCas9结构域包含SEQ ID NO:58的D1134E、R1334Q和T1336R突变，或在SEQ ID NO:108-35中提供的任何氨基酸序列中的相应的突变中的一个或多个。在一些实施方案中，SpCas9结构域包含SEQID NO:58的D1134E、R1334Q和T1336R突变或在SEQ ID NO:108-35中提供的任何氨基酸序列中的相应的突变。在一些实施方案中，SpCas9结构域包含SEQ ID NO:58的D1134X、R1334X和T1336X突变或SEQ ID NO:108-35中提供的任何氨基酸序列中的相应的突变中的一个或多个，其中X是任何氨基酸。在一些实施方案中，SpCas9结构域包含SEQ ID NO:58的D1134V、R1334Q和T1336R突变，或在SEQ ID NO:108-35中提供的任何氨基酸序列中的相应的突变中的一个或多个。在一些实施方案中，SpCas9结构域包含SEQ ID NO:58的D1134V、R1334Q和T1336R突变，或在SEQ ID NO:108-35中提供的任何氨基酸序列中的相应的突变。在一些实施方案中，SpCas9结构域包含SEQ ID NO:58的D1134X、G1217X、R1334X和T1336X突变，或SEQID NO:108-35中提供的任何氨基酸序列中的相应的突变中的一个或多个，其中X是任何氨基酸。在一些实施方案中，SpCas9结构域包含SEQ ID NO:58的D1134V、G1217R、R1334Q和T1336R突变，或在SEQ ID NO:108-35中提供的任何氨基酸序列中的相应的突变中的一个或多个。在一些实施方案中，SpCas9结构域包含SEQ ID NO:58的D1134V、G1217R、R1334Q和T1336R突变或在SEQ ID NO:108-35中提供的任何氨基酸序列中的相应的突变。
在一些实施方案中，本文提供的任何融合蛋白的Cas9结构域包含与SEQ ID NO:58-62至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同的氨基酸序列。在一些实施方案中，本文提供的任何融合蛋白的Cas9结构域包含SEQ ID NO:58-62的任一个的氨基酸序列。在一些实施方案中，本文提供的任何融合蛋白的Cas9结构域由SEQ ID NO:58-62的任一个的氨基酸序列组成。
示例性的SpCas9
DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(SEQ ID NO:58)
示例性的SpCas9n
DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(SEQ ID NO:59)
示例性的SpEQR Cas9
Figure BDA0002539874730000691
SEQ ID NO:60的残基E1134、Q1334和R1336(其可以从SEQ ID NO:58的D1134、R1334和T1336突变以产生SpEQR Cas9)是加下划线且粗体的。
示例性的SpVQR Cas9
Figure BDA0002539874730000692
Figure BDA0002539874730000701
SEQ ID NO:61的残基V1134、Q1334和R1336(其可以从SEQ ID NO:58的D1134、R1334和T1336突变以产生SpVQR Cas9)是加下划线且粗体的。
示例性的SpVRER Cas9
Figure BDA0002539874730000702
SEQ ID NO:62的残基V1134、R1217、Q1334和R1336(其可以从SEQ ID NO:58的D1134、G1217、R1334和T1336突变以产生SpVRER Cas9)是加下划线且粗体的。
高保真性Cas9结构域
本公开的一些方面提供了本文提供的核碱基编辑器的高保真性Cas9结构域。在一些实施方案中，与相应的野生型Cas9结构域相比，高保真性Cas9结构域是包含一个或多个突变的经工程化的Cas9结构域，所述突变降低Cas9结构域和DNA的糖-磷酸主链之间的静电相互作用。不希望受任何特定理论的束缚，具有降低的与DNA的糖-磷酸主链的静电相互作用的高保真性Cas9结构域可以具有较少的脱靶效应。在一些实施方案中，Cas9结构域(例如，野生型Cas9结构域)包含一个或多个降低Cas9结构域与DNA的糖-磷酸主链之间的缔合的突变。在一些实施方案中，Cas9结构域包含一个或多个将Cas9结构域与DNA的糖-磷酸主链之间的缔合降低至少1％、至少2％、至少3％、至少4％、至少5％、至少10％、至少15％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％或更多的突变。
在一些实施方案中，本文提供的任何Cas9融合蛋白包含SEQ ID NO:52中提供的氨基酸序列的N497X、R661X、Q695X和/或Q926X突变，或在SEQ ID NO:108-357中提供的任何氨基酸序列中的相应的突变中的一个或多个，其中X是任何氨基酸。在一些实施方案中，本文提供的任何Cas9融合蛋白包含SEQ ID NO:52中提供的氨基酸序列的N497A、R661A、Q695A和/或Q926A突变，或在SEQ ID NO:108-357中提供的任何氨基酸序列中的相应的突变中的一个或多个。在一些实施方案中，Cas9结构域包含SEQ ID NO:52中提供的氨基酸序列的D10A突变，或在SEQ ID NO:108-357中提供的任何氨基酸序列中的相应的突变。在一些实施方案中，Cas9结构域(例如本文提供的任何融合蛋白的)包含如SEQ ID NO:62中所示的氨基酸序列。具有高保真度的Cas9结构域是本领域已知的，并且对于本领域技术人员而言是显而易见的。例如，具有高保真度的Cas9结构域已经描述于Kleinstiver,B.P.,et al.“High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-targeteffects.”Nature 529,490-495(2016)；和Slaymaker,I.M.,et al.“Rationallyengineered Cas9 nucleases with improved specificity.”Science 351,84-88(2015)；每篇的全部内容通过引用并入本文。
应当理解，本文提供的任何碱基编辑器，例如，本文提供的任何腺苷脱氨酶碱基编辑器，可以通过如本文所述修饰Cas9结构域而转换成高保真碱基编辑器以产生高保真碱基编辑器，例如，高保真腺苷碱基编辑器。在一些实施方案中，高保真性Cas9结构域是dCas9结构域。在一些实施方案中，高保真性Cas9结构域是nCas9结构域。
高保真性Cas9结构域，其中相对于SEQ ID NO:10的Cas9的突变以粗体和下划线显示
Figure BDA0002539874730000721
核酸可编程DNA结合蛋白
本公开的一些方面提供了核酸可编程DNA结合蛋白，其可以用于将蛋白质(例如碱基编辑器)引导至特定核酸(例如DNA或RNA)序列。核酸可编程DNA结合蛋白包括但不限于Cas9(例如dCas9和nCas9)、CasX、CasY、Cpf1、C2c1、C2c2、C2C3和Argonaute。具有与Cas9不同的PAM特异性的核酸可编程DNA结合蛋白的一个实例是来自普雷沃氏菌(Prevotella)和弗朗西斯菌(Francisella)1(Cpf1)的聚簇规则间隔短回文重复。与Cas9类似，Cpf1也是2类CRISPR效应物。已经显示，Cpf1介导了强大的DNA干扰，其具有与Cas9不同的特征。Cpf1是缺乏tracrRNA的单RNA引导的内切核酸酶，并且它利用富含T的前间隔区相邻基序(TTN、TTTN或YTN)。此外，Cpf1经由交错的DNA双链断裂切割DNA。在16种Cpf1家族蛋白中，来自氨基酸球菌(Acidaminococcus)和毛螺菌(Lachnospiraceae)的两种酶显示在人类细胞中具有有效的基因组编辑活性。Cpf1蛋白是本领域已知的并且先前已有描述，例如Yamano et al.,“Crystal structure of Cpf1 in complex with guide RNA and target DNA.”Cell(165)2016,p.949-962；其全部内容在此通过引用并入。
在一些实施方案中，核酸可编程DNA结合蛋白(napDNAbp)是微生物CRISPR-Cas系统的单一效应物。微生物CRISPR-Cas系统的单一效应物包括但不限于Cas9、Cpf1、C2c1、C2c2和C2c3。通常，微生物CRISPR-Cas系统分为1类和2类系统。1类系统具有多亚基效应物复合物，而2类系统具有单一蛋白质效应物。例如，Cas9和Cpf1是2类效应物。除了Cas9和Cpf1之外，Shmakov et al.,“Discovery and Functional Characterization of DiverseClass 2CRISPR Cas Systems”,Mol.Cell,2015Nov 5；60(3):385–397已经描述了三种不同的2类CRISPR-Cas系统(C2c1、C2c2和C2c3)，其全部内容在此通过引用并入。
包含核酸酶可编程DNA结合蛋白和腺苷脱氨酶的融合蛋白
本公开的一些方面提供了包含核酸可编程DNA结合蛋白(napDNAbp)和腺苷脱氨酶的融合蛋白。在一些实施方案中，本文提供的任何融合蛋白是碱基编辑器。在一些实施方案中，napDNAbp是Cas9结构域、Cpf1结构域、CasX结构域、CasY结构域、C2c1结构域、C2c2结构域、C2c3结构域或Argonaute结构域。在一些实施方案中，napDNAbp是本文提供的任何napDNAbp。本公开的一些方面提供了包含Cas9结构域和腺苷脱氨酶的融合蛋白。Cas9结构域可以是本文提供的任何Cas9结构域或Cas9蛋白(例如，dCas9或nCas9)。在一些实施方案中，本文提供的任何Cas9结构域或Cas9蛋白(例如，dCas9或nCas9)可以与本文提供的任何腺苷脱氨酶融合。在一些实施方案中，融合蛋白包含以下结构：
NH2-[腺苷脱氨酶]-[napDNAbp]-COOH；或
NH2-[napDNAbp]-[腺苷脱氨酶]-COOH
在一些实施方案中，包含腺苷脱氨酶和napDNAbp(例如，Cas9结构域)的融合蛋白不包括接头序列。在一些实施方案中，接头存在于腺苷脱氨酶结构域和napDNAbp之间。在一些实施方案中，上文一般构造中使用的“-”表示存在任选的接头。在一些实施方案中，腺苷脱氨酶和napDNAbp经由本文提供的任何接头融合。例如，在一些实施方案中，腺苷脱氨酶和napDNAbp经由下文标题为“接头”的部分中提供的任何接头融合。在一些实施方案中，腺苷脱氨酶和napDNAbp经由包含1和200个氨基酸之间的接头融合。在一些实施方案中，腺苷脱氨酶和napDNAbp经由包含长度为1至5、1至10、1至20、1至30、1至40、1至50、1至60、1至80、1至100、1至150、1至200、5至10、5至20、5至30、5至40、5至60、5至80、5至100、5至150、5至200、10至20、10至30、10至40、10至50、10至60、10至80、10至100、10至150、10至200、20至30、20至40、20至50、20至60、20至80、20至100、20至150、20至200、30至40、30至50、30至60、30至80、30至100、30至150、30至200、40至50、40至60、40至80、40至100、40至150、40至200、50至6050至80、50至100、50至150、50至200、60至80、60至100、60至150、60至200、80至100、80至150、80至200、100至150、100至200或150至200个氨基酸的接头融合。在一些实施方案中，腺苷脱氨酶和napDNAbp经由包含长度为3、4、16、24、32、64、100或104个氨基酸的接头融合。在一些实施方案中，腺苷脱氨酶和napDNAbp经由包含以下的氨基酸序列的接头融合：SGSETPGTSESATPES(SEQ ID NO:10)、SGGS(SEQ ID NO:37)、SGGSSGSETPGTSESATPESSGGS(SEQ ID NO:384)、SGGSSGGSSGSETPGTSESATPESSGGSSGGS(SEQ ID NO:385)或GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS(SEQ ID NO:386)。在一些实施方案中，腺苷脱氨酶和napDNAbp经由包含氨基酸序列SGSETPGTSESATPES(SEQ ID NO:10)的接头融合，其也可以称为XTEN接头。在一些实施方案中，接头长度为24个氨基酸。在一些实施方案中，接头包含氨基酸序列SGGSSGGSSGSETPGTSESATPES(SEQ ID NO:685)。在一些实施方案中，接头长度为32个氨基酸。在一些实施方案中，接头包含氨基酸序列(SGGS)2-SGSETPGTSESATPES-(SGGS)2(SEQ IDNO:800)，其也可以称为(SGGS)2-XTEN-(SGGS)2。在一些实施方案中，接头包含氨基酸序列(SGGS)n-SGSETPGTSESATPES-(SGGS)n(SEQ ID NO:801)，其中n是0、1、2、3、4、5、6、7、8、9或10。在一些实施方案中，接头长度为40个氨基酸。在一些实施方案中，接头包含氨基酸序列SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS(SEQ ID NO:686)。在一些实施方案中，接头长度为64个氨基酸。在一些实施方案中，接头包含氨基酸序列SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESAT PESSGGSSGGS(SEQ ID NO:687)。在一些实施方案中，接头长度为92个氨基酸。在一些实施方案中，接头包含氨基酸序列PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS(SEQID NO:688)。
包含核定位序列(NLS)的融合蛋白
在一些实施方案中，本文提供的融合蛋白进一步包含一个或多个核靶向序列，例如核定位序列(NLS)。在一些实施方案中，NLS包含促进包含NLS的蛋白质输入细胞核中(例如，通过核转运)的氨基酸序列。在一些实施方案中，本文提供的任何融合蛋白进一步包含核定位序列(NLS)。在一些实施方案中，NLS与融合蛋白的N端融合。在一些实施方案中，NLS与融合蛋白的C端融合。在一些实施方案中，NLS与IBR(例如，dISN)的N端融合。在一些实施方案中，NLS与IBR(例如，dISN)的C端融合。在一些实施方案中，NLS与napDNAbp的N端融合。在一些实施方案中，NLS与napDNAbp的C端融合。在一些实施方案中，NLS与腺苷脱氨酶的N端融合。在一些实施方案中，NLS与腺苷脱氨酶的C端融合。在一些实施方案中，NLS经由一个或多个接头与融合蛋白融合。在一些实施方案中，NLS与融合蛋白在没有接头的情况下融合。在一些实施方案中，NLS包含本文提供或引用的NLS序列的任一个的氨基酸序列。在一些实施方案中，NLS包含如SEQ ID NO:4或SEQ ID NO:5中所示的氨基酸序列。另外的核定位序列是本领域已知的并且对于技术人员是显而易见的。例如，NLS序列描述于Plank et al.,PCT/EP2000/011690中，其内容通过引用并入本文，用于其对示例性的核定位序列的公开。在一些实施方案中，NLS包含氨基酸序列PKKKRKV(SEQ ID NO:4)、MDSLLMNRRKFLYQFKNVRWAKGRRETYLC(SEQ ID NO:5)、MKRTADGSEFEPKKKRKV(SEQ ID NO:342)或KRTADGSEFEPKKKRKV(SEQ ID NO:343)。
在一些实施方案中，具有腺苷脱氨酶和napDNAbp的示例性融合蛋白的一般构造包含以下结构中的任一种，其中NLS是核定位序列(例如，本文提供的任何NLS)，NH2是融合蛋白的N端，并且COOH是融合蛋白的C端。
包含腺苷脱氨酶、napDNAbp和NLS的融合蛋白。
NH2-[NLS]-[腺苷脱氨酶]-[napDNAbp]-COOH；
NH2-[腺苷脱氨酶]-[NLS]-[napDNAbp]-COOH；
NH2-[腺苷脱氨酶]-[napDNAbp]-[NLS]-COOH；
NH2-[NLS]-[napDNAbp]-[腺苷脱氨酶]-COOH；
NH2-[napDNAbp]-[NLS]-[腺苷脱氨酶]-COOH；和
NH2-[napDNAbp]-[腺苷脱氨酶]-[NLS]-COOH。
在一些实施方案中，本文提供的融合蛋白不包含接头。在一些实施方案中，接头存在于一个或多个结构域或蛋白质(例如，腺苷脱氨酶、napDNAbp和/或NLS)之间。在一些实施方案中，上文一般构造中使用的“-”表示存在任选的接头。
本公开的一些方面提供了融合蛋白，其包含核酸可编程DNA结合蛋白(napDNAbp)和至少两个腺苷脱氨酶结构域。不希望受任何特定理论的束缚，腺苷脱氨酶的二聚化(例如，顺式或反式)可以改善融合蛋白修饰核酸碱基(例如使腺嘌呤脱氨基)的能力(例如，效率)。在一些实施方案中，任何融合蛋白可以包含2、3、4或5个腺苷脱氨酶结构域。在一些实施方案中，本文提供的任何融合蛋白包含两个腺苷脱氨酶。在一些实施方案中，本文提供的任何融合蛋白仅含有两个腺苷脱氨酶。在一些实施方案中，腺苷脱氨酶是相同的。在一些实施方案中，腺苷脱氨酶是本文提供的任何腺苷脱氨酶。在一些实施方案中，腺苷脱氨酶是不同的。在一些实施方案中，第一腺苷脱氨酶是本文提供的任何腺苷脱氨酶，并且第二腺苷是本文提供的任何腺苷脱氨酶，但与第一腺苷脱氨酶不相同。作为一个实例，融合蛋白可以包含第一腺苷脱氨酶和第二腺苷脱氨酶，两者均包含SEQ ID NO:72的氨基酸序列，其含有来自ecTadA(SEQ ID NO:1)的A106V、D108N、D147Y和E155V突变。在一些实施方案中，融合蛋白可以包含：包含SEQ ID NO:802的氨基酸序列的第一腺苷脱氨酶，其含有来自SEQ ID NO:1的H36L、P48S、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、E155V、I156F和K157N突变，以及包含野生型ecTadA的氨基酸序列(SEQ ID NO:1)的第二腺苷脱氨酶结构域。在一些实施方案中，融合蛋白可以包含：包含SEQ ID NO:803的氨基酸序列的第一腺苷脱氨酶，其含有来自SEQ ID NO:1的W23L、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、E155V、I156F和K157N突变，以及包含野生型ecTadA的氨基酸序列(SEQ IDNO:1)的第二腺苷脱氨酶结构域。在一些实施方案中，融合蛋白可以包含：包含SEQ ID NO:804的氨基酸序列的第一腺苷脱氨酶，其含有来自SEQ ID NO:1的W23L、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、R152P、E155V、I156F和K157N突变，以及包含野生型ecTadA的氨基酸序列(SEQ ID NO:1)的第二腺苷脱氨酶结构域。在一些实施方案中，融合蛋白可以包含：包含SEQ ID NO:805的氨基酸序列的第一腺苷脱氨酶，其含有来自SEQ ID NO:1的W23R、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、R152P、E155V、I156F和K157N突变，以及包含野生型ecTadA的氨基酸序列(SEQ ID NO:1)的第二腺苷脱氨酶结构域。包含两个腺苷脱氨酶结构域的另外的融合蛋白构建体如图7中所示。
在一些实施方案中，融合蛋白包含两个腺苷脱氨酶(例如，第一腺苷脱氨酶和第二腺苷脱氨酶)。在一些实施方案中，融合蛋白包含第一腺苷脱氨酶和第二腺苷脱氨酶。在一些实施方案中，第一腺苷脱氨酶在融合蛋白中第二腺苷脱氨酶的N端。在一些实施方案中，第一腺苷脱氨酶在融合蛋白中第二腺苷脱氨酶的C端。在一些实施方案中，第一腺苷脱氨酶和第二脱氨酶直接地或经由接头融合。在一些实施方案中，接头是本文提供的任何接头，例如，“接头”部分中描述的任何接头。在一些实施方案中，接头包含SEQ ID NO:10、37-40、384-386、685-688或800-801的任一个的氨基酸序列。在一些实施方案中，接头长度为32个氨基酸。在一些实施方案中，接头包含氨基酸序列(SGGS)2-SGSETPGTSESATPES-(SGGS)2(SEQID NO:800)，其也可以称为(SGGS)2-XTEN-(SGGS)2。在一些实施方案中，接头包含氨基酸序列(SGGS)n-SGSETPGTSESATPES-(SGGS)n(SEQ ID NO:801)，其中n是0、1、2、3、4、5、6、7、8、9或10。在一些实施方案中，第一腺苷脱氨酶与第二腺苷脱氨酶相同。在一些实施方案中，第一腺苷脱氨酶和第二腺苷脱氨酶是本文所述的任何腺苷脱氨酶。在一些实施方案中，第一腺苷脱氨酶和第二腺苷脱氨酶不同。在一些实施方案中，第一腺苷脱氨酶是本文提供的任何腺苷脱氨酶。在一些实施方案中，第二腺苷脱氨酶是本文提供的任何腺苷脱氨酶，但与第一腺苷脱氨酶不相同。在一些实施方案中，第一腺苷脱氨酶是ecTadA腺苷脱氨酶。在一些实施方案中，第一腺苷脱氨酶包含与SEQ ID NO:1、64-84、420-437、672-684的任一个中所示的氨基酸序列的任一个或与本文提供的任何腺苷脱氨酶至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同的氨基酸序列。在一些实施方案中，第一腺苷脱氨酶包含SEQ ID NO:1的氨基酸序列。在一些实施方案中，第二腺苷脱氨酶包含与SEQ ID NO:1、64-84、420-437、672-684的任一个中所示的氨基酸序列的任一个或与本文提供的任何腺苷脱氨酶至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同的氨基酸序列。在一些实施方案中，第二腺苷脱氨酶包含SEQ ID NO:1的氨基酸序列。在一些实施方案中，融合蛋白的第一腺苷脱氨酶和第二腺苷脱氨酶包含ecTadA(SEQ ID NO:1)中的突变，或另一种腺苷脱氨酶中的相应的突变，如表4中提供的构建体的任一个中所示(例如，pNMG-371、pNMG-477、pNMG-576、pNMG-586和pNMG-616)。在一些实施方案中，融合蛋白包含表4中的构建体(例如，pNMG-371、pNMG-477、pNMG-576、pNMG-586和pNMG-616)的任一个的两个腺苷脱氨酶(例如，第一腺苷脱氨酶和第二腺苷脱氨酶)。
在一些实施方案中，具有第一腺苷脱氨酶、第二腺苷脱氨酶和napDNAbp的示例性融合蛋白的一般构造包含以下结构中的任一种，其中NLS是核定位序列(例如，本文提供的任何NLS)，NH2是融合蛋白的N端，并且COOH是融合蛋白的C端。
包含第一腺苷脱氨酶、第二腺苷脱氨酶和napDNAbp的融合蛋白。
NH2-[第一腺苷脱氨酶]-[第二腺苷脱氨酶]-[napDNAbp]-COOH；
NH2-[第一腺苷脱氨酶]-[napDNAbp]-[第二腺苷脱氨酶]-COOH；
NH2-[napDNAbp]-[第一腺苷脱氨酶]-[第二腺苷脱氨酶]-COOH；
NH2-[第二腺苷脱氨酶]-[第一腺苷脱氨酶]-[napDNAbp]-COOH；
NH2-[第二腺苷脱氨酶]-[napDNAbp]-[第一腺苷脱氨酶]-COOH；
NH2-[napDNAbp]-[第二腺苷脱氨酶]-[第一腺苷脱氨酶]-COOH；
在一些实施方案中，本文提供的融合蛋白不包含接头。在一些实施方案中，接头存在于一个或多个结构域或蛋白质(例如，第一腺苷脱氨酶、第二腺苷脱氨酶和/或napDNAbp)之间。在一些实施方案中，上文一般构造中使用的“-”表示存在任选的接头。
包含第一腺苷脱氨酶、第二腺苷脱氨酶、napDNAbp和NLS的融合蛋白。
NH2-[NLS]-[第一腺苷脱氨酶]-[第二腺苷脱氨酶]-[napDNAbp]-COOH；
NH2-[第一腺苷脱氨酶]-[NLS]-[第二腺苷脱氨酶]-[napDNAbp]-COOH；
NH2-[第一腺苷脱氨酶]-[第二腺苷脱氨酶]-[NLS]-[napDNAbp]-COOH；
NH2-[第一腺苷脱氨酶]-[第二腺苷脱氨酶]-[napDNAbp]-[NLS]-COOH；
NH2-[NLS]-[第一腺苷脱氨酶]-[napDNAbp]-[第二腺苷脱氨酶]-COOH；
NH2-[第一腺苷脱氨酶]-[NLS]-[napDNAbp]-[第二腺苷脱氨酶]-COOH；
NH2-[第一腺苷脱氨酶]-[napDNAbp]-[NLS]-[第二腺苷脱氨酶]-COOH；
NH2-[第一腺苷脱氨酶]-[napDNAbp]-[第二腺苷脱氨酶]-[NLS]-COOH；
NH2-[NLS]-[napDNAbp]-[第一腺苷脱氨酶]-[第二腺苷脱氨酶]-COOH；
NH2-[napDNAbp]-[NLS]-[第一腺苷脱氨酶]-[第二腺苷脱氨酶]-COOH；
NH2-[napDNAbp]-[第一腺苷脱氨酶]-[NLS]-[第二腺苷脱氨酶]-COOH；
NH2-[napDNAbp]-[第一腺苷脱氨酶]-[第二腺苷脱氨酶]-[NLS]-COOH；
NH2-[NLS]-[第二腺苷脱氨酶]-[第一腺苷脱氨酶]-[napDNAbp]-COOH；
NH2-[第二腺苷脱氨酶]-[NLS]-[第一腺苷脱氨酶]-[napDNAbp]-COOH；
NH2-[第二腺苷脱氨酶]-[第一腺苷脱氨酶]-[NLS]-[napDNAbp]-COOH；
NH2-[第二腺苷脱氨酶]-[第一腺苷脱氨酶]-[napDNAbp]-[NLS]-COOH；
NH2-[NLS]-[第二腺苷脱氨酶]-[napDNAbp]-[第一腺苷脱氨酶]-COOH；
NH2-[第二腺苷脱氨酶]-[NLS]-[napDNAbp]-[第一腺苷脱氨酶]-COOH；
NH2-[第二腺苷脱氨酶]-[napDNAbp]-[NLS]-[第一腺苷脱氨酶]-COOH；
NH2-[第二腺苷脱氨酶]-[napDNAbp]-[第一腺苷脱氨酶]-[NLS]-COOH；
NH2-[NLS]-[napDNAbp]-[第二腺苷脱氨酶]-[第一腺苷脱氨酶]-COOH；
NH2-[napDNAbp]-[NLS]-[第二腺苷脱氨酶]-[第一腺苷脱氨酶]-COOH；
NH2-[napDNAbp]-[第二腺苷脱氨酶]-[NLS]-[第一腺苷脱氨酶]-COOH；
NH2-[napDNAbp]-[第二腺苷脱氨酶]-[第一腺苷脱氨酶]-[NLS]-COOH；
在一些实施方案中，本文提供的融合蛋白不包含接头。在一些实施方案中，接头存在于一个或多个结构域或蛋白质(例如，第一腺苷脱氨酶、第二腺苷脱氨酶、napDNAbp和/或NLS)之间。在一些实施方案中，上文一般构造中使用的“-”表示存在任选的接头。
应当理解，本公开的融合蛋白可以包含一个或多个另外的特征。例如，在一些实施方案中，融合蛋白可以包含细胞质定位序列、输出序列(例如核输出序列)或其他定位序列，以及可用于融合蛋白的溶解、纯化或检测的序列标签。本文提供的合适的蛋白质标签包括但不限于生物素羧化酶载体蛋白(BCCP)标签、myc标签、钙调蛋白标签、FLAG标签、血凝素(HA)标签、多组氨酸标签(也称为组氨酸标签或His标签)、麦芽糖结合蛋白(MBP)-标签、nus标签、谷胱甘肽-S-转移酶(GST)标签、绿色荧光蛋白(GFP)标签、硫氧还蛋白标签、S标签、Softag(例如，Softag 1、Softag 3)、strep标签、生物素连接酶标签、FlAsH标签、V5标签和SBP标签。另外的合适的序列对于本领域技术人员而言是显而易见的。在一些实施方案中，融合蛋白包含一个或多个His标签。
接头
在某些实施方案中，接头可以用于连接本文所述的任何蛋白质或蛋白质结构域。接头可以简单地为共价键，或者它可以是长度为许多原子的聚合物接头。在某些实施方案中，接头是多肽或基于氨基酸。在其他实施方案中，接头不是肽样的。在某些实施方案中，接头是共价键(例如，碳-碳键、二硫键、碳-杂原子键等)。在某些实施方案中，接头是酰胺连接的碳-氮键。在某些实施方案中，接头是环状或非环状，取代或未取代的，分支或未分支的脂肪族或杂脂肪族接头。在某些实施方案中，接头是聚合物(例如，聚乙烯、聚乙二醇、聚酰胺、聚酯等)。在某些实施方案中，接头包含氨基烷酸的单体、二聚体或聚合物。在某些实施方案中，接头包含氨基烷酸(例如甘氨酸、乙酸、丙氨酸、β-丙氨酸、3-氨基丙酸、4-氨基丁酸、5-戊酸等)。在某些实施方案中，接头包含氨基己酸(Ahx)的单体、二聚体或聚合物。在某些实施方案中，接头基于碳环部分(例如，环戊烷、环己烷)。在其他实施方案中，接头包含聚乙二醇部分(PEG)。在其他实施方案中，接头包含氨基酸。在某些实施方案中，接头包含肽。在某些实施方案中，接头包含芳基或杂芳基部分。在某些实施方案中，接头基于苯环。接头可以包括官能化部分以促进亲核试剂(例如，硫醇、氨基)从肽附接至接头。任何亲电子试剂都可以用作接头的一部分。示例性亲电子试剂包括但不限于活化酯、活化酰胺、迈克尔受体、烷基卤化物、芳基卤化物、酰基卤化物和异硫氰酸酯。
在一些实施方案中，接头是一个氨基酸或多个氨基酸(例如肽或蛋白质)。在一些实施方案中，接头是键(例如，共价键)、有机分子、基团、聚合物或化学部分。在一些实施方案中，接头的长度为5-100个氨基酸，例如长度为5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35-40、40-45、45-50、50-60、60-70、70-80、80-90、90-100、100-110、110-120、120-130、130-140、140-150或150-200个氨基酸。也考虑了更长或更短的接头。在一些实施方案中，接头包含氨基酸序列SGSETPGTSESATPES(SEQ ID NO:10)，其也可以称为XTEN接头。在一些实施方案中，接头长度为32个氨基酸。在一些实施方案中，接头包含氨基酸序列(SGGS)2-SGSETPGTSESATPES-(SGGS)2(SEQ ID NO:800)，其也可以称为(SGGS)2-XTEN-(SGGS)2。在一些实施方案中，接头包含氨基酸序列，其中n是0、1、2、3、4、5、6、7、8、9或10。在一些实施方案中，接头包含氨基酸序列SGGS(SEQ ID NO:37)。在一些实施方案中，接头包含(SGGS)n(SEQ ID NO:37)、(GGGS)n(SEQ ID NO:38)、(GGGGS)n(SEQ ID NO:39)、(G)n、(EAAAK)n(SEQ ID NO:40)、(SGGS)n-SGSETPGTSESATPES-(SGGS)n(SEQ ID NO:801)、(GGS)n、SGSETPGTSESATPES(SEQ ID NO:10)或(XP)n基序，或这些的任何的组合，其中n独立地是1和30之间的整数，并且其中X是任何氨基酸。在一些实施方案中，n是1、2、3、4、5、6、7、8、9、10、11、12、13、14或15。在一些实施方案中，接头包含SGSETPGTSESATPES(SEQ ID NO:10)和SGGS(SEQ ID NO:37)。在一些实施方案中，接头包含SGGSSGSETPGTSESATPESSGGS(SEQ ID NO:384)。在一些实施方案中，接头包含SGGSSGGSSGSETPGTSESATPESSGGSSGGS(SEQ ID NO:385)。在一些实施方案中，接头包含GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS(SEQ ID NO:386)。在一些实施方案中，接头长度为24个氨基酸。在一些实施方案中，接头包含氨基酸SGGSSGGSSGSETPGTSESATPES(SEQ ID NO:685)。在一些实施方案中，接头长度为40个氨基酸。在一些实施方案中，接头包含氨基酸序列SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS(SEQ ID NO:686)。在一些实施方案中，接头长度为64个氨基酸。在一些实施方案中，接头包含氨基酸序列SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESAT PESSGGSSGGS(SEQ ID NO:687)。在一些实施方案中，接头长度为92个氨基酸。在一些实施方案中，接头包含氨基酸序列PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS(SEQ IDNO:688)。应当理解，本文提供的任何接头可以用于连接第一腺苷脱氨酶和第二腺苷脱氨酶；腺苷脱氨酶(例如，第一或第二腺苷脱氨酶)和napDNAbp；napDNAbp和NLS；或腺苷脱氨酶(例如，第一或第二腺苷脱氨酶)和NLS。
在一些实施方案中，本文提供的任何融合蛋白包含经由接头彼此融合的腺苷脱氨酶和napDNAbp。在一些实施方案中，本文提供的任何融合蛋白包含经由接头彼此融合的第一腺苷脱氨酶和第二腺苷脱氨酶。在一些实施方案中，本文提供的任何融合蛋白包含NLS，其可以与腺苷脱氨酶(例如，第一和/或第二腺苷脱氨酶)、核酸可编程DNA结合蛋白(napDNAbp)和或碱基修复抑制剂(IBR)融合。可以采用腺苷脱氨酶(例如，工程化的ecTadA)和napDNAbp(例如，Cas9结构域)之间和/或第一腺苷脱氨酶和第二腺苷脱氨酶之间的各种接头长度和柔性(例如，范围为从形式(GGGGS)n(SEQ ID NO:38)、(GGGGS)n(SEQ ID NO:39)和(G)n的非常柔性接头到形式(EAAAK)n(SEQ ID NO:40)、(SGGS)n(SEQ ID NO:37)、SGSETPGTSESATPES(SEQ ID NO:10)(参见例如，Guilinger JP,Thompson DB,LiuDR.Fusion of catalytically inactive Cas9 to FokI nuclease improves thespecificity of genome modification.Nat.Biotechnol.2014；32(6):577-82；其全部内容通过引用并入本文)和(XP)n的更刚性的接头)，以达到用于特定应用的脱氨酶活性的最佳长度。在一些实施方案中，n是1、2、3、4、5、6、7、8、9、10、11、12、13、14或15。在一些实施方案中，接头包含(GGS)n基序，其中n是1、3或7。在一些实施方案中，本文提供的任何融合蛋白的腺苷脱氨酶和napDNAbp，和/或第一腺苷脱氨酶和第二腺苷脱氨酶经由接头融合，所述接头包含氨基酸序列SGSETPGTSESATPES(SEQ ID NO:10),SGGS(SEQ ID NO:37)、SGGSSGSETPGTSESATPESSGGS(SEQ ID NO:384)、SGGSSGGSSGSETPGTSESATPESSGGSSGGS(SEQID NO:385)或GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS(SEQ ID NO:386)。在一些实施方案中，接头长度为24个氨基酸。在一些实施方案中，接头包含氨基酸SGGSSGGSSGSETPGTSESATPES(SEQ ID NO:685)。在一些实施方案中，接头长度为32个氨基酸。在一些实施方案中，接头长度为32个氨基酸。在一些实施方案中，接头包含氨基酸序列(SGGS)2-SGSETPGTSESATPES-(SGGS)2(SEQ ID NO:800)，其也可以称为(SGGS)2-XTEN-(SGGS)2。在一些实施方案中，接头包含氨基酸序列，其中n是0、1、2、3、4、5、6、7、8、9或10。在一些实施方案中，接头长度为40个氨基酸。在一些实施方案中，接头包含氨基酸序列SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS(SEQ ID NO:686)。在一些实施方案中，接头长度为64个氨基酸。在一些实施方案中，接头包含氨基酸序列SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS(SEQ ID NO:687)。在一些实施方案中，接头长度为92个氨基酸。在一些实施方案中，接头包含氨基酸序列PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS(SEQ ID NO:688)。
本公开的一些方面提供了包含Cas9结构域和腺苷脱氨酶的融合蛋白。示例性融合蛋白包括但不限于以下融合蛋白(为了清楚起见，腺苷脱氨酶结构域以粗体显示；ecTadA脱氨酶结构域的突变以粗体下划线显示；XTEN接头以斜体显示；UGI/AAG/EndoV结构域以粗体斜体显示；NLS以下划线斜体显示)：ecTadA(wt)-XTEN-nCas9-NLS:
Figure BDA0002539874730000831
Figure BDA0002539874730000841
ecTadA(D108N)-XTEN-nCas9-NLS:(哺乳动物构建体，对DNA有活性，A至G编辑):
Figure BDA0002539874730000842
Figure BDA0002539874730000851
ecTadA(D108G)-XTEN-nCas9-NLS:(哺乳动物构建体，对DNA上活性，A至G编辑):
Figure BDA0002539874730000852
ecTadA(D108V)-XTEN-nCas9-NLS:(哺乳动物构建体，对DNA有活性，A至G编辑):
Figure BDA0002539874730000853
Figure BDA0002539874730000861
从第一轮演化(细菌中)所得的变体ecTadA(H8Y_D108N_N127S)-XTEN-dCas9:
Figure BDA0002539874730000862
来自第二轮演化(细菌中)的富集的变体ecTadA(H8Y_D108N_N127S_E155X)-XTEN-dCas9；X＝D,G或V:
Figure BDA0002539874730000871
ABE7.7:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS
Figure BDA0002539874730000872
Figure BDA0002539874730000881
pNMG-624氨基酸序列:ecTadA(野生型)-32a.a.接头-ecTadA(W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-24a.a.接头_nCas9_SGGS_NLS
Figure BDA0002539874730000882
Figure BDA0002539874730000891
ABE3.2:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)-(SGGS)2-XTEN-(SGGS)2 _nCas9_SGGS_NLS
Figure BDA0002539874730000892
ABE5.3:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS
Figure BDA0002539874730000893
Figure BDA0002539874730000901
pNMG-558氨基酸序列:ecTadA(野生型)-32a.a.接头-ecTadA(H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)-24a.a.接头_nCas9_SGGS_NLS
Figure BDA0002539874730000902
Figure BDA0002539874730000911
pNMG-576氨基酸序列:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS
Figure BDA0002539874730000912
pNMG-577氨基酸序列:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS
Figure BDA0002539874730000921
pNMG-586氨基酸序列:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS
Figure BDA0002539874730000922
Figure BDA0002539874730000931
ABE7.2:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS
Figure BDA0002539874730000932
Figure BDA0002539874730000941
pNMG-620氨基酸序列:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS
Figure BDA0002539874730000942
pNMG-617氨基酸序列:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS
Figure BDA0002539874730000951
pNMG-618氨基酸序列:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS
Figure BDA0002539874730000952
Figure BDA0002539874730000961
pNMG-620氨基酸序列:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS
Figure BDA0002539874730000962
Figure BDA0002539874730000971
pNMG-621氨基酸序列:ecTadA(野生型)-32a.a.接头-ecTadA(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-24a.a.接头_nCas9_GGS_NLS
Figure BDA0002539874730000972
pNMG-622氨基酸序列:ecTadA(野生型)-32a.a.接头-ecTadA(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N)-24a.a.接头_nCas9_GGS_NLS
Figure BDA0002539874730000973
Figure BDA0002539874730000981
pNMG-623氨基酸序列:ecTadA(野生型)-32a.a.接头-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-24a.a.接头_nCas9_GGS_NLS
Figure BDA0002539874730000982
Figure BDA0002539874730000991
ABE6.3:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS
Figure BDA0002539874730000992
Figure BDA0002539874730001001
ABE6.4:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS
Figure BDA0002539874730001002
ABE7.8:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS
Figure BDA0002539874730001003
Figure BDA0002539874730001011
ABE7.9:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS
Figure BDA0002539874730001012
Figure BDA0002539874730001021
ABE7.10:ecTadA(野生型)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS
Figure BDA0002539874730001022
Figure BDA0002539874730001031
在一些实施方案中，融合蛋白包含与SEQ ID NO:11-28、387、388、440、691-711的任一个中所示的氨基酸序列的任一个，或者与本文提供的任何融合蛋白至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同。在一些实施方案中，融合蛋白包含与SEQ ID NO:11-28、387、388、440、691-711中所示的氨基酸序列的任一个，或本文提供的任何融合蛋白相比，具有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、21、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50个或更多个突变的氨基酸序列。在一些实施方案中，融合蛋白包含与SEQ ID NO:11-28、387、388、440、691-711中所示的氨基酸序列的任一个，或本文提供的任何融合蛋白相比，具有至少5个、至少10个、至少15个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个、至少60个、至少70个、至少80个、至少90个、至少100个、至少110个、至少120个、至少130个、至少140个、至少150个、至少160个、至少170个、至少200个、至少300个、至少400个、至少500个、至少600个、至少700个、至少800个、至少900个、至少1000个、至少1100个、至少1200个、至少1300个、至少1400个、至少1500个、至少1600个、至少1700个、至少1750个或至少1800个相同的连续氨基酸残基的氨基酸序列。
具有引导核酸的核酸可编程DNA结合蛋白(napDNAbp)的复合物
本公开的一些方面提供了包含本文提供的任何融合蛋白(例如本文提供的任何碱基编辑器)，以及与融合蛋白的napDNAbp结合的引导核酸的复合物。在一些实施方案中，本公开提供了与本文提供的任何引导RNA结合的本文提供的任何融合蛋白(例如腺苷碱基编辑器)。在一些实施方案中，融合蛋白(例如腺苷碱基编辑器)的napDNAbp是与引导RNA结合的Cas9结构域(例如，dCas9、核酸酶活性Cas9或Cas9切口酶)。在一些实施方案中，本文提供的复合物配置为在核酸中产生突变，例如以校正基因(例如，HFE、HBB或F8)中的点突变，或突变启动子(例如，HBG1或HBG2启动子)以调控在该启动子控制下的一种或多种蛋白质(例如，gamma珠蛋白蛋白质)的表达。
在一些实施方案中，引导RNA包含以下引导序列，所述引导序列包含与靶序列(例如靶DNA序列)100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。在一些实施方案中，引导RNA包含以下引导序列，所述引导序列包含与HBG1或HBG2基因的启动子区域(例如人HBG1或HBG2基因的启动子区域)中的DNA序列100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。在一些实施方案中，人血红蛋白亚基gamma 1(HBG1)是基因ID:3047的HBG1。在一些实施方案中，人血红蛋白亚基gamma 2(HBG2)是基因ID:3048的HBG2。在一些实施方案中，HBG1或HBG2启动子是人、黑猩猩、猿、猴、狗、小鼠或大鼠启动子。在一些实施方案中，HBG1或HBG2启动子是人HBG1或HBG2启动子。在一些实施方案中，HBG1或HBG2启动子是HBG1或HBG2基因上游的100至300个核苷酸。在一些实施方案中，HBG1或HBG2启动子是HBG1或HBG2基因上游的100至300、110至290、120至280、130至270、140至260、150至250、160至240、160至230、170至220、180至210或190至200个核苷酸。在一些实施方案中，驱动HBG1表达的启动子包含HBG1上游的198核苷酸的T(-198T)。在一些实施方案中，将与本文提供的任何融合蛋白(例如腺苷碱基编辑器)复合的gRNA设计为靶向相对于HBG1或HBG2位置-198处的T，引起该T至C的突变。示例性HBG1和HBG2启动子序列分别作为SEQ ID NO:344和345提供。应当理解的是，示例性HBG1和HBG2启动子区域是示例性的且并不意图于限制。
在一些实施方案中，HBG1或HBG2启动子包含以下序列的任一个的核酸序列，如SEQID NO:838-846、297-323和在核酸的5′末端处具有CCT的SEQ ID NO:838-846。在一些实施方案中，靶向以突变为C的T在以下序列中以粗体表示。
5′-CTTGGGGGCCCCTTCCCCACACTA-3′(SEQ ID NO:838)；
5′-CTTGGGGGCCCCTTCCCCACACT-3′(SEQ ID NO:839)；
5′-CTTGGGGGCCCCTTCCCCACAC-3′(SEQ ID NO:840)；
5′-CTTGGGGGCCCCTTCCCCACA-3′(SEQ ID NO:841)；
5′-CTTGGGGGCCCCTTCCCCAC-3′(SEQ ID NO:842)；
5′-CTTGGGGGCCCCTTCCCCA-3′(SEQ ID NO:843)；
5′-CTTGGGGGCCCCTTCCCC-3′(SEQ ID NO:844)；
5′-CTTGGGGGCCCCTTCCC-3′(SEQ ID NO:845)；
5′-CCTCTTGGGGGCCCCTTCCCCACACTA-3′(SEQ ID NO:6)；
5′-CCTCTTGGGGGCCCCTTCCCCACACT-3′(SEQ ID NO:7)；
5′-CCTCTTGGGGGCCCCTTCCCCACAC-3′(SEQ ID NO:15)；
5′-CCTCTTGGGGGCCCCTTCCCCACA-3′(SEQ ID NO:16)；
5′-CCTCTTGGGGGCCCCTTCCCCAC-3′(SEQ ID NO:17)；
5′-CCTCTTGGGGGCCCCTTCCCCA-3′(SEQ ID NO:18)；
5′-CCTCTTGGGGGCCCCTTCCCC-3′(SEQ ID NO:19)；或
5′-CCTCTTGGGGGCCCCTTCCC-3′(SEQ ID NO:20)。
在一些实施方案中，本文提供的任何复合物包含具有以下引导序列的gRNA，所述引导序列包含与以上提供的核酸序列(例如，SEQ ID NO:838-845、297-323和进一步包含5′末端处的CCT的SEQ ID NO:838-845)的任一个100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。应当理解的是，gRNA的引导序列可以包含一个或多个不与靶序列互补的核苷酸。在一些实施方案中，gRNA的引导序列处于gRNA的5′末端。在一些实施方案中，gRNA的引导序列进一步包含gRNA的5′末端处的G。在一些实施方案中，gRNA的5′末端处的G不与靶序列互补。在一些实施方案中，gRNA的引导序列包含不与靶序列(例如，本文提供的任何靶序列)互补的1、2、3、4、5、6、7或8个核苷酸。应当理解的是，启动子序列在个体的基因组之间可能有所差异。因此，本公开提供了具有以下引导序列的gRNA，所述引导序列包含与人的基因组中HBG1或HBG2启动子靶序列100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。
在一些实施方案中，gRNA包含引导序列，所述引导序列包含如下提供的SEQ IDNO:846-853和254-280的任一个。
5′-UCAUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:846)；
5′-CAUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:847)；
5′-AUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:848)；
5′-UGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:849).
5′-GUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:850)；
5′-UGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:851)；
5′-GGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:852)；
5′-GGGAAGGGGCCCCCAAG-3′(SEQ ID NO:853)；
5′-GACAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:254)；
5′-ACAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:255)；
5′-CAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:256)；
5′-AGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:257)；
5′-GAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:258)；
5′-AUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:259)；
5′-AUGCAAAUAUCUGUCUGAAACGG-3′(SEQ ID NO:260)；
5′-GCAAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:261)；
5′-CAAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:262)；
5′-AAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:263)；
5′-AAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:264)；
5′-AUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:265)；
5′-UAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:266)；
5′-AGAUAUUUGCAUUGAGAUAGUGU-3′(SEQ ID NO:267)；
5′-ACAGAUAUUUGCAUUGAGAUAGU-3′(SEQ ID NO:268)；
5′-GUGGGGAAGGGGCCCCCAAGAGG-3′(SEQ ID NO:269)；
5′-CUUGACCAAUAGCCUUGACAAGG-3′(SEQ ID NO:270)；
5′-CUUGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:271)；
5′-UUGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:272)；
5′-UGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:273)；
5′-GUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:274)；
5′-UCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:275)；
5′-CAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:276)；
5′-UUGUCAAGGCUAUUGGUCAAGGC-3′(SEQ ID NO:277)；
5′-CUUGUCAAGGCUAUUGGUCAAGG-3′(SEQ ID NO:278)；
5′-UUGACCAAUAGCCUUGACAAGGC-3′(SEQ ID NO:279)；或
5′-UAGCCUUGACAAGGCAAACUUGA-3′(SEQ ID NO:280)
考虑到个体基因组中的靶序列可能有所差异，应当理解，SEQ ID NO:846-853和254-280中提供的RNA序列可以有一个或多个核碱基的差异。在一些实施方案中，本文提供的任何引导RNA序列的引导序列可以包括相对于SEQ ID NO:846-853和254-280的任一个的1、2、3、4、5、6、7、8、9或10个核碱基变化。在一些实施方案中，gRNA的引导序列进一步包含gRNA的5′末端处的G。因此，本申请提供了SEQ ID NO:846-853和254-280，其进一步包含其5′末端处的G。
在一些实施方案中，引导RNA包含以下引导序列，所述引导序列包含与靶序列例如血色素沉着症(HFE)基因中的靶DNA序列100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。在一些实施方案中，引导RNA包含引导序列，所述引导序列包含与人HFE基因中的DNA序列100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。在一些实施方案中，HFE基因是人、黑猩猩、猿、猴、狗、小鼠或大鼠HFE基因。在一些实施方案中，HFE基因是人HFE基因。在一些实施方案中，HFE基因是基因ID:3077的HFE基因，其也曾称为HH、HFE1、HLA-H、MVCD7和TFQTL2。不希望受任何特定理论的束缚，由该基因编码的HFE蛋白是与MHC I类型蛋白相似的膜蛋白，并与beta2-微球蛋白(beta2M)缔合。据认为该蛋白质通过调节转铁蛋白受体与转铁蛋白的相互作用来发挥调节铁的吸收的功能。铁贮积病症(遗传性血色素沉着症)是由该基因中的缺陷引起的隐性遗传病。对于该基因已经描述了至少九个可变剪接变体。也发现了另外的变体。
HFE基因的示例性编码序列如下所示，其中突变为A从而导致氨基酸残基282处的Cys(C)至Tyr(Y)突变(C282Y)的野生型G以粗体和下划线表示。在一些实施方案中，该突变引起血色素沉着症(例如遗传性血色素沉着症)：ATGGGCCCGCGAGCCAGGCCGGCGCTTCTCCTCCTGATGCTTTTGCAGACCGCGGTCCTGCAGGGGCGCTTGCTGCGTTCACACTCTCTGCACTACCTCTTCATGGGTGCCTCAGAGCAGGACCTTGGTCTTTCCTTGTTTGAAGCTTTGGGCTACGTGGATGACCAGCTGTTCGTGTTCTATGATCATGAGAGTCGCCGTGTGGAGCCCCGAACTCCATGGGTTTCCAGTAGAATTTCAAGCCAGATGTGGCTGCAGCTGAGTCAGAGTCTGAAAGGGTGGGATCACATGTTCACTGTTGACTTCTGGACTATTATGGAAAATCACAACCACAGCAAGGAGTCCCACACCCTGCAGGTCATCCTGGGCTGTGAAATGCAAGAAGACAACAGTACCGAGGGCTACTGGAAGTACGGGTATGATGGGCAGGACCACCTTGAATTCTGCCCTGACACACTGGATTGGAGAGCAGCAGAACCCAGGGCCTGGCCCACCAAGCTGGAGTGGGAAAGGCACAAGATTCGGGCCAGGCAGAACAGGGCCTACCTGGAGAGGGACTGCCCTGCACAGCTGCAGCAGTTGCTGGAGCTGGGGAGAGGTGTTTTGGACCAACAAGTGCCTCCTTTGGTGAAGGTGACACATCATGTGACCTCTTCAGTGACCACTCTACGGTGTCGGGCCTTGAACTACTACCCCCAGAACATCACCATGAAGTGGCTGAAGGATAAGCAGCCAATGGATGCCAAGGAGTTCGAACCTAAAGACGTATTGCCCAATGGGGATGGGACCTACCAGGGCTGGATAACCTTGGCTGTACCCCCTGGGGAAGAGCAGAGATATACGTGCCAGGTGGAGCACCCAGGCCTGGATCAGCCCCTCATTGTGATCTGGGAGCCCTCACCGTCTGGCACCCTAGTCATTGGAGTCATCAGTGGAATTGCTGTTTTTGTCGTCATCTTGTTCATTGGAATTTTGTTCATAATATTAAGGAAGAGGCAGGGTTCAAGAGGAGCCATGGGGCACTACGTCTTAGCTGAACGTGAGTGA(SEQ ID NO:871)
由上述HFE核酸编码序列编码的示例性HFE氨基酸序列如下(SEQ ID NO:750)所示，其中在血色素沉着症中突变为Y的位置282处的C以粗体和下划线表示：
MGPRARPALLLLMLLQTAVLQGRLLRSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDFWTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIWEPSPSGTLVIGVISGIAVFVVILFIGILFIILRKRQGSRGAMGHYVLAER(SEQ ID NO:750)。
在一些实施方案中，HFE基因包含HFE基因的编码序列中的G至A突变，其导致HFE蛋白中的C至Y突变。例如，SEQ ID NO:750中的C282Y突变。在一些实施方案中，HFE基因包含SEQ ID NO:871的核酸残基845中的G至A突变，其导致编码的HFE蛋白中的C至Y突变。在一些实施方案中，本文提供的复合物设计为校正引起血色素沉着症的HFE中的C至Y突变(例如，SEQ ID NO:750的C282Y突变)。应当理解，HFE的编码序列可以在个体之间有所差异。因此，本文提供的任何gRNA的引导序列可以经工程化以解决此类差异，以校正引起血色素沉着症的HFE中的C至Y突变。
在一些实施方案中，HFE基因包含以下序列的任一个的核酸序列，如SEQ ID NO:854-861和在核酸的5′末端处具有CCT的SEQ ID NO:854-861。在一些实施方案中，靶向以突变为C的T在以下序列中以粗体表示。可以使用本文提供的任何复合物使与靶向的T相对的A脱氨基。以下提供的序列是HFE基因的编码序列的部分的反向互补。
5′-GGGTGCTCCACCTGGTACGTATAT-3′(SEQ ID NO:854)；
5′-GGGTGCTCCACCTGGTACGTATA-3′(SEQ ID NO:855)；
5′-GGGTGCTCCACCTGGTACGTAT-3′(SEQ ID NO:856)；
5′-GGGTGCTCCACCTGGTACGTA-3′(SEQ ID NO:857)；
5′-GGGTGCTCCACCTGGTACGT-3′(SEQ ID NO:858)；
5′-GGGTGCTCCACCTGGTACG-3′(SEQ ID NO:859)；
5′-GGGTGCTCCACCTGGTAC-3′(SEQ ID NO:860)；
5′-GGGTGCTCCACCTGGTA-3′(SEQ ID NO:861)；
5′-CCTGGGTGCTCCACCTGGTACGTATAT-3′(SEQ ID NO:21)；
5′-CCTGGGTGCTCCACCTGGTACGTATA-3′(SEQ ID NO:22)；
5′-CCTGGGTGCTCCACCTGGTACGTAT-3′(SEQ ID NO:23)；
5′-CCTGGGTGCTCCACCTGGTACGTA-3′(SEQ ID NO:24)；
5′-CCTGGGTGCTCCACCTGGTACGT-3′(SEQ ID NO:25)；
5′-CCTGGGTGCTCCACCTGGTACG-3′(SEQ ID NO:26)；
5′-CCTGGGTGCTCCACCTGGTAC-3′(SEQ ID NO:29)；或
5′-CCTGGGTGCTCCACCTGGTA-3′(SEQ ID NO:30)。
在一些实施方案中，本文提供的任何复合物包含具有以下引导序列的gRNA，所述引导序列包含与以上提供的核酸序列(例如，SEQ ID NO:854-861和具有5′末端处的CCT的SEQ ID NO:854-861)的任一个100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。应当理解的是，gRNA的引导序列可以包含一个或多个不与靶序列互补的核苷酸。在一些实施方案中，gRNA的引导序列处于gRNA的5′末端。在一些实施方案中，gRNA的引导序列进一步包含gRNA的5′末端处的G。在一些实施方案中，gRNA的5′末端处的G不与靶序列互补。在一些实施方案中，gRNA的引导序列包含不与靶序列(例如，本文提供的任何靶序列)互补的1、2、3、4、5、6、7或8个核苷酸。应当理解的是，HFE基因序列在个体的基因组之间可能有所差异。因此，本公开提供了具有以下引导序列的gRNA，所述引导序列包含与人的基因组中HFE基因靶序列100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。
在一些实施方案中，gRNA包含引导序列，所述引导序列包含如下提供的SEQ IDNO:862-869的任一个。
5′-AUAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:862)；
5′-UAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:863)；
5′-AUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:864)；
5′-UACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:865)；
5′-ACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:866)；
5′-CGUACCAGGUGGAGCACCC-3′(SEQ ID NO:867)；
5′-GUACCAGGUGGAGCACCC-3′(SEQ ID NO:868)；或
5′-UACCAGGUGGAGCACCC-3′(SEQ ID NO:869)。
考虑到个体基因组中的靶序列可能有所差异，应当理解，SEQ ID NO:862-869中提供的RNA序列可以有一个或多个核碱基的差异。在一些实施方案中，本文提供的任何引导RNA序列的引导序列可以包括相对于SEQ ID NO:862-869的任一个的1、2、3、4、5、6、7、8、9或10个核碱基变化。在一些实施方案中，gRNA的引导序列进一步包含gRNA的5′末端处的G。因此，本申请提供了SEQ ID NO:862-869，其进一步包含其5′末端处的G。
在一些实施方案中，引导RNA包含引导序列，所述引导序列包含与靶序列例如beta珠蛋白(HBB)基因中的靶DNA序列100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。在一些实施方案中，引导RNA包含引导序列，所述引导序列包含与人HBB基因中的DNA序列100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。在一些实施方案中，HBB基因是人、黑猩猩、猿、猴、狗、小鼠或大鼠HBB基因。在一些实施方案中，HBB基因是人HBB基因。在一些实施方案中，HBB基因是基因ID:3043的HBB基因，其也曾称为ECYT6、CD113t-C和beta珠蛋白。不希望受任何理论的束缚，血红蛋白亚基beta是珠蛋白蛋白质，其与alpha珠蛋白(HBA)一起构成成年人类中血红蛋白的最常见形式。基因中的突变产生蛋白质的几种变体，这些变体与遗传病症有关，如镰状细胞病和beta地中海贫血，包括血红蛋白C病(HbC)和血红蛋白E病(Hb E)。
HBB基因的示例性编码序列如下所示。在一些实施方案中，该序列经突变(例如A至T突变)以导致镰状细胞病(蛋白质中的E6V突变)。在一些实施方案中，该序列经突变(例如G至A突变)以导致Hb C(蛋白质中的E6K突变)。在一些实施方案中，该序列经突变(例如G至A突变)以导致HbE(蛋白质中的E26K突变)。
示例性HBB基因：
ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC(SEQ ID NO:346)
示例性HBB氨基酸序列如下(SEQ ID NO:340)所示，其中在镰状细胞病中突变为V或在Hb C中突变为K的位置6处的E以粗体和下划线表示。在Hb E中突变为K的位置26处的E也以粗体和下划线表示。
Figure BDA0002539874730001111
在一些实施方案中，HBB基因包含HBB基因的编码序列中的G至A或A至T突变，其导致HBB中的E6V、E6K或E26K突变。例如，SEQ ID NO:340中的E6V、E6K或E26K突变。在一些实施方案中，本文提供的复合物设计为分别校正引起镰状细胞病、Hb C和Hb E的HBB中的E6V(将致病性V突变改变为非致病性A突变)、E6K或E26K突变。应当理解，HBB的编码序列可以在个体之间有所差异。因此，本文提供的任何gRNA的引导序列可以经工程化以解决此类差异，以校正引起本文提供的突变。
在一些实施方案中，HBB基因包含以下序列(如SEQ ID NO:324-337)的任一个的核酸序列，或其反向互补。在一些实施方案中，靶向以突变为C的T在以下序列中以粗体表示。可以使用本文提供的任何复合物使与靶向的T相对的A脱氨基。
5′-GTGCATCTGACTCCTGTGGAGAA-3′(SEQ ID NO:324)；
5′-GGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:325)；
5′-CCATGGTGCATCTGACTCCTGTGGAGAA-3′(SEQ ID NO:326)；
5′-CCATGGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:327)；
5′-CCATGGTGCATCTGACTCCTGTGGAG-3′(SEQ ID NO:328)；
5′-CCATGGTGCATCTGACTCCTGTGGA-3′(SEQ ID NO:329)；
5′-CCATGGTGCATCTGACTCCTGTGG-3′(SEQ ID NO:330)；
5′-CCATGGTGCATCTGACTCCTGTG-3′(SEQ ID NO:331)；
5′-GCATCTGACTCCTGTGGAGAAGT-3′(SEQ ID NO:332)；
5′-ACCATGGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:333)；
5′-ACGGCAGACTTCTCCTTAGGAGT-3′(SEQ ID NO:334)；
5′-CCTGCCCAGGGCCTTACCACCAA-3′(SEQ ID NO:335)；
5′-ACCTGCCCAGGGCCTTACCACCA-3′(SEQ ID NO:336)；或
5′-CCAACCTGCCCAGGGCCTTACCA-3′(SEQ ID NO:337)
在一些实施方案中，本文提供的任何复合物包含具有以下引导序列的gRNA，所述引导序列包含与以上提供的核酸序列(例如，SEQ ID NO:324-337)的任一个100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。应当理解的是，gRNA的引导序列可以包含一个或多个不与靶序列互补的核苷酸。在一些实施方案中，gRNA的引导序列处于gRNA的5′末端。在一些实施方案中，gRNA的引导序列进一步包含gRNA的5′末端处的G。在一些实施方案中，gRNA的5′末端处的G不与靶序列互补。在一些实施方案中，gRNA的引导序列包含不与靶序列(例如，本文提供的任何靶序列)互补的1、2、3、4、5、6、7或8个核苷酸。应当理解的是，HBB基因序列在个体的基因组之间可能有所差异。因此，本公开提供了具有以下引导序列的gRNA，所述引导序列包含与人的基因组中HBB基因靶序列100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。
在一些实施方案中，gRNA包含以下引导序列，所述引导序列包含如下提供的SEQID NO:281-294的任一个。SEQ ID NO:281-290经设计以治疗镰状细胞病(例如，将E6V改变为具有位置6处的A，其是非致病性的)。SEQ ID NO:291经设计以治疗Hb C(例如E6K突变)。SEQ ID NOs:292-294经设计以治疗Hb E(例如E26K突变)。
5′-UUCUCCACAGGAGUCAGAUGCAC-3′(SEQ ID NO:281)；
5′-UCUCCACAGGAGUCAGAUGCACC-3′(SEQ ID NO:282)；
5′-UUCUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:283)；
5′-UCUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:284)；
5′-CUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:285)；
5′-UCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:286)；
5′-CCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:287)；
5′-CACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:288)；
5′-ACUUCUCCACAGGAGUCAGAUGC-3′(SEQ ID NO:289)；
5′-UCUCCACAGGAGUCAGAUGCACCAUGGU-3′(SEQ ID NO:290)；
5′-ACUCCUAAGGAGAAGUCUGCCGU-3′(SEQ ID NO:291)；
5′-UUGGUGGUAAGGCCCUGGGCAGG-3′(SEQ ID NO:292)；
5′-UGGUGGUAAGGCCCUGGGCAGGU-3′(SEQ ID NO:293)；或
5′-UGGUAAGGCCCUGGGCAGGUUGG-3′(SEQ ID NO:294)。
考虑到个体基因组中的靶序列可能有所差异，应当理解，SEQ ID NO:281-294中提供的RNA序列可以有一个或多个核碱基的差异。在一些实施方案中，本文提供的任何引导RNA序列的引导序列可以包括相对于SEQ ID NO:281-294的任一个的1、2、3、4、5、6、7、8、9或10个核碱基变化。在一些实施方案中，gRNA的引导序列进一步包含gRNA的5′末端处的G。因此，本申请提供了SEQ ID NO:281-294，其进一步包含其5′末端处的G。
在一些实施方案中，引导RNA包含以下引导序列，所述引导序列包含与靶序列(例如凝血因子VIII(F8)基因中的靶DNA序列)100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。在一些实施方案中，引导RNA包含以下引导序列，所述引导序列包含与人F8基因中的DNA序列100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。在一些实施方案中，F8基因是人、黑猩猩、猿、猴、狗、小鼠或大鼠F8基因。在一些实施方案中，F8基因是人F8基因。在一些实施方案中，F8基因是基因ID:2157的F8基因，其也曾称为AHF、F8B、F8C、HEMA、FVIII和DXS1253E。不希望受任何特定理论的束缚，F8编码参与血液凝固内在途径的凝血因子VIII；因子VIII是因子IXa的辅因子，其在存在Ca+2和磷脂的情况下将因子X转化为活化形式Xa。该基因的缺陷导致血友病A，这是一种常见的隐性X连锁凝血障碍。
F8基因的示例性编码序列如SEQ ID NO:347所提供。在一些实施方案中，该序列经突变(例如C至T突变)以导致血友病A(蛋白质中的R612C突变)。
示例性凝血因子VIII氨基酸序列如下(SEQ ID NO：341)所示，其中血友病A中经突变为C的位置612处的R以粗体和下划线表示。
Figure BDA0002539874730001141
Figure BDA0002539874730001151
在一些实施方案中，F8基因包含F8基因的编码序列中的C至T突变，其导致凝血因子VIII中的R至C突变。例如，SEQ ID NO:341中的R612C突变。在一些实施方案中，本文提供的复合物设计为校正引起血友病A的F8中的R至C突变(例如R612C)。应当理解，F8的编码序列可以在个体之间有所差异。因此，本文提供的任何gRNA的引导序列可以经工程化以解决此类差异，以校正引起本文提供的突变。
在一些实施方案中，F8基因包含以下序列(如SEQ ID NO:338-339)的任一个的核酸序列，或其反向互补。在一些实施方案中，靶向以突变为C的T在以下序列中以粗体表示。可以使用本文提供的任何复合物使与靶向的T相对的A脱氨基。
5′-CCTCACAGAGAATATACAATGCT-3′(SEQ ID NO:338)；或
5′-TCACAGAGAATATACAATGCTTT-3′(SEQ ID NO:339)。
在一些实施方案中，本文提供的任何复合物包含具有以下引导序列的gRNA，所述引导序列包含与以上提供的核酸序列(例如，SEQ ID NO:338-339)的任一个100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。应当理解的是，gRNA的引导序列可以包含一个或多个不与靶序列互补的核苷酸。在一些实施方案中，gRNA的引导序列处于gRNA的5′末端。在一些实施方案中，gRNA的引导序列进一步包含gRNA的5′末端处的G。在一些实施方案中，gRNA的5′末端处的G不与靶序列互补。在一些实施方案中，gRNA的引导序列包含不与靶序列(例如，本文提供的任何靶序列)互补的1、2、3、4、5、6、7或8个核苷酸。应当理解的是，F8基因序列在个体的基因组之间可能有所差异。因此，本公开提供了具有以下引导序列的gRNA，所述引导序列包含与人的基因组中F8基因靶序列100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续核酸。
在一些实施方案中，gRNA包含以下引导序列，所述引导序列包含如下提供的SEQID NO:295-296的任一个，其经设计以治疗血友病A(例如SEQ ID NO:341的例如R612C突变)。
5′-AGCAUUGUAUAUUCUCUGUGAGG-3′(SEQ ID NO:295)；或
5′-AAAGCAUUGUAUAUUCUCUGUGA-3′(SEQ ID NO:296)
考虑到个体基因组中的靶序列可能有所差异，应当理解，SEQ ID NO:295-296中提供的RNA序列可以有一个或多个核碱基的差异。在一些实施方案中，本文提供的任何引导RNA序列的引导序列可以包括相对于SEQ ID NO:295-296的任一个的1、2、3、4、5、6、7、8、9或10个核碱基变化。在一些实施方案中，gRNA的引导序列进一步包含gRNA的5′末端处的G。因此，本申请提供了SEQ ID NO:295-296，其进一步包含其5′末端处的G。
在一些实施方案中，引导核酸(例如引导RNA)为15-150个核苷酸长且包含与靶序列互补的至少10个连续核苷酸的引导序列。在一些实施方案中，引导RNA为至少15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、60、70、80、90、100、110、120、130、140或150个核苷酸长。在一些实施方案中，引导RNA包含与靶序列(例如本文提供的HBG1或HBG2启动子序列的任一个，或本文提供的HFE、HBB或F8靶序列的任一个)互补的15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39或40个连续核苷酸的引导序列。在一些实施方案中，靶序列是DNA序列。在一些实施方案中，靶序列是哺乳动物的基因组中的序列。在一些实施方案中，靶序列是人的基因组中的序列。在一些实施方案中，靶序列的3′末端与规范PAM序列(NGG)紧邻。在一些实施方案中，引导核酸(例如引导RNA)与和疾病或病症相关的序列互补，所述疾病或病症例如遗传性胎儿血红蛋白持续存在症(HPFH)、beta地中海贫血、遗传性血色素沉着症(HHC)、镰状细胞病、Hb C、Hb E或血友病(例如血友病A)。
使用包含腺苷脱氨酶和核酸可编程DNA结合蛋白(napDNAbp)结构域的融合蛋白的方法
本公开的一些方面提供了使用融合蛋白或包含引导核酸(例如gRNA)和本文提供的核碱基编辑器的复合物的方法。例如，本公开的一些方面提供了包括使DNA或RNA分子与本文提供的任何融合蛋白接触，以及与至少一种引导核酸(例如引导RNA)接触的方法，其中引导核酸(例如引导RNA)包含与靶序列互补的至少10个(例如至少10、15、20、25或30个)连续核苷酸的序列(例如与DNA靶序列结合的引导序列)。在一些实施方案中，靶序列的3’末端与规范PAM序列(NGG)紧邻。在一些实施方案中，靶序列的3’末端不与规范PAM序列(NGG)直接相邻。在一些实施方案中，靶序列的3’末端与AGC、GAG、TTT、GTG或CAA序列紧邻。
表达血红蛋白
本公开的一些方面提供了在细胞(例如哺乳动物细胞)中引入抑制疾病的突变的方法。在一些实施方案中，本公开提供了使用碱基编辑器(例如，本文提供的任何融合蛋白)和gRNA来调控血红蛋白基因(例如，HBG1和/或HBG2)的表达的方法。在一些实施方案中，本公开提供了使用碱基编辑器(例如，本文提供的任何融合蛋白)和gRNA来生成HBG1和/或HBG2的启动子区域中的A至T和/或T至C突变的方法。
患有罕见良性状况遗传性胎儿血红蛋白持续存在症(HPFH)的人类对一些β-珠蛋白疾病(包括镰状细胞贫血)有抗性。在某些患者中，这种表型由γ-珠蛋白基因HBG1和HBG2的启动子中的突变介导，所述突变使胎儿血红蛋白能够持续表达，所述胎儿血红蛋白通常在人出生后沉默。不希望受任何特定理论的束缚，在HBG1或HBG2的启动子区域中生成一个或多个突变可以增加HBG1和/或HBG2的表达以治疗β-珠蛋白疾病。
在一些实施方案中，方法包括通过使启动子与碱基编辑器和与该碱基编辑器结合的引导RNA接触来使HBG1或HBG2基因的启动子中的腺苷核碱基(A)脱氨基，其中引导RNA(gRNA)包含与HBG1和/或HBG2基因的启动子中的靶核酸序列互补的引导序列。应当理解，HBG1和HBG2基因的启动子可以包括本文所述的HBG1和HBG2启动子的任何序列。在一些实施方案中，gRNA的引导序列包含与HBG1或HBG2的启动子序列中的靶核酸序列100％互补的至少5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40个或更多个连续核酸。
在一些实施方案中，方法进一步包括对靶序列进行切口，其可以通过使用核酸可编程结合蛋白(napDNAbp)，如对与gRNA的引导序列互补的靶序列进行切口的Cas9切口酶(nCas9)来实现。在一些实施方案中，启动子中的靶核酸序列包含核酸序列
5′-CTTGGGGGCCCCTTCCCCACACTA-3′(SEQ ID NO:838)；
5′-CTTGGGGGCCCCTTCCCCACACT-3′(SEQ ID NO:839)；
5′-CTTGGGGGCCCCTTCCCCACAC-3′(SEQ ID NO:840)；
5′-CTTGGGGGCCCCTTCCCCACA-3′(SEQ ID NO:841)；
5′-CTTGGGGGCCCCTTCCCCAC-3′(SEQ ID NO:842)；
5′-CTTGGGGGCCCCTTCCCCA-3′(SEQ ID NO:843)；
5′-CTTGGGGGCCCCTTCCCC-3′(SEQ ID NO:844)；
5′-CTTGGGGGCCCCTTCCC-3′(SEQ ID NO:845)；或
SEQ ID NO:297-323的任一个。
应当理解，SEQ ID NO:838-845的核酸的任一个可以进一步在其5′末端处包含5′-CCT-3′。
在一些实施方案中，gRNA的引导序列包含核酸序列
5′-UCAUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:846)；
5′-CAUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:847)；
5′-AUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:848)；
5′-UGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:849).
5′-GUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:850)；
5′-UGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:851)；
5′-GGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:852)；
5′-GGGAAGGGGCCCCCAAG-3′(SEQ ID NO:853)或
SEQ ID NO:254-280的任一个。
应当理解，SEQ ID NO:846-853或254-280的核酸的任一个可以进一步在其5′末端处包含G。
在一些实施方案中，使HBG1或HBG2的启动子中的腺苷核碱基脱氨基导致启动子中的T-A碱基对突变为启动子中的C-G碱基对。在一些实施方案中，使启动子中的腺苷核碱基脱氨基导致与遗传性胎儿血红蛋白持续存在症(HPFH)相关的序列。在一些实施方案中，遗传性胎儿血红蛋白持续存在症表征为良性状况，其中胎儿血红蛋白(例如血红蛋白F)在成年期表达。在一些实施方案中，HPFH的特征在于在2岁或以上、5岁或以上、10岁或以上、15岁或以上、20岁或以上、25岁或以上或30岁或以上的受试者中表达胎儿血红蛋白。在一些实施方案中，若HPFH以比2岁或以上、5岁或以上、10岁或以上、15岁或以上、20岁或以上、25岁或以上或30岁或以上的正常受试者中至少5％、10％、15％、20％、25％、30％、40％、50％、60％、70％、80％或更多更高的水平表达，则认为HPFH在成年人中表达。
在一些实施方案中，使HBG1基因的启动子中的腺苷核碱基脱氨基导致HBG1基因的转录相对于表达HBG1的健康成年人或胎儿增加至少5％、10％、15％、20％、25％、30％、40％、50％、60％、70％、80％、90％、100％、200％、500％、1000％或更多。在一些实施方案中，使HBG2基因的启动子中的腺苷核碱基脱氨基导致HBG2基因的转录相对于表达HBG1的健康成年人或胎儿增加至少5％、10％、15％、20％、25％、30％、40％、50％、60％、70％、80％、90％、100％、200％、500％、1000％或更多。在一些实施方案中，使HBG1基因的启动子中的腺苷核碱基脱氨基导致HBG1蛋白水平相对于表达HBG1的健康成年人或胎儿增加至少5％、10％、15％、20％、25％、30％、40％、50％、60％、70％、80％、90％、100％、200％、500％、1000％或更多。在一些实施方案中，使HBG2基因的启动子中的腺苷核碱基脱氨基导致HBG2蛋白水平相对于表达HBG2的健康成年人或胎儿增加至少5％、10％、15％、20％、25％、30％、40％、50％、60％、70％、80％、90％、100％、200％、500％、1000％或更多。
在一些实施方案中，方法在体外进行，例如在培养的细胞中。在一些实施方案中，方法在体内进行。在一些实施方案中，方法离体进行。在一些实施方案中，方法在受试者的细胞中进行。在一些实施方案中，受试者患有或疑似患有血液的疾病或病症。在一些实施方案中，疾病或病症是贫血。在一些实施方案中，贫血是是缺铁性贫血、再生障碍性贫血、溶血性贫血、地中海贫血、镰状细胞贫血、恶性贫血或范科尼贫血(fanconi anemia)。在一些实施方案中，疾病或病症由编码珠蛋白蛋白质的基因或基因的启动子中的突变引起，所述基因例如CYGB、HBA1、HBA2、HBB、HBD、HBE1、HBG1、HBG2、HBM、HBQ1、HBZ或MB。
校正HFE基因中的突变
本公开的一些方面提供了使用碱基编辑器(例如本文提供的任何融合蛋白)和gRNA以校正HFE基因中的点突变的方法。在一些实施方案中，本公开提供了使用碱基编辑器(例如本文提供的任何融合蛋白)和gRNA以生成HFE基因中的A至G和/或T至C突变的方法。在一些实施方案中，本公开提供了用于使HFE基因中的腺苷核碱基(A)脱氨基的方法，该方法包括使HFE基因与碱基编辑器和与该碱基编辑器结合的引导RNA接触，其中引导RNA包含与HFE基因中的靶核酸序列互补的引导序列。在一些实施方案中，HFE基因包含C至T或G至A突变。在一些实施方案中，HFE基因中的C至T或G至A突变损害由HFE基因编码的HFE蛋白的功能。在一些实施方案中，HFE基因中的C至T或G至A突变损害由HFE基因编码的HFE蛋白的功能至少1％、2％、5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％或至少99％。在一些实施方案中，HFE蛋白的功能是铁吸收。
在一些实施方案中，使与T互补的腺苷(A)核碱基脱氨基校正HFE基因中的C至T或G至A突变。在一些实施方案中，HFE基因中的C至T或G至A突变导致由HFE基因编码的HFE蛋白中的Cys(C)至Tyr(Y)突变。在一些实施方案中，使与T互补的腺苷核碱基脱氨基校正HFE蛋白中的Cys至Tyr突变。
在一些实施方案中，gRNA的引导序列包含与HFE基因的靶核酸序列100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或35个连续核酸。在一些实施方案中，碱基编辑器对与引导序列互补的靶序列进行切口。在一些实施方案中，HFE基因中的靶核酸序列包含核酸序列：
5′-GGGTGCTCCACCTGGTACGTATAT-3′(SEQ ID NO:854)；
5′-GGGTGCTCCACCTGGTACGTATA-3′(SEQ ID NO:855)；
5′-GGGTGCTCCACCTGGTACGTAT-3′(SEQ ID NO:856)；
5′-GGGTGCTCCACCTGGTACGTA-3′(SEQ ID NO:857)；
5′-GGGTGCTCCACCTGGTACGT-3′(SEQ ID NO:858)；
5′-GGGTGCTCCACCTGGTACG-3′(SEQ ID NO:859)；
5′-GGGTGCTCCACCTGGTAC-3′(SEQ ID NO:860)；或
5′-GGGTGCTCCACCTGGTA-3′(SEQ ID NO:861)。
应当理解，SEQ ID NO:854-861的核酸的任一个可以进一步在其5′末端处包含5′-CCT-3′。
在一些实施方案中，gRNA的引导序列包含核酸序列：
5′-AUAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:862)；
5′-UAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:863)；
5′-AUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:864)；
5′-UACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:865)；
5′-ACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:866)；
5′-CGUACCAGGUGGAGCACCC-3′(SEQ ID NO:867)；
5′-GUACCAGGUGGAGCACCC-3′(SEQ ID NO:868)；或
5′-UACCAGGUGGAGCACCC-3′(SEQ ID NO:869)。
应当理解，SEQ ID NO:862-869的核酸的任一个可以进一步在其5′末端处包含G。
在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基导致HFE基因中的T-A碱基对突变为HFE基因中的C-G碱基对。在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基导致校正与遗传性血色素沉着症(HHC)相关的序列。在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基导致HFE蛋白功能增加。在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基导致HFE蛋白功能与野生型HFE蛋白功能相比增加至至少20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或至少100％。在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基导致减少血色素沉着症的一种或多种症状。在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基导致铁吸收功能的增加。在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基导致铁吸收功能增加至正常铁吸收水平(例如不具有血色素沉着症的受试者中的铁吸收的水平)的至少1％、2％、5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％或至少99％。在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基引起从HFE基因转录的HFE蛋白的功能增加。在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基引起HFE稳定性或半衰期增加，例如增加至少5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％或至少99％。
在一些实施方案中，HFE基因在细胞中，如培养中的细胞(例如，永生化的淋巴样干细胞(LCL))或受试者中的细胞。在一些实施方案中，HFE基因编码包含Cys至Tyr突变的HFE蛋白。在一些实施方案中，HFE蛋白包含氨基酸序列SEQ ID NO:750的残基282处的Cys至Tyr突变(C282Y)，其中位置282处的Cys以粗体显示：
Figure BDA0002539874730001221
在一些实施方案中，方法在体内进行。在一些实施方案中，方法在体内进行。在一些实施方案中，方法离体进行。在一些实施方案中，方法在受试者的细胞中进行。在一些实施方案中，受试者患有或疑似患有铁贮积病症。在一些实施方案中，受试者患有或疑似患有血色素沉着症。在一些实施方案中，受试者患有或疑似患有遗传性血色素沉着症(HHC)。在一些实施方案中，受试者具有HFE基因中的突变，其中突变为A的野生型G(例如SEQ ID NO:871中的G845A突变)导致Cys(C)至Tyr(Y)突变，例如SEQ ID NO:750的氨基酸残基282处(C282Y)。在一些实施方案中，该突变导致血色素沉着症(例如遗传性血色素沉着症)。在一些实施方案中，使HFE基因中的腺苷核碱基脱氨基改善受试者中铁贮积病症的一种或多种症状。
校正/生成HBB基因中的突变
本公开的一些方面提供了使用碱基编辑器(例如本文提供的任何融合蛋白)和gRNA以校正HBB基因中的点突变或生成非致病性的点突变的方法。在一些实施方案中，本公开提供了使用碱基编辑器(例如本文提供的任何融合蛋白)和gRNA以生成HBB基因中的A至G和/或T至C突变的方法。在一些实施方案中，本公开提供了用于使HBB基因中的腺苷核碱基(A)脱氨基的方法，该方法包括使HBB基因与碱基编辑器和与该碱基编辑器结合的引导RNA接触，其中引导RNA包含与HBB基因中的靶核酸序列互补的引导序列。在一些实施方案中，HBB基因包含A至T或G至A突变。在一些实施方案中，HBB基因中的A至T或G至A突变损害由HBB基因编码的beta珠蛋白蛋白质的功能。在一些实施方案中，HBB基因中的A至T或G至A突变损害由HBB基因编码的HBB蛋白的功能至少1％、2％、5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％或至少99％。在一些实施方案中，HBB蛋白的功能是携氧能力。在一些实施方案中，A至T突变导致镰状细胞病，并且将T:A碱基对改变为C:G碱基对产生非致病性点突变，所述点突变降低血红蛋白聚合的能力，例如至少1％、2％、5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％或至少99％。
在一些实施方案中，使与T互补的腺苷(A)核碱基脱氨基校正HBB基因中的C至T或G至A突变，或生成非致病性突变。在一些实施方案中，HBB基因中的A至T或G至A突变引起由HBB基因编码的HBB蛋白中的E至V突变或E至K突变。在一些实施方案中，使与T互补的腺苷核碱基脱氨基校正HBB蛋白中的E至K突变。例如，使与T互补的腺苷(A)核碱基脱氨基校正HBB中的E6K(Hb C)或E26K(Hb E)突变，例如与SEQ ID NO:340相比。在一些实施方案中，使与T互补的腺苷核碱基脱氨基将致病性突变改变为非致病性突变。例如，HBB中的E6V突变(例如与SEQ ID NO:340相比)可以经突变以生成位置6处的A(例如V6A突变)以产生非致病性突变。
在一些实施方案中，gRNA的引导序列包含与HBB基因的靶核酸序列100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或35个连续核酸。在一些实施方案中，碱基编辑器对与引导序列互补的靶序列进行切口。在一些实施方案中，HBB基因中的靶核酸序列包含核酸序列：
5′-GTGCATCTGACTCCTGTGGAGAA-3′(SEQ ID NO:324)；
5′-GGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:325)；
5′-CCATGGTGCATCTGACTCCTGTGGAGAA-3′(SEQ ID NO:326)；
5′-CCATGGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:327)；
5′-CCATGGTGCATCTGACTCCTGTGGAG-3′(SEQ ID NO:328)；
5′-CCATGGTGCATCTGACTCCTGTGGA-3′(SEQ ID NO:329)；
5′-CCATGGTGCATCTGACTCCTGTGG-3′(SEQ ID NO:330)；
5′-CCATGGTGCATCTGACTCCTGTG-3′(SEQ ID NO:331)；
5′-GCATCTGACTCCTGTGGAGAAGT-3′(SEQ ID NO:332)；
5′-ACCATGGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:333)；
5′-ACGGCAGACTTCTCCTTAGGAGT-3′(SEQ ID NO:334)；
5′-CCTGCCCAGGGCCTTACCACCAA-3′(SEQ ID NO:335)；
5′-ACCTGCCCAGGGCCTTACCACCA-3′(SEQ ID NO:336)；或
5′-CCAACCTGCCCAGGGCCTTACCA-3′(SEQ ID NO:337)。
在一些实施方案中，gRNA的引导序列包含核酸序列：
5′-UUCUCCACAGGAGUCAGAUGCAC-3′(SEQ ID NO:281)；
5′-UCUCCACAGGAGUCAGAUGCACC-3′(SEQ ID NO:282)；
5′-UUCUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:283)；
5′-UCUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:284)；
5′-CUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:285)；
5′-UCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:286)；
5′-CCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:287)；
5′-CACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:288)；
5′-ACUUCUCCACAGGAGUCAGAUGC-3′(SEQ ID NO:289)；
5′-UCUCCACAGGAGUCAGAUGCACCAUGGU-3′(SEQ ID NO:290)；
5′-ACUCCUAAGGAGAAGUCUGCCGU-3′(SEQ ID NO:291)；
5′-UUGGUGGUAAGGCCCUGGGCAGG-3′(SEQ ID NO:292)；
5′-UGGUGGUAAGGCCCUGGGCAGGU-3′(SEQ ID NO:293)；或
5′-UGGUAAGGCCCUGGGCAGGUUGG-3′(SEQ ID NO:294)。
应当理解，SEQ ID NO:281-294的核酸的任一个可以进一步在其5′末端处包含G。
在一些实施方案中，使HBB基因中的腺苷核碱基脱氨基导致HBB基因中的T-A碱基对突变为HBB基因中的C-G碱基对。在一些实施方案中，使HBB基因中的腺苷核碱基脱氨基导致校正与Hb C或Hb E相关的序列；或导致生成非致病性突变。在一些实施方案中，使HBB基因中的腺苷核碱基脱氨基导致HBB蛋白功能增加。在一些实施方案中，使HBB基因中的腺苷核碱基脱氨基导致与野生型HBB蛋白功能相比HBB蛋白功能增加至至少20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或至少100％。在一些实施方案中，使HBB基因中的腺苷核碱基脱氨基导致减少镰状细胞病、HbC或Hb E的一种或多种症状。在一些实施方案中，使HBB基因中的腺苷核碱基脱氨基导致携氧功能的增加，或beta珠蛋白聚合的降低，或细胞镰状的减少。在一些实施方案中，使HBB基因中的腺苷核碱基脱氨基导致携氧功能增加至正常携氧功能水平(例如不具有镰状细胞病、Hb C和/或Hb E的受试者中的携氧功能(例如氧饱和)的水平)的至少1％、2％、5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％或至少99％。在一些实施方案中，使HBB基因中的腺苷核碱基脱氨基引起从HBB基因转录的HBB蛋白的功能增加。在一些实施方案中，使HBB基因中的腺苷核碱基脱氨基引起HBB稳定性或半衰期增加，例如增加至少5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％或至少99％。
在一些实施方案中，HBB基因在细胞中，如培养中的细胞或受试者中的细胞。在一些实施方案中，HBB基因编码包含E至V或E至K突变的HBB蛋白。在一些实施方案中，HBB蛋白包含氨基酸序列SEQ ID NO:340的残基6处的E至V突变(E6V)(镰状细胞病)，其中位置6处的E以粗体显示。在一些实施方案中，HBB蛋白包含氨基酸序列SEQ ID NO:340的残基6处的E至K突变(E6K)(Hb C)，其中位置6处的E以粗体显示。在一些实施方案中，HBB蛋白包含氨基酸序列SEQ ID NO:340的残基6处的E至K突变(E26K)(Hb E)，其中位置26处的E以粗体和下划线显示：
Figure BDA0002539874730001261
在一些实施方案中，方法在体内进行。在一些实施方案中，方法在体内进行。在一些实施方案中，方法离体进行。在一些实施方案中，方法在受试者的细胞中进行。在一些实施方案中，受试者患有或疑似患有铁贮积病症。在一些实施方案中，受试者患有或疑似患有镰状细胞病或beta地中海贫血。在一些实施方案中，受试者患有或疑似患有Hb C或Hb E。在一些实施方案中，受试者具有HBB基因中的突变，其中野生型A突变为T，或野生型G突变为A。在一些实施方案中，使HBB基因中的腺苷核碱基脱氨基改善受试者中镰状细胞病、Hb C或HbE的一种或多种症状。
校正F8基因中的突变
本公开的一些方面提供了使用碱基编辑器(例如本文提供的任何融合蛋白)和gRNA以校正F8基因中的点突变的方法。在一些实施方案中，本公开提供了使用碱基编辑器(例如本文提供的任何融合蛋白)和gRNA以生成F8基因中的A至G和/或T至C突变的方法。在一些实施方案中，本公开提供了用于使F8基因中的腺苷核碱基(A)脱氨基的方法，该方法包括使F8基因与碱基编辑器和与该碱基编辑器结合的引导RNA接触，其中引导RNA包含与F8基因中的靶核酸序列互补的引导序列。在一些实施方案中，F8基因包含C至T或G至A突变。在一些实施方案中，F8基因中的C至T或G至A突变损害由F8基因编码的凝血因子VIII蛋白质的功能。在一些实施方案中，F8基因中的C至T或G至A突变损害由F8基因编码的凝血因子VIII蛋白质的功能至少1％、2％、5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％或至少99％。在一些实施方案中，凝血因子VIII蛋白质的功能是血液凝结。
在一些实施方案中，使与T互补的腺苷(A)核碱基脱氨基校正F8基因中的C至T或G至A突变。在一些实施方案中，F8基因中的C至T或G至A突变引起由F8基因编码的因子VIII蛋白质中的R至C突变。在一些实施方案中，使与T互补的腺苷核碱基脱氨基校正因子VIII蛋白质中的R至C突变。
在一些实施方案中，gRNA的引导序列包含与F8基因的靶核酸序列100％互补的至少8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或35个连续核酸。在一些实施方案中，碱基编辑器对与引导序列互补的靶序列进行切口。在一些实施方案中，F8基因中的靶核酸序列包含核酸序列：
5′-CCTCACAGAGAATATACAATGCT-3′(SEQ ID NO:338)；或
5′-TCACAGAGAATATACAATGCTTT-3′(SEQ ID NO:339)。
在一些实施方案中，gRNA的引导序列包含核酸序列：
5′-AGCAUUGUAUAUUCUCUGUGAGG-3′(SEQ ID NO:295)；或
5′-AAAGCAUUGUAUAUUCUCUGUGA-3′(SEQ ID NO:296)。
应当理解，SEQ ID NO:295-296的核酸的任一个可以进一步在其5′末端处包含G。
在一些实施方案中，使F8基因中的腺苷核碱基脱氨基导致F8基因中的T-A碱基对突变为F8基因中的C-G碱基对。在一些实施方案中，使F8基因中的腺苷核碱基脱氨基导致校正与血友病(例如血友病A)相关的序列。在一些实施方案中，使F8基因中的腺苷核碱基脱氨基导致因子VIII蛋白质功能增加。在一些实施方案中，使F8基因中的腺苷核碱基脱氨基导致与野生型因子VIII蛋白质功能相比因子VIII蛋白质功能增加至至少20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或至少100％。在一些实施方案中，使F8基因中的腺苷核碱基脱氨基导致减少血友病的一种或多种症状。在一些实施方案中，使F8基因中的腺苷核碱基脱氨基导致血液凝结功能的增加。在一些实施方案中，使F8基因中的腺苷核碱基脱氨基导致血液凝结功能增加至正常血液凝结功能水平(例如不具有血友病的受试者中的血液凝结功能的水平)的至少1％、2％、5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％或至少99％。在一些实施方案中，使F8基因中的腺苷核碱基脱氨基引起从F8基因转录的因子VIII蛋白质的功能增加。在一些实施方案中，使F8基因中的腺苷核碱基脱氨基引起因子VIII稳定性或半衰期增加，例如增加至少5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％或至少99％。
在一些实施方案中，F8基因在细胞中，如培养中的细胞或受试者中的细胞。在一些实施方案中，F8基因编码包含R至C突变的因子VIII蛋白质。在一些实施方案中，因子VIII蛋白质包含氨基酸序列SEQ ID NO:341的残基612处的R至C突变(R612C)，其中位置612处的R以粗体显示：
Figure BDA0002539874730001281
Figure BDA0002539874730001291
在一些实施方案中，方法在体内进行。在一些实施方案中，方法在体内进行。在一些实施方案中，方法离体进行。在一些实施方案中，方法在受试者的细胞中进行。在一些实施方案中，受试者患有或疑似患有血友病。在一些实施方案中，受试者患有或疑似患有血友病A。在一些实施方案中，受试者具有F8基因中的突变，其中野生型G突变为A导致R至C突变，例如SEQ ID NO:341的氨基酸残基612处(R612C)。在一些实施方案中，该突变导致血友病(例如血友病A)。在一些实施方案中，使F8基因中的腺苷核碱基脱氨基改善受试者中血友病的一种或多种症状。
在一些实施方案中，靶DNA序列包含与疾病或病症相关的序列。在一些实施方案中，靶DNA序列包含与疾病或病症相关的点突变。在一些实施方案中，融合蛋白(例如，包含腺苷脱氨酶和Cas9结构域)或复合物的活性导致点突变的校正。在一些实施方案中，靶DNA序列包含与疾病或病症相关的G→A或C→T点突变，并且其中突变体A碱基的脱氨基作用产生与疾病或病症无关的序列。在一些实施方案中，靶DNA序列编码蛋白质，并且点突变在密码子中并导致与野生型密码子相比由突变体密码子编码的氨基酸中的变化。在一些实施方案中，突变体A的脱氨基作用导致由突变体密码子编码的氨基酸的变化。在一些实施方案中，突变体A的脱氨基作用产生编码野生型氨基酸的密码子。在一些实施方案中，接触在受试者体内。在一些实施方案中，受试者具有或已经诊断患有疾病或病症。
一些实施方案提供了使用本文提供的DNA编辑融合蛋白的方法。在一些实施方案中，融合蛋白用于通过使靶核碱基(例如A残基)脱氨基而将点突变引入到核酸中。在一些实施方案中，靶核碱基的脱氨基作用导致遗传缺陷的校正，例如在校正导致基因产物中功能丧失的点突变中。在一些实施方案中，遗传缺陷与疾病或病症例如遗传性血色素沉着症相关。在一些实施方案中，本文提供的方法用于将点突变引入到调控(例如增加或减少)基因表达的基因(例如HBG1或HBG2)的启动子中。例如，在一些实施方案中，本文提供了采用DNA编辑融合蛋白将点突变引入到HBG1或HBG2的启动子区域中以增加HBG1或HBG2的表达的方法。在一些实施方案中，此类点突变可以增加受试者中HBG1或HBG2的表达，这可以用于治疗β-珠蛋白疾病包括镰状细胞贫血。
在一些实施方案中，本文中提供的方法的目的是经由基因组编辑恢复功能失调基因的功能。可以验证本文提供的核碱基编辑蛋白以用于体外基于基因编辑的人治疗学，例如通过校正人细胞培养物中的疾病相关的突变。本领域技术人员将理解，本文提供的核碱基编辑蛋白，例如包含核酸可编程DNA结合蛋白(例如，Cas9)和腺苷脱氨酶结构域的融合蛋白可以用于校正任何单点G至A或C至T突变。在第一种情况下，突变体A脱氨基为I校正突变，而在后一种情况下，与突变体T碱基配对的A的脱氨基作用及随后的一轮复制或随后的碱基编辑修复活性校正突变。
本公开提供了用于治疗诊断患有与点突变相关或由点突变引起的疾病的受试者的方法，所述点突变可以通过本文提供的DNA编辑融合蛋白进行校正。例如，在一些实施方案中，提供的方法包括向患有此类疾病(例如，贫血或血色素沉着症)的受试者施用有效量的校正基因(例如HFE)中的点突变或将突变引入到基因的启动子区域(例如HBG1或HBG2的启动子区域)中。在一些实施方案中，疾病是遗传疾病。在一些实施方案中，疾病是与贫血相关疾病。在一些实施方案中，疾病是铁贮积疾病(例如遗传性血色素沉着症)。在一些实施方案中，疾病是溶酶体贮积病。可以通过校正点突变或将失活性突变引入到疾病相关基因中来治疗的其他疾病对于本领域技术人员来说是已知的，并且本公开内容在这方面不受限制。
在一些实施方案中，融合蛋白识别规范PAM，并因此可以在侧翼序列中具有规范PAM(例如NGG)的情况下校正致病性G至A或C至T突变。例如，识别规范PAM的Cas9蛋白包含与如SEQ ID NO:52提供的酿脓链球菌的氨基酸序列，或与包含SEQ ID NO:52的RuvC和HNH结构域的其片段至少80％、85％、90％、95％、97％、98％或99％相同的氨基酸序列。
对于本领域技术人员显而易见的是，为了将如本文所公开的包含Cas9结构域和腺苷脱氨酶的任何融合蛋白靶向到靶位点，例如包含待编辑的点突变的位点，通常必需将融合蛋白与引导RNA(例如sgRNA)一起共表达。如本文其他地方更详细解释的，引导RNA通常包含允许Cas9结合的tracrRNA框架和引导序列，其将序列特异性赋予Cas9:核酸编辑酶/结构域融合蛋白。在一些实施方案中，引导RNA包含结构5’-[引导序列]-guuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu-3’(SEQ ID NO:389)，其中引导序列包含与靶序列互补的序列。在一些实施方案中，引导序列包含表2中提供的任何核苷酸序列。引导序列通常为20个核苷酸长。基于本公开，用于将Cas9:核酸编辑酶/结构域融合蛋白靶向至特定基因组靶位点的合适的引导RNA的序列对于本领域技术人员而言将是显而易见的。此类合适的引导RNA序列通常包含与待编辑的靶核苷酸的上游或下游50个核苷酸内的核酸序列互补的引导序列。本文提供了适合于将任何提供的融合蛋白靶向到特定靶序列的一些示例性引导RNA序列。另外的引导序列如下显示于表3中，包括它们的基因座。
碱基编辑器效率
本公开的一些方面基于认识到本文提供的任何碱基编辑器能够修饰特定的核苷酸碱基而不产生显著比例的插入/缺失。如本文所用，“插入/缺失”是指核酸内的核苷酸碱基的插入或缺失。此类插入或缺失可以导致基因编码区内的移码突变。在一些实施方案中，期望产生有效修饰(例如突变或脱氨基)核酸内的特定核苷酸而不在核酸中产生大量插入或缺失(即插入/缺失)的碱基编辑器。在某些实施方案中，本文提供的任何碱基编辑器能够相对于插入/缺失产生更大比例的意图修饰(例如，点突变或脱氨基作用)。在一些实施方案中，本文提供的碱基编辑器能够生成大于1:1的意图点突变与插入/缺失的比率。在一些实施方案中，本文提供的碱基编辑器能够产生意图点突变与插入/缺失的比率，其为至少1.5:1、至少2:1、至少2.5:1、至少3:1、至少3.5:1、至少4:1、至少4.5:1、至少5:1、至少5.5:1、至少6:1、至少6.5:1、至少7:1、至少7.5:1、至少8:1、至少10:1、至少12:1、至少15:1、至少20:1、至少25:1、至少30:1、至少40:1、至少50:1、至少100:1、至少200:1、至少300:1、至少400:1、至少500:1、至少600:1、至少700:1、至少800:1、至少900:1或至少1000:1或更多。可以使用任何合适的方法，例如以下实施例中使用的方法来测定意图的突变和插入/缺失的数目。在一些实施方案中，为了计算插入/缺失频率，扫描测序读段以与两个10-bp序列精确匹配，所述两个10-bp序列在可能发生插入/缺失的窗口的两侧侧翼。若未定位精确匹配，则从分析中排除读段。若该插入/缺失窗口的长度与参照序列精确匹配，则读段分类为不含有插入/缺失。若插入/缺失窗口比参照序列长或短两个或更多个碱基，则测序读段分别分类为插入或缺失。
在一些实施方案中，本文提供的碱基编辑器能够限制核酸的区域中的插入/缺失的形成。在一些实施方案中，区域在由碱基编辑器靶向的核苷酸处或由碱基编辑器靶向的核苷酸的2、3、4、5、6、7、8、9或10个核苷酸内的区域。在一些实施方案中，本文提供的任何碱基编辑器能够将核酸的区域处的插入/缺失的形成限制至小于1％、小于1.5％、小于2％、小于2.5％、小于3％、小于3.5％、小于4％、小于4.5％、小于5％、小于6％、小于7％、小于8％、小于9％、小于10％、小于12％、小于15％或小于20％。在核酸区域处形成的缺失/缺失的数目可以取决于核酸(例如细胞的基因组内的核酸)暴露于碱基编辑器的时间的量。在一些实施方案中，在将核酸(例如，细胞的基因组内的核酸)暴露于碱基编辑器的至少1小时、至少2小时、至少6小时、至少12小时、至少24小时、至少36小时、至少48小时、至少3天、至少4天、至少5天、至少7天、至少10天或至少14天后测定插入/缺失的数目或比例。
本公开的一些方面基于认识到本文提供的任何碱基编辑器能够有效地在核酸(例如受试者的基因组内的核酸)中产生意图的突变(如点突变)而不产生大量的非意图突变(如非意图的点突变)。在一些实施方案中，意图的突变是由与gRNA结合的特定碱基编辑器产生的突变，所述碱基编辑器特别设计为产生意图的突变。在一些实施方案中，意图的突变是与疾病或病症相关的突变。在一些实施方案中，意图的突变是与疾病或病症相关的腺嘌呤(A)至鸟嘌呤(G)点突变。在一些实施方案中，意图的突变是与疾病或病症相关的胸腺嘧啶(T)至胞嘧啶(C)点突变。在一些实施方案中，意图的突变是基因的编码区内的腺嘌呤(A)至鸟嘌呤(G)点突变。在一些实施方案中，意图的突变是基因的编码区内的胸腺嘧啶(T)至胞嘧啶(C)点突变。在一些实施方案中，意图的突变是产生终止密码子，例如基因的编码区内的提前终止密码子的点突变。在一些实施方案中，意图的突变是消除终止密码子的突变。在一些实施方案中，意图的突变是改变基因的剪接的突变。在一些实施方案中，意图的突变是改变基因的调控序列(例如，基因启动子或基因阻抑物)的突变。在一些实施方案中，本文提供的任何碱基编辑器能够产生大于1:1的意图突变与非意图突变(例如，意图的点突变:非意图的点突变)的比率。在一些实施方案中，本文提供的任何碱基编辑器能够产生意图突变与非意图突变的比率(例如，意图的点突变:非意图的点突变)，其为至少1.5:1、至少2:1、至少2.5:1、至少3:1、至少3.5:1、至少4:1、至少4.5:1、至少5:1、至少5.5:1、至少6:1、至少6.5:1、至少7:1、至少7.5:1、至少8:1、至少10:1、至少12:1、至少15:1、至少20:1、至少25:1、至少30:1、至少40:1、至少50:1、至少100:1、至少150:1、至少200:1、至少250:1、至少500:1或至少1000:1或更多。应当理解，本文“碱基编辑器效率”部分中描述的碱基编辑器的特性可以应用于任何融合蛋白或使用本文提供的融合蛋白的方法。
用于编辑核酸的方法
本公开的一些方面提供了用于编辑核酸的方法。在一些实施方案中，该方法是用于编辑核酸的核碱基(例如，双链DNA序列的碱基对)的方法。在一些实施方案中，方法包括以下步骤：a)使核酸(例如双链DNA序列)的靶区域与包含碱基编辑器(例如与腺苷脱氨酶融合的Cas9结构域)和引导核酸(例如gRNA)的复合物接触，其中所述靶区域包含靶向的核碱基对，b)诱导所述靶区域的链分离，c)将靶区域的单链中的所述靶核碱基对的第一核碱基转换成第二核碱基，以及d)切割所述靶区域的不超过一条链，其中与所述第一核碱基碱基互补的第三核碱基被与第二核碱基互补的第四核碱基替换。在一些实施方案中，该方法导致核酸中小于20％的插入/缺失形成。应当理解的是，在一些实施方案中，省略步骤b。在一些实施方案中，第一核碱基是腺嘌呤。在一些实施方案中，第二核碱基是脱氨基的腺嘌呤或肌苷。在一些实施方案中，第三核碱基是胸腺嘧啶。在一些实施方案中，第四核碱基是胞嘧啶。在一些实施方案中，方法导致小于19％、18％、16％、14％、12％、10％、8％、6％、4％、2％、1％、0.5％、0.2％或小于0.1％插入/缺失形成。在一些实施方案中，方法进一步包括用与第四核碱基互补的第五核碱基替换第二核碱基，由此产生意图的经编辑的碱基对(例如A:T至G:C)。在一些实施方案中，第五核碱基是鸟嘌呤。在一些实施方案中，编辑至少5％的意图碱基对。在一些实施方案中，编辑至少10％、15％、20％、25％、30％、35％、40％、45％或50％的意图碱基对。
在一些实施方案中，靶核苷酸中意图产物与非意图产物的比率为至少2:1、5:1、10:1、20:1、30:1、40:1、50:1、60:1、70:1、80:1、90:1、100:1或200:1或更多。在一些实施方案中，意图点突变与插入/缺失形成的比率大于1:1、10:1、50:1、100:1、500:1、或1000:1或更多。在一些实施方案中，切割单链(切口链)与引导核酸杂交。在一些实施方案中，切割单链与包含第一核碱基的链相反。在一些实施方案中，碱基编辑器包括Cas9结构域。在一些实施方式中，第一碱基是腺嘌呤，而第二碱基不是G、C、A或T。在一些实施方式中，第二碱基是肌苷。在一些实施方案中，第一碱基是腺嘌呤。在一些实施方案中，第二碱基不是G、C、A或T。在一些实施方案中，第二碱基是肌苷。在一些实施方案中，碱基编辑器抑制编辑链的碱基切除修复。在一些实施方案中，碱基编辑器保护或结合非编辑的链。在一些实施方案中，碱基编辑器包含UGI活性。在一些实施方案中，碱基编辑器包含催化无活性的肌苷特异性核酸酶。在一些实施方案中，碱基编辑器包含切口酶活性。在一些实施方案中，意图的经编辑的碱基对在PAM位点的上游。在一些实施方案中，意图的经编辑的碱基对在PAM位点的上游1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个核苷酸。在一些实施方案中，意图的经编辑的碱基对在PAM位点的下游。在一些实施方案中，意图的经编辑的碱基对在PAM位点的下游1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个核苷酸。在一些实施方案中，方法不需要规范(例如，NGG)PAM位点。在一些实施方案中，核碱基编辑器包含接头。在一些实施方案中，接头的长度为1-25个氨基酸。在一些实施方案中，接头的长度为5-20个氨基酸。在一些实施方案中，接头的长度为10、11、12、13、14、15、16、17、18、19或20个氨基酸。在一些实施方案中，靶区域包含靶窗口，其中靶窗口包含靶核碱基对。在一些实施方案中，靶窗口包含1-10个核苷酸。在一些实施方案中，靶窗口的长度为1-9、1-8、1-7、1-6、1-5、1-4、1-3、1-2或1个核苷酸。在一些实施方案中，靶窗口的长度为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个核苷酸。在一些实施方案中，意图的经编辑的碱基对在靶窗口内。在一些实施方案中，靶窗口包含意图的经编辑的碱基对。在一些实施方案中，使用本文提供的任何碱基编辑器进行方法。在一些实施方案中，靶窗口是脱氨基作用窗口。
在一些实施方案中，本公开提供了用于编辑核苷酸的方法。在一些实施方案中，本公开提供了用于编辑双链DNA序列的核碱基对的方法。在一些实施方案中，方法包括a)使双链DNA序列的靶区域与包含碱基编辑器和引导核酸(例如gRNA)的复合物接触，其中靶区域包含靶核碱基对，b)诱导所述靶区域的链分离，c)将靶区域的单链中的所述靶核碱基对的第一核碱基转换为第二核碱基，d)切割所述靶区域的不超过一条链，其中与第一核碱基碱基互补的第三核碱基被与第二核碱基互补的第四核碱基替换，并且第二核碱基被与第四核碱基互补的第五核碱基替换，由此产生意图的经编辑的碱基对，其中产生意图的经编辑的碱基对的效率是至少5％。应当理解的是，在一些实施方案中，省略步骤b。在一些实施方案中，编辑至少5％的意图碱基对。在一些实施方案中，编辑至少10％、15％、20％、25％、30％、35％、40％、45％或50％的意图碱基对。在一些实施方案中，方法引起小于19％、18％、16％、14％、12％、10％、8％、6％、4％、2％、1％、0.5％、0.2％或小于0.1％的插入/缺失形成。在一些实施方案中，靶核苷酸处的意图产物与非意图产物的比率为至少2:1、5:1、10:1、20:1、30:1、40:1、50:1、60:1、70:1、80:1、90:1、100:1或200:1或更多。在一些实施方案中，意图点突变与插入/缺失形成的比率大于1:1、10:1、50:1、100:1、500:1或1000:1或更多。在一些实施方案中，切割单链与引导核酸杂交。在一些实施方案中，切割单链与包含第一核碱基的链相对。在一些实施方案中，第一碱基是腺嘌呤。在一些实施方案中，第二核碱基不是G、C、A或T。在一些实施方案中，第二碱基是肌苷。在一些实施方案中，碱基编辑器抑制编辑链的碱基切除修复。在一些实施方案中，碱基编辑器保护(例如，形成碱基切除修复)或结合非编辑的链。在一些实施方案中，核碱基编辑器包含UGI活性。在一些实施方案中，碱基编辑器包含催化无活性的肌苷特异性核酸酶。在一些实施方案中，核碱基编辑器包含切口酶活性。在一些实施方案中，意图的经编辑的碱基对在PAM位点的上游。在一些实施方案中，意图的经编辑的碱基对在PAM位点的上游1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个核苷酸。在一些实施方案中，意图的经编辑的碱基对在PAM位点的下游。在一些实施方案中，意图的经编辑的碱基对在PAM位点的下游1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个核苷酸。在一些实施方案中，该方法不需要规范(例如，NGG)PAM位点。在一些实施方案中，核碱基编辑器包含接头。在一些实施方案中，接头的长度为1-25个氨基酸。在一些实施方案中，接头的长度为5-20个氨基酸。在一些实施方案中，接头的长度为10、11、12、13、14、15、16、17、18、19或20个氨基酸。在一些实施方案中，靶区域包含靶窗口，其中靶窗口包含靶核碱基对。在一些实施方案中，靶窗口包含1-10个核苷酸。在一些实施方案中，靶窗口的长度为1-9、1-8、1-7、1-6、1-5、1-4、1-3、1-2或1个核苷酸。在一些实施方案中，靶窗口的长度为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个核苷酸。在一些实施方案中，意图的经编辑的碱基对发生在靶窗口内。在一些实施方案中，靶窗口包含意图的经编辑的碱基对。在一些实施方案中，核碱基编辑器是本文提供的碱基编辑器的任一个。
药物组合物
本公开的其他方面涉及药物组合物，其包含本文所述的腺苷脱氨酶、融合蛋白或融合蛋白-gRNA复合物中的任一种。如本文所用，术语“药物组合物”是指配制用于药物用途的组合物。在一些实施方案中，药物组合物进一步包含药学上可接受的载体。在一些实施方案中，药物组合物包含另外的试剂(例如用于特异性递送，增加半衰期或其他治疗性化合物)。
如本文所用，术语“药学上可接受的载体”是指药学上可接受的材料、组合物或媒介物，例如液体或固体填充剂、稀释剂、赋形剂、制造助剂(例如，润滑剂、滑石粉、硬脂酸镁、钙或锌或硬脂酸)或溶剂包封材料，涉及将化合物从身体的一个部位(例如，递送部位)运送或运输到另一个部位(例如，器官、组织或身体的一部分)。药学上可接受的载体是“可接受的”，意思是与制剂的其他成分相容并且对受试者的组织无害(例如，生理学相容的、无菌的、生理学的pH等)。可以充当药学上可接受的载体的材料的一些实例包括：(1)糖，例如乳糖、葡萄糖和蔗糖；(2)淀粉，如玉米淀粉和马铃薯淀粉；(3)纤维素及其衍生物，如羧甲基纤维素钠、甲基纤维素、乙基纤维素、微晶纤维素和醋酸纤维素；(4)粉末黄蓍胶；(5)麦芽；(6)明胶；(7)润滑剂，如硬脂酸镁、十二烷基硫酸钠和滑石粉；(8)赋形剂，如可可脂和栓剂蜡；(9)油，如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油和豆油；(10)二醇，如丙二醇；(11)多元醇，如甘油、山梨糖醇、甘露醇和聚乙二醇(PEG)；(12)酯类，如油酸乙酯和月桂酸乙酯；(13)琼脂；(14)缓冲剂，如氢氧化镁和氢氧化铝；(15)海藻酸；(16)无热原水；(17)等渗盐水；(18)林格氏液；(19)乙醇；(20)pH缓冲溶液；(21)聚酯，聚碳酸酯和/或聚酸酐；(22)增量剂(bulking agent)，如多肽和氨基酸(23)血清成分，如血清白蛋白、HDL和LDL；(22)C2-C12醇，如乙醇；和(23)药物制剂中采用的其他无毒相容物质。润湿剂、着色剂、脱模剂、包衣剂、甜味剂、调味剂、芳香剂、防腐剂和抗氧化剂也可以存在于制剂中。诸如“赋形剂”、“载体”、“药学上可接受的载体”等术语在本文中可互换使用。
在一些实施方案中，配制药物组合物用于递送至受试者，例如用于基因编辑。施用本文所述药物组合物的合适途径包括但不限于：局部、皮下、经皮、皮内、病灶内、关节内、腹膜内、膀胱内、经粘膜、牙龈、牙内、耳蜗内、经鼓室、器官内、硬膜外、鞘内、肌肉内、静脉内、血管内、骨内(intraosseus)、眼周、肿瘤内、脑内和脑室内施用。
在一些实施方案中，将本文所述的药物组合物局部施用于患病部位(例如，肿瘤部位)。在一些实施方案中，本文所述的药物组合物通过注射、通过导管的方法、通过栓剂的方法或通过植入物的方法施用于受试者，所述植入物是多孔的、无孔的或凝胶状的材料，包括膜(例如硅橡胶膜(sialastic membrane))或纤维。
在其他实施方案中，本文所述的药物组合物在控释系统中递送。在一个实施方案中，可以使用泵(参见例如，Langer,1990,Science 249:1527-1533；Sefton,1989,CRCCrit.Ref.Biomed.Eng.14:201；Buchwald et al.,1980,Surgery 88:507；Saudek et al.,1989,N.Engl.J.Med.321:574)。在另一个实施方案中，可以使用聚合物材料。(参见例如，Medical Applications of Controlled Release(Langer and Wise eds.,CRC Press,Boca Raton,Fla.,1974)；ControlledDrug Bioavailability,Drug Product Design andPerformance(Smolen and Ball eds.,Wiley,New York,1984)；Ranger and Peppas,1983,Macromol.Sci.Rev.Macromol.Chem.23:61.还参见Levy et al.,1985,Science 228:190；During et al.,1989,Ann.Neurol.25:351；Howard et al.,1989,J.Neurosurg.71:105.)其他控释系统讨论于例如Langer，同上中。
在一些实施方案中，药物组合物根据常规规程配制为适合于对受试者(例如人)进行静脉内或皮下施用的组合物。在一些实施方案中，用于通过注射施用的药物组合物是无菌等渗水性缓冲液中的溶液。必要时，药物还可以包括增溶剂和局部麻醉剂如利多卡因，以缓解注射部位的疼痛。通常，成分单独供应或以单位剂量形式混合在一起，例如，作为干燥的冻干粉末或无水浓缩物，在密封容器如安瓿或小药囊中，其表明活性剂的量。当药物通过输注施用时，可以用含有无菌药用级水或盐水的输液瓶分配。当药物组合物通过注射施用时，可以提供安瓿的无菌注射用水或盐水，以便在施用前混合成分。
用于全身施用的药物组合物可以是液体，例如无菌盐水、乳酸林格氏液或汉克氏液。此外，药物组合物可以是固体形式，并在使用前立即重新溶解或悬浮。还考虑了冻干形式。
药物组合物可以包含在脂质颗粒或囊泡(例如脂质体或微晶)内，其也适用于肠胃外施用。颗粒可以是任何合适的结构的，例如单层或多层，只要其中含有组合物即可。化合物可以包埋在含有融合脂质二油酰磷脂酰乙醇胺(DOPE)、低水平(5-10mol％)的阳离子脂质的“稳定质粒-脂质颗粒”(SPLP)中，并通过聚乙二醇(PEG)涂层稳定(Zhang Y.P.et al.,Gene Ther.1999,6:1438-47)。对于此类颗粒和囊泡，特别优选带正电荷的脂质如N-[1-(2,3-二油酰氧基)丙基]-N,N,N-三甲基-甲基硫酸铵或“DOTAP”。此类脂质颗粒的制备是众所周知的。参见例如，美国专利号4,880,635；4,906,477；4,911,928；4,917,951；4,920,016；和4,921,757；其每一个通过引用并入本文。
例如，本文所述的药物组合物可以作为单位剂量施用或包装。当用于提及本公开的药物组合物时，术语“单位剂量”是指适合作为受试者的单一剂量的物理上离散的单位，每个单位含有预定量的活性物质，其经计算与所需稀释剂联合产生所需治疗效果；即载体(carrier)或媒介物(vehicle)。
此外，药物组合物可以作为药物试剂盒提供，其包含(a)含有冻干形式的本发明的化合物的容器和(b)含有用于注射的药学上可接受的稀释剂(例如无菌水)的第二容器。药学上可接受的稀释剂可以用于重构或稀释本发明的冻干化合物。任选地与这种容器结合的可以是由管理药物或生物制品的制造、使用或销售的政府机构规定的形式的通知，该通知反映了制造、使用或销售机构对人类施用的批准。
在另一方面，包括含有可用于治疗上述疾病的材料的制品。在一些实施方案中，制品包含容器和标签。合适的容器包括例如瓶子、小瓶、注射器和试管。容器可以由多种材料形成，例如玻璃或塑料。在一些实施方案中，容器容纳有效治疗本文所述疾病的组合物，并且可以具有无菌进入口。例如，容器可以是静脉内溶液袋或具有可由皮下注射针刺穿的塞子的小瓶。组合物中的活性剂是本发明的化合物。在一些实施方案中，容器上或与容器结合的标签表明组合物用于治疗所选择的疾病。制品可以进一步包含第二容器，其包含药学上可接受的缓冲液，例如磷酸盐缓冲盐水、林格氏溶液或右旋糖溶液。它可以进一步包括从商业和用户角度所需的其他材料，包括其他缓冲剂、稀释剂、过滤器、针头、注射器和具有使用说明的包装说明书。
试剂盒、载体、细胞
本公开的一些方面提供了包含核酸构建体的试剂盒，所述核酸构建体包含编码能够使脱氧核糖核酸(DNA)分子中的腺苷脱氨基的腺苷脱氨酶的核苷酸序列。在一些实施方案中，核苷酸序列编码本文提供的任何腺苷脱氨酶。在一些实施方案中，核苷酸序列包含驱动腺苷脱氨酶的表达的异源启动子。
本公开的一些方面提供了包含核酸构建体的试剂盒，所述核酸构建体包含(a)编码与腺苷脱氨酶融合的napDNAbp(例如Cas9结构域)的核苷酸序列，或包含如本文提供的napDNAbp(例如Cas9结构域)和腺苷脱氨酶的融合蛋白；和(b)驱动(a)的序列的表达的异源启动子。在一些实施方案中，试剂盒进一步包含编码引导核酸主链(例如引导RNA主链)的表达构建体，其中构建体包含克隆位点，所述克隆位点定位为允许将与靶序列相同或互补的核酸序列克隆到引导核酸(例如引导RNA主链)中。
本公开的一些方面提供了包含本文提供的任何腺苷脱氨酶、融合蛋白或复合物的细胞。在一些实施方案中，细胞包含编码本文提供的任何腺苷脱氨酶或融合蛋白的核苷酸。在一些实施方案中，细胞包含本文提供的任何核苷酸或载体。
提供以上报告物系统的示例性实施方案的描述仅用于说明的目的，而不意味着限制。本公开涵盖另外的报告物系统，例如上面详细描述的示例性系统的变体。
然而，应当理解，基于本公开和本领域的知识，另外的融合蛋白对于技术人员将是显而易见的。
从以下实施例将更全面地理解本发明的这些和其他实施方案的功能和优点。以下实施例旨在说明本发明的益处并描述特定实施方案，但并不旨在举例说明本发明的全部范围。因此，应当理解，实施例不意味着限制本发明的范围。
实施例
以下实施例中提供的数据描述了能够在DNA的背景下催化腺苷的水解脱氨基作用(形成肌苷，其像鸟嘌呤(G)一样碱基配对)的碱基编辑器的工程化。没有已知的天然存在的作用于DNA的腺苷脱氨酶。相反，已知的腺苷脱氨酶作用于RNA(例如，tRNA或mRNA)。第一脱氧腺苷脱氨酶演化为接受DNA底物并使脱氧腺苷(dA)脱氨基为脱氧肌苷。作为一个实例，使用来自大肠杆菌的作用于tRNA的腺苷脱氨酶(ADAT)(TadA，代表tRNA腺苷脱氨酶A)进行演化实验，以工程化改造作用于DNA的腺苷脱氨酶。简而言之，ecTadA与dCas9结构域共价融合，并且组装该融合物的文库，其在构建体的脱氨酶部分中含有突变。在下面描述的演化实验中，发现ecTadA中的几个突变改进了ecTadA使DNA中的腺苷脱氨基的能力。在此报道了介导细菌和人细胞中A·T至G·C碱基对的可编程转化的腺嘌呤碱基编辑器(ABE)的定向演化、工程化和表征。实际上，已知致病性单核苷酸多态性的大约一半是C·G至T·A的转换。因此，以可编程、高效和精确的方式将A·T碱基对转换为G·C碱基对的能力可以大大推动研究和治疗遗传疾病的工作。广泛的演化和工程化以使ABE效率和序列通用性最大化产生了第七代腺嘌呤碱基编辑器，如ABE7.10，其有效地将靶A·T转化为G·C碱基对(在人细胞中的17个基因组位点中平均53％)，其中具有非常高的产物纯度(通常>99％)和与未经处理的细胞相比非常低的插入缺失率(通常≤0.1％)。在随后的实施例中显示，与目前的Cas9核酸酶介导的HDR方法相比，ABE7变体更有效且更干净地引入点突变，比Cas9核酸酶诱导更少的脱靶基因组修饰，并且可以用于校正与疾病相关的SNP，并在培养的人细胞中引入抑制疾病的SNP。
在人中1，从胞嘧啶和5-甲基胞嘧啶分别自发水解脱氨基形成尿嘧啶和胸腺嘧啶1,2，估计每天每细胞发生100-500次，并可以导致C·G至T·A突变，约占所有已知致病性SNP的一半(图1A)。因此，在未经修饰的细胞的基因组DNA中的靶基因座处将A·T碱基对转化为G·C碱基对的能力可以能够校正很大一部分与疾病相关的人SNP。
碱基编辑是基因组编辑的一种形式，其使得在靶基因组基因座处的一个碱基对能够直接、不可逆地转化为另一个碱基对，而无需双链DNA断裂(DSB)、同源性定向修复(HDR)过程或供体DNA模板3-5。与引入点突变的标准基因组编辑方法相比，碱基编辑可以更有效地进行6，并且具有少得多的不期望的产物如随机插入或缺失(indel)或易位。4,6-8
最常用的碱基编辑器是第三代设计(BE3)，其由以下组成：(i)不能造成DSB的催化受损的CRISPR-Cas9突变体，(ii)在Cas9产生的单链DNA泡(bubble)中的小窗口(约5个核苷酸)内将C转化为尿嘧啶(U)的单链特异性胞苷脱氨酶，(iii)阻止降低碱基编辑效率及产物纯度的尿嘧啶切除和下游过程的尿嘧啶糖基化酶抑制剂(UGI)9，和(iv)切口酶活性以对未编辑的DNA链进行切口，从而指导细胞错配修复以取代含G的DNA链6,9。这些组分一起使细菌、酵母4,10、植物11,12、斑马鱼13、哺乳动物细胞14,15、小鼠16,17以及甚至人胚胎18,19中的C·G能够高效且永久转化为T·A碱基对。碱基编辑能力通过开发具有不同前间隔区(PAM)兼容性7、缩小的编辑窗口7、增强的DNA特异性8和小分子依赖性20的碱基编辑器而得到了扩展。第四代碱基编辑器(BE4和BE4-Gam)进一步改进了C·G至T·A编辑效率和产物纯度。9
迄今为止，所有报道的碱基编辑器均介导C·G至T·A转化。在这项研究中，蛋白质演化和工程化用于开发一类新的腺嘌呤碱基编辑器(ABE)，其将细菌和人细胞中DNA中的A·T转化为G·C碱基对。第七代ABE如ABE7.10(图7)在人细胞中广泛的靶基因组基因座处将A·T转化为G·C，其中典型效率为约50％，且产物纯度非常高(>99％)，超过了BE3的典型性能特征。ABE大大扩展了碱基编辑的范围，并且与先前描述的碱基编辑器一起能够在活细胞的基因组中可编程安装所有四种转换突变(C至T、A至G、T至C和G至A)。
实施例1-对DNA起作用的腺嘌呤脱氨酶的演化
腺苷的水解脱氨基产生肌苷(图1B)。在聚合酶活性位点的限制内，肌苷与C最稳定配对，因此被读作G或作为G复制21。理论上，用腺嘌呤脱氨酶替换现有碱基编辑器的胞苷脱氨酶结构域可以提供ABE(图1C)，但尚无已知的酶使DNA中的腺嘌呤脱氨基。尽管所有报道的腺嘌呤脱氨酶的实例均可以处理RNA或错配的RNA:DNA异源双链体中的游离腺嘌呤、游离腺苷、腺苷22，或者奇怪的是，催化单链DNA上C至U的形成23，但目前的工作通过用天然腺嘌呤脱氨酶(包括大肠杆菌TadA24-26、人ADAR27,28、小鼠ADA29和人ADAT230,31(补充序列1))替换BE3的APOBEC1组分开始以测试单链DNA的高有效摩尔浓度可以克服其对DNA不良活性的可能性。不幸的是，当将编码这些腺嘌呤脱氨酶-Cas9 D10A切口酶融合物的质粒DNA构建体与相应的单一引导RNA(sgRNA)一起转染到HEK293T细胞中时，与未经处理的细胞相比，没有观察到显著的A·T至G·C编辑(图8A)。这些结果表明，测试的天然腺嘌呤脱氨酶不能处理DNA，排除了它们直接用于ABE中。
鉴于这些结果，寻求演化起始于天然存在的RNA腺嘌呤脱氨酶的接受DNA为底物的腺嘌呤脱氨酶变体。通过创建在关键位置处含有点突变的抗生素抗性基因，开发了用于碱基编辑的细菌选择(表8和补充序列2)。通过碱基编辑器回复这些突变使细菌能够在存在抗生素的情况下存活。为了验证选择，使用了细菌密码子优化版本的BE26(与dCas9和UGI融合的APOBEC1胞嘧啶脱氨酶)，因为细菌缺少能够使BE3进行更有效的碱基编辑的切口导向的错配修复机制32。优化靶突变选择、启动子强度、选择质粒拷贝数、温育时间和抗生素选择严格性后，观察到成功拯救了在催化残基处含有A·T至G·C突变(H193R)的有缺陷的氯霉素乙酰转移酶(CamR)，其通过BE2和sgRNA经编程以将碱基编辑导向失活突变，从而导致氯霉素的最小抑菌浓度(MIC)从1μg/mL增加到32μg/mL。DNA测序证实，从选择存活的细菌细胞含有恢复CamR功能的C·G至T·A突变。
接下来，通过在CamR基因中引入C·G至T·A突变，使选择质粒适应ABE活性，产生赋予1μg/mL氯霉素的MIC的H193Y取代(表8和补充序列2)。H193Y突变处的A·T至G·C转化应恢复氯霉素抗性，从而在存在氯霉素的情况下将ABE活性与细菌存活联系起来。
先前描述的碱基编辑器6-9利用对单链DNA起作用并排斥双链DNA的胞苷脱氨酶的使用。这种单链DNA要求对于将脱氨酶活性集中在由Cas9产生的单链泡内的核苷酸的小窗口上(从而将超出靶核苷酸的不期望的脱氨基事件最小化)至关重要。TadA是在tRNAArg的单链反密码子环中将腺嘌呤转化为肌苷(I)的tRNA腺嘌呤脱氨酶24。大肠杆菌TadA与原始碱基编辑器中使用的APOBEC酶家族共享同源性33，并且结构研究表明，一些ABOBEC以类似于tRNA结合TadA的构象结合单链DNA33。TadA不需要小分子激活物(与ADAR相反34)，并且可以作用于多核酸(与ADA不同29)。基于这些考虑，选择大肠杆菌TadA作为工作的起点以演化DNA腺嘌呤脱氨酶。
创建了仅在构建体的腺嘌呤脱氨酶部分中含有突变以避免改变编辑器的Cas9部分的有利特性的的ecTadA-dCas9融合物的无偏质粒文库。将所得的质粒文库转化到带有CamR H193Y选择的大肠杆菌中，并将约5.0x 106个转化体铺板到含有2至16μg/mL氯霉素的培养基上(图2A)。存活的菌落对于TadA突变A106V和D108N强力地富集(图2B)。经演化的大肠杆菌TadA与金黄色葡萄球菌TadA(据报道是与tRNAArg复合的结构的同源物35)的序列比对预示D108的侧链与紧邻底物腺苷上游的尿苷核苷酸中核糖的2’-OH基团形成氢键(图2C)。D108处的突变可能消除该氢键，且从而降低在底物结合位点中接受DNA的能量机会成本。DNA测序证实，所有从选择存活的细菌克隆在CamR中的靶定位点处均显示出大量的A·T至G·C回复。总的来说，这些结果表明TadA D108处或附近的突变使TadA能够在DNA底物上进行腺嘌呤脱氨基。
将TadA A106V和D108N突变掺入哺乳动物密码子优化的TadA-Cas9切口酶融合构建体中，该构建体用BE3中使用的Cas9 D10A切口酶替换细菌演化中使用的dCas9，以操纵细胞DNA错配修复以促进所期望的碱基编辑结果，并添加C端核定位信号(NLS)。所得的TadA*-XTEN-nCas9-NLS构建体命名为ABE1.2，其中TadA*代表经演化的TadA变体，且XTEN是BE3中使用的16个氨基酸的接头6。表达靶向六个人类基因组位点的ABE1.2和sgRNA的质粒的转染(图3A)导致在人基因组中的六个不同的靶位点上(图2A)在前间隔区位置5处或附近非常低但可观察到的A至G编辑效率(3.2±0.88％；所有编辑效率均报道为三个生物学重复的平均值±SD，除非另有说明，否则不富集转染细胞)，将PAM计算为位置21-23(图3B)。这些数据证实，能够催化低水平的A·T至G·C转化的ABE从第一轮蛋白质演化和工程化出现。
实施例2-改进的脱氨酶变体和ABE构造
通过第二轮演化寻求改进ABE1.2的编辑效率。如前所述生成ABE1.2变体的无偏文库，并且用比第1轮中使用的更高浓度的氯霉素(16至128μg/mL)在细菌中攻击所得的TadA*1.2–dCas9突变体(表7和8)。从该第二轮演化中，鉴定了预计位于与TadA中底物邻接的螺旋中的两个突变(D147Y和E155V)(图2C)。在哺乳动物细胞中，在所测试的六个基因组位点处，ABE2.1(ABE1.2+D147Y+E155V)展现出比ABE1.2 2至7倍更高的活性，导致A·T至G·C碱基编辑的平均值为11±2.9％(图3B)。
接下来，通过另外的蛋白质工程化寻求改进ABE2.1编辑效率。将TadA(2.1)*结构域与Cas9切口酶的C端而不是N端融合导致编辑活性完全丧失(图7和8B)，这与BE3以前的发现一致6。接头长度在TadA(2.1)*和Cas9切口酶之间也有所不同。具有ABE2.1中原始16个残基的XTEN接头两倍长的接头(32个氨基酸，(SGGS)2-XTEN-(SGGS)2)的ABE2变体(ABE2.6)与ABE2.1相比，提供了略高的编辑效率，现在在所测试的六个基因组位点上平均为14±2.4％(图7和8B)。
类似于尿嘧啶N-糖基化酶(UNG)催化从DNA去除尿嘧啶并启动碱基切除修复的机制，烷基腺嘌呤DNA糖基化酶(AAG)催化DNA中肌苷的糖苷键的切割36,37。为了测试肌苷切除是否阻碍ABE性能，创建了经设计以最小化肌苷碱基切除修复(BER)潜在来源的ABE2变体。鉴于不存在已知的AAG的蛋白质抑制剂，试图通过分别将参与肌苷结合或去除的酶催化失活形式(人AAG(用E125Q突变失活38)或大肠杆菌Endo V(用D35A突变失活39))与ABE2.1的C端融合来阻断内源性AAG接近肌苷中间体。与ABE2.1相比，HEK293T细胞中ABE2.1-AAG(E125Q)(ABE2.2)和ABE2.1-Endo V(D35A)(ABE2.3)均未表现出改变的A·T至G·C编辑效率(图7和8C)。实际上，与含有野生型AAG的Hap1细胞相比，在缺少AAG的Hap1细胞中使用ABE2.1不导致碱基编辑效率的增加，产物纯度的增加或插入/缺失频率的降低(图8D)。此外，在所测试的六个基因座处，ABE2.1几乎没有诱导插入/缺失(≤0.1％)或A·T至非G·C产物(≤0.1％)，这与肌苷中间体的低效切除效率一致(图11A至11B)。综上所述，这些观察结果强烈表明，由ABE产生的肌苷中间体的细胞修复效率低下，从而消除了颠覆过程如BER的需要。该情况与BE3和BE4形成对比，BE3和BE4强烈依赖于抑制尿嘧啶切除，以最大化碱基编辑效率和产物纯度，并抑制插入/缺失形成6,9。
作为最终的ABE2工程化研究，探索了TadA*二聚化状态对人细胞中碱基编辑效率的作用。TadA以其天然形式作为同二聚体起作用，其中一个单体催化A至I的脱氨基作用，而另一个单体充当tRNA底物的对接站(docking station)40。在大肠杆菌中选择期间，推测内源性TadA充当非催化单体。假设在哺乳动物细胞中，拴系另外的野生型或经演化的TadA单体可以通过最小化对分子间ABE二聚化的依赖来改进编辑效率。实际上，与ABE2.1共表达野生型TadA或TadA*2.1以促进反式ABE2.1:TadA或ABE2.1:TadA*2.1二聚体形成(分别为ABE2.7和ABE2.8)以及将经演化的或野生型TadA与ABE2.1的N端直接融合(分别为ABE2.9和ABE2.10)大大改进了编辑效率(图3B、7和10A)。鉴定了融合的TadA–ABE2.1构造(ABE2.9)提供最高的编辑效率(六个基因组基因座上平均为20±3.8％，与ABE1.2相比，每个位点处的平均改进为7.6±2.6倍)并且其用于所有后续实验中(图2B和3B)。含有两个野生型TadA结构域且无经演化的TadA*结构域的对照ABE变体在所测试的六个基因组位点处未导致A·T至G·C编辑(图10A)，证实仅二聚化不足以介导ABE活性。
由于这些结果暗示TadA二聚化为ABE编辑效率的重要组成部分，因此确定了TadA-ABE2融合物内两个TadA亚基中的哪一个负责A至I催化。将失活的E59A突变24引入到ABE2.9的N端或内部TadA单体中，分别生成ABE2.11或ABE2.12。具有失活的N端TadA亚基的变体(ABE2.11)表现出与ABE2相当的编辑效率，而具有失活的内部TadA亚基的变体丧失了所有编辑活性(图7和10A)。这些结果证明内部的TadA亚基负责A至I的催化。
实施例3-有效编辑靶标亚组的ABE
接下来，以TadA*2.1–dCas9(依赖于与内源性大肠杆菌TadA或其自身进行反式二聚化)开始进行第三轮的细菌演化，以进一步增加A·T至G·C的编辑效率。通过在卡那霉素抗性基因(KanR，氨基糖苷磷酸转移酶，表8和补充序列2)中引入两个早期终止密码子(Q4终止和W15终止)来增加选择严格性。每个突变都需要A·T至G·C回复以校正提前终止密码子。携带演化第3轮选择质粒的宿主细胞的MIC为约8μg/mL卡那霉素，并且需要相同质粒上的两个KanR突变的碱基编辑来恢复卡那霉素抗性(表8)。在存在16至128μg/mL卡那霉素的情况下，对含有TadA结构域中突变的TadA*2.1–dCas9变体的文库进行了该更高严格性的选择，从而导致以下三个新TadA突变的强烈富集：L84F，H123Y和I157F。将这些突变导入ABE2.9哺乳动物构建体中以生成ABE3.1(图2B)。在HEK293T细胞中，ABE3.1导致六个测试位点上的编辑效率平均为29±2.6％，每个位点处的A·T至G·C转化比ABE 2.9增加平均1.6倍，并且比ABE1.2改进平均11倍(图3C)。还测试了两个TadA单体之间，或TadA*和Cas9切口酶之间更长的(64个或100个氨基酸)接头，并观察到与ABE3.1相比对编辑效率的负面影响(图7和10B)。
尽管在一些位点处ABE3.1介导的碱基编辑效率高，如位点1(65±4.2％转化)，其将经编辑的A置于CAC背景中的前间隔区位置5处，对于其他位点，如将经编辑的A置于GAG背景中的位点5，编辑效率低得多(8.3±0.67％转化)(图3C)。来自围绕靶标A的具有不同序列背景的六个基因组基因座的结果表明，来自第1-3轮的ABE强烈优先靶向YAC的背景，其中Y＝T或C。该序列背景偏好可能是从天然大肠杆菌TadA的底物特异性继承的，其在tRNAArg的反密码子环的UAC背景中使A脱氨基。然而，通过此类靶序列限制，ABE用于碱基编辑应用的效用将受到极大的限制。
为了克服ABE3.1的YAC序列偏好，启动了第四演化运动，其集中于预计与靶标A的上游和下游的核苷酸相互作用的TadA残基处的诱变。对金黄色葡萄球菌TadA·tRNA共晶体结构的检查35显示了直接接触tRNA底物的反密码子环的残基，其对应于大肠杆菌TadA E25、R26、R107、A142和A143。对在这些位置处含有随机残基的TadA*2.1–dCas9文库(表7)进行新的细菌选择，其中非YAC靶标的A·T至G·C转化(GAT，其在壮观霉素抗性蛋白中导致T89I突变)恢复抗生素抗性(表8和补充序列2)。用高浓度的壮观霉素(64至512μg/mL)攻击携带选择质粒的细胞中的文库成员(MIC为约32μg/mL壮观霉素，表8)。存活的细菌强烈聚集在TadA突变A142N上。尽管通过壮观霉素MIC判断，具有TadA*4.3–dCas9(TadA*3.1+A142N–dCas9)的细菌细胞中明显的A·T至G·C碱基编辑效率高于具有TadA*3.1–dCas9的细菌细胞(图10C)，ABE4.3与哺乳动物细胞中的ABE3.1相比，一般显示出降低的碱基编辑效率(平均16±5.8％)(图2B和3C)。假设A142N突变可能以背景依赖的方式使碱基编辑受益，并在以后的几轮演化中重新考虑了它的纳入(参见下文)。
进行了第五轮演化以增加ABE催化性能并拓宽靶序列兼容性。如前所述生成了TadA*3.1–dCas9变体的文库，其含有整个TadA*结构域中的无偏突变(表7)。为了支持具有更快动力学的ABE构建体，在允许ABE表达仅前轮次演化持续时间(约14h)的一半(7h)后，对该文库用高剂量的氯霉素(≥128μg/mL)的CamR H193Y选择。存活的克隆在TadA的N和C端结构域附近包含各种突变。出人意料的是，将这些突变(H36L、R51L、S146C和K157N)的共有组导入ABE3.1，创建ABE5.1，使HEK293T细胞中的总体编辑效率降低了1.7±0.29倍(图2B和3C)。
由于ABE5.1包括自从先前在ABE2.1上的二聚化状态实验以来的七个突变，因此推测这些新突变的积累可能损害非催化性N端TadA亚基在哺乳动物细胞中发挥其结构作用的能力。在大肠杆菌中，反式提供内源性野生型TadA单体，这可能解释了细菌选择表型与哺乳动物细胞编辑效率之间的脱节。因此，研究了在ABE5变体的N端非催化TadA结构域中使用野生型TadA代替经演化的TadA*变体的作用。这些研究表明，与具有两个相同的经演化的TadA*结构域的同二聚体ABE5.1相比，由与内部演化的TadA*融合的野生型大肠杆菌TadA组成的异二聚体构建体(ABE5.3)显示出大大改进的编辑效率。六个基因组测试位点上的ABE5.3编辑效率平均为39±5.9％，相对于ABE5.1，每个位点处平均改进2.9±0.78倍(图2B和3C)。与ABE3.1相比，ABE5.3在每个测试位点处的编辑效率平均增加1.8±0.39倍。重要的是，ABE5.3还显示出更广泛的序列兼容性，其现在能够对非YAC靶标(包括位点3(AAG)、位点4(CAA)、位点5(GAG)和位点6(GAC))进行22-33％的编辑(图3C)。
同时，对第5轮文库进行了第4轮中使用的非YAC壮观霉素选择。尽管没有出现高度富集的突变，但出现自该选择的两种基因型的新突变N72D+G125A；和P48S+S97C(图7)包括在后续文库生成步骤中。将这些突变简单添加至ABE3.1(分别生成ABE5.13和ABE5.14)并未改进编辑效率(图7和11A)。
由于ABE3接头的研究表明比32个氨基酸长得多的接头降低ABE活性(图7和10B)，因此采用了更为完善的方法来优化ABE5接头。在HEK293T细胞中测试了八个异二聚体野生型TadA-TadA*ABE5.3变体(ABE5.5至ABE5.12)，其在TadA结构域之间或TadA与Cas9切口酶之间含有24、32或40个残基的接头，没有导致碱基编辑效率明显改进(图7和11B)。因此，所有后续研究均使用含有具有两个32个残基接头的异二聚体wtTadA–TadA*–Cas9切口酶的ABE5.3构造。
实施例4-具有广泛序列兼容性的高活性ABE
第六轮演化旨在通过DNA改组去除任何非有益的突变，并重新检查来自先前轮次演化的突变，其一旦从具有其他突变的负异位显性(negative epistasis)释放后，可能对ABE性能具有不同的影响。将来自第1轮至第5轮的经演化的TadA*–dCas9变体与野生型大肠杆菌TadA一起改组，转化到带有壮观霉素抗性T89I选择质粒的大肠杆菌中，并在补充有384μg/mL壮观霉素的培养基上进行选择。从该选择强烈地富集了两个突变：P48S/T和A142N(第4轮首次出现)。将这些突变单独或一起添加至ABE5.3，形成ABE6.1至ABE6.6(图7)。在六个测试的基因组位点的每一个处，相对于ABE5.3，ABE6.3(ABE5.3+P48S)导致1.3±0.28倍更高的平均A·T至G·C编辑，以及47±5.8％的平均转化效率(图2B和4A)。预测P48距TadA晶体结构中的底物腺嘌呤核苷碱基和2’-羟基约5A(图2C)，据推测，将该残基突变为Ser可以改进与脱氧腺苷底物的兼容性。尽管在大多数位点，含有A142N突变的ABE6变体的活性比缺少该突变的ABE少，但由ABE6.4(ABE6.3+A142N)在位点6(其在前间隔区中的位置7处含有靶标A)处进行的编辑比ABE6.3的编辑效率高1.5±0.13倍，并且比ABE5.3的编辑效率高1.8±0.16倍(图4A)。这些结果表明，A142N变体可以提供比位置5更接近PAM的靶腺嘌呤的编辑。
尽管六轮演化和工程化产生了实质性的改进，但是ABE6编辑器仍然在靶A附近含有多个腺嘌呤的靶序列处(如位点3(AAG)和位点4(CAA))编辑效率降低(约20-40％)(图4A)。为了应对这一挑战，进行了第七轮演化，其中将新鲜生成的TadA*6-dCas9变体的文库靶向卡那霉素抗性基因中的两个单独位点：需要编辑TAT基序的第三轮中使用的Q4终止突变，以及需要编辑TAA序列的新D208N突变(表7和8，补充序列2)。带有第7轮选择质粒的宿主细胞的MIC为8μg/mL卡那霉素(表8)。将突变的TadA*6-dCas9变体的无偏文库转化到大肠杆菌中，并在含有64μg/mL至384μg/mL卡那霉素的培养基上进行选择。存活的克隆包含三组富集的突变：W23L/R、P48A和R152H/P。
将这些突变单独或组合引入到哺乳动物细胞ABE(ABE7.1至ABE7.10)中，导致A·T至G·C编辑效率大大增加，特别是在含有多个A残基的靶标处(图2B、4A、4B、7、12A和12B)。ABE7.10编辑了六个基因组测试位点，其中平均效率为58±4.0％，并且相对于ABE6.3，每个位点的平均改进为1.3±0.20倍(图4A)，且与ABE1.2相比为29±7.4倍。尽管突变剖析揭示了所有这三个新突变都有助于增加编辑效率(图7、12A和12B)，但R152P取代尤其值得注意。经比对的ecTadA晶体结构预测R152位于C端螺旋中，并与tRNA底物的UAC反密码子环中的C接触(图2B和2C)。据推测，用Arg取代Pro破坏该螺旋结构，并可能消除碱基特异性酶:DNA相互作用。
实施例5-晚期ABE的表征
对来自第5-7轮的最有希望的ABE进行深入表征。选择扩展的一组17个人基因组靶标，其将靶A置于前间隔区的位置5或7处，并共同包括所有可能的NAN序列背景(图3A)。总体而言，在从ABE5至ABE7变体进展期间，在HEK293T细胞中观察到A·T至G·C编辑效率的显著改进(图4B)。总体上最具活性的编辑器ABE7.10的碱基编辑效率在所测试的17个位点处平均为53±3.7％，在这些位点的11个处超过50％，且范围为34-68％(图4A和4B)。这些效率与BE3的典型C·G至T·A编辑效率6相比具有优势。
接下来，寻求进一步表征晚期ABE的碱基编辑活性窗口。选择了可以用两个sgRNA的任一个靶向的含有交替的5’-A-N-A-N-A-N-3’序列的人基因组位点，使得A可以位于前间隔区中2至9的每个奇数位置(位点18)或每个偶数位置(位点19)(图3A)。所得的编辑结果(图5A)，以及对所有19个测试的位点上每个前间隔区位置处的编辑效率的分析(图5B)表明，晚期变体的活性窗口为约4-6个核苷酸宽，对于ABE7.10距前间隔区位置约4-7，并且对于ABE6.3、ABE7.8和ABE7.9距位置约4-9，将PAM视为位置21-23(图5A至5C)。注意，精确的编辑窗口边界可以以靶标依赖性的方式变化(表1)，与BE3和BE4的情况一样。还在位点1-6处，在U2OS细胞中测试了ABE7.8、ABE7.9和ABE7.10，并且观察到与HEK293T细胞中相似的编辑结果(图12C)，表明ABE活性不仅限于HEK293T细胞。
对来自HEK293T细胞中6至17个基因组位点处的ABE编辑的单个高通量DNA测序读出的分析表明编辑窗口内邻近的腺嘌呤处的碱基编辑结果不是统计独立事件。随着ABE演化的进行，邻近的靶腺嘌呤之间的平均标准化连锁不平衡(LD)稳步增加，因此ABE1.2、ABE3.1、ABE5.3和ABE7.10的标准化LD平均分别为0.17±0.12、0.56±0.27、0.67±0.25和0.94±0.08(图14A和14B)。因此，早期ABE更独立地编辑邻近的腺嘌呤，而晚期ABE可以更进行性地编辑邻近的腺嘌呤，并且如果还编辑相同DNA链中的邻近的A，则更可能编辑A。这些发现表明，在演化过程期间，TadA可能已经演化了动力学变化，其降低了在编辑窗口内另外的A转化之前底物释放的可能性，从而导致类似于BE36的行为的持续合成能力。
与由BE3介导的胞苷碱基编辑产生的C至非T编辑和插入/缺失的形成不同，ABE在HEK293T和U2OS细胞中非常干净地将A·T转化为G·C，其中平均为<0.1％插入/缺失，与未经处理的对照细胞相似，并且在所测试的17个基因组NAN位点中，未观察到高于未经处理细胞的A至非G编辑(图5C和表1)。最近发现，尿嘧啶切除和下游修复过程产生不期望的BE3产物9。与BE3相比，所有测试的ABE变体的非凡的产物纯度表明与UNG相比，从DNA去除肌苷的酶的活性或丰度可能较低，从而导致腺嘌呤碱基编辑后的碱基切除修复最少。
将ABE7.10催化的A·T至G·C编辑效率与现有技术的Cas9核酸酶介导的HDR方法CORRECT41进行了比较。在优化的48小时条件下，在HEK293T细胞中使用CORRECT HDR方法，观察到在HEK293T细胞中的五个基因组基因座处，平均靶突变频率范围为0.47％至4.2％，其中插入/缺失为3.3％至10.6％(图6A)。在这五个相同的基因组基因座处，ABE7.10导致平均靶突变频率在48小时后为10-35％，且在120小时后为55-68％(图6A)，其中插入/缺失<0.1％(图6B)。靶突变:插入/缺失比率对于CORRECT HDR平均为0.43，对于ABE7.10为>500，代表产物选择性改进了>1,000倍，有利于ABE7.10。这些结果表明，与目前的Cas9核酸酶介导的HDR方法相比，ABE7.10可以以更高效率且少得多的不期望的产物引入A·T至G·C点突变。
接下来，检查了ABE7变体的脱靶活性。由于尚无方法全面描述ABE的脱靶活性，因此假定脱靶ABE编辑主要发生在当Cas9核酸酶与特定引导RNA复合时所编辑的相同的脱靶位点，如在BE36,8,9,42观察到的情况。用三个充分表征的引导RNA(靶向HEK位点2、3和4)43和Cas9核酸酶或ABE7变体处理HEK293T细胞，并对与通过全基因组GUIDE-Seq方法43鉴定的引导RNA相关的在靶基因座和12个最具活性的脱靶人基因组基因座进行测序。Cas9在靶插入/缺失效率和ABE7.10的在靶碱基编辑效率两者均平均为54％(表2-4)。在12个已知的脱靶基因座中的9个(75％)中观察到Cas9核酸酶的可检测的修饰(≥0.2％插入/缺失)(图6C和表2-4)。相反，当与相同的sgRNA复合时，ABE7.10、ABE7.9或ABE 7.8导致12个已知的Cas9脱靶位点中仅四个(33％)处≥0.2％脱靶碱基编辑。此外，以14％插入/缺失的平均效率对九个已确认的Cas9脱靶基因座进行修饰，而以仅1.3％A·T至G·C突变的平均对四个已确认的ABE脱靶基因座进行修饰(表2-4)。尽管九个已确认的Cas9脱靶基因座中的七个在ABE活性窗口内含有至少一个A，但是这七个脱靶基因座中的三个未被ABE7.8、7.9或7.10可检测地编辑。总之，这些数据表明，即使对于含有可编辑A的脱靶位点，ABE7变体也可以比Cas9核酸酶更不易脱靶基因组修饰。此外，在ABE处理后，没有检测到ABE诱导的A·T至G·C编辑在在靶或脱靶前间隔区之外的证据。
实施例6-ABE介导的与人疾病相关的突变的校正和安装
最后，测试了ABE校正致病性突变并在哺乳动物细胞中引入抑制疾病的突变的潜力。b-珠蛋白基因中的突变导致多种血液疾病。伴罕见良性状况HPFH(遗传性胎儿血红蛋白持续存在症)的人对一些b-珠蛋白疾病(包括镰状细胞贫血)有抗性。在某些患者中，这种表型由g-珠蛋白基因HBG1和HBG2的启动子中的突变介导，这些突变使胎儿血红蛋白能够持续表达，所述胎儿血红蛋白通常在人出生后沉默44,45。sgRNA经设计，通过将靶A·T碱基对置于前间隔区位置7处对ABE进行编程，使其同时将驱动HBG1表达的启动子中的-198T突变为C，并将驱动HBG2表达的启动子中的-198T突变为C。已知这些突变赋予英国型HPFH，并能够使成年人中产生胎儿血红蛋白46。ABE7.10在HEK293T细胞中分别以29％和30％的效率在HBG1和HBG2启动子中安装了所期望的T·A至C·G突变(图6C和14)。
铁贮积病症遗传性血色素沉着症(HHC)是常染色体隐性遗传病症，通常由人HFE基因中核苷酸845处的G至A突变引起，导致HFE蛋白中的C282Y突变47,48。这种突变导致肝铁激素铁调素(hepcidin)的产生不足，从而导致肠道铁吸收过多，并导致可能危及生命的血清铁蛋白升高。将编码ABE7.10的DNA和将靶A置于前间隔区位置5处的引导RNA转染到带有HFEC282Y基因组突变的永生化的淋巴样干细胞(LCL)中。由于LCL细胞对转染具有极强的抗性，因此分离了经转染的细胞，并通过对所得基因组DNA进行HTS来测量编辑效率。在28％的来自转染细胞的总DNA测序读出中，观察到了Tyr282至Cys282密码子的干净转化，没有在靶基因座处有不期望的编辑或插入/缺失的证据(图6C)。尽管需要进行大量另外的研究以将这些ABE编辑策略发展为针对珠蛋白病、HHC和其他具有遗传组分的疾病的潜在未来临床疗法，但这些实施例共同证明了ABE在人细胞中校正驱动疾病的突变并安装已知抑制遗传疾病表型的突变的潜力。
总之，七轮演化和工程化将最初在DNA中的靶基因座处上未表现出使腺嘌呤脱氨基的能力的蛋白质(野生型TadA-dCas9融合物)转化为弱编辑DNA的形式(ABE1和ABE2)，有效地编辑位点的有限亚组的变体(ABE3、ABE4和ABE5)，以及最终具有广泛序列兼容性的高活性腺嘌呤碱基编辑器(ABE6和ABE7)。ABE的开发极大地扩展了碱基编辑的能力和可以通过基因组编辑而无需引入双链DNA断裂来解决的致病性SNP的部分(图1A)。另外，ABE还可以用于进行广泛效用的精确遗传变化，包括63个非同义密码子变化、起始密码子的破坏或创建、提前终止密码子的破坏、剪接供体或受体位点的修复，以及调节序列的修饰。对于一般的A·T至G·C碱基编辑，推荐ABE7.10。当靶A位于前间隔区位置8-10时，ABE7.9、ABE7.8或ABE6.3可以提供比ABE7.10更高的编辑效率，尽管这些位置处的转化效率通常低于前间隔区位置4-7。与BE3和BE49一起，通过将所有四个转换突变直接安装在活细胞中的靶基因座处，具有最少的不期望的副产物，这些ABE推动了基因组编辑领域的发展。
数据可用性。可从Addgene获得编码ABE6.3、ABE7.8、ABE7.9和ABE7.10的表达载体。高通量DNA测序数据将保存在NCBI Sequence Read Archive中。
方法
一般方法。除非另有说明，否则使用Phusion U Green多重PCR预混液(ThermoFisher Scientific)或Q5热启动高保真2x预混液(New England BioLabs)通过PCR进行DNA扩增。使用如前所述的USER克隆方法49组装了这项工作中生成的所有哺乳动物细胞和细菌质粒，并以细菌或哺乳动物密码子优化的gBlock基因片段(Integrated DNATechnologies)的方式合成了起始材料基因模板。所有sgRNA表达质粒均通过PCR产物的1片平末端连接而构建，该PCR产物含有对应于所期望的sgRNA靶定位点的可变20nt序列。表5中列出了用于合成本工作中使用的所有sgRNA质粒的引物和模板。所有哺乳动物ABE构建体sgRNA质粒和细菌构建体均在Mach1 T1R感受态细胞(其是recA–)(Thermo FisherScientific)中经转化并以甘油储备液的形式在-80℃下储存。分子生物学级的Hyclone水(GE Healthcare Life Sciences)用于所有测定法和PCR反应中。使用ZympPURE质粒Midiprep(Zymo Research Corportion)纯化了演化实验和哺乳动物细胞测定法中使用的所有载体，其包括去除内毒素。用于演化期间质粒维持或选择的抗生素购自GoldBiotechnology。
细菌TadA*文库的生成(第1-3、5和7轮演化)。简而言之，细菌ABE构建体的文库是由含有诱变的大肠杆菌TadA基因的PCR产物和含有编辑器质粒的其余部分(包括XTEN接头、dCas9、sgRNA、可选择标志物、复制起点和启动子)的PCR产物的两片USER组装生成的。具体而言，遵循制造商的方案，使用Mutazyme II(Agilent Technologies)以及引物NMG-823和824(表6)，在含有75ng-1.2μg的模板的8x 25μLPCR反应中将突变引入起始模板(表7)中。扩增后，将所得的PCR产物合并在一起，并使用MinElute PCR纯化试剂盒(Qiagen)从聚合酶和反应缓冲液中纯化。将PCR产物在37℃下用Dpn1(NEB)处理2小时以消化任何残留的模板质粒。随后，使用含有0.5μg/mL溴化乙锭的1％琼脂糖凝胶，通过凝胶电泳纯化所期望的PCR产物。使用QIAquick凝胶提取试剂盒(Qiagen)从凝胶中提取PCR产物，并用30μL的H2O洗脱。凝胶纯化后，使用Phusion U Green多重PCR预混液(8x 50μLPCR反应，66℃退火，20-s延伸)用引物NMG-825和NMG-826(表6)扩增诱变的ecTadA DNA片段以将适当的USER接合序列安装到片段的5’和3’末端上。通过凝胶电泳纯化所得的PCR产物。接下来，遵循制造商的方案，用引物NMG-799和NMG-824(表6)和Phusion U Green多重PCR预混液(98孔PCR板中每孔100μL，总共5-6个板，Tm 66℃，4.5分钟延伸)扩增细菌碱基编辑器质粒模板的主链(表7)。将每个PCR反应与300mL的PB DNA结合缓冲液(Qiagen)组合，并将25mL的溶液加样到HiBind DNA Midi柱(Omega Bio-Tek)上。用5倍柱体积的PE洗涤缓冲液(Qiagen)洗涤结合的DNA，并用每柱800μL的H2O洗脱DNA片段。使用NanoDrop 1000分光光度计(Themo Fisher Scientific)对两个DNA片段进行定量。
根据先前报道的USER组装规程49，在以下条件下组装TadA*文库：将每10μL的USER组装混合物中0.22pmol的ecTadA诱变的DNA片段1，0.22pmol的质粒主链片段2，1U的USER(尿嘧啶特异性切除试剂，New England Biolabs)和1U的DpnI酶(New England Biolabs)在50mM乙酸钾、20Mm Tris-乙酸盐、10mM乙酸镁、100μg/mL BSA，pH7.9中(1x CutSmartBuffer，New England Biolabs)中组合。通常，每轮演化需要约1mL的USER组装混合物(每个DNA装配片段为22nmol)，将其在多个8孔PCR条上分装为10μL等分试样。将反应加热至37℃60分钟，然后加热至80℃ 3分钟以使两种酶变性。将组装混合物在热循环仪中以0.1℃/s的速度缓慢冷却至12℃，以促进两个USER接合的新生成末端的退火。
用手中的构建体文库，通过向组装反应混合物中添加5体积的PB缓冲液(Qiagen)，并将材料结合到MinElute柱上(每柱480μL)，从组装混合物中除去变性的酶和反应缓冲液。将ABE杂交文库构建体在每柱30μL的H2O中洗脱，并将2μL的该洗脱的材料添加到20μL的NEB10-beta电感受态大肠杆菌中，并使用细菌程序5在16孔Nucleocuvette条中用Lonza4D-核转染系统进行电穿孔。典型的演化轮次使用约300次电穿孔来生成5-10百万个菌落形成单位(cfu)。在37℃的200mL预热的Davis Rich Media(DRM)中回收新鲜电穿孔的大肠杆菌，并在500mL通风有挡板(vented baffled)的烧瓶中以200rpm摇动温育15分钟，然后再加入羧苄青霉素(用于质粒维持)至30μg/mL。将培养物在37℃下以200rpm摇动温育18小时。遵循制造商的规程，用ZympPURE质粒Midiprep试剂盒分离质粒文库(每DNA柱50mL培养物)，除了质粒文库在每柱200μL预热的水中洗脱。遵循此规程进行第1-3、5和7轮演化，以生成具有较小变化的相应的文库(表7)。
位点饱和的细菌TadA*文库的生成(演化第4轮)。使用ecTadA*(2.1)-dCas9作为模板并用经适当设计的简并的含NNK引物(表6)进行扩增来实现ecTadA的Arg24、Glu25、Arg107、Ala142和Ala143处的诱变。简而言之，使用Phusion U Green多重PCR预混液，用两组引物：NMG-1197+NMG-1200和NMG-1199+NMG-1200分别扩增ecTadA*(2.1)-dCas9模板，从而分别形成PCR产物1和PCR产物2。两种PCR产物均使用PB结合缓冲液和MiniElute柱分别纯化，并且以每200μL的PCR反应用20μL的H2O洗脱。在第三次PCR反应中，将1μL的PCR产物1和1μL PCR产物2与外部含尿嘧啶的引物NMG-1202和NMG-1197组合，并通过Phusion U Green多重PCR预混液进行扩增以形成所期望的具有含尿嘧啶USER接合侧翼的延伸重叠PCR产物。在第四次PCR反应中，用NMG-1201和NMG-1198扩增ecTadA*(2.1)-dCas9，以生成用于USER组装的主链DNA片段。在对两个USER组装片段进行DpnI消化和凝胶纯化后，如上所述，通过USER组装将延伸重叠PCR产物(在ecTadA中含有所期望的NNK突变)掺入到ecTadA*(2.1)-dCas9主链中。将新鲜生成的NNK文库转化到NEB10-beta电感受态大肠杆菌中，并如上所述收获DNA。
DNA改组的细菌TadA*文库的生成(演化第6轮)。DNA改组是通过核苷酸交换和切除技术(NExT)DNA改组方法50的修改版本实现的。新鲜制备dATP、dCTP、dGTP和dTTP/dUTP(3份dUTP:7份dTTP)各10mM的溶液。接下来，使用Taq DNA聚合酶(NEB)，引物NMG-822和NMG-823(表6)和1x ThermoPol反应缓冲液中各400μM的dATP、dCTP、dGTP和dUTP/dTTP(3:7)中以等摩尔浓度从演化第1-5轮中分离的20fmol的TadA*-XTEN-dCas9细菌构建体的库扩增TadA*片段(Tm 63℃，1.5分钟延伸时间)。通过凝胶电泳纯化新鲜生成的含尿嘧啶的DNA文库片段，并用QIAquick凝胶提取试剂盒(Qiagen)提取，每提取柱用20μL的H2O洗脱。在1xCutSmart缓冲液中，每40μL用2U的USER酶在37℃下消化纯化的DNA产物，并通过分析琼脂糖凝胶电泳进行监测，直到消化完成。当不再观察到起始材料时(通常在37℃下3-4小时)，用10体积的PN1结合缓冲液(Qiagen)淬灭反应。如果需要，将另外的USER酶加入反应中。使用制造商的方案用QiaexII试剂盒(Qiagen)纯化消化的材料，并在每柱50μL的预热的H2O中洗脱DNA片段。
通过内部引物延伸规程，将纯化的改组的TadA*片段重新组装为全长TadA*-XTENdCas9产物。将洗脱的消化的DNA片段(25μL)与4U的Vent聚合酶(NEB)，各800μM的dATP、dCTP、dGTP和dTTP，1U的Taq DNA聚合酶在补充有0.5mM MgSO4的1x ThermoPol缓冲液中组合。重新组装规程的热循环程序如下：94℃ 3分钟，40个循环的92℃下变性30s，在起始于30℃的逐渐增加的温度下退火60s以上，每循环增加1℃(冷却坡度＝1℃/s)，和72℃下延伸60s，每循环另外的4s，以及以最后一个循环的72℃10分钟结束。在以下条件下用PCR扩增重新组装的产物：将15μL的未纯化的内部组装与各1μM的USER引物NMG-825和NMG-826，100μL的Phusion U Green多重PCR预混液和H2O组合至200μL的最终体积，63℃退火，延伸时间30s。通过凝胶电泳纯化PCR产物，并使用USER方法将其组装到相应的ecTadA*-XTEN-dCas9主链中，该主链具有如之前通过用USER引物NMG-799和NMG-824扩增主链而生成的相应的侧翼USER接合。在将杂交文库转化到NEB10-beta电感受到大肠杆菌中后，遵循制造商的规程，使用ZymoPURE质粒Midiprep试剂盒分离了演化6构建体的文库。
TadA变体的细菌演化。在所有演化实验中均使用了先前描述的菌株S103051，并如先前所述49制备了细菌的电感受态形式，中带有针对每轮演化的适当的选择质粒(表7)。简而言之，将如上所述制备的2μL的新鲜生成的TadA*文库(300-600ng/μL)添加到22μL的含有靶选择质粒的新鲜制备的电感受态S1030细胞中，并使用细菌程序5在16孔Nucleocuvette条中用Lonza4D-核转染系统进行电穿孔。典型的选择使用5-10x 106cfu。电穿孔后，将新鲜转化的S1030细胞在总共250mL的预热的DRM培养基中于37℃以200rpm摇动15分钟进行恢复。在短暂的恢复温育后，加入羧苄青霉素至终浓度为30μg/mL以维持文库质粒，以及适当的抗生素以维持选择质粒；选择条件的列表(包括每轮使用的抗生素)参见表7。添加质粒维持抗生素后，立即向培养物中添加100mM的L-阿拉伯糖以诱导TadA*–dCas9融合物文库成员的翻译，其从PBAD启动子表达。将培养物在37℃下以200rpm摇动生长18h至饱和，除了第5轮演化的温育时间仅为7h。
通过将10mL的饱和培养物铺板到四个500-cm2方形培养皿中的每一个上来攻击文库成员，培养皿包含1.8％琼脂-2xYT，30μg/mL的质粒维持抗生素和一定浓度的预确定高于仅带有抗生素的S1030菌株的MIC的选择抗生素(表8)。将板在37℃下温育2天，并分离出约500个存活的菌落。用引物NMG-822和NMG-823(表6)通过PCR扩增来自这些菌落的TadA*基因，并提交与DNA测序。同时，将菌落分别接种在96深孔板中的1-mL DRM培养物中，并在37℃，200r.p.m下生长过夜。合并各过夜培养物的等分试样(100μL)，分离质粒DNA，并用USER引物NMG-825和NMG-826(表6)扩增TadA*基因。用上述USER组装方案将TadA*基因亚克隆回到质粒主链(含有XTEN接头–dCas9和适当的引导RNA)。将该富集的文库转化到合适的S1030(+选择质粒)电感受态细胞中，与维持抗生素和L-Ara温育，并用选择条件再次攻击。温育2天后，如上所述分离出300-400个存活克隆，并对其TadA*基因进行测序。如下所述，将从每个选择轮产生的突变导入到哺乳动物ABE构建体中，并在哺乳动物细胞中进行测试。
一般哺乳动物细胞培养条件。HEK293T(ATCC CRL-3216)和U2OS(ATTC HTB-96)购自ATCC，并在补充有10％(v/v)胎牛血清(FBS)的Dulbecco改良Eagle培养基(DMEM)加GlutaMax(ThermoFisher Scientific)中培养并传代。Hap1(Horizon Discovery，C631)和Hap1 AAG细胞(Horizon Discovery，HZGHC001537c002)在补充有10％(v/v)FBS的Iscove的改良Dulbecco培养基(IMDM)加GlutaMax(ThermoFisher Scientific)中维持。含有HFE基因的C282Y突变的永生化的淋巴样干细胞(LCL)(Coriell Biorepository，GM14620)在补充有20％FBS的Roswell Park Memorial Institute Medium1640(RPMI-1640)加GlutaMax(ThermoFisher Scientific)中维持。所有细胞类型在37℃及5％CO2中温育、维持个培养。
HEK293T组织培养转染方案和基因组DNA制备。将在不存在抗生素的情况下生长的HEK293T细胞接种到48孔聚D-赖氨酸包被的板(Corning)上。接种后12-14小时，根据制造商的方案，用1.5μL的Lipofectamine 2000(Thermo Fisher Scientific)、750ng的ABE质粒、250ng的sgRNA表达和10ng的GFP表达质粒(Lonza)转染处于约70％汇合的细胞。除非另有说明，否则将细胞培养5天，第3天更换培养基。去除培养基，用1x PBS溶液(Thermo FisherScientific)洗涤细胞，并通过将100μL新鲜制备的裂解缓冲液(10mM Tris-HCl，pH 7.0，0.05％SDS，25μg/mL蛋白酶K(ThermoFisher Scientific))直接添加到组织培养板的每个孔中来提取基因组DNA。将基因组DNA混合物转移到96孔PCR板中，在37℃温育1h，然后80℃酶变性步骤30分钟。表9中列出了用于哺乳动物细胞基因组DNA扩增的引物。
HAP1和HAP1 AAG-细胞的核转染和基因组DNA提取。根据制造商的方案，使用SE细胞系4D-Nucleofector X试剂盒S对HAP1和HAP1 AAG-细胞进行了核转染。简而言之，使用4D-Nucleofector程序DZ-113，用300ng的ABE质粒和100ng的sgRNA表达质粒对4x 105个细胞进行核转染，并在48孔聚D-赖氨酸包被的培养板中的250μL的培养基中培养3天。如上所述提取DNA。
U2OS细胞的核转染和基因组DNA提取。根据制造商的方案，使用SE细胞系4D-Nucleofector X试剂盒(Lonza)对U2OS细胞进行了核转染。简而言之，使用4D-Nucleofector程序EH-100在16孔Nucleocuvette条(每孔20μL的细胞)中将1.25x 105个细胞与500ng的ABE质粒和100ng的sgRNA表达质粒一起在20μL的SG缓冲液中进行核转染。将新鲜经核转染的细胞转移到48孔聚D-赖氨酸包被的培养板中的250μL的培养基中。将细胞温育5天，标签每天更换培养基。如上所述提取DNA。
LCL HFE C828Y细胞的电穿孔。使用Gene Pulser Xcell电穿孔仪(BioRad)和0.4cm间隙的Gene Pulser电穿孔比色皿(BioRad)对LCL细胞进行电穿孔。简而言之，将1x107个LCL细胞重悬于250μL RPMI-160加GlutaMax中。向该培养基中添加65μg的表达ABE7.10、GFP的质粒，以及靶向HFE基因中C282Y突变的相应sgRNA。将混合物添加到预冷的0.4cm间隙电穿孔比色皿中，并将细胞/DNA混合物在比色皿中冰上温育10分钟。以250V和950μF对细胞进行脉冲3ms。将细胞转移回冰上10分钟，然后转移到T-75烧瓶中15mL补充有20％FBS的预热的RPMI-160中。第二天，将另外的5mL的培养基加入到烧瓶中，并且将细胞温育总共5天。温育后，通过离心分离细胞，将其重悬于400μL的培养基中，通过40μm滤网(Thermo Fisher Scientific)过滤，并使用FACSAria III流式细胞仪(Becton DickensonBiosciences)分选GFP荧光。GFP阳性细胞收集在含有500μL的培养基的1.5mL试管中。离心后，除去培养基，并用600μL的1x PBS(Thermo Fisher Scientific)洗涤细胞两次。如上所述提取基因组DNA。
ABE 7.10和使用“CORRECT”方法52的同源性定向修复之间的比较。将在不存在抗生素的情况下生长的HEK293T细胞接种在48孔聚-D赖氨酸包被的板(Corning)上。12-14小时后，用750ng的Cas9或碱基编辑质粒、250ng的sgRNA表达质粒、1.5μL的Lipofectamine 3000(Thermo Fisher Scientific)转染处于约70％汇合的细胞，标签用于HDR测定法时，根据制造商的说明，用0.7μg的单链供体DNA模板(100nt，从IDT进行PAGE纯化)。表10中列出了100-mer单链寡核苷酸供体模板。
根据制造商的说明，使用Agencourt DNAdvance基因组DNA分离试剂盒(BeckmanCoulter)，在转染后48小时收获基因组DNA(如Tessier-Lavigne等人在开发CORRECT方法期间所述52)。大小选择性的DNA分离步骤确保了在随后的PCR扩增和测序步骤中不存在单链供体DNA模板污染的风险。重新设计了扩增引物，以确保扩增供体寡核苷酸模板的风险最小。
基因组DNA样品的高通量DNA测序(HTS)。用含有与感兴趣的区域的同源性的引物和适当的Illumina正向和反向衔接物，通过PCR扩增感兴趣的基因组位点(表9)。在表9中可以找到该工作中讨论的所有基因组位点在第一轮PCR(PCR 1)中使用的引物对。具体而言，组装了25μL的给定PCR 1反应，其中含有正向引物和反向引物各0.5μM，1μL的基因组DNA提取物和12.5μL的Phusion U Green多重PCR预混液。PCR反应如下进行：95℃ 2分钟，然后30个循环的[95℃ 15s，62℃ 20s和72℃ 20s]，然后最终72℃延伸2分钟。通过在补充有溴化乙锭的2％琼脂糖凝胶上与DNA标准品(Quick-Load 100bp DNA ladder)进行比较来验证PCR产物。在次级PCR反应(PCR 2)中，将独特的Illumina条形码引物对添加到每个样品中。具体而言，组装25μL的给定的PCR 2反应，其中含有独特的正向和反向illumine条形码对引物对各0.5μM，2μL的未纯化的PCR 1反应混合物和12.5μL的Q5热启动高保真2x预混液。条形码PCR 2反应如下进行：95℃ 2分钟，然后15个循环的[95℃ 15s，61℃ 20s和72℃ 20s]，然后最终72℃延伸2分钟。使用QIAquick凝胶提取试剂盒用2％琼脂糖凝胶通过电泳纯化PCR产物，并用30μL的H2O洗脱。用KAPA文库定量试剂盒-Illumina(KAPA Biosystems)对DNA浓度定量，并根据制造商的方案在Illumina MiSeq仪器上进行测序。
一般HTS数据分析。测序读出在MiSeq Reporter(Illumina)中解复用。如前所述，使用具有改进的输出格式的Matlab脚本，进行了扩增子序列与参考序列的比对(补充说明1)。简而言之，使用Smith-Waterman算法将没有插入/缺失的序列与参考序列进行比对；质量得分低于30的碱基转化为“N”，以防止由于测序错误而导致碱基错误判定(miscall)。使用先前描述的Matlab脚本的修改版本，分别对插入/缺失进行定量，其中滤除了超过一半碱基判定(call)低于Q30的质量得分的测序读出(补充说明2)。将插入/缺失视为包含在预测的Cas9切割位点周围的30bp窗口内大于或等于1bp的插入或缺失的读出。
由于HBG1和HBG2基因座中的同源性，引物经设计将在单个PCR反应中扩增两个基因座。为了从计算上分离这两个基因组位点的序列，使用单独的Python脚本处理了涉及该扩增子的测序实验(补充说明3)。简而言之，如果超过一半的碱基判定低于Q30，并且质量得分低于Q30的碱基判定转化为“N”，则忽略读出。HBG1或HBG2读出经鉴定为与含有在位点之间不同的两个SNP的37bp序列完全匹配。碱基判定和插入/缺失窗口是通过与以前间隔区序列为中心的43bp窗口两侧的10bp侧翼序列精确匹配来定义的。插入/缺失视为其中该碱基判定窗口的长度相差≥1bp的读出。该Python脚本产生具有相同的质量的输出(估计的碱基判定错误率<1,000中的1)，但是由于不存在比对步骤，因此产生的时间要少得多。
为了讲编辑的读出的总数计算为成功测序的读取读出的总数的比例，将通过比对算法测量的编辑的读出的分数乘以[1–含有插入/缺失的读出的分数]。
连锁不平衡分析。使用定制的Python脚本(补充说明4)来评价主要靶A(P1)，次要靶A(P2)以及主要和次要靶A(P1,2)两者的编辑概率。然后将连锁不平衡(LD)评估为P1,2–(P1x P2)。用Min(P1(1–P2),(1–P1)P2)的标准化因子对LD值进行标准化。此标准化控制等位基因频率，并产生从0至1的标准化LD值。
实施例7-ABE介导的HBG1/2启动子中突变的安装以治疗人类疾病，如镰状细胞和beta地中海贫血
图15中指示的sgRNA靶向HBG1/2启动子中的位点，主要集中在已知赋予成年人中胎儿血红蛋白上调的突变处或附近。将ABE 7.10和单个sgRNA质粒(750ng的编辑器，250ng的引导)脂转染到HEK293T细胞中。在HEK293T细胞中表达三天后，吸出培养基并添加新鲜培养基。再生长两天后，提取DNA。进行PCR以扩增HBG1/2启动子区域。使用Illumina MiSeq对PCR产物进行测序，并对每个样品的编辑进行定量。在图15中绘制了用指定的引导处理的细胞的窗口内观察到的最高编辑。这些结果表明可以引入可以在成年人中上调胎儿血红蛋白的突变。引入此类突变可以治疗镰状细胞病和beta地中海贫血。
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表1.未经处理的HEK293T细胞和在17个基因组位点处用与相应的sgRNA表达质粒共转染的ABE6.3、ABE7.8、ABE7.9或ABE7.10处理的HEK293T细胞的HTS测序结果以及％插入/缺失。显示了一个任意选择的重复；可从NCBI测序读出档案中获得所有重复的数据。
Figure BDA0002539874730001671
Figure BDA0002539874730001681
Figure BDA0002539874730001691
Figure BDA0002539874730001701
Figure BDA0002539874730001711
Figure BDA0002539874730001721
Figure BDA0002539874730001731
Figure BDA0002539874730001741
Figure BDA0002539874730001751
Figure BDA0002539874730001761
Figure BDA0002539874730001771
Figure BDA0002539874730001781
Figure BDA0002539874730001791
Figure BDA0002539874730001801
Figure BDA0002539874730001811
Figure BDA0002539874730001821
Figure BDA0002539874730001831
表2.ABE7.8、ABE7.9和ABE7.10在以前针对酿脓链球菌Cas9核酸酶1表征的HEK2在靶和脱靶位点处的活性。
Figure BDA0002539874730001841
表3.ABE7.8、ABE7.9和ABE7.10在以前针对酿脓链球菌Cas9核酸酶1的在靶和脱靶修饰表征的HEK3位点处的活性。
Figure BDA0002539874730001851
Figure BDA0002539874730001852
表4.ABE7.8、ABE7.9和ABE7.10在以前针对酿脓链球菌Cas9核酸酶1的在靶和脱靶修饰表征的HEK4位点处的活性。尽管HEK4脱靶位点3在ABE处理后显示出明显的插入/缺失形成，但该基因座也显示出Cas9核酸酶异常高的插入/缺失形成(89％)，并且是唯一经测试在用Cas9切口酶处理后显示出插入/缺失形成的脱靶位点。据推测，该基因座异常脆弱，并且此处插入/缺失形成的原因仅是对位点进行切口引起，而不是由ABE介导的腺嘌呤脱氨基引起的。
Figure BDA0002539874730001861
表5.用于生成sgRNA质粒的引物。20nt靶前间隔区以红色显示。由于人类U6启动子在转录起始位点处偏好‘G’2-4，当靶DNA序列不以‘G’起始时在引物的5’末端添加了‘G’。先前描述的pFYF sgRNA质粒5用作PCR扩增的模板。
Figure BDA0002539874730001871
表6.用于生成细菌TadA*文库的引物。
Figure BDA0002539874730001872
表7.用于每轮TadA*诱变和在大肠杆菌中选择的起始构建体。所有质粒均含有SC101复制起点、β-内酰胺酶基因(用于用羧苄青霉素维持质粒)、驱动TadA*–dCas9表达的PBAD启动子以及一个驱动sgRNA转录的lac启动子。在细菌选择期间使用的碱基编辑器的构造为：TadA*-接头(16aa)-dCas9。
Figure BDA0002539874730001881
表8.抗生素选择质粒及其对应的大肠杆菌抗生素最低抑制浓度(MIC)。
Figure BDA0002539874730001882
表9.用于哺乳动物细胞基因组DNA扩增的引物。
Figure BDA0002539874730001891
Figure BDA0002539874730001901
表10.HDR实验中使用的100-mer单链寡核苷酸供体模板(ssODN)。
Figure BDA0002539874730001902
补充序列1.该研究中使用的腺嘌呤脱氨基酶的DNA序列。
细菌密码子优化的ecTadA(野生型)：
ATGTCTGAAGTCGAATTTAGCCACGAATACTGGATGCGTCACGCGCTGACGCTGGCGAAACGTGCCTGGGATGAGCGGGAAGTGCCGGTCGGCGCGGTATTAGTGCATAACAATCGGGTAATCGGCGAAGGCTGGAACCGCCCGATTGGTCGCCATGATCCCACCGCACATGCAGAAATCATGGCCCTGCGGCAGGGTGGTCTGGTGATGCAAAATTATCGTCTGATCGACGCCACGTTGTATGTCACGCTTGAACCATGTGTAATGTGTGCCGGAGCGATGATCCACAGTCGCATTGGTCGCGTGGTCTTTGGTGCGCGTGACGCGAAAACTGGCGCTGCGGGATCTTTAATGGATGTGCTGCATCATCCGGGTATGAATCACCGAGTGGAAATTACGGAAGGAATACTGGCGGATGAGTGCGCGGCGTTGCTCAGTGACTTCTTTCGCATGCGCCGCCAGGAAATTAAAGCGCAGAAAAAAGCGCAATCCTCGACGGAT(SEQ ID NO:31)
哺乳动物密码子优化的ecTadA(野生型)：
ATGTCCGAAGTCGAGTTTTCCCATGAGTACTGGATGAGACACGCATTGACTCTCGCAAAGAGGGCTTGGGATGAACGCGAGGTGCCCGTGGGGGCAGTACTCGTGCATAACAATCGCGTAATCGGCGAAGGTTGGAATAGGCCGATCGGACGCCACGACCCCACTGCACATGCGGAAATCATGGCCCTTCGACAGGGAGGGCTTGTGATGCAGAATTATCGACTTATCGATGCGACGCTGTACGTCACGCTTGAACCTTGCGTAATGTGCGCGGGAGCTATGATTCACTCCCGCATTGGACGAGTTGTATTCGGTGCCCGCGACGCCAAGACGGGTGCCGCAGGTTCACTGATGGACGTGCTGCATCACCCAGGCATGAACCACCGGGTAGAAATCACAGAAGGCATATTGGCGGACGAATGTGCGGCGCTGTTGTCCGACTTTTTTCGCATGCGGAGGCAGGAGATCAAGGCCCAGAAAAAAGCACAATCCTCTACTGAC(SEQ ID NO:32)
哺乳动物密码子优化的mADA：
ATGGCCCAGACACCCGCATTCAACAAACCCAAAGTAGAGTTACACGTCCACCTGGATGGAGCCATCAAGCCAGAAACCATCTTATACTTTGGCAAGAAGAGAGGCATCGCCCTCCCGGCAGATACAGTGGAGGAGCTGCGCAACATTATCGGCATGGACAAGCCCCTCTCGCTCCCAGGCTTCCTGGCCAAGTTTGACTACTACATGCCTGTGATTGCGGGCTGCAGAGAGGCCATCAAGAGGATCGCCTACGAGTTTGTGGAGATGAAGGCAAAGGAGGGCGTGGTCTATGTGGAAGTGCGCTATAGCCCACACCTGCTGGCCAATTCCAAGGTGGACCCAATGCCCTGGAACCAGACTGAAGGGGACGTCACCCCTGATGACGTTGTGGATCTTGTGAACCAGGGCCTGCAGGAGGGAGAGCAAGCATTTGGCATCAAGGTCCGGTCCATTCTGTGCTGCATGCGCCACCAGCCCAGCTGGTCCCTTGAGGTGTTGGAGCTGTGTAAGAAGTACAATCAGAAGACCGTGGTGGCTATGGACTTGGCTGGGGATGAGACCATTGAAGGAAGTAGCCTCTTCCCAGGCCACGTGGAAGCCTATGAGGGCGCAGTAAAGAATGGCATTCATCGGACCGTCCACGCTGGCGAGGTGGGCTCTCCTGAGGTTGTGCGTGAGGCTGTGGACATCCTCAAGACAGAGAGGGTGGGACATGGTTATCACACCATCGAGGATGAAGCTCTCTACAACAGACTACTGAAAGAAAACATGCACTTTGAGGTCTGCCCCTGGTCCAGCTACCTCACAGGCGCCTGGGATCCCAAAACGACGCATGCGGTTGTTCGCTTCAAGAATGATAAGGCCAACTACTCACTCAACACAGACGACCCCCTCATCTTCAAGTCCACCCTAGACACTGACTACCAGATGACCAAGAAAGACATGGGCTTCACTGAGGAGGAGTTCAAGCGACTGAACATCAACGCAGCGAAGTCAAGCTTCCTCCCAGAGGAAGAGAAGAAGGAACTTCTGGAACGGCTCTACAGAGAATACCAA(SEQ ID NO:33)
哺乳动物密码子优化的hADAR2(催化结构域)：
ATGCATCTCGATCAAACCCCGAGCCGCCAACCAATCCCGAGTGAAGGCCTGCAACTGCATCTGCCACAAGTTCTGGCGGATGCCGTTAGCCGCCTGGTCTTGGGTAAGTTCGGTGATCTGACAGACAACTTTTCTAGTCCACATGCTCGCCGTAAGGTGCTGGCTGGCGTTGTGATGACCACAGGTACAGACGTCAAAGATGCTAAAGTGATTTCTGTGTCTACTGGCACGAAGTGCATTAACGGCGAATATATGTCTGACCGTGGCTTAGCGCTTAACGATTGTCATGCCGAAATCATCTCCCGTCGTTCATTGCTTCGCTTCCTGTACACGCAGTTGGAACTGTATCTGAATAACAAAGACGATCAGAAGCGTTCTATTTTCCAGAAGTCTGAGCGCGGCGGGTTCCGTCTTAAAGAGAATGTGCAGTTTCACCTTTATATTTCAACCTCTCCTTGTGGTGATGCCCGTATTTTTTCACCACACGAACCTATTTTAGAGGAACCGGCCGATCGTCATCCGAACCGCAAAGCCCGTGGGCAGCTGCGTACGAAAATCGAATCAGGTGAAGGCACCATTCCCGTCCGCTCCAATGCGAGCATTCAAACGTGGGACGGTGTGTTACAGGGCGAACGCCTGTTAACCATGAGCTGCTCAGACAAAATTGCACGTTGGAACGTGGTAGGCATCCAGGGCTCGTTATTGAGCATTTTCGTGGAGCCGATTTATTTTAGTTCCATCATTTTGGGCTCACTCTACCACGGCGATCACCTTAGCCGCGCGATGTACCAGCGCATTAGTAACATCGAAGATTTACCGCCCCTGTATACCCTGAACAAACCACTGTTAAGCGGTATTTCTAACGCGGAGGCGCGTCAGCCTGGTAAAGCCCCGAACTTCAGTGTGAACTGGACTGTGGGTGATTCTGCAATTGAGGTAATTAACGCGACGACGGGTAAAGATGAACTGGGCCGTGCCTCTCGTCTGTGTAAACACGCGCTGTACTGTCGTTGGATGCGCGTGCACGGTAAAGTTCCCAGTCATCTGTTACGTAGCAAGATCACCAAGCCAAATGTCTACCACGAATCGAAGCTGGCCGCGAAAGAATACCAAGCGGCTAAGGCGCGTCTGTTCACCGCCTTTATTAAGGCTGGCTTAGGGGCCTGGGTGGAAAAACCAACCGAGCAAGATCAATTCAGTCTGACCCCG(SEQ ID NO:41)
哺乳动物密码子优化的hADAT2：
ATGGAGGCGAAGGCGGCACCCAAGCCAGCTGCAAGCGGCGCGTGCTCGGTGTCGGCAGAGGAGACCGAAAAGTGGATGGAGGAGGCGATGCACATGGCCAAAGAAGCCCTCGAAAATACTGAAGTTCCTGTTGGCTGTCTTATGGTCTACAACAATGAAGTTGTAGGGAAGGGGAGAAATGAAGTTAACCAAACCAAAAATGCTACTCGACATGCAGAAATGGTGGCCATCGATCAGGTCCTCGATTGGTGTCGTCAAAGTGGCAAGAGTCCCTCTGAAGTATTTGAACACACTGTGTTGTATGTCACTGTGGAGCCGTGCATTATGTGTGCAGCTGCTCTCCGCCTGATGAAAATCCCGCTGGTTGTATATGGCTGTCAGAATGAACGATTTGGTGGTTGTGGCTCTGTTCTAAATATTGCCTCTGCTGACCTACCAAACACTGGGAGACCATTTCAGTGTATCCCTGGATATCGGGCTGAGGAAGCAGTGGAAATGTTAAAGACCTTCTACAAACAAGAAAATCCAAATGCACCAAAATCGAAAGTTCGGAAAAAGGAATGTCAGAAATCT(SEQ ID NO:42)
补充序列2.该研究中使用的抗生素抗性基因的DNA序列。失活突变以粗体显示。
氯霉素抗性基因(CamR)H193Y：
Figure BDA0002539874730001921
Figure BDA0002539874730001931
卡那霉素抗性基因(KanR)Q4终止和W15终止：
Figure BDA0002539874730001932
壮观霉素抗性基因(SpectR)T89I：
Figure BDA0002539874730001933
卡那霉素抗性基因(KanR)Q4终止和D208N：
Figure BDA0002539874730001934
Figure BDA0002539874730001941
补充说明1.碱基判定的Matlab脚本。
function basecall(WTnuc,directory)
％cycle through fastq files for different samples cd directory
files＝dir('*.fastq')；for d＝1:2
filename＝files(d).name；
％read fastq file
[header,seqs,qscore]＝fastqread(filename)；
seqsLength＝length(seqs)；％number of sequences seqsFile＝strrep(filename,'.fastq',”)；％trims off.fastq
％create a directory with the same name as fastq file if exist(seqsFile,'dir')；
error('Directory already exists.Please rename or move it beforemoving on.')；
end
mkdir(seqsFile)；％make directory
wtLength＝length(WTnuc)；％length of wildtype sequence
％％aligning back to the wildtype nucleotide sequence
％
％AlN is a matrix of the nucleotide alignment window＝1:wtLength；
sBLength＝length(seqs)；％number of sequences
％counts number of skips nSkips＝0；
ALN＝repmat(”,[sBLength wtLength])；
％iterate through each sequencing read for i＝1:sBLength
％If you only have forward read fastq files leave as is
％If you have R1 foward and R2 is reverse fastq files uncomment the
％next four lines of code and the subsequent end statement
％if mod(d,2)＝＝0；
％reverse＝seqrcomplement(seqs{i})；
％[score,alignment,start]＝swalign(reverse,WTnuc,'Alphabet','NT')；
％else
[score,alignment,start]＝swalign(seqs{i},WTnuc,'Alphabet','NT')；
％end
％length of the sequencing read len＝length(alignment(3,:))；
％if there is a gap in the alignment,skip＝1 and we will
％throw away the entire read skip＝0；
for j＝1:len
if(alignment(3,j)＝＝'-'||alignment(1,j)＝＝'-')skip＝1；
break；
letters)
end
％in addition if the qscore for any given base in the read is
％below 31 the nucleotide is turned into an N(fastq qscores that arenot
if isletter(qscore{i}(start(1)+j-1))else
alignment(1,j)＝'N'；
end
end
if skip＝＝0&&len>10
ALN(i,start(2):(start(2)+length(alignment)-1))＝alignment(1,:)；
end
end
％with the alignment matrices we can simply tally up the occurrencesof
％each nucleotide at each column in the alignment these
％tallies ignore bases annotated as N
％due to low qscores TallyNTD＝zeros(5,wtLength)；FreqNTD＝zeros(4,wtLength)；
SUM＝zeros(1,wtLength)；
for i＝1:wtLength
TallyNTD(:,i)＝[sum(ALN(:,i)＝＝'A'),sum(ALN(:,i)＝＝'C'),sum(ALN(:,i)＝＝'G'),sum
(ALN(:,i)＝＝'T'
),sum(ALN(:,i)＝＝'N')]；
end
for i＝1:wtLength FreqNTD(:,i)＝100*TallyNTD(1:4,i)/sum(TallyNTD(1:4,i))；
end
for i＝1:wtLength SUM(:,i)＝sum(TallyNTD(1:4,i))；
end
％we then save these tally matrices in the respective folder for
％further processing
save(strcat(seqsFile,'/TallyNTD'),'TallyNTD')；
dlmwrite(strcat(seqsFile,'/TallyNTD.csv'),TallyNTD,'precision','％.3f','newline','pc')；
save(strcat(seqsFile,'/FreqNTD'),'FreqNTD')；
dlmwrite(strcat(seqsFile,'/FreqNTD.csv'),FreqNTD,'precision','％.3f','newline','pc')；
fid＝fopen('FrequencySummary.csv','a')；fprintf(fid,'\n\n')；
fprintf(fid,filename)；fprintf(fid,'\n\n')；
dlmwrite('FrequencySummary.csv',FreqNTD,'precision','％.3f','newline','pc','-append')；
dlmwrite('FrequencySummary.csv',SUM,'precision','％.3f','newline','pc','-append')；
end
％set up queue of basecalling runs
％change directory to folder of fastq files for a given target sitecd('/Users/michaelpacker/Documents/MATLAB/BaseCallingWithSummary')cdPUTFOLDERNAMEHERE
％call upon the basecall program basecall(PUTWTSEQUENCEHERE)
％and repeatcd('/Users/michaelpacker/Documents/MATLAB/BaseCallingWithSummary')cdPUTFOLDERNAMEHERE
basecall(PUTWTSEQUENCEHERE)
％and repeat…
补充说明2.插入/缺失分析的Matlab脚本。
％WTnuc＝'CGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGAGATCCGCGGCCGCTAATACGACTCAC
CCTAGGGAGAGCCGCCACCGTGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA
ACGGCCACAAGTTCAGCGTGTCCGGCGAG'；
％cycle through fastq files for different samples files＝dir('*.fastq')；
indelstart＝55；width＝30；flank＝10；
(SEQ ID NO:85)
for d＝1:2
filename＝files(d).name；
％read fastq file
[header,seqs,qscore]＝fastqread(filename)；
seqsLength＝length(seqs)；％number of sequences
seqsFile＝strcat(strrep(filename,'.fastq',”),'_INDELS')；％trimsoff.fastq
％create a directory with the same name as fastq file+_INDELS ifexist(seqsFile,'dir')；
error('Directory already exists.Please rename or move it beforemoving on.')；
end
mkdir(seqsFile)；％make directory
wtLength＝length(WTnuc)；％length of wildtype sequence sBLength＝length(seqs)；％number of sequences
％initialize counters and cell arrays nSkips＝0；
notINDEL＝0；
ins＝{}；
dels＝{}；NumIns＝0；NumDels＝0；
％iterate through each sequencing read for i＝1:sBLength
％search for 10BP sequences that should flank both sides of the"INDELWINDOW"windowstart＝strfind(seqs{i},WTnuc(indelstart-flank:indelstart))；windowend＝strfind(seqs{i},WTnuc(indelstart+width:indelstart+width+flank))；
％if these flanks are found and more than half of base calls
％are above Q31 THEN proceed OTHERWISE save as a skip if length(windowstart)＝＝1&&length(windowend)＝＝1&&(sum(isletter(qscore{i}))/length(qscore{i}))>＝0.5
％if the sequence length matches the INDEL window length save as
％not INDEL
if windowend-windowstart＝＝width+flank notINDEL＝notINDEL+1；
％if the sequence is ONE or more baseslonger than the INDEL
％window length save as an Insertion
elseif windowend-windowstart>＝width+flank+1 NumIns＝NumIns+1；
ins{NumIns}＝seqs{i}；
％if the sequence is ONE or more bases shorter than the INDEL
％window length save as a Deletion
elseif windowend-windowstart<＝width+flank-1 NumDels＝NumDels+1；dels{NumDels}＝seqs{i}；
end
％keep track of skipped sequences that do not posess matching flank
％sequences and do not pass quality cutoff else
nSkips＝nSkips+1；
end
end INDELrate＝(NumIns+NumDels)/(NumIns+NumDels+notINDEL)*100.；FID
＝fopen('INDELSummary.csv','a')；
fprintf(FID,'\n\n')；fprintf(FID,filename)；fprintf(FID,'\n')；
fprintf(FID,num2str(INDELrate))；
fid＝fopen(strcat(seqsFile,'/summary.txt'),'wt')；
fprintf(fid,'Skipped reads％i\n not INDEL ％i\n Insertions％i\nDeletions％i\n INDEL percent％e\n',[nSkips,notINDEL,NumIns,NumDels,INDELrate])；
fclose(fid)；
save(strcat(seqsFile,'/nSkips'),'nSkips')；save(strcat(seqsFile,'/notINDEL'),'notINDEL')；save(strcat(seqsFile,'/NumIns'),'NumIns')；save(strcat(seqsFile,'/NumDels'),'NumDels')；save(strcat(seqsFile,'/INDELrate'),'INDELrate')；save(strcat(seqsFile,'/dels'),'dels')；
C＝dels；
fid＝fopen(strcat(seqsFile,'/dels.txt'),'wt')；fprintf(fid,'"％s"\n',C{:})；
fclose(fid)；
save(strcat(seqsFile,'/ins'),'ins')；C＝ins；
fid＝fopen(strcat(seqsFile,'/ins.txt'),'wt')；fprintf(fid,'"％s"\n',C{:})；
fclose(fid)；
补充说明3.分析HBG1和HBG2碱基编辑和插入/缺失的Python脚本。
％matplotlib inline import numpy as np import scipy as sp importmatplotlib as mpl
import matplotlib.cm as cm import matplotlib.pyplot as plt importpandas as pd pd.set_option('display.width',500)
pd.set_option('display.max_columns',100)
pd.set_option('display.notebook_repr_html',True)import seaborn as snssns.set_style("whitegrid")sns.set_context("poster")import requests importtime
from bs4 import BeautifulSoup import regeximport re import os
from Bio import SeqIO import Bio
from Bio import motifs from Bio import pairwise2
from Bio.pairwise2 import format_alignment from Bio.Alphabet importIUPAC
from sklearn import preprocessing
basecall analysis with 50％Q31 cutoff on protospacer region(asdefined by flanks)
#includes a check for match with two HBG1 SNPs
#inputs:
#directory,working directory folder containing all fastq files
#site,genomic site name as it appears in the fastq filenames
#orientation,'FWD'if you want output in the same direction as thesequencingread or'REV'if you want reverse complement output,
#flank1,sequence that is used to define the 5'end of protospacer inthe sequencingread direction,
#flank2,sequence that is used to define the 5'end of protospacer inthe sequencingread direction,
#width,expected bp length of basecalling window
#
#outputs:
#'_counts.csv',all base editing product sequences with correspondingnumber ofoccurences
#'_rawsummary.csv',summarizes base call counts for all samples
#'_normalizedsummary.csv',summarizes base call percentages for allsamples def basecallhbg1(directory,site,orientation,flank1,flank2,width):
indir＝directory outdir＝directory filenames＝os.listdir(indir)
for i in range(len(filenames)):seqs＝{}
if(filenames[i][-5:]＝＝'fastq')and(site in filenames[i]):for recordin SeqIO.parse(indir+filenames[i],"fastq"):
recordqual＝[x>31 for x in record.letter_annotations['phred_quality']]
#only process reads that have more than half of basecalls>Q31 andcontain two HBG1 specific SNPs at 3'end of read
if(record.seq.find('GTTTTTCTCTAATTTATTCTTCCCTTTAGCTAGTTTC')>0)
and
(float(sum(recordqual))/float(len(recordqual))>＝.5):
recordseq＝"".join([y if x else'N'for(x,y)in zip(recordqual,record.seq)])
(SEQ ID NO:86)
record.seq)])
first item
recordseq＝"".join([y if x else'N'for(x,y)in zip(recordqual,
#split prior to spacer window split1＝recordseq.split(flank1)if len(split1)＝＝2:
#take second item in first split
#split again at the sequence right after the protospacer and take
split2＝split1[1].split(flank2)[0]
#keep only entries with exact width if(len(split2)＝＝width):
if orientation＝＝'FWD':seqs[record.id]＝split2
elif orientation＝＝'REV':seqs[record.id]＝Bio.Seq.reverse_complement(split2)
frame＝pd.DataFrame({'Spacer':seqs.values()},index＝seqs.keys())
Motif＝motifs.create(frame.Spacer.values,alphabet＝IUPAC.IUPACAmbiguousDNA())raw＝pd.DataFrame(Motif.counts,index＝[str(s+1)fors in
range(width)])[['A','C','G','T','N']].transpose()
normalized＝pd.DataFrame(Motif.counts,index＝[str(s+1)for s in range(width)])[['A','C','G','T']].transpose()
normalized＝normalized/normalized.sum(axis＝0)*100.
normalized＝normalized.round(2)Counts＝pd.DataFrame(seqs.items(),columns＝['ID','Window'])Counts＝Counts[['N'not in x for x in Counts.Window]]
Counts＝Counts.groupby('Window').count().sort_values('ID',ascending＝False)
Counts.to_csv(outdir+filenames[i].strip('.fastq')+'_hbg1.csv')
fd＝open(directory+site+'_normalizedsummary_hbg1.csv','a')
fd.write('\n'+filenames[i]+'\n')
normalized.to_csv(fd)fd.close()
fd＝open(directory+site+'_rawsummary_hbg1.csv','a')
fd.write('\n'+filenames[i]+'\n')
raw.to_csv(fd)fd.close()
return
#basecall analysis with 50％Q31 cutoff on protospacer region(asdefined by flanks)
#includes a check for match with two HBG2 SNPs
#inputs:
#directory,working directory folder containing all fastq files
#site,genomic site name as it appears in the fastq filenames
#orientation,'FWD'if you want output in the same direction as thesequencing read or'REV'if you want reverse complement output,
#flank1,sequence that is used to define the 5'end of protospacer inthe sequencing read direction,
#flank2,sequence that is used to define the 5'end of protospacer inthe sequencing read direction,
#width,expected bp length of basecalling window
#
#outputs:
#'_counts.csv',all base editing product sequences with correspondingnumber of occurences
#'_rawsummary.csv',summarizes base call counts for all samples
#'_normalizedsummary.csv',summarizes base call percentages for allsamples def basecallhbg2(directory,site,orientation,flank1,flank2,width):
indir＝directory outdir＝directory filenames＝os.listdir(indir)
for i in range(len(filenames)):seqs＝{}
if(filenames[i][-5:]＝＝'fastq')and(site in filenames[i]):
for record in SeqIO.parse(indir+filenames[i],"fastq"):
recordqual＝[x>31 for x in record.letter_annotations['phred_quality']]
#only process reads that have more than half of basecalls>Q31 andcontain two HBG2 specific SNPs at 3'end of read
if(record.seq.find('ATTTTTCTCTAATTTATTCTTCCCTTTAGCTAGTTTT')>0)
and
(float(sum(recordqual))/float(len(recordqual))>＝.5):
recordseq＝"".join([y if x else'N'for(x,y)in zip(recordqual,
(SEQ ID NO:87)
record.seq)])
first item
#split prior to spacer window split1＝recordseq.split(flank1)if len(split1)＝＝2:
#take second item in first split
#split again at the sequence right after the protospacer and take
split2＝split1[1].split(flank2)[0]
#keep only entries with exact width if(len(split2)＝＝width):
if orientation＝＝'FWD':seqs[record.id]＝split2
elif orientation＝＝'REV':seqs[record.id]＝Bio.Seq.reverse_complement(split2)
frame＝pd.DataFrame({'Spacer':seqs.values()},index＝seqs.keys())
Motif＝motifs.create(frame.Spacer.values,alphabet＝IUPAC.IUPACAmbiguousDNA())raw＝pd.DataFrame(Motif.counts,index＝[str(s+1)fors in
range(width)])[['A','C','G','T','N']].transpose()
normalized＝pd.DataFrame(Motif.counts,index＝[str(s+1)for s inrange(width)])[['A','C','G','T']].transpose()
normalized＝normalized/normalized.sum(axis＝0)*100.
normalized＝normalized.round(2)Counts＝pd.DataFrame(seqs.items(),columns＝['ID','Window'])Counts＝Counts[['N'not in x for x in Counts.Window]]
Counts＝Counts.groupby('Window').count().sort_values('ID',ascending＝False)
Counts.to_csv(outdir+filenames[i].strip('.fastq')+'_hbg2.csv')
fd＝open(directory+site+'_normalizedsummary_hbg2.csv','a')
fd.write('\n'+filenames[i]+'\n')
normalized.to_csv(fd)fd.close()
fd＝open(directory+site+'_rawsummary_hbg2.csv','a')
fd.write('\n'+filenames[i]+'\n')
raw.to_csv(fd)fd.close()
return
#indel analysis
#includes a check for match with two HBG1 SNPs
#inputs:
#directory,working directory folder containing all fastq files
#site,genomic site name as it appears in the fastq filenames
#orientation,'FWD'if you want output in the same direction as thesequencing read or'REV'if you want reverse complement output,
#flank1,sequence that is used to define the 5'end of protospacer inthe sequencing read direction,
#flank2,sequence that is used to define the 5'end of protospacer inthe sequencing read direction,
#width,expected bp length of basecalling window
#ouputs:
#"_Insertions_hbg1.csv",sequences of all insertion reads
#"_deletions_hbg1.csv",sequences of all deletion reads
#'indelsummary_hbg1.csv',contains all indel stats for all fastq filesdef indelshbg1(directory,site,flank1,flank2,width):
indir＝directory outdir＝directory
filenames＝os.listdir(indir)
for i in range(len(filenames)):seqs＝{}
if(filenames[i][-5:]＝＝'fastq')and(site in filenames[i]):skips＝0
ins＝0 insertions＝[]dels＝0 deletions＝[]notindel＝0
for record in SeqIO.parse(indir+filenames[i],"fastq"):
recordqual＝[x>31 for x in record.letter_annotations['phred_quality']]
#only process reads that have more than half of basecalls>Q31 andcontain two HBG1 specific SNPs at 3'end of read
if(record.seq.find('GTTTTTCTCTAATTTATTCTTCCCTTTAGCTAGTTTC')>0)(SEQ IDNO:88)and
(float(sum(recordqual))/float(len(recordqual))>＝.5):
#split prior to indel window split1＝record.seq.split(flank1)if len(split1)＝＝2:
#take second item in first split
#split again at the sequence right after the indel window if len(split1[1].split(flank2))＝＝2:
split2＝split1[1].split(flank2)[0]
#if INDEL window is+1 add to Insertions if(len(split2)>＝width+1):
ins＝ins+1 insertions.append(split2)
#if INDEL window is-1 add to Deletions if(len(split2)<＝width-1):
dels＝dels+1 deletions.append(split2)
if len(split2)＝＝width:notindel＝notindel+1
else:
skips＝skips+1
else:
skips＝skips+1
else:
skips＝skips+1 fd＝open(directory+'indelsummary_hbg1.csv','a')
fd.write('\n'+filenames[i]+'\n')fd.write('skipped reads:'+str(skips)+'\n')
fd.write('insertions:'+str(ins)+'\n')fd.write('deletions:'+str(dels)+'\n')
fd.write('notindels:'+str(notindel)+'\n')fd.write('indel rate:'+str(float(ins+dels)/float(ins+dels+notindel)*100.)+'％'+'\n')fd.close()
pd.DataFrame(insertions).to_csv(directory+filenames[i]+'Insertions_hbg1.csv')
pd.DataFrame(deletions).to_csv(directory+filenames[i]+'Deletions_hbg1.csv')return
#indel analysis
#includes a check for match with two HBG2 SNPs
#inputs:
#directory,working directory folder containing all fastq files
#site,genomic site name as it appears in the fastq filenames
#orientation,'FWD'if you want output in the same direction as thesequencing read or'REV'if you want reverse complement output,
#flank1,sequence that is used to define the 5'end of protospacer inthe sequencing read direction,
#flank2,sequence that is used to define the 5'end of protospacer inthe sequencing read direction,
#width,expected bp length of basecalling window
#ouputs:
#"_Insertions_hbg2.csv",sequences of all insertion reads
#"_deletions_hbg2.csv",sequences of all deletion reads
#'indelsummary_hbg2.csv',contains all indel stats for all fastq filesdef indelshbg2(directory,site,flank1,flank2,width):
indir＝directory outdir＝directory filenames＝os.listdir(indir)
for i in range(len(filenames)):seqs＝{}
if(filenames[i][-5:]＝＝'fastq')and(site in filenames[i]):skips＝0
ins＝0 insertions＝[]dels＝0 deletions＝[]notindel＝0
for record in SeqIO.parse(indir+filenames[i],"fastq"):
recordqual＝[x>31 for x in record.letter_annotations['phred_quality']]
#only process reads that have more than half of basecalls>Q31 andcontain two HBG2 specific SNPs at 3'end of read
if(record.seq.find('ATTTTTCTCTAATTTATTCTTCCCTTTAGCTAGTTTT')>0)(SEQ IDNO:89)and
(float(sum(recordqual))/float(len(recordqual))>＝.5):
#split prior to indel window split1＝record.seq.split(flank1)if len(split1)＝＝2:
#take second item in first split
#split again at the sequence right after the indel window iflen(split1[1].split(flank2))＝＝2:
split2＝split1[1].split(flank2)[0]
#if INDEL window is+1 add to Insertions if(len(split2)>＝width+1):
ins＝ins+1 insertions.append(split2)
#if INDEL window is-1 add to Deletions if(len(split2)<＝width-1):
dels＝dels+1 deletions.append(split2)
if len(split2)＝＝width:notindel＝notindel+1
else:
skips＝skips+1
else:
skips＝skips+1
else:
skips＝skips+1 fd＝open(directory+'indelsummary_hbg2.csv','a')
fd.write('\n'+filenames[i]+'\n')fd.write('skipped reads:'+str(skips)+'\n')
fd.write('insertions:'+str(ins)+'\n')fd.write('deletions:'+str(dels)+'\n')
fd.write('notindels:'+str(notindel)+'\n')fd.write('indel rate:'+str(float(ins+dels)/float(ins+dels+notindel)*100.)+'％'+'\n')fd.close()
pd.DataFrame(insertions).to_csv(directory+filenames[i]+'Insertions_hbg2.csv')
pd.DataFrame(deletions).to_csv(directory+filenames[i]+'Deletions_hbg2.csv')return
directory1＝'/Users/michaelpacker/Desktop/Liu_Lab/MiSeqData/y-globin_632/'
basecallhbg1(directory1,'632','FWD','ATTTGCA','TTAATTTTTT'(SEQ ID NO:90),43)
basecallhbg2(directory1,'632','FWD','ATTTGCA','TTAATTTTTT'(SEQ ID NO:90),43)indelshbg1(directory1,'632','ATTTGCA','TTAATTTTTT'(SEQ ID NO:90),43)indelshbg2(directory1,'632','ATTTGCA','TTAATTTTTT'(SEQ ID NO:90),43
等同实施方案和范围，通过引用并入
本领域技术人员将认识到或能够使用不超过常规的实验确定本文所述的本发明具体实施方案的许多等同实施方案。本发明的范围不意图限于以上说明书，而是如所附权利要求中所述。
在权利要求中，诸如“一种”、“一个”和“该”的冠词可以表示一个或超出一个，除非相反地指出或者从上下文中显而易见。如果一个、超出一个或所有组成员在给定产物或过程中存在、使用或以其他方式相关，则认为在组中的一个或多个成员之间包括“或”的权利要求或说明书是满足的，除非另有说明或从上下文中显而易见。本发明包括实施方案，其中组的恰好一个成员在给定产物或过程中存在、使用或以其他方式相关。本发明还包括实施方案，其中超出一个或所有组成员在给定产物或过程中存在、使用或以其他方式相关。
此外，应理解，本发明涵盖所有变型、组合和置换，其中来自一个或多个权利要求或来自说明书的相关部分的一个或多个限制、元素、条款、描述性术语等被引入另一个权利要求中。例如，可以修改依赖于另一个权利要求的任何权利要求以包括在依赖于相同基本权利要求的任何其他权利要求中找到的一个或多个限制。此外，在权利要求叙述组合物的情况下，应当理解包括将组合物用于本文公开的任何目的的方法，并且包括根据本文公开的任何制备方法或本领域中已知的其他方法制备组合物的方法，除非另有说明或者除非本领域普通技术人员明白会出现矛盾或不一致。
在将元素呈现为列表(例如，以马库什群组格式)的情况下，应当理解，还公开了元素的每个子群，并且可以从群组中移除任何元素。还应注意，术语“包含”旨在是开放的并且允许包含另外的元素或步骤。应当理解，通常，在本发明或本发明的方面称为包含特定元素、特征、步骤等的情况下，本发明或本发明的方面的某些实施方案由此类元素、特征、步骤等组成，或基本上由之组成。出于简化的目的，这些实施方案未在本文中具体阐述。因此，对于包含一个或多个元素、特征、步骤等的本发明的每个实施方案，本发明还提供了由这些元素、特征、步骤等组成或基本上由之组成的实施方案。
在给出范围的情况下，端点包括在内。此外，应当理解，除非另有说明或从上下文和/或本领域普通技术人员的理解中明显看出，否则表示为范围的值可以假定在本发明的不同实施方案中的所述范围内的任何特定值，至该范围下限的单位的十分之一，除非上下文另有明确规定。还应当理解，除非另有说明或从上下文和/或本领域普通技术人员的理解中明显看出，否则表示为范围的值可以假定给定范围内的任何子范围，其中子范围的端点表示为与范围的下限的单位的十分之一相同的精度。
此外，应当理解，本发明的任何具体实施方案可以明确地从任何一个或多个权利要求中排除。在给出范围的情况下，该范围内的任何值可以明确地从任何一个或多个权利要求中排除。本发明的组合物和/或方法的任何实施方案、元素、特征、应用或方面可以从任何一个或多个权利要求中排除。出于简洁的目的，本文未明确阐述其中排除一个或多个元素、特征、目的或方面的所有实施方案。
本文提及的所有出版物、专利和序列数据库条目，包括上面列出的那些项，通过引用整体并入本文，如同每个单独的出版物或专利被具体和单独地指出通过引用并入。在冲突的情况下，以本申请(包括本文中的任何定义)为准。
序列表
<110> 布罗德研究所股份有限公司
哈佛大学的校长及成员们
比姆医疗股份有限公司
<120> 腺苷碱基编辑器的用途
<130> B1195.70048WO00
<140> Not Yet Assigned
<141> Concurrently Herewith
<150> US 62/573,127
<151> 2017-10-16
<160> 871
<170> PatentIn version 3.5
<210> 1
<211> 167
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 1
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp
165
<210> 2
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 2
gtggggaagg ggcccccaag agg 23
<210> 3
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 3
tatacgtacc aggtggagca cccagg 26
<210> 4
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 4
Pro Lys Lys Lys Arg Lys Val
1 5
<210> 5
<211> 30
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 5
Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys
20 25 30
<210> 6
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 6
cctcttgggg gccccttccc cacacta 27
<210> 7
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 7
cctcttgggg gccccttccc cacact 26
<210> 8
<211> 160
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 8
Met Gly Ser His Met Thr Asn Asp Ile Tyr Phe Met Thr Leu Ala Ile
1 5 10 15
Glu Glu Ala Lys Lys Ala Ala Gln Leu Gly Glu Val Pro Ile Gly Ala
20 25 30
Ile Ile Thr Lys Asp Asp Glu Val Ile Ala Arg Ala His Asn Leu Arg
35 40 45
Glu Thr Leu Gln Gln Pro Thr Ala His Ala Glu His Ile Ala Ile Glu
50 55 60
Arg Ala Ala Lys Val Leu Gly Ser Trp Arg Leu Glu Gly Cys Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Thr Ile Val Met
85 90 95
Ser Arg Ile Pro Arg Val Val Tyr Gly Ala Asp Asp Pro Lys Gly Gly
100 105 110
Cys Ser Gly Ser Leu Met Asn Leu Leu Gln Gln Ser Asn Phe Asn His
115 120 125
Arg Ala Ile Val Asp Lys Gly Val Leu Lys Glu Ala Cys Ser Thr Leu
130 135 140
Leu Thr Thr Phe Phe Lys Asn Leu Arg Ala Asn Lys Lys Ser Thr Asn
145 150 155 160
<210> 9
<211> 161
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 9
Met Thr Gln Asp Glu Leu Tyr Met Lys Glu Ala Ile Lys Glu Ala Lys
1 5 10 15
Lys Ala Glu Glu Lys Gly Glu Val Pro Ile Gly Ala Val Leu Val Ile
20 25 30
Asn Gly Glu Ile Ile Ala Arg Ala His Asn Leu Arg Glu Thr Glu Gln
35 40 45
Arg Ser Ile Ala His Ala Glu Met Leu Val Ile Asp Glu Ala Cys Lys
50 55 60
Ala Leu Gly Thr Trp Arg Leu Glu Gly Ala Thr Leu Tyr Val Thr Leu
65 70 75 80
Glu Pro Cys Pro Met Cys Ala Gly Ala Val Val Leu Ser Arg Val Glu
85 90 95
Lys Val Val Phe Gly Ala Phe Asp Pro Lys Gly Gly Cys Ser Gly Thr
100 105 110
Leu Met Asn Leu Leu Gln Glu Glu Arg Phe Asn His Gln Ala Glu Val
115 120 125
Val Ser Gly Val Leu Glu Glu Glu Cys Gly Gly Met Leu Ser Ala Phe
130 135 140
Phe Arg Glu Leu Arg Lys Lys Lys Lys Ala Ala Arg Lys Asn Leu Ser
145 150 155 160
Glu
<210> 10
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 10
Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser
1 5 10 15
<210> 11
<211> 1561
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 11
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Ser Glu Thr Pro Gly Thr Ser
165 170 175
Glu Ser Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala
180 185 190
Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys
195 200 205
Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser
210 215 220
Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr
225 230 235 240
Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg
245 250 255
Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met
260 265 270
Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu
275 280 285
Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile
290 295 300
Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu
305 310 315 320
Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile
325 330 335
Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile
340 345 350
Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile
355 360 365
Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn
370 375 380
Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys
385 390 395 400
Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys
405 410 415
Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro
420 425 430
Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu
435 440 445
Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile
450 455 460
Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp
465 470 475 480
Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys
485 490 495
Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln
500 505 510
Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys
515 520 525
Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr
530 535 540
Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro
545 550 555 560
Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn
565 570 575
Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile
580 585 590
Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln
595 600 605
Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys
610 615 620
Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly
625 630 635 640
Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr
645 650 655
Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser
660 665 670
Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys
675 680 685
Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn
690 695 700
Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala
705 710 715 720
Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys
725 730 735
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys
740 745 750
Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg
755 760 765
Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys
770 775 780
Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp
785 790 795 800
Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu
805 810 815
Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
820 825 830
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu
835 840 845
Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe
850 855 860
Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His
865 870 875 880
Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser
885 890 895
Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser
900 905 910
Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu
915 920 925
Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
930 935 940
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg
945 950 955 960
Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln
965 970 975
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys
980 985 990
Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
995 1000 1005
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile
1010 1015 1020
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1025 1030 1035
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro
1040 1045 1050
Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu
1055 1060 1065
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
1070 1075 1080
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe
1085 1090 1095
Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
1100 1105 1110
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1115 1120 1125
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
1130 1135 1140
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
1145 1150 1155
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
1160 1165 1170
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1175 1180 1185
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1190 1195 1200
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1205 1210 1215
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1220 1225 1230
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1235 1240 1245
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1250 1255 1260
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1265 1270 1275
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1280 1285 1290
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1295 1300 1305
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1310 1315 1320
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1325 1330 1335
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1340 1345 1350
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1355 1360 1365
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1370 1375 1380
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1385 1390 1395
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1400 1405 1410
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1415 1420 1425
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1430 1435 1440
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1445 1450 1455
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1460 1465 1470
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1475 1480 1485
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1490 1495 1500
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1505 1510 1515
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1520 1525 1530
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1535 1540 1545
Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1550 1555 1560
<210> 12
<211> 1561
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 12
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asn Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Ser Glu Thr Pro Gly Thr Ser
165 170 175
Glu Ser Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala
180 185 190
Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys
195 200 205
Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser
210 215 220
Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr
225 230 235 240
Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg
245 250 255
Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met
260 265 270
Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu
275 280 285
Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile
290 295 300
Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu
305 310 315 320
Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile
325 330 335
Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile
340 345 350
Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile
355 360 365
Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn
370 375 380
Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys
385 390 395 400
Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys
405 410 415
Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro
420 425 430
Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu
435 440 445
Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile
450 455 460
Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp
465 470 475 480
Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys
485 490 495
Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln
500 505 510
Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys
515 520 525
Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr
530 535 540
Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro
545 550 555 560
Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn
565 570 575
Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile
580 585 590
Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln
595 600 605
Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys
610 615 620
Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly
625 630 635 640
Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr
645 650 655
Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser
660 665 670
Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys
675 680 685
Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn
690 695 700
Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala
705 710 715 720
Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys
725 730 735
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys
740 745 750
Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg
755 760 765
Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys
770 775 780
Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp
785 790 795 800
Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu
805 810 815
Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
820 825 830
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu
835 840 845
Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe
850 855 860
Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His
865 870 875 880
Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser
885 890 895
Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser
900 905 910
Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu
915 920 925
Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
930 935 940
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg
945 950 955 960
Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln
965 970 975
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys
980 985 990
Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
995 1000 1005
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile
1010 1015 1020
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1025 1030 1035
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro
1040 1045 1050
Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu
1055 1060 1065
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
1070 1075 1080
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe
1085 1090 1095
Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
1100 1105 1110
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1115 1120 1125
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
1130 1135 1140
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
1145 1150 1155
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
1160 1165 1170
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1175 1180 1185
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1190 1195 1200
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1205 1210 1215
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1220 1225 1230
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1235 1240 1245
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1250 1255 1260
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1265 1270 1275
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1280 1285 1290
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1295 1300 1305
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1310 1315 1320
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1325 1330 1335
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1340 1345 1350
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1355 1360 1365
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1370 1375 1380
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1385 1390 1395
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1400 1405 1410
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1415 1420 1425
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1430 1435 1440
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1445 1450 1455
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1460 1465 1470
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1475 1480 1485
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1490 1495 1500
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1505 1510 1515
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1520 1525 1530
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1535 1540 1545
Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1550 1555 1560
<210> 13
<211> 1561
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 13
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Gly Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Ser Glu Thr Pro Gly Thr Ser
165 170 175
Glu Ser Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala
180 185 190
Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys
195 200 205
Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser
210 215 220
Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr
225 230 235 240
Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg
245 250 255
Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met
260 265 270
Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu
275 280 285
Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile
290 295 300
Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu
305 310 315 320
Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile
325 330 335
Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile
340 345 350
Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile
355 360 365
Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn
370 375 380
Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys
385 390 395 400
Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys
405 410 415
Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro
420 425 430
Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu
435 440 445
Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile
450 455 460
Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp
465 470 475 480
Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys
485 490 495
Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln
500 505 510
Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys
515 520 525
Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr
530 535 540
Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro
545 550 555 560
Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn
565 570 575
Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile
580 585 590
Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln
595 600 605
Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys
610 615 620
Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly
625 630 635 640
Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr
645 650 655
Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser
660 665 670
Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys
675 680 685
Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn
690 695 700
Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala
705 710 715 720
Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys
725 730 735
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys
740 745 750
Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg
755 760 765
Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys
770 775 780
Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp
785 790 795 800
Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu
805 810 815
Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
820 825 830
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu
835 840 845
Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe
850 855 860
Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His
865 870 875 880
Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser
885 890 895
Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser
900 905 910
Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu
915 920 925
Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
930 935 940
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg
945 950 955 960
Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln
965 970 975
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys
980 985 990
Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
995 1000 1005
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile
1010 1015 1020
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1025 1030 1035
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro
1040 1045 1050
Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu
1055 1060 1065
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
1070 1075 1080
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe
1085 1090 1095
Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
1100 1105 1110
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1115 1120 1125
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
1130 1135 1140
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
1145 1150 1155
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
1160 1165 1170
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1175 1180 1185
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1190 1195 1200
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1205 1210 1215
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1220 1225 1230
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1235 1240 1245
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1250 1255 1260
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1265 1270 1275
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1280 1285 1290
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1295 1300 1305
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1310 1315 1320
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1325 1330 1335
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1340 1345 1350
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1355 1360 1365
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1370 1375 1380
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1385 1390 1395
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1400 1405 1410
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1415 1420 1425
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1430 1435 1440
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1445 1450 1455
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1460 1465 1470
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1475 1480 1485
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1490 1495 1500
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1505 1510 1515
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1520 1525 1530
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1535 1540 1545
Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1550 1555 1560
<210> 14
<211> 1561
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 14
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Val Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Ser Glu Thr Pro Gly Thr Ser
165 170 175
Glu Ser Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala
180 185 190
Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys
195 200 205
Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser
210 215 220
Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr
225 230 235 240
Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg
245 250 255
Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met
260 265 270
Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu
275 280 285
Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile
290 295 300
Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu
305 310 315 320
Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile
325 330 335
Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile
340 345 350
Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile
355 360 365
Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn
370 375 380
Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys
385 390 395 400
Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys
405 410 415
Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro
420 425 430
Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu
435 440 445
Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile
450 455 460
Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp
465 470 475 480
Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys
485 490 495
Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln
500 505 510
Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys
515 520 525
Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr
530 535 540
Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro
545 550 555 560
Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn
565 570 575
Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile
580 585 590
Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln
595 600 605
Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys
610 615 620
Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly
625 630 635 640
Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr
645 650 655
Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser
660 665 670
Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys
675 680 685
Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn
690 695 700
Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala
705 710 715 720
Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys
725 730 735
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys
740 745 750
Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg
755 760 765
Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys
770 775 780
Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp
785 790 795 800
Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu
805 810 815
Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
820 825 830
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu
835 840 845
Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe
850 855 860
Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His
865 870 875 880
Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser
885 890 895
Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser
900 905 910
Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu
915 920 925
Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
930 935 940
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg
945 950 955 960
Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln
965 970 975
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys
980 985 990
Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
995 1000 1005
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile
1010 1015 1020
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1025 1030 1035
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro
1040 1045 1050
Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu
1055 1060 1065
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
1070 1075 1080
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe
1085 1090 1095
Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
1100 1105 1110
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1115 1120 1125
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
1130 1135 1140
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
1145 1150 1155
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
1160 1165 1170
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1175 1180 1185
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1190 1195 1200
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1205 1210 1215
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1220 1225 1230
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1235 1240 1245
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1250 1255 1260
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1265 1270 1275
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1280 1285 1290
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1295 1300 1305
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1310 1315 1320
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1325 1330 1335
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1340 1345 1350
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1355 1360 1365
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1370 1375 1380
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1385 1390 1395
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1400 1405 1410
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1415 1420 1425
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1430 1435 1440
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1445 1450 1455
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1460 1465 1470
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1475 1480 1485
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1490 1495 1500
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1505 1510 1515
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1520 1525 1530
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1535 1540 1545
Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1550 1555 1560
<210> 15
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 15
cctcttgggg gccccttccc cacac 25
<210> 16
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 16
cctcttgggg gccccttccc caca 24
<210> 17
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 17
cctcttgggg gccccttccc cac 23
<210> 18
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 18
cctcttgggg gccccttccc ca 22
<210> 19
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 19
cctcttgggg gccccttccc c 21
<210> 20
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 20
cctcttgggg gccccttccc 20
<210> 21
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 21
cctgggtgct ccacctggta cgtatat 27
<210> 22
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 22
cctgggtgct ccacctggta cgtata 26
<210> 23
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 23
cctgggtgct ccacctggta cgtat 25
<210> 24
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 24
cctgggtgct ccacctggta cgta 24
<210> 25
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 25
cctgggtgct ccacctggta cgt 23
<210> 26
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 26
cctgggtgct ccacctggta cg 22
<210> 27
<211> 1550
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 27
Met Ser Glu Val Glu Phe Ser Tyr Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asn Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Ser His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Ser Glu Thr Pro Gly Thr Ser
165 170 175
Glu Ser Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala
180 185 190
Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys
195 200 205
Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser
210 215 220
Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr
225 230 235 240
Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg
245 250 255
Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met
260 265 270
Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu
275 280 285
Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile
290 295 300
Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu
305 310 315 320
Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile
325 330 335
Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile
340 345 350
Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile
355 360 365
Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn
370 375 380
Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys
385 390 395 400
Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys
405 410 415
Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro
420 425 430
Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu
435 440 445
Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile
450 455 460
Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp
465 470 475 480
Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys
485 490 495
Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln
500 505 510
Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys
515 520 525
Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr
530 535 540
Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro
545 550 555 560
Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn
565 570 575
Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile
580 585 590
Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln
595 600 605
Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys
610 615 620
Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly
625 630 635 640
Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr
645 650 655
Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser
660 665 670
Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys
675 680 685
Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn
690 695 700
Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala
705 710 715 720
Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys
725 730 735
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys
740 745 750
Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg
755 760 765
Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys
770 775 780
Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp
785 790 795 800
Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu
805 810 815
Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
820 825 830
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu
835 840 845
Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe
850 855 860
Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His
865 870 875 880
Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser
885 890 895
Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser
900 905 910
Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu
915 920 925
Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
930 935 940
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg
945 950 955 960
Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln
965 970 975
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys
980 985 990
Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
995 1000 1005
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp Ala Ile
1010 1015 1020
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1025 1030 1035
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro
1040 1045 1050
Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu
1055 1060 1065
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
1070 1075 1080
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe
1085 1090 1095
Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
1100 1105 1110
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1115 1120 1125
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
1130 1135 1140
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
1145 1150 1155
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
1160 1165 1170
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1175 1180 1185
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1190 1195 1200
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1205 1210 1215
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1220 1225 1230
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1235 1240 1245
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1250 1255 1260
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1265 1270 1275
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1280 1285 1290
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1295 1300 1305
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1310 1315 1320
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1325 1330 1335
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1340 1345 1350
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1355 1360 1365
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1370 1375 1380
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1385 1390 1395
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1400 1405 1410
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1415 1420 1425
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1430 1435 1440
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1445 1450 1455
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1460 1465 1470
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1475 1480 1485
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1490 1495 1500
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1505 1510 1515
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1520 1525 1530
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1535 1540 1545
Gly Asp
1550
<210> 28
<211> 1550
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<220>
<221> misc_feature
<222> (155)..(155)
<223> Xaa可以是任何天然存在的氨基酸
<400> 28
Met Ser Glu Val Glu Phe Ser Tyr Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asn Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Ser His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Xaa Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Ser Glu Thr Pro Gly Thr Ser
165 170 175
Glu Ser Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala
180 185 190
Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys
195 200 205
Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser
210 215 220
Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr
225 230 235 240
Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg
245 250 255
Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met
260 265 270
Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu
275 280 285
Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile
290 295 300
Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu
305 310 315 320
Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile
325 330 335
Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile
340 345 350
Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile
355 360 365
Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn
370 375 380
Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys
385 390 395 400
Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys
405 410 415
Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro
420 425 430
Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu
435 440 445
Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile
450 455 460
Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp
465 470 475 480
Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys
485 490 495
Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln
500 505 510
Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys
515 520 525
Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr
530 535 540
Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro
545 550 555 560
Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn
565 570 575
Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile
580 585 590
Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln
595 600 605
Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys
610 615 620
Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly
625 630 635 640
Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr
645 650 655
Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser
660 665 670
Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys
675 680 685
Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn
690 695 700
Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala
705 710 715 720
Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys
725 730 735
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys
740 745 750
Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg
755 760 765
Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys
770 775 780
Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp
785 790 795 800
Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu
805 810 815
Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
820 825 830
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu
835 840 845
Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe
850 855 860
Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His
865 870 875 880
Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser
885 890 895
Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser
900 905 910
Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu
915 920 925
Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
930 935 940
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg
945 950 955 960
Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln
965 970 975
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys
980 985 990
Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
995 1000 1005
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp Ala Ile
1010 1015 1020
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1025 1030 1035
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro
1040 1045 1050
Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu
1055 1060 1065
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
1070 1075 1080
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe
1085 1090 1095
Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
1100 1105 1110
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1115 1120 1125
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
1130 1135 1140
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
1145 1150 1155
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
1160 1165 1170
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1175 1180 1185
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1190 1195 1200
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1205 1210 1215
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1220 1225 1230
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1235 1240 1245
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1250 1255 1260
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1265 1270 1275
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1280 1285 1290
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1295 1300 1305
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1310 1315 1320
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1325 1330 1335
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1340 1345 1350
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1355 1360 1365
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1370 1375 1380
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1385 1390 1395
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1400 1405 1410
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1415 1420 1425
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1430 1435 1440
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1445 1450 1455
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1460 1465 1470
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1475 1480 1485
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1490 1495 1500
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1505 1510 1515
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1520 1525 1530
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1535 1540 1545
Gly Asp
1550
<210> 29
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 29
cctgggtgct ccacctggta c 21
<210> 30
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 30
cctgggtgct ccacctggta 20
<210> 31
<211> 501
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 31
atgtctgaag tcgaatttag ccacgaatac tggatgcgtc acgcgctgac gctggcgaaa 60
cgtgcctggg atgagcggga agtgccggtc ggcgcggtat tagtgcataa caatcgggta 120
atcggcgaag gctggaaccg cccgattggt cgccatgatc ccaccgcaca tgcagaaatc 180
atggccctgc ggcagggtgg tctggtgatg caaaattatc gtctgatcga cgccacgttg 240
tatgtcacgc ttgaaccatg tgtaatgtgt gccggagcga tgatccacag tcgcattggt 300
cgcgtggtct ttggtgcgcg tgacgcgaaa actggcgctg cgggatcttt aatggatgtg 360
ctgcatcatc cgggtatgaa tcaccgagtg gaaattacgg aaggaatact ggcggatgag 420
tgcgcggcgt tgctcagtga cttctttcgc atgcgccgcc aggaaattaa agcgcagaaa 480
aaagcgcaat cctcgacgga t 501
<210> 32
<211> 501
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 32
atgtccgaag tcgagttttc ccatgagtac tggatgagac acgcattgac tctcgcaaag 60
agggcttggg atgaacgcga ggtgcccgtg ggggcagtac tcgtgcataa caatcgcgta 120
atcggcgaag gttggaatag gccgatcgga cgccacgacc ccactgcaca tgcggaaatc 180
atggcccttc gacagggagg gcttgtgatg cagaattatc gacttatcga tgcgacgctg 240
tacgtcacgc ttgaaccttg cgtaatgtgc gcgggagcta tgattcactc ccgcattgga 300
cgagttgtat tcggtgcccg cgacgccaag acgggtgccg caggttcact gatggacgtg 360
ctgcatcacc caggcatgaa ccaccgggta gaaatcacag aaggcatatt ggcggacgaa 420
tgtgcggcgc tgttgtccga cttttttcgc atgcggaggc aggagatcaa ggcccagaaa 480
aaagcacaat cctctactga c 501
<210> 33
<211> 1056
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 33
atggcccaga cacccgcatt caacaaaccc aaagtagagt tacacgtcca cctggatgga 60
gccatcaagc cagaaaccat cttatacttt ggcaagaaga gaggcatcgc cctcccggca 120
gatacagtgg aggagctgcg caacattatc ggcatggaca agcccctctc gctcccaggc 180
ttcctggcca agtttgacta ctacatgcct gtgattgcgg gctgcagaga ggccatcaag 240
aggatcgcct acgagtttgt ggagatgaag gcaaaggagg gcgtggtcta tgtggaagtg 300
cgctatagcc cacacctgct ggccaattcc aaggtggacc caatgccctg gaaccagact 360
gaaggggacg tcacccctga tgacgttgtg gatcttgtga accagggcct gcaggaggga 420
gagcaagcat ttggcatcaa ggtccggtcc attctgtgct gcatgcgcca ccagcccagc 480
tggtcccttg aggtgttgga gctgtgtaag aagtacaatc agaagaccgt ggtggctatg 540
gacttggctg gggatgagac cattgaagga agtagcctct tcccaggcca cgtggaagcc 600
tatgagggcg cagtaaagaa tggcattcat cggaccgtcc acgctggcga ggtgggctct 660
cctgaggttg tgcgtgaggc tgtggacatc ctcaagacag agagggtggg acatggttat 720
cacaccatcg aggatgaagc tctctacaac agactactga aagaaaacat gcactttgag 780
gtctgcccct ggtccagcta cctcacaggc gcctgggatc ccaaaacgac gcatgcggtt 840
gttcgcttca agaatgataa ggccaactac tcactcaaca cagacgaccc cctcatcttc 900
aagtccaccc tagacactga ctaccagatg accaagaaag acatgggctt cactgaggag 960
gagttcaagc gactgaacat caacgcagcg aagtcaagct tcctcccaga ggaagagaag 1020
aaggaacttc tggaacggct ctacagagaa taccaa 1056
<210> 34
<211> 1367
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 34
Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 35
<211> 1367
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 35
Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 36
<211> 1367
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 36
Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 37
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<220>
<221> MISC_FEATURE
<222> (5)..(120)
<223> 可以不存在
<400> 37
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
1 5 10 15
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
20 25 30
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
35 40 45
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
50 55 60
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
65 70 75 80
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
85 90 95
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
100 105 110
Ser Gly Gly Ser Ser Gly Gly Ser
115 120
<210> 38
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<220>
<221> MISC_FEATURE
<222> (5)..(120)
<223> 可以不存在
<400> 38
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
20 25 30
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
35 40 45
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
50 55 60
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
65 70 75 80
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
85 90 95
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
100 105 110
Gly Gly Gly Ser Gly Gly Gly Ser
115 120
<210> 39
<211> 150
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<220>
<221> MISC_FEATURE
<222> (6)..(150)
<400> 39
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
20 25 30
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
50 55 60
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
85 90 95
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
100 105 110
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser
145 150
<210> 40
<211> 150
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<220>
<221> MISC_FEATURE
<222> (6)..(150)
<400> 40
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
1 5 10 15
Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala
20 25 30
Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala
35 40 45
Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala
50 55 60
Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
65 70 75 80
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
85 90 95
Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala
100 105 110
Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala
115 120 125
Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala
130 135 140
Lys Glu Ala Ala Ala Lys
145 150
<210> 41
<211> 1209
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 41
atgcatctcg atcaaacccc gagccgccaa ccaatcccga gtgaaggcct gcaactgcat 60
ctgccacaag ttctggcgga tgccgttagc cgcctggtct tgggtaagtt cggtgatctg 120
acagacaact tttctagtcc acatgctcgc cgtaaggtgc tggctggcgt tgtgatgacc 180
acaggtacag acgtcaaaga tgctaaagtg atttctgtgt ctactggcac gaagtgcatt 240
aacggcgaat atatgtctga ccgtggctta gcgcttaacg attgtcatgc cgaaatcatc 300
tcccgtcgtt cattgcttcg cttcctgtac acgcagttgg aactgtatct gaataacaaa 360
gacgatcaga agcgttctat tttccagaag tctgagcgcg gcgggttccg tcttaaagag 420
aatgtgcagt ttcaccttta tatttcaacc tctccttgtg gtgatgcccg tattttttca 480
ccacacgaac ctattttaga ggaaccggcc gatcgtcatc cgaaccgcaa agcccgtggg 540
cagctgcgta cgaaaatcga atcaggtgaa ggcaccattc ccgtccgctc caatgcgagc 600
attcaaacgt gggacggtgt gttacagggc gaacgcctgt taaccatgag ctgctcagac 660
aaaattgcac gttggaacgt ggtaggcatc cagggctcgt tattgagcat tttcgtggag 720
ccgatttatt ttagttccat cattttgggc tcactctacc acggcgatca ccttagccgc 780
gcgatgtacc agcgcattag taacatcgaa gatttaccgc ccctgtatac cctgaacaaa 840
ccactgttaa gcggtatttc taacgcggag gcgcgtcagc ctggtaaagc cccgaacttc 900
agtgtgaact ggactgtggg tgattctgca attgaggtaa ttaacgcgac gacgggtaaa 960
gatgaactgg gccgtgcctc tcgtctgtgt aaacacgcgc tgtactgtcg ttggatgcgc 1020
gtgcacggta aagttcccag tcatctgtta cgtagcaaga tcaccaagcc aaatgtctac 1080
cacgaatcga agctggccgc gaaagaatac caagcggcta aggcgcgtct gttcaccgcc 1140
tttattaagg ctggcttagg ggcctgggtg gaaaaaccaa ccgagcaaga tcaattcagt 1200
ctgaccccg 1209
<210> 42
<211> 573
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 42
atggaggcga aggcggcacc caagccagct gcaagcggcg cgtgctcggt gtcggcagag 60
gagaccgaaa agtggatgga ggaggcgatg cacatggcca aagaagccct cgaaaatact 120
gaagttcctg ttggctgtct tatggtctac aacaatgaag ttgtagggaa ggggagaaat 180
gaagttaacc aaaccaaaaa tgctactcga catgcagaaa tggtggccat cgatcaggtc 240
ctcgattggt gtcgtcaaag tggcaagagt ccctctgaag tatttgaaca cactgtgttg 300
tatgtcactg tggagccgtg cattatgtgt gcagctgctc tccgcctgat gaaaatcccg 360
ctggttgtat atggctgtca gaatgaacga tttggtggtt gtggctctgt tctaaatatt 420
gcctctgctg acctaccaaa cactgggaga ccatttcagt gtatccctgg atatcgggct 480
gaggaagcag tggaaatgtt aaagaccttc tacaaacaag aaaatccaaa tgcaccaaaa 540
tcgaaagttc ggaaaaagga atgtcagaaa tct 573
<210> 43
<211> 660
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 43
atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa 60
cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat 120
attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt 180
cacattcttg cccgcctgat gaatgctcat ccggagttcc gtatggcaat gaaagacggt 240
gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa 300
acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat 360
tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag 420
aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg 480
gccaatatgg acaacttctt cgcccccgtt ttcactatgg gcaaatatta tacgcaaggc 540
gacaaggtgc tgatgccgct ggccatccag gtgcactacg ccgtatgcga cggcttccat 600
gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa 660
<210> 44
<211> 795
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 44
atgatcgaat aagatggatt gcacgcaggt tctccggccg cttaggtgga gcgcctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct tctaa 795
<210> 45
<211> 792
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 45
atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc 60
gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc 120
ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa 180
acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc 240
gagattctcc gcgctgtaga agtcatcatt gttgtgcacg acgacatcat tccgtggcgt 300
tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt 360
atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa 420
catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag 480
gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct 540
ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc 600
aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat 660
cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc 720
tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta 780
gtcggcaaat aa 792
<210> 46
<211> 815
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 46
atgatcgaat aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat agccggccac agttaatgaa 300
tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat 360
ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg 420
accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg 480
atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc 540
tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc 600
cgaatatcat ggtggaaaat ggccgctttt ctggattcat taactgtggc cggctgggtg 660
tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg 720
gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca 780
tcgccttcta tcgccttctt gacgagttct tctaa 815
<210> 47
<211> 4104
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 47
atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg 60
atcactgatg attataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120
cacagtatca aaaaaaatct tataggggct cttttatttg gcagtggaga gacagcggaa 180
gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240
tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300
cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360
aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420
aaattggcag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480
atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540
gtggacaaac tatttatcca gttggtacaa atctacaatc aattatttga agaaaaccct 600
attaacgcaa gtagagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660
cgattagaaa atctcattgc tcagctcccc ggtgagaaga gaaatggctt gtttgggaat 720
ctcattgctt tgtcattggg attgacccct aattttaaat caaattttga tttggcagaa 780
gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 840
caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900
ttactttcag atatcctaag agtaaatagt gaaataacta aggctcccct atcagcttca 960
atgattaagc gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020
caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080
ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140
gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200
aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260
gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320
gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380
cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440
gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500
aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560
tataacgaat tgacaaaggt caaatatgtt actgagggaa tgcgaaaacc agcatttctt 1620
tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680
gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740
tcaggagttg aagatagatt taatgcttca ttaggcgcct accatgattt gctaaaaatt 1800
attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860
ttaacattga ccttatttga agataggggg atgattgagg aaagacttaa aacatatgct 1920
cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980
cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040
gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100
agtttgacat ttaaagaaga tattcaaaaa gcacaggtgt ctggacaagg ccatagttta 2160
catgaacaga ttgctaactt agctggcagt cctgctatta aaaaaggtat tttacagact 2220
gtaaaaattg ttgatgaact ggtcaaagta atggggcata agccagaaaa tatcgttatt 2280
gaaatggcac gtgaaaatca gacaactcaa aagggccaga aaaattcgcg agagcgtatg 2340
aaacgaatcg aagaaggtat caaagaatta ggaagtcaga ttcttaaaga gcatcctgtt 2400
gaaaatactc aattgcaaaa tgaaaagctc tatctctatt atctacaaaa tggaagagac 2460
atgtatgtgg accaagaatt agatattaat cgtttaagtg attatgatgt cgatcacatt 2520
gttccacaaa gtttcattaa agacgattca atagacaata aggtactaac gcgttctgat 2580
aaaaatcgtg gtaaatcgga taacgttcca agtgaagaag tagtcaaaaa gatgaaaaac 2640
tattggagac aacttctaaa cgccaagtta atcactcaac gtaagtttga taatttaacg 2700
aaagctgaac gtggaggttt gagtgaactt gataaagctg gttttatcaa acgccaattg 2760
gttgaaactc gccaaatcac taagcatgtg gcacaaattt tggatagtcg catgaatact 2820
aaatacgatg aaaatgataa acttattcga gaggttaaag tgattacctt aaaatctaaa 2880
ttagtttctg acttccgaaa agatttccaa ttctataaag tacgtgagat taacaattac 2940
catcatgccc atgatgcgta tctaaatgcc gtcgttggaa ctgctttgat taagaaatat 3000
ccaaaacttg aatcggagtt tgtctatggt gattataaag tttatgatgt tcgtaaaatg 3060
attgctaagt ctgagcaaga aataggcaaa gcaaccgcaa aatatttctt ttactctaat 3120
atcatgaact tcttcaaaac agaaattaca cttgcaaatg gagagattcg caaacgccct 3180
ctaatcgaaa ctaatgggga aactggagaa attgtctggg ataaagggcg agattttgcc 3240
acagtgcgca aagtattgtc catgccccaa gtcaatattg tcaagaaaac agaagtacag 3300
acaggcggat tctccaagga gtcaatttta ccaaaaagaa attcggacaa gcttattgct 3360
cgtaaaaaag actgggatcc aaaaaaatat ggtggttttg atagtccaac ggtagcttat 3420
tcagtcctag tggttgctaa ggtggaaaaa gggaaatcga agaagttaaa atccgttaaa 3480
gagttactag ggatcacaat tatggaaaga agttcctttg aaaaaaatcc gattgacttt 3540
ttagaagcta aaggatataa ggaagttaaa aaagacttaa tcattaaact acctaaatat 3600
agtctttttg agttagaaaa cggtcgtaaa cggatgctgg ctagtgccgg agaattacaa 3660
aaaggaaatg agctggctct gccaagcaaa tatgtgaatt ttttatattt agctagtcat 3720
tatgaaaagt tgaagggtag tccagaagat aacgaacaaa aacaattgtt tgtggagcag 3780
cataagcatt atttagatga gattattgag caaatcagtg aattttctaa gcgtgttatt 3840
ttagcagatg ccaatttaga taaagttctt agtgcatata acaaacatag agacaaacca 3900
atacgtgaac aagcagaaaa tattattcat ttatttacgt tgacgaatct tggagctccc 3960
gctgctttta aatattttga tacaacaatt gatcgtaaac gatatacgtc tacaaaagaa 4020
gttttagatg ccactcttat ccatcaatcc atcactggtc tttatgaaac acgcattgat 4080
ttgagtcagc taggaggtga ctga 4104
<210> 48
<211> 1367
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 48
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Asp Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Gly Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Ala Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Ile Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Arg Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Arg Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Ser Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Ala Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Gly Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly His Ser Leu
705 710 715 720
His Glu Gln Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Ile Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Ile Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 49
<211> 4212
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 49
atggataaaa agtattctat tggtttagac atcggcacta attccgttgg atgggctgtc 60
ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 120
cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 180
gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 240
tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 300
ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 360
aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 420
aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 480
atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 540
gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 600
ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 660
cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 720
cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 780
gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 840
caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 900
ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 960
atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 1020
cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 1080
ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 1140
gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 1200
aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 1260
gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 1320
gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 1380
cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 1440
gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 1500
aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 1560
tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 1620
agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 1680
gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 1740
tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 1800
attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 1860
ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 1920
cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 1980
cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 2040
gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 2100
tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 2160
cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 2220
gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 2280
atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 2340
atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 2400
gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 2460
gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 2520
attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 2580
gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 2640
aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 2700
actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 2760
ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 2820
acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 2880
aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 2940
taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 3000
tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 3060
atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 3120
aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 3180
cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 3240
gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 3300
cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc 3360
gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 3420
tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 3480
aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 3540
ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 3600
tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 3660
caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 3720
cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 3780
cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 3840
atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 3900
cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 3960
ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag 4020
gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 4080
gatttgtcac agcttggggg tgacggatcc cccaagaaga agaggaaagt ctcgagcgac 4140
tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac 4200
aaggctgcag ga 4212
<210> 50
<211> 1368
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 50
Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 51
<211> 4107
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 51
atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg 60
atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120
cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 180
gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240
tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300
cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360
aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420
aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480
atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540
gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 600
attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660
cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 720
ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa 780
gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 840
caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900
ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca 960
atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020
caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080
ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140
gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200
aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260
gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320
gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380
cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440
gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500
aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560
tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1620
tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680
gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740
tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1800
attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860
ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct 1920
cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980
cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040
gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100
agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160
catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220
gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280
attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt 2340
atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct 2400
gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga 2460
gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac 2520
attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct 2580
gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2640
aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2700
acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2760
ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2820
actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct 2880
aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2940
taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa 3000
tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3060
atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct 3120
aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3180
cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3240
gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300
cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt 3360
gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct 3420
tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt 3480
aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540
tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa 3600
tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3660
caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3720
cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3780
cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3840
attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3900
ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960
cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020
gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080
gatttgagtc agctaggagg tgactga 4107
<210> 52
<211> 1368
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 52
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 53
<211> 1368
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 53
Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 54
<211> 1368
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 54
Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 55
<211> 1052
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 55
Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser Val Gly
1 5 10 15
Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly Val
20 25 30
Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg Ser
35 40 45
Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile Gln
50 55 60
Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His Ser
65 70 75 80
Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu Ser
85 90 95
Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu Ala
100 105 110
Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr Gly
115 120 125
Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala Leu
130 135 140
Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys Asp
145 150 155 160
Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr Val
165 170 175
Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln Leu
180 185 190
Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg Arg
195 200 205
Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys Asp
210 215 220
Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe Pro
225 230 235 240
Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr Asn
245 250 255
Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn Glu
260 265 270
Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe Lys
275 280 285
Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu Val
290 295 300
Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys Pro
305 310 315 320
Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr Ala
325 330 335
Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala Lys
340 345 350
Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu Thr
355 360 365
Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser Asn
370 375 380
Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile Asn
385 390 395 400
Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala Ile
405 410 415
Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln Gln
420 425 430
Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro Val
435 440 445
Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile Ile
450 455 460
Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg Glu
465 470 475 480
Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys Arg
485 490 495
Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr Gly
500 505 510
Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp Met
515 520 525
Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu Asp
530 535 540
Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro Arg
545 550 555 560
Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys Gln
565 570 575
Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu Ser
580 585 590
Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile Leu
595 600 605
Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu Tyr
610 615 620
Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp Phe
625 630 635 640
Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu Met
645 650 655
Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys Val
660 665 670
Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp Lys
675 680 685
Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp Ala
690 695 700
Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys Leu
705 710 715 720
Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys Gln
725 730 735
Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu Ile
740 745 750
Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp Tyr
755 760 765
Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile Asn
770 775 780
Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu Ile
785 790 795 800
Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu Lys
805 810 815
Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His Asp
820 825 830
Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly Asp
835 840 845
Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr Leu
850 855 860
Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile Lys
865 870 875 880
Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp Tyr
885 890 895
Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr Arg
900 905 910
Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val Lys
915 920 925
Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser Lys
930 935 940
Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala Glu
945 950 955 960
Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly Glu
965 970 975
Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile Glu
980 985 990
Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met Asn
995 1000 1005
Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys Thr
1010 1015 1020
Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1040 1045 1050
<210> 56
<211> 1052
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 56
Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser Val Gly
1 5 10 15
Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly Val
20 25 30
Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg Ser
35 40 45
Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile Gln
50 55 60
Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His Ser
65 70 75 80
Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu Ser
85 90 95
Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu Ala
100 105 110
Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr Gly
115 120 125
Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala Leu
130 135 140
Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys Asp
145 150 155 160
Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr Val
165 170 175
Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln Leu
180 185 190
Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg Arg
195 200 205
Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys Asp
210 215 220
Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe Pro
225 230 235 240
Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr Asn
245 250 255
Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn Glu
260 265 270
Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe Lys
275 280 285
Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu Val
290 295 300
Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys Pro
305 310 315 320
Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr Ala
325 330 335
Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala Lys
340 345 350
Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu Thr
355 360 365
Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser Asn
370 375 380
Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile Asn
385 390 395 400
Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala Ile
405 410 415
Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln Gln
420 425 430
Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro Val
435 440 445
Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile Ile
450 455 460
Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg Glu
465 470 475 480
Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys Arg
485 490 495
Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr Gly
500 505 510
Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp Met
515 520 525
Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu Asp
530 535 540
Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro Arg
545 550 555 560
Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys Gln
565 570 575
Glu Glu Ala Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu Ser
580 585 590
Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile Leu
595 600 605
Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu Tyr
610 615 620
Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp Phe
625 630 635 640
Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu Met
645 650 655
Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys Val
660 665 670
Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp Lys
675 680 685
Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp Ala
690 695 700
Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys Leu
705 710 715 720
Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys Gln
725 730 735
Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu Ile
740 745 750
Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp Tyr
755 760 765
Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile Asn
770 775 780
Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu Ile
785 790 795 800
Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu Lys
805 810 815
Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His Asp
820 825 830
Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly Asp
835 840 845
Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr Leu
850 855 860
Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile Lys
865 870 875 880
Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp Tyr
885 890 895
Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr Arg
900 905 910
Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val Lys
915 920 925
Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser Lys
930 935 940
Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala Glu
945 950 955 960
Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly Glu
965 970 975
Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile Glu
980 985 990
Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met Asn
995 1000 1005
Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys Thr
1010 1015 1020
Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1040 1045 1050
<210> 57
<211> 1052
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 57
Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser Val Gly
1 5 10 15
Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly Val
20 25 30
Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg Ser
35 40 45
Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile Gln
50 55 60
Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His Ser
65 70 75 80
Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu Ser
85 90 95
Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu Ala
100 105 110
Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr Gly
115 120 125
Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala Leu
130 135 140
Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys Asp
145 150 155 160
Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr Val
165 170 175
Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln Leu
180 185 190
Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg Arg
195 200 205
Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys Asp
210 215 220
Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe Pro
225 230 235 240
Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr Asn
245 250 255
Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn Glu
260 265 270
Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe Lys
275 280 285
Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu Val
290 295 300
Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys Pro
305 310 315 320
Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr Ala
325 330 335
Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala Lys
340 345 350
Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu Thr
355 360 365
Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser Asn
370 375 380
Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile Asn
385 390 395 400
Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala Ile
405 410 415
Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln Gln
420 425 430
Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro Val
435 440 445
Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile Ile
450 455 460
Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg Glu
465 470 475 480
Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys Arg
485 490 495
Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr Gly
500 505 510
Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp Met
515 520 525
Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu Asp
530 535 540
Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro Arg
545 550 555 560
Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys Gln
565 570 575
Glu Glu Ala Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu Ser
580 585 590
Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile Leu
595 600 605
Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu Tyr
610 615 620
Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp Phe
625 630 635 640
Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu Met
645 650 655
Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys Val
660 665 670
Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp Lys
675 680 685
Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp Ala
690 695 700
Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys Leu
705 710 715 720
Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys Gln
725 730 735
Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu Ile
740 745 750
Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp Tyr
755 760 765
Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Lys Leu Ile Asn
770 775 780
Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu Ile
785 790 795 800
Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu Lys
805 810 815
Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His Asp
820 825 830
Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly Asp
835 840 845
Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr Leu
850 855 860
Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile Lys
865 870 875 880
Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp Tyr
885 890 895
Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr Arg
900 905 910
Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val Lys
915 920 925
Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser Lys
930 935 940
Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala Glu
945 950 955 960
Phe Ile Ala Ser Phe Tyr Lys Asn Asp Leu Ile Lys Ile Asn Gly Glu
965 970 975
Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile Glu
980 985 990
Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met Asn
995 1000 1005
Asp Lys Arg Pro Pro His Ile Ile Lys Thr Ile Ala Ser Lys Thr
1010 1015 1020
Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1040 1045 1050
<210> 58
<211> 1367
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 58
Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 59
<211> 1367
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 59
Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 60
<211> 1367
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 60
Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Glu Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Gln Tyr Arg Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 61
<211> 1367
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 61
Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Val Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Gln Tyr Arg Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 62
<211> 1367
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 62
Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Val Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Arg Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Glu Tyr Arg Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 63
<211> 1367
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 63
Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Ala
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Ala Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Ala Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Ala Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 64
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 64
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 65
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 65
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 66
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 66
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Gly Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 67
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 67
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Val Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 68
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 68
Ser Glu Val Glu Phe Ser Tyr Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Ser His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 69
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 69
Ser Glu Val Glu Phe Ser Tyr Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Ser His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Asp Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 70
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 70
Ser Glu Val Glu Phe Ser Tyr Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Ser His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Gly Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 71
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 71
Ser Glu Val Glu Phe Ser Tyr Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Ser His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Val Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 72
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 72
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 73
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 73
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 74
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 74
Ala Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Phe
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 75
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 75
Ser Glu Val Glu Phe Ser Tyr Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Thr Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Ser His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Ser Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 76
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 76
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Gly Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val His Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Asp Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 77
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 77
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Gly Gly Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val His Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Asp Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 78
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 78
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Asp Gly Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Lys Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Gly Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 79
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 79
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Gln Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 80
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 80
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Met Gly Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Pro Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Asp Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 81
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 81
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Cys Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val His Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 82
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 82
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Leu Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 83
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 83
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Gly Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 84
<211> 178
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 84
Met Arg Arg Ala Phe Ile Thr Gly Val Phe Phe Leu Ser Glu Val Glu
1 5 10 15
Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg
20 25 30
Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val His Asn
35 40 45
Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile Gly Arg His Asp
50 55 60
Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val
65 70 75 80
Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Leu Glu
85 90 95
Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg
100 105 110
Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala Ala Gly Ser Leu
115 120 125
Met Asp Val Leu His His Pro Gly Met Asn His Arg Val Glu Ile Thr
130 135 140
Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Ser Asp Phe Phe
145 150 155 160
Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys Ala Gln Ser Ser
165 170 175
Thr Asp
<210> 85
<211> 199
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 85
cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg ctagagatcc 60
gcggccgcta atacgactca ccctagggag agccgccacc gtggtgagca agggcgagga 120
gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa 180
gttcagcgtg tccggcgag 199
<210> 86
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 86
gtttttctct aatttattct tccctttagc tagtttc 37
<210> 87
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 87
atttttctct aatttattct tccctttagc tagtttt 37
<210> 88
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 88
gtttttctct aatttattct tccctttagc tagtttc 37
<210> 89
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 89
atttttctct aatttattct tccctttagc tagtttt 37
<210> 90
<211> 10
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 90
ttaatttttt 10
<210> 91
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 91
gaacacaaag catagactgc ggg 23
<210> 92
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 92
gagtatgagg catagactgc agg 23
<210> 93
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 93
gtcaagaaag cagagactgc cgg 23
<210> 94
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 94
gagcaaagac aatagactgt agg 23
<210> 95
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 95
gatgagataa tgatgagtca ggg 23
<210> 96
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 96
ggattgaccc aggccagggc tgg 23
<210> 97
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 97
gaatactaag catagactcc agg 23
<210> 98
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 98
gtaaacaaag catagactga ggg 23
<210> 99
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 99
gaagaccaag gatagactgc tgg 23
<210> 100
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 100
gaacataaag aatagaatga tgg 23
<210> 101
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 101
ggacaggcag catagactgt ggg 23
<210> 102
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 102
gtagaaaaag tatagactgc agg 23
<210> 103
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 103
gaagatagag aatagactgc tgg 23
<210> 104
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 104
ggctaaagac catagactgt ggg 23
<210> 105
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 105
gtctagaaag cttagactgc tgg 23
<210> 106
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 106
gggaataatc atagaatcct gg 22
<210> 107
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 107
gacaaagagg aagagagacg ggg 23
<210> 108
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 108
acacacacac ttagaatctg tgg 23
<210> 109
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 109
cacacacact tagaatctgt ggg 23
<210> 110
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 110
acacacacac ttagaatctg tgg 23
<210> 111
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 111
cacacacact tagaatctgt ggg 23
<210> 112
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 112
ggtgtttcgt cctttccaca ag 22
<210> 113
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 113
gaacacaaag catagactgc gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 114
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 114
gagtatgagg catagactgc gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 115
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 115
gtcaagaaag cagagactgc gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 116
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 116
gagcaaagag aatagactgt gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 117
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 117
gatgagataa tgatgagtca gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 118
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 118
ggattgaccc aggccagggc gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 119
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 119
gaatactaag catagactcc gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 120
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 120
gtaaacaaag catagactga gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 121
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 121
gaagaccaag gatagactgc gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 122
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 122
gaacataaag aatagaatga gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 123
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 123
ggacaggcag catagactgt gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 124
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 124
gtagaaaaag tatagactgc gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 125
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 125
gaagatagag aatagactgc gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 126
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 126
ggctaaagac catagactgt gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 127
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 127
gtctagaaag cttagactgc gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 128
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 128
gggaataaat catagaatcc gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 129
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 129
gacaaagagg aagagagacg gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 130
<211> 56
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 130
gacacacaca cttagaatct ggttttagag ctagaaatag caagttaaaa taaggc 56
<210> 131
<211> 56
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 131
gcacacacac ttagaatctg tgttttagag ctagaaatag caagttaaaa taaggc 56
<210> 132
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 132
gtggggaagg ggcccccaag gttttagagc tagaaatagc aagttaaaat aaggc 55
<210> 133
<211> 56
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 133
gacgtaccag gtggagcacc cgttttagag ctagaaatag caagttaaaa taaggc 56
<210> 134
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (12)..(12)
<223> U是ideoxyU
<400> 134
agttgtacgc guccaaaaaa acggg 25
<210> 135
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 135
agattagcgg atcctacctg ac 22
<210> 136
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 136
gcggtctgta tttcccagaa c 21
<210> 137
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (17)..(17)
<223> U是ideoxyU
<400> 137
accggggact tcagaaucgg c 21
<210> 138
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (17)..(17)
<223> U是ideoxyU
<400> 138
attctgaagt ccccggugtt tcg 23
<210> 139
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (12)..(12)
<223> U是ideoxyU
<400> 139
acgcgtacaa cucaaaggag gaaaaaaaaa tg 32
<210> 140
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (15)..(15)
<223> U是ideoxyU
<220>
<221> misc_feature
<222> (25)..(26)
<223> n是a, c, g, t或u
<220>
<221> misc_feature
<222> (28)..(29)
<223> n是a, c, g, t或u
<400> 140
acgctggcga aacgugcctg ggatnnknnk gaagtgccgg tcggcgc 47
<210> 141
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (15)..(15)
<223> U是ideoxyU
<400> 141
acgtttcgcc agcgucagcg cgtgacg 27
<210> 142
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (17)..(17)
<223> U是ideoxyU
<400> 142
acgcgaaaac tggcgcugcg 20
<210> 143
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (17)..(17)
<223> U是ideoxyU
<220>
<221> misc_feature
<222> (20)..(21)
<223> n是a, c, g, t或u
<400> 143
agcgccagtt ttcgcgutmn ncacaccaaa gaccacgcga cc 42
<210> 144
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (14)..(14)
<223> U是ideoxyU
<220>
<221> misc_feature
<222> (17)..(18)
<223> n是a, c, g, t或u
<220>
<221> misc_feature
<222> (20)..(21)
<223> n是a, c, g, t或u
<400> 144
agtggcggat gagugcnnkn nkttgctcag ttacttcttt cgcatgcg 48
<210> 145
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (14)..(14)
<223> U是ideoxyU
<400> 145
actcatccgc caguattcct tccg 24
<210> 146
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 146
tacggcgtag tgcacctgga 20
<210> 147
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 147
tacggcgtag tgcacctgga 20
<210> 148
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 148
atcttattcg atcatgcgaa 20
<210> 149
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 149
caatgatgac ttctacagcg 20
<210> 150
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 150
caatgatgac ttctacagcg 20
<210> 151
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 151
caatgatgac ttctacagcg 20
<210> 152
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 152
ttcattaact gtggccggct 20
<210> 153
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 153
gcttaggtgg agcgcctatt 20
<210> 154
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 154
tacggcgtag tgcacctgga 20
<210> 155
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 155
tacggcgtag tgcacctgga 20
<210> 156
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 156
atcttattcg atcatgcgaa 20
<210> 157
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 157
tacggcgtag tgcacctgga 20
<210> 158
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 158
tacggcgtag tgcacctgga 20
<210> 159
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 159
atcttattgg agcgcctatt 20
<210> 160
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 160
gcttaggtgg agcgcctatt 20
<210> 161
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 161
caatgatgac ttctacagcg 20
<210> 162
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 162
tacggcgtag tgcacctgga 20
<210> 163
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 163
caatgatgac ttctacagcg 20
<210> 164
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 164
tacggcgtag tgcacctgga 20
<210> 165
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 165
caatgatgac ttctacagcg 20
<210> 166
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 166
tacggcgtag tgcacctgga 20
<210> 167
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 167
atcttattcg atcatgcgaa 20
<210> 168
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 168
ttcattaact gtggccggct 20
<210> 169
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 169
acactctttc cctacacgac gctcttccga tctnnnncca gccccatctg tcaaact 57
<210> 170
<211> 54
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 170
tggagttcag acgtgtgctc ttccgatctt gaatggattc cttggaaaca atga 54
<210> 171
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 171
acactctttc cctacacgac gctcttccga tctnnnnaga gactgattgc gtggagt 57
<210> 172
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 172
tggagttcag acgtgtgctc ttccgatctc actccagcct aggcaacaa 49
<210> 173
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 173
acactctttc cctacacgac gctcttccga tctnnnnccg acagccagtg gttaagt 57
<210> 174
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 174
tggagttcag acgtgtgctc ttccgatctg cttttcaccg actgcacag 49
<210> 175
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 175
acactctttc cctacacgac gctcttccga tctnnnnctg cacctagcct ccatgtc 57
<210> 176
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 176
tggagttcag acgtgtgctc ttccgatctc cttgcactga gaccgtgaa 49
<210> 177
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 177
acactctttc cctacacgac gctcttccga tctnnnngtc tgaggtcaca cagtggg 57
<210> 178
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 178
tggagttcag acgtgtgctc ttccgatctc tgagagcagg gaccacatc 49
<210> 179
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 179
acactctttc cctacacgac gctcttccga tctnnnnatg tgggctgcct agaaagg 57
<210> 180
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 180
tggagttcag acgtgtgctc ttccgatctc ccagccaaac ttgtcaacc 49
<210> 181
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 181
acactctttc cctacacgac gctcttccga tctnnnngat gccctccatc ttctccg 57
<210> 182
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 182
tggagttcag acgtgtgctc ttccgatcta ggtttgcata gacctgccc 49
<210> 183
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 183
acactctttc cctacacgac gctcttccga tctnnnnccc tgttcctaaa gcccacc 57
<210> 184
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 184
tggagttcag acgtgtgctc ttccgatcta ctggttctgt ttgtggcca 49
<210> 185
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 185
acactctttc cctacacgac gctcttccga tctnnnnttg cttattgctg aggggca 57
<210> 186
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 186
tggagttcag acgtgtgctc ttccgatcta cctctctcct ccagctgag 49
<210> 187
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 187
acactctttc cctacacgac gctcttccga tctnnnntcc acctccccac ttctctt 57
<210> 188
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 188
tggagttcag acgtgtgctc ttccgatctg gtgaaatgag caaggcaca 49
<210> 189
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 189
acactctttc cctacacgac gctcttccga tctnnnnccc taaaccacct gcagagg 57
<210> 190
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 190
tggagttcag acgtgtgctc ttccgatctc agccccagcc acattctat 49
<210> 191
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 191
acactctttc cctacacgac gctcttccga tctnnnnacc catgtgcctg acatagg 57
<210> 192
<211> 51
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 192
tggagttcag acgtgtgctc ttccgatctt ggtgattatg gttacacagc g 51
<210> 193
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 193
acactctttc cctacacgac gctcttccga tctnnnntca cttcagccca ggagtat 57
<210> 194
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 194
tggagttcag acgtgtgctc ttccgatctt ctctttctct cccccaccc 49
<210> 195
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 195
acactctttc cctacacgac gctcttccga tctnnnngaa cctgaagcct ttcccca 57
<210> 196
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 196
tggagttcag acgtgtgctc ttccgatcta acctgtgtga cacttggca 49
<210> 197
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 197
acactctttc cctacacgac gctcttccga tctnnnngca gacacccaca actgtct 57
<210> 198
<211> 52
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 198
tggagttcag acgtgtgctc ttccgatctg cactcagcta gacttaactc cc 52
<210> 199
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 199
acactctttc cctacacgac gctcttccga tctnnnnggg aggtggagag aggatgt 57
<210> 200
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 200
tggagttcag acgtgtgctc ttccgatctt cctgaggtct aggaacccg 49
<210> 201
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 201
acactctttc cctacacgac gctcttccga tctnnnncgc gggctgaagt agatcaa 57
<210> 202
<211> 52
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 202
tggagttcag acgtgtgctc ttccgatctc ctgtctctgc tcctttgtcc cc 52
<210> 203
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 203
acactctttc cctacacgac gctcttccga tctnnnngca ttacctggga gcctgtt 57
<210> 204
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 204
tggagttcag acgtgtgctc ttccgatcta acttcagcgg gcatcagaa 49
<210> 205
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 205
acactctttc cctacacgac gctcttccga tctnnnngtg gagtttagcc agggacc 57
<210> 206
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 206
tggagttcag acgtgtgctc ttccgatctt acaggcctca ctggagcta 49
<210> 207
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 207
acactctttc cctacacgac gctcttccga tctnnnnggc tggataacct tggctgt 57
<210> 208
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 208
tggagttcag acgtgtgctc ttccgatctt cctcaggcac tcctctcaa 49
<210> 209
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 209
acactctttc cctacacgac gctcttccga tctnnnncca gccccatctg tcaaact 57
<210> 210
<211> 54
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 210
tggagttcag acgtgtgctc ttccgatctt gaatggattc cttggaaaca atga 54
<210> 211
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 211
acactctttc cctacacgac gctcttccga tctnnnnatg tgggctgcct agaaagg 57
<210> 212
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 212
tggagttcag acgtgtgctc ttccgatctc ccagccaaac ttgtcaacc 49
<210> 213
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 213
acactctttc cctacacgac gctcttccga tctnnnngaa cccaggtagc cagagac 57
<210> 214
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 214
tggagttcag acgtgtgctc ttccgatctt cctttcaacc cgaacggag 49
<210> 215
<211> 58
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 215
acactctttc cctacacgac gctcttccga tctnnnngtg tggagagtga gtaagcca 58
<210> 216
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 216
tggagttcag acgtgtgctc ttccgatcta cggtaggatg atttcaggca 50
<210> 217
<211> 58
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 217
acactctttc cctacacgac gctcttccga tctnnnncac aaagcagtgt agctcagg 58
<210> 218
<211> 54
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 218
tggagttcag acgtgtgctc ttccgatctt ttttggtact cgagtgttat tcag 54
<210> 219
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 219
acactctttc cctacacgac gctcttccga tctnnnntcc cctgttgacc tggagaa 57
<210> 220
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 220
tggagttcag acgtgtgctc ttccgatctc actgtacttg ccctgacca 49
<210> 221
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 221
acactctttc cctacacgac gctcttccga tctnnnnttg gtgttgacag ggagcaa 57
<210> 222
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 222
tggagttcag acgtgtgctc ttccgatctc tgagatgtgg gcagaaggg 49
<210> 223
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 223
acactctttc cctacacgac gctcttccga tctnnnntga gagggaacag aagggct 57
<210> 224
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 224
tggagttcag acgtgtgctc ttccgatctg tccaaaggcc caagaacct 49
<210> 225
<211> 58
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 225
acactctttc cctacacgac gctcttccga tctnnnntcc tagcactttg gaaggtcg 58
<210> 226
<211> 51
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 226
tggagttcag acgtgtgctc ttccgatctg ctcatcttaa tctgctcagc c 51
<210> 227
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 227
acactctttc cctacacgac gctcttccga tctnnnnaaa ggagcagctc ttcctgg 57
<210> 228
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 228
tggagttcag acgtgtgctc ttccgatctg tctgcaccat ctcccacaa 49
<210> 229
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 229
acactctttc cctacacgac gctcttccga tctnnnnggc atggcttctg agactca 57
<210> 230
<211> 53
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 230
tggagttcag acgtgtgctc ttccgatctg tctcccttgc actccctgtc ttt 53
<210> 231
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 231
acactctttc cctacacgac gctcttccga tctnnnnttt ggcaatggag gcattgg 57
<210> 232
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 232
tggagttcag acgtgtgctc ttccgatctg aagaggctgc ccatgagag 49
<210> 233
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 233
acactctttc cctacacgac gctcttccga tctnnnnggt ctgaggctcg aatcctg 57
<210> 234
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 234
tggagttcag acgtgtgctc ttccgatctc tgtggcctcc atatccctg 49
<210> 235
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 235
acactctttc cctacacgac gctcttccga tctnnnnttt ccaccagaac tcagccc 57
<210> 236
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 236
tggagttcag acgtgtgctc ttccgatctc ctcggttcct ccacaacac 49
<210> 237
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 237
acactctttc cctacacgac gctcttccga tctnnnncac gggaaggaca ggagaag 57
<210> 238
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 238
tggagttcag acgtgtgctc ttccgatctg caggggaggg ataaagcag 49
<210> 239
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 239
acactctttc cctacacgac gctcttccga tctnnnncca gccccatctg tcaaact 57
<210> 240
<211> 54
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 240
tggagttcag acgtgtgctc ttccgatctt gaatggattc cttggaaaca atga 54
<210> 241
<211> 59
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 241
acactctttc cctacacgac gctcttccga tctnnnnccc tgagatacag tcacgaggt 59
<210> 242
<211> 51
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 242
tggagttcag acgtgtgctc ttccgatctc ctgaaatgct gtgcgtgtct a 51
<210> 243
<211> 59
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 243
acactctttc cctacacgac gctcttccga tctnnnngcc cacattacct tggtgcata 59
<210> 244
<211> 51
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 244
tggagttcag acgtgtgctc ttccgatctg gcaggcagat tatcattccc a 51
<210> 245
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 245
acactctttc cctacacgac gctcttccga tctnnnnaaa gtgctgcgat tacaggc 57
<210> 246
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 246
tggagttcag acgtgtgctc ttccgatctg tggcatccag agacatggt 49
<210> 247
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (34)..(37)
<223> n是a, c, g或t
<400> 247
acactctttc cctacacgac gctcttccga tctnnnnatg tgggctgcct agaaagg 57
<210> 248
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 248
tggagttcag acgtgtgctc ttccgatctc ccagccaaac ttgtcaacc 49
<210> 249
<211> 100
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 249
ttttccagcc cgctggccct gtaaaggaaa ctggaacgca aagcatagac tgcgcggcgg 60
gccagcctga atagctgcaa acaagtgcag aatatctgat 100
<210> 250
<211> 99
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 250
catgaaaaag agactgattg cgtggagttc atggagtgtg aggcatagac tgcacgagac 60
ataaaccatg acttgcagat gaagaagcat tttaaaagt 99
<210> 251
<211> 100
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 251
gacagccagt ggttaagtca gaacccgact caggtcagga aagcagagac tgcccggggt 60
tgggaaggcg gtgaactcag agatagaaac agggtgggtg 100
<210> 252
<211> 100
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 252
attttaagct gtagtattat gaagggaaat ctggagcgaa gagaatagac tgtacggaaa 60
ccagttaaga aataggacat ggaggctagg tgcagtggct 100
<210> 253
<211> 100
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 253
cctctgccat cacgtgctca gtctgggccc caaggattgg cccaggccag ggctcgagaa 60
gcagaaaaaa agcatcaagc ctacaaatgc atgcttactt 100
<210> 254
<211> 28
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 254
gacagauauu ugcauugaga uagugugg 28
<210> 255
<211> 27
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 255
acagauauuu gcauugagau agugugg 27
<210> 256
<211> 26
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 256
cagauauuug cauugagaua gugugg 26
<210> 257
<211> 25
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 257
agauauuugc auugagauag ugugg 25
<210> 258
<211> 24
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 258
gauauuugca uugagauagu gugg 24
<210> 259
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 259
auauuugcau ugagauagug ugg 23
<210> 260
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 260
augcaaauau cugucugaaa cgg 23
<210> 261
<211> 28
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 261
gcaaauaucu gucugaaacg gucccugg 28
<210> 262
<211> 27
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 262
caaauaucug ucugaaacgg ucccugg 27
<210> 263
<211> 26
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 263
aaauaucugu cugaaacggu cccugg 26
<210> 264
<211> 25
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 264
aauaucuguc ugaaacgguc ccugg 25
<210> 265
<211> 24
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 265
auaucugucu gaaacggucc cugg 24
<210> 266
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 266
uaucugucug aaacgguccc ugg 23
<210> 267
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 267
agauauuugc auugagauag ugu 23
<210> 268
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 268
acagauauuu gcauugagau agu 23
<210> 269
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 269
guggggaagg ggcccccaag agg 23
<210> 270
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 270
cuugaccaau agccuugaca agg 23
<210> 271
<211> 28
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 271
cuugucaagg cuauugguca aggcaagg 28
<210> 272
<211> 27
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 272
uugucaaggc uauuggucaa ggcaagg 27
<210> 273
<211> 26
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 273
ugucaaggcu auuggucaag gcaagg 26
<210> 274
<211> 25
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 274
gucaaggcua uuggucaagg caagg 25
<210> 275
<211> 24
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 275
ucaaggcuau uggucaaggc aagg 24
<210> 276
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 276
caaggcuauu ggucaaggca agg 23
<210> 277
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 277
uugucaaggc uauuggucaa ggc 23
<210> 278
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 278
cuugucaagg cuauugguca agg 23
<210> 279
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 279
uugaccaaua gccuugacaa ggc 23
<210> 280
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 280
uagccuugac aaggcaaacu uga 23
<210> 281
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 281
uucuccacag gagucagaug cac 23
<210> 282
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 282
ucuccacagg agucagaugc acc 23
<210> 283
<211> 28
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 283
uucuccacag gagucagaug caccaugg 28
<210> 284
<211> 27
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 284
ucuccacagg agucagaugc accaugg 27
<210> 285
<211> 26
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 285
cuccacagga gucagaugca ccaugg 26
<210> 286
<211> 25
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 286
uccacaggag ucagaugcac caugg 25
<210> 287
<211> 24
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 287
ccacaggagu cagaugcacc augg 24
<210> 288
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 288
cacaggaguc agaugcacca ugg 23
<210> 289
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 289
acuucuccac aggagucaga ugc 23
<210> 290
<211> 28
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 290
ucuccacagg agucagaugc accauggu 28
<210> 291
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 291
acuccuaagg agaagucugc cgu 23
<210> 292
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 292
uuggugguaa ggcccugggc agg 23
<210> 293
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 293
uggugguaag gcccugggca ggu 23
<210> 294
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 294
ugguaaggcc cugggcaggu ugg 23
<210> 295
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 295
agcauuguau auucucugug agg 23
<210> 296
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 296
aaagcauugu auauucucug uga 23
<210> 297
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 297
ccacactatc tcaatgcaaa tatctgtc 28
<210> 298
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 298
ccacactatc tcaatgcaaa tatctgt 27
<210> 299
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 299
ccacactatc tcaatgcaaa tatctg 26
<210> 300
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 300
ccacactatc tcaatgcaaa tatct 25
<210> 301
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 301
ccacactatc tcaatgcaaa tatc 24
<210> 302
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 302
ccacactatc tcaatgcaaa tat 23
<210> 303
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 303
ccgtttcaga cagatatttg cat 23
<210> 304
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 304
ccagggaccg tttcagacag atatttgc 28
<210> 305
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 305
ccagggaccg tttcagacag atatttg 27
<210> 306
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 306
ccagggaccg tttcagacag atattt 26
<210> 307
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 307
ccagggaccg tttcagacag atatt 25
<210> 308
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 308
ccagggaccg tttcagacag atat 24
<210> 309
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 309
ccagggaccg tttcagacag ata 23
<210> 310
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 310
acactatctc aatgcaaata tct 23
<210> 311
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 311
actatctcaa tgcaaatatc tgt 23
<210> 312
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 312
cctcttgggg gccccttccc cac 23
<210> 313
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 313
ccttgtcaag gctattggtc aag 23
<210> 314
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 314
ccttgccttg accaatagcc ttgacaag 28
<210> 315
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 315
ccttgccttg accaatagcc ttgacaa 27
<210> 316
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 316
ccttgccttg accaatagcc ttgaca 26
<210> 317
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 317
ccttgccttg accaatagcc ttgac 25
<210> 318
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 318
ccttgccttg accaatagcc ttga 24
<210> 319
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 319
ccttgccttg accaatagcc ttg 23
<210> 320
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 320
gccttgacca atagccttga caa 23
<210> 321
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 321
ccttgaccaa tagccttgac aag 23
<210> 322
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 322
gccttgtcaa ggctattggt caa 23
<210> 323
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 323
tcaagtttgc cttgtcaagg cta 23
<210> 324
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 324
gtgcatctga ctcctgtgga gaa 23
<210> 325
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 325
ggtgcatctg actcctgtgg aga 23
<210> 326
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 326
ccatggtgca tctgactcct gtggagaa 28
<210> 327
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 327
ccatggtgca tctgactcct gtggaga 27
<210> 328
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 328
ccatggtgca tctgactcct gtggag 26
<210> 329
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 329
ccatggtgca tctgactcct gtgga 25
<210> 330
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 330
ccatggtgca tctgactcct gtgg 24
<210> 331
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 331
ccatggtgca tctgactcct gtg 23
<210> 332
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 332
gcatctgact cctgtggaga agt 23
<210> 333
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 333
accatggtgc atctgactcc tgtggaga 28
<210> 334
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 334
acggcagact tctccttagg agt 23
<210> 335
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 335
cctgcccagg gccttaccac caa 23
<210> 336
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 336
acctgcccag ggccttacca cca 23
<210> 337
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 337
ccaacctgcc cagggcctta cca 23
<210> 338
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 338
cctcacagag aatatacaat gct 23
<210> 339
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 339
tcacagagaa tatacaatgc ttt 23
<210> 340
<211> 146
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 340
Val His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp Gly
1 5 10 15
Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu Leu
20 25 30
Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp Leu
35 40 45
Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His Gly
50 55 60
Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp Asn
65 70 75 80
Leu Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys Leu
85 90 95
His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val Cys
100 105 110
Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro Val Gln Ala
115 120 125
Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala His Lys
130 135 140
Tyr His
145
<210> 341
<211> 2351
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 341
Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe
1 5 10 15
Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser
20 25 30
Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg
35 40 45
Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val
50 55 60
Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile
65 70 75 80
Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln
85 90 95
Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser
100 105 110
His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser
115 120 125
Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp
130 135 140
Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu
145 150 155 160
Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser
165 170 175
Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile
180 185 190
Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr
195 200 205
Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly
210 215 220
Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp
225 230 235 240
Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr
245 250 255
Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val
260 265 270
Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile
275 280 285
Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser
290 295 300
Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met
305 310 315 320
Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His
325 330 335
Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro
340 345 350
Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp
355 360 365
Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser
370 375 380
Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr
385 390 395 400
Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro
405 410 415
Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn
420 425 430
Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met
435 440 445
Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu
450 455 460
Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu
465 470 475 480
Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
485 490 495
His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys
500 505 510
Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe
515 520 525
Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp
530 535 540
Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg
545 550 555 560
Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu
565 570 575
Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val
580 585 590
Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu
595 600 605
Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp
610 615 620
Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val
625 630 635 640
Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp
645 650 655
Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe
660 665 670
Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr
675 680 685
Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro
690 695 700
Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly
705 710 715 720
Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp
725 730 735
Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys
740 745 750
Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro
755 760 765
Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp
770 775 780
Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys
785 790 795 800
Ile Gln Asn Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser
805 810 815
Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr
820 825 830
Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn
835 840 845
Ser Leu Ser Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly
850 855 860
Asp Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu
865 870 875 880
Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys
885 890 895
Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn
900 905 910
Leu Ala Ala Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met
915 920 925
Pro Val His Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys
930 935 940
Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu
945 950 955 960
Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu
965 970 975
Ser Ser Trp Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe
980 985 990
Lys Gly Lys Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala
995 1000 1005
Leu Phe Lys Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser
1010 1015 1020
Asn Asn Ser Ala Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser
1025 1030 1035
Leu Leu Ile Glu Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu
1040 1045 1050
Ser Asp Thr Glu Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg
1055 1060 1065
Met Leu Met Asp Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met
1070 1075 1080
Ser Asn Lys Thr Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln
1085 1090 1095
Lys Lys Glu Gly Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met
1100 1105 1110
Ser Phe Phe Lys Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile
1115 1120 1125
Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro
1130 1135 1140
Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu
1145 1150 1155
Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys
1160 1165 1170
Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro
1175 1180 1185
Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu
1190 1195 1200
Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu
1205 1210 1215
Lys Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile
1220 1225 1230
His Thr Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu
1235 1240 1245
Leu Ser Thr Arg Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr
1250 1255 1260
Ala Pro Val Leu Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn
1265 1270 1275
Arg Thr Lys Lys His Thr Ala His Phe Ser Lys Lys Gly Glu Glu
1280 1285 1290
Glu Asn Leu Glu Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu
1295 1300 1305
Lys Tyr Ala Cys Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln
1310 1315 1320
Asn Phe Val Thr Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg
1325 1330 1335
Leu Pro Leu Glu Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp
1340 1345 1350
Asp Thr Ser Thr Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro
1355 1360 1365
Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala
1370 1375 1380
Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser
1385 1390 1395
Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser
1400 1405 1410
Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe
1415 1420 1425
Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys
1430 1435 1440
Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys
1445 1450 1455
Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly
1460 1465 1470
Asp Gln Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser
1475 1480 1485
Val Thr Tyr Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp
1490 1495 1500
Leu Pro Lys Thr Ser Gly Lys Val Glu Leu Leu Pro Lys Val His
1505 1510 1515
Ile Tyr Gln Lys Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser
1520 1525 1530
Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr
1535 1540 1545
Glu Gly Ala Ile Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val
1550 1555 1560
Pro Phe Leu Arg Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser
1565 1570 1575
Lys Leu Leu Asp Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln
1580 1585 1590
Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys
1595 1600 1605
Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys
1610 1615 1620
Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys
1625 1630 1635
Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg
1640 1645 1650
Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu
1655 1660 1665
Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr
1670 1675 1680
Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile
1685 1690 1695
Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys
1700 1705 1710
Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr
1715 1720 1725
Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser
1730 1735 1740
Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr
1745 1750 1755
Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu
1760 1765 1770
His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp
1775 1780 1785
Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser
1790 1795 1800
Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly
1805 1810 1815
Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr
1820 1825 1830
Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu
1835 1840 1845
Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu
1850 1855 1860
Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His
1865 1870 1875
Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln
1880 1885 1890
Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp
1895 1900 1905
Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn
1910 1915 1920
Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His
1925 1930 1935
Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met
1940 1945 1950
Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser
1955 1960 1965
Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr
1970 1975 1980
Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr
1985 1990 1995
Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly
2000 2005 2010
Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly
2015 2020 2025
Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro
2030 2035 2040
Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala
2045 2050 2055
Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His
2060 2065 2070
Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser
2075 2080 2085
Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile
2090 2095 2100
Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser
2105 2110 2115
Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr
2120 2125 2130
Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn
2135 2140 2145
Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile
2150 2155 2160
Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg
2165 2170 2175
Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys
2180 2185 2190
Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln
2195 2200 2205
Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser
2210 2215 2220
Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp
2225 2230 2235
Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe
2240 2245 2250
Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys
2255 2260 2265
Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser
2270 2275 2280
Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys
2285 2290 2295
Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val
2300 2305 2310
Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His
2315 2320 2325
Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu
2330 2335 2340
Gly Cys Glu Ala Gln Asp Leu Tyr
2345 2350
<210> 342
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 342
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys Arg
1 5 10 15
Lys Val
<210> 343
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 343
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys
1 5 10 15
Val
<210> 344
<211> 343
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 344
ctgacaaaag aagtcctggt atcctctatg atgggagaag gaaactagct aaagggaaga 60
ataaattaga gaaaaactgg aatgactgaa tcggaacaag gcaaaggcta taaaaaaaat 120
tagcagtatc ctcttggggg ccccttcccc acactatctc aatgcaaata tctgtctgaa 180
acggtccctg gctaaactcc acccatgggt tggccagcct tgccttgacc aatagccttg 240
acaaggcaaa cttgaccaat agtcttagag tatccagtga ggccaggggc cggcggctgg 300
ctagggatga agaataaaag gaagcaccct tcagcagttc cac 343
<210> 345
<211> 318
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 345
ggtgggagaa gaaaactagc taaagggaag aataaattag agaaaaattg gaatgactga 60
atcggaacaa ggcaaaggct ataaaaaaaa ttaagcagca gtatcctctt gggggcccct 120
tccccacact atctcaatgc aaatatctgt ctgaaacggt ccctggctaa actccaccca 180
tgggttggcc agccttgcct tgaccaatag ccttgacaag gcaaacttga ccaatagtct 240
tagagtatcc agtgaggcca ggggccggcg gctggctagg gatgaagaat aaaaggaagc 300
acccttcagc agttccac 318
<210> 346
<211> 1606
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 346
acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc atggtgcatc 60
tgactcctga ggagaagtct gccgttactg ccctgtgggg caaggtgaac gtggatgaag 120
ttggtggtga ggccctgggc aggttggtat caaggttaca agacaggttt aaggagacca 180
atagaaactg ggcatgtgga gacagagaag actcttgggt ttctgatagg cactgactct 240
ctctgcctat tggtctattt tcccaccctt aggctgctgg tggtctaccc ttggacccag 300
aggttctttg agtcctttgg ggatctgtcc actcctgatg ctgttatggg caaccctaag 360
gtgaaggctc atggcaagaa agtgctcggt gcctttagtg atggcctggc tcacctggac 420
aacctcaagg gcacctttgc cacactgagt gagctgcact gtgacaagct gcacgtggat 480
cctgagaact tcagggtgag tctatgggac gcttgatgtt ttctttcccc ttcttttcta 540
tggttaagtt catgtcatag gaaggggata agtaacaggg tacagtttag aatgggaaac 600
agacgaatga ttgcatcagt gtggaagtct caggatcgtt ttagtttctt ttatttgctg 660
ttcataacaa ttgttttctt ttgtttaatt cttgctttct ttttttttct tctccgcaat 720
ttttactatt atacttaatg ccttaacatt gtgtataaca aaaggaaata tctctgagat 780
acattaagta acttaaaaaa aaactttaca cagtctgcct agtacattac tatttggaat 840
atatgtgtgc ttatttgcat attcataatc tccctacttt attttctttt atttttaatt 900
gatacataat cattatacat atttatgggt taaagtgtaa tgttttaata tgtgtacaca 960
tattgaccaa atcagggtaa ttttgcattt gtaattttaa aaaatgcttt cttcttttaa 1020
tatacttttt tgtttatctt atttctaata ctttccctaa tctctttctt tcagggcaat 1080
aatgatacaa tgtatcatgc ctctttgcac cattctaaag aataacagtg ataatttctg 1140
ggttaaggca atagcaatat ctctgcatat aaatatttct gcatataaat tgtaactgat 1200
gtaagaggtt tcatattgct aatagcagct acaatccagc taccattctg cttttatttt 1260
atggttggga taaggctgga ttattctgag tccaagctag gcccttttgc taatcatgtt 1320
catacctctt atcttcctcc cacagctcct gggcaacgtg ctggtctgtg tgctggccca 1380
tcactttggc aaagaattca ccccaccagt gcaggctgcc tatcagaaag tggtggctgg 1440
tgtggctaat gccctggccc acaagtatca ctaagctcgc tttcttgctg tccaatttct 1500
attaaaggtt cctttgttcc ctaagtccaa ctactaaact gggggatatt atgaagggcc 1560
ttgagcatct ggattctgcc taataaaaaa catttatttt cattgc 1606
<210> 347
<211> 9036
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 347
gcttagtgct gagcacatcc agtgggtaaa gttccttaaa atgctctgca aagaaattgg 60
gacttttcat taaatcagaa attttacttt tttcccctcc tgggagctaa agatatttta 120
gagaagaatt aaccttttgc ttctccagtt gaacatttgt agcaataagt catgcaaata 180
gagctctcca cctgcttctt tctgtgcctt ttgcgattct gctttagtgc caccagaaga 240
tactacctgg gtgcagtgga actgtcatgg gactatatgc aaagtgatct cggtgagctg 300
cctgtggacg caagatttcc tcctagagtg ccaaaatctt ttccattcaa cacctcagtc 360
gtgtacaaaa agactctgtt tgtagaattc acggatcacc ttttcaacat cgctaagcca 420
aggccaccct ggatgggtct gctaggtcct accatccagg ctgaggttta tgatacagtg 480
gtcattacac ttaagaacat ggcttcccat cctgtcagtc ttcatgctgt tggtgtatcc 540
tactggaaag cttctgaggg agctgaatat gatgatcaga ccagtcaaag ggagaaagaa 600
gatgataaag tcttccctgg tggaagccat acatatgtct ggcaggtcct gaaagagaat 660
ggtccaatgg cctctgaccc actgtgcctt acctactcat atctttctca tgtggacctg 720
gtaaaagact tgaattcagg cctcattgga gccctactag tatgtagaga agggagtctg 780
gccaaggaaa agacacagac cttgcacaaa tttatactac tttttgctgt atttgatgaa 840
gggaaaagtt ggcactcaga aacaaagaac tccttgatgc aggataggga tgctgcatct 900
gctcgggcct ggcctaaaat gcacacagtc aatggttatg taaacaggtc tctgccaggt 960
ctgattggat gccacaggaa atcagtctat tggcatgtga ttggaatggg caccactcct 1020
gaagtgcact caatattcct cgaaggtcac acatttcttg tgaggaacca tcgccaggcg 1080
tccttggaaa tctcgccaat aactttcctt actgctcaaa cactcttgat ggaccttgga 1140
cagtttctac tgttttgtca tatctcttcc caccaacatg atggcatgga agcttatgtc 1200
aaagtagaca gctgtccaga ggaaccccaa ctacgaatga aaaataatga agaagcggaa 1260
gactatgatg atgatcttac tgattctgaa atggatgtgg tcaggtttga tgatgacaac 1320
tctccttcct ttatccaaat tcgctcagtt gccaagaagc atcctaaaac ttgggtacat 1380
tacattgctg ctgaagagga ggactgggac tatgctccct tagtcctcgc ccccgatgac 1440
agaagttata aaagtcaata tttgaacaat ggccctcagc ggattggtag gaagtacaaa 1500
aaagtccgat ttatggcata cacagatgaa acctttaaga ctcgtgaagc tattcagcat 1560
gaatcaggaa tcttgggacc tttactttat ggggaagttg gagacacact gttgattata 1620
tttaagaatc aagcaagcag accatataac atctaccctc acggaatcac tgatgtccgt 1680
cctttgtatt caaggagatt accaaaaggt gtaaaacatt tgaaggattt tccaattctg 1740
ccaggagaaa tattcaaata taaatggaca gtgactgtag aagatgggcc aactaaatca 1800
gatcctcggt gcctgacccg ctattactct agtttcgtta atatggagag agatctagct 1860
tcaggactca ttggccctct cctcatctgc tacaaagaat ctgtagatca aagaggaaac 1920
cagataatgt cagacaagag gaatgtcatc ctgttttctg tatttgatga gaaccgaagc 1980
tggtacctca cagagaatat acaacgcttt ctccccaatc cagctggagt gcagcttgag 2040
gatccagagt tccaagcctc caacatcatg cacagcatca atggctatgt ttttgatagt 2100
ttgcagttgt cagtttgttt gcatgaggtg gcatactggt acattctaag cattggagca 2160
cagactgact tcctttctgt cttcttctct ggatatacct tcaaacacaa aatggtctat 2220
gaagacacac tcaccctatt cccattctca ggagaaactg tcttcatgtc gatggaaaac 2280
ccaggtctat ggattctggg gtgccacaac tcagactttc ggaacagagg catgaccgcc 2340
ttactgaagg tttctagttg tgacaagaac actggtgatt attacgagga cagttatgaa 2400
gatatttcag catacttgct gagtaaaaac aatgccattg aaccaagaag cttctcccag 2460
aattcaagac accctagcac taggcaaaag caatttaatg ccaccacaat tccagaaaat 2520
gacatagaga agactgaccc ttggtttgca cacagaacac ctatgcctaa aatacaaaat 2580
gtctcctcta gtgatttgtt gatgctcttg cgacagagtc ctactccaca tgggctatcc 2640
ttatctgatc tccaagaagc caaatatgag actttttctg atgatccatc acctggagca 2700
atagacagta ataacagcct gtctgaaatg acacacttca ggccacagct ccatcacagt 2760
ggggacatgg tatttacccc tgagtcaggc ctccaattaa gattaaatga gaaactgggg 2820
acaactgcag caacagagtt gaagaaactt gatttcaaag tttctagtac atcaaataat 2880
ctgatttcaa caattccatc agacaatttg gcagcaggta ctgataatac aagttcctta 2940
ggacccccaa gtatgccagt tcattatgat agtcaattag ataccactct atttggcaaa 3000
aagtcatctc cccttactga gtctggtgga cctctgagct tgagtgaaga aaataatgat 3060
tcaaagttgt tagaatcagg tttaatgaat agccaagaaa gttcatgggg aaaaaatgta 3120
tcgtcaacag agagtggtag gttatttaaa gggaaaagag ctcatggacc tgctttgttg 3180
actaaagata atgccttatt caaagttagc atctctttgt taaagacaaa caaaacttcc 3240
aataattcag caactaatag aaagactcac attgatggcc catcattatt aattgagaat 3300
agtccatcag tctggcaaaa tatattagaa agtgacactg agtttaaaaa agtgacacct 3360
ttgattcatg acagaatgct tatggacaaa aatgctacag ctttgaggct aaatcatatg 3420
tcaaataaaa ctacttcatc aaaaaacatg gaaatggtcc aacagaaaaa agagggcccc 3480
attccaccag atgcacaaaa tccagatatg tcgttcttta agatgctatt cttgccagaa 3540
tcagcaaggt ggatacaaag gactcatgga aagaactctc tgaactctgg gcaaggcccc 3600
agtccaaagc aattagtatc cttaggacca gaaaaatctg tggaaggtca gaatttcttg 3660
tctgagaaaa acaaagtggt agtaggaaag ggtgaattta caaaggacgt aggactcaaa 3720
gagatggttt ttccaagcag cagaaaccta tttcttacta acttggataa tttacatgaa 3780
aataatacac acaatcaaga aaaaaaaatt caggaagaaa tagaaaagaa ggaaacatta 3840
atccaagaga atgtagtttt gcctcagata catacagtga ctggcactaa gaatttcatg 3900
aagaaccttt tcttactgag cactaggcaa aatgtagaag gttcatatga cggggcatat 3960
gctccagtac ttcaagattt taggtcatta aatgattcaa caaatagaac aaagaaacac 4020
acagctcatt tctcaaaaaa aggggaggaa gaaaacttgg aaggcttggg aaatcaaacc 4080
aagcaaattg tagagaaata tgcatgcacc acaaggatat ctcctaatac aagccagcag 4140
aattttgtca cgcaacgtag taagagagct ttgaaacaat tcagactccc actagaagaa 4200
acagaacttg aaaaaaggat aattgtggat gacacctcaa cccagtggtc caaaaacatg 4260
aaacatttga ccccgagcac cctcacacag atagactaca atgagaagga gaaaggggcc 4320
attactcagt ctcccttatc agattgcctt acgaggagtc atagcatccc tcaagcaaat 4380
agatctccat tacccattgc aaaggtatca tcatttccat ctattagacc tatatatctg 4440
accagggtcc tattccaaga caactcttct catcttccag cagcatctta tagaaagaaa 4500
gattctgggg tccaagaaag cagtcatttc ttacaaggag ccaaaaaaaa taacctttct 4560
ttagccattc taaccttgga gatgactggt gatcaaagag aggttggctc cctggggaca 4620
agtgccacaa attcagtcac atacaagaaa gttgagaaca ctgttctccc gaaaccagac 4680
ttgcccaaaa catctggcaa agttgaattg cttccaaaag ttcacattta tcagaaggac 4740
ctattcccta cggaaactag caatgggtct cctggccatc tggatctcgt ggaagggagc 4800
cttcttcagg gaacagaggg agcgattaag tggaatgaag caaacagacc tggaaaagtt 4860
ccctttctga gagtagcaac agaaagctct gcaaagactc cctccaagct attggatcct 4920
cttgcttggg ataaccacta tggtactcag ataccaaaag aagagtggaa atcccaagag 4980
aagtcaccag aaaaaacagc ttttaagaaa aaggatacca ttttgtccct gaacgcttgt 5040
gaaagcaatc atgcaatagc agcaataaat gagggacaaa ataagcccga aatagaagtc 5100
acctgggcaa agcaaggtag gactgaaagg ctgtgctctc aaaacccacc agtcttgaaa 5160
cgccatcaac gggaaataac tcgtactact cttcagtcag atcaagagga aattgactat 5220
gatgatacca tatcagttga aatgaagaag gaagattttg acatttatga tgaggatgaa 5280
aatcagagcc cccgcagctt tcaaaagaaa acacgacact attttattgc tgcagtggag 5340
aggctctggg attatgggat gagtagctcc ccacatgttc taagaaacag ggctcagagt 5400
ggcagtgtcc ctcagttcaa gaaagttgtt ttccaggaat ttactgatgg ctcctttact 5460
cagcccttat accgtggaga actaaatgaa catttgggac tcctggggcc atatataaga 5520
gcagaagttg aagataatat catggtaact ttcagaaatc aggcctctcg tccctattcc 5580
ttctattcta gccttatttc ttatgaggaa gatcagaggc aaggagcaga acctagaaaa 5640
aactttgtca agcctaatga aaccaaaact tacttttgga aagtgcaaca tcatatggca 5700
cccactaaag atgagtttga ctgcaaagcc tgggcttatt tctctgatgt tgacctggaa 5760
aaagatgtgc actcaggcct gattggaccc cttctggtct gccacactaa cacactgaac 5820
cctgctcatg ggagacaagt gacagtacag gaatttgctc tgtttttcac catctttgat 5880
gagaccaaaa gctggtactt cactgaaaat atggaaagaa actgcagggc tccctgcaat 5940
atccagatgg aagatcccac ttttaaagag aattatcgct tccatgcaat caatggctac 6000
ataatggata cactacctgg cttagtaatg gctcaggatc aaaggattcg atggtatctg 6060
ctcagcatgg gcagcaatga aaacatccat tctattcatt tcagtggaca tgtgttcact 6120
gtacgaaaaa aagaggagta taaaatggca ctgtacaatc tctatccagg tgtttttgag 6180
acagtggaaa tgttaccatc caaagctgga atttggcggg tggaatgcct tattggcgag 6240
catctacatg ctgggatgag cacacttttt ctggtgtaca gcaataagtg tcagactccc 6300
ctgggaatgg cttctggaca cattagagat tttcagatta cagcttcagg acaatatgga 6360
cagtgggccc caaagctggc cagacttcat tattccggat caatcaatgc ctggagcacc 6420
aaggagccct tttcttggat caaggtggat ctgttggcac caatgattat tcacggcatc 6480
aagacccagg gtgcccgtca gaagttctcc agcctctaca tctctcagtt tatcatcatg 6540
tatagtcttg atgggaagaa gtggcagact tatcgaggaa attccactgg aaccttaatg 6600
gtcttctttg gcaatgtgga ttcatctggg ataaaacaca atatttttaa ccctccaatt 6660
attgctcgat acatccgttt gcacccaact cattatagca ttcgcagcac tcttcgcatg 6720
gagttgatgg gctgtgattt aaatagttgc agcatgccat tgggaatgga gagtaaagca 6780
atatcagatg cacagattac tgcttcatcc tactttacca atatgtttgc cacctggtct 6840
ccttcaaaag ctcgacttca cctccaaggg aggagtaatg cctggagacc tcaggtgaat 6900
aatccaaaag agtggctgca agtggacttc cagaagacaa tgaaagtcac aggagtaact 6960
actcagggag taaaatctct gcttaccagc atgtatgtga aggagttcct catctccagc 7020
agtcaagatg gccatcagtg gactctcttt tttcagaatg gcaaagtaaa ggtttttcag 7080
ggaaatcaag actccttcac acctgtggtg aactctctag acccaccgtt actgactcgc 7140
taccttcgaa ttcaccccca gagttgggtg caccagattg ccctgaggat ggaggttctg 7200
ggctgcgagg cacaggacct ctactgaggg tggccactgc agcacctgcc actgccgtca 7260
cctctccctc ctcagctcca gggcagtgtc cctccctggc ttgccttcta cctttgtgct 7320
aaatcctagc agacactgcc ttgaagcctc ctgaattaac tatcatcagt cctgcatttc 7380
tttggtgggg ggccaggagg gtgcatccaa tttaacttaa ctcttaccta ttttctgcag 7440
ctgctcccag attactcctt ccttccaata taactaggca aaaagaagtg aggagaaacc 7500
tgcatgaaag cattcttccc tgaaaagtta ggcctctcag agtcaccact tcctctgttg 7560
tagaaaaact atgtgatgaa actttgaaaa agatatttat gatgttaaca tttcaggtta 7620
agcctcatac gtttaaaata aaactctcag ttgtttatta tcctgatcaa gcatggaaca 7680
aagcatgttt caggatcaga tcaatacaat cttggagtca aaaggcaaat catttggaca 7740
atctgcaaaa tggagagaat acaataacta ctacagtaaa gtctgtttct gcttccttac 7800
acatagatat aattatgtta tttagtcatt atgaggggca cattcttatc tccaaaacta 7860
gcattcttaa actgagaatt atagatgggg ttcaagaatc cctaagtccc ctgaaattat 7920
ataaggcatt ctgtataaat gcaaatgtgc atttttctga cgagtgtcca tagatataaa 7980
gccatttggt cttaattctg accaataaaa aaataagtca ggaggatgca attgttgaaa 8040
gctttgaaat aaaataacaa tgtcttcttg aaatttgtga tggccaagaa agaaaatgat 8100
gatgacatta ggcttctaaa ggacatacat ttaatatttc tgtggaaata tgaggaaaat 8160
ccatggttat ctgagatagg agatacaaac tttgtaattc taataatgca ctcagtttac 8220
tctctccctc tactaatttc ctgctgaaaa taacacaaca aaaatgtaac aggggaaatt 8280
atataccgtg actgaaaact agagtcctac ttacatagtt gaaatatcaa ggaggtcaga 8340
agaaaattgg actggtgaaa acagaaaaaa cactccagtc tgccatatca ccacacaata 8400
ggatccccct tcttgccctc cacccccata agattgtgaa gggtttactg ctccttccat 8460
ctgcctgacc ccttcactat gactacacag aatctcctga tagtaaaggg ggctggagac 8520
aaggataagt tatagagcag ttggaggaag catccaaaga ttgcaaccca gggcaaatgg 8580
aaaacaggag atcctaatat gaaagaaaaa tggatcccaa tctgagaaaa ggcaaaagaa 8640
tggctacttt tttctatgct ggagtatttt ctaataatcc tgcttgaccc ttatctgacc 8700
tctttggaaa ctataacata gctgtcacag tatagtcaca atccacaaat gatgcaggtg 8760
caaatggttt atagccctgt gaagttctta aagtttagag gctaacttac agaaatgaat 8820
aagttgtttt gttttatagc ccggtagagg agttaacccc aaaggtgata tggttttatt 8880
tcctgttatg tttaacttga taatcttatt ttggcattct tttcccattg actatataca 8940
tctctatttc tcaaatgttc atggaactag ctcttttatt ttcctgctgg tttcttcagt 9000
aatgagttaa ataaaacatt gacacataca aacaaa 9036
<210> 348
<400> 348
000
<210> 349
<400> 349
000
<210> 350
<400> 350
000
<210> 351
<400> 351
000
<210> 352
<400> 352
000
<210> 353
<400> 353
000
<210> 354
<400> 354
000
<210> 355
<400> 355
000
<210> 356
<400> 356
000
<210> 357
<400> 357
000
<210> 358
<400> 358
000
<210> 359
<400> 359
000
<210> 360
<400> 360
000
<210> 361
<400> 361
000
<210> 362
<400> 362
000
<210> 363
<400> 363
000
<210> 364
<400> 364
000
<210> 365
<400> 365
000
<210> 366
<400> 366
000
<210> 367
<400> 367
000
<210> 368
<400> 368
000
<210> 369
<400> 369
000
<210> 370
<400> 370
000
<210> 371
<211> 183
<212> PRT
<213> 鼠伤寒沙门氏菌
<400> 371
Met Pro Pro Ala Phe Ile Thr Gly Val Thr Ser Leu Ser Asp Val Glu
1 5 10 15
Leu Asp His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg
20 25 30
Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val His Asn
35 40 45
His Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile Gly Arg His Asp
50 55 60
Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val
65 70 75 80
Leu Gln Asn Tyr Arg Leu Leu Asp Thr Thr Leu Tyr Val Thr Leu Glu
85 90 95
Pro Cys Val Met Cys Ala Gly Ala Met Val His Ser Arg Ile Gly Arg
100 105 110
Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala Ala Gly Ser Leu
115 120 125
Ile Asp Val Leu His His Pro Gly Met Asn His Arg Val Glu Ile Ile
130 135 140
Glu Gly Val Leu Arg Asp Glu Cys Ala Thr Leu Leu Ser Asp Phe Phe
145 150 155 160
Arg Met Arg Arg Gln Glu Ile Lys Ala Leu Lys Lys Ala Asp Arg Ala
165 170 175
Glu Gly Ala Gly Pro Ala Val
180
<210> 372
<211> 164
<212> PRT
<213> 腐败希瓦氏菌
<400> 372
Met Asp Glu Tyr Trp Met Gln Val Ala Met Gln Met Ala Glu Lys Ala
1 5 10 15
Glu Ala Ala Gly Glu Val Pro Val Gly Ala Val Leu Val Lys Asp Gly
20 25 30
Gln Gln Ile Ala Thr Gly Tyr Asn Leu Ser Ile Ser Gln His Asp Pro
35 40 45
Thr Ala His Ala Glu Ile Leu Cys Leu Arg Ser Ala Gly Lys Lys Leu
50 55 60
Glu Asn Tyr Arg Leu Leu Asp Ala Thr Leu Tyr Ile Thr Leu Glu Pro
65 70 75 80
Cys Ala Met Cys Ala Gly Ala Met Val His Ser Arg Ile Ala Arg Val
85 90 95
Val Tyr Gly Ala Arg Asp Glu Lys Thr Gly Ala Ala Gly Thr Val Val
100 105 110
Asn Leu Leu Gln His Pro Ala Phe Asn His Gln Val Glu Val Thr Ser
115 120 125
Gly Val Leu Ala Glu Ala Cys Ser Ala Gln Leu Ser Arg Phe Phe Lys
130 135 140
Arg Arg Arg Asp Glu Lys Lys Ala Leu Lys Leu Ala Gln Arg Ala Gln
145 150 155 160
Gln Gly Ile Glu
<210> 373
<211> 173
<212> PRT
<213> 流感嗜血杆菌
<400> 373
Met Asp Ala Ala Lys Val Arg Ser Glu Phe Asp Glu Lys Met Met Arg
1 5 10 15
Tyr Ala Leu Glu Leu Ala Asp Lys Ala Glu Ala Leu Gly Glu Ile Pro
20 25 30
Val Gly Ala Val Leu Val Asp Asp Ala Arg Asn Ile Ile Gly Glu Gly
35 40 45
Trp Asn Leu Ser Ile Val Gln Ser Asp Pro Thr Ala His Ala Glu Ile
50 55 60
Ile Ala Leu Arg Asn Gly Ala Lys Asn Ile Gln Asn Tyr Arg Leu Leu
65 70 75 80
Asn Ser Thr Leu Tyr Val Thr Leu Glu Pro Cys Thr Met Cys Ala Gly
85 90 95
Ala Ile Leu His Ser Arg Ile Lys Arg Leu Val Phe Gly Ala Ser Asp
100 105 110
Tyr Lys Thr Gly Ala Ile Gly Ser Arg Phe His Phe Phe Asp Asp Tyr
115 120 125
Lys Met Asn His Thr Leu Glu Ile Thr Ser Gly Val Leu Ala Glu Glu
130 135 140
Cys Ser Gln Lys Leu Ser Thr Phe Phe Gln Lys Arg Arg Glu Glu Lys
145 150 155 160
Lys Ile Glu Lys Ala Leu Leu Lys Ser Leu Ser Asp Lys
165 170
<210> 374
<211> 161
<212> PRT
<213> 新月柄杆菌
<400> 374
Met Arg Thr Asp Glu Ser Glu Asp Gln Asp His Arg Met Met Arg Leu
1 5 10 15
Ala Leu Asp Ala Ala Arg Ala Ala Ala Glu Ala Gly Glu Thr Pro Val
20 25 30
Gly Ala Val Ile Leu Asp Pro Ser Thr Gly Glu Val Ile Ala Thr Ala
35 40 45
Gly Asn Gly Pro Ile Ala Ala His Asp Pro Thr Ala His Ala Glu Ile
50 55 60
Ala Ala Met Arg Ala Ala Ala Ala Lys Leu Gly Asn Tyr Arg Leu Thr
65 70 75 80
Asp Leu Thr Leu Val Val Thr Leu Glu Pro Cys Ala Met Cys Ala Gly
85 90 95
Ala Ile Ser His Ala Arg Ile Gly Arg Val Val Phe Gly Ala Asp Asp
100 105 110
Pro Lys Gly Gly Ala Val Val His Gly Pro Lys Phe Phe Ala Gln Pro
115 120 125
Thr Cys His Trp Arg Pro Glu Val Thr Gly Gly Val Leu Ala Asp Glu
130 135 140
Ser Ala Asp Leu Leu Arg Gly Phe Phe Arg Ala Arg Arg Lys Ala Lys
145 150 155 160
Ile
<210> 375
<211> 179
<212> PRT
<213> 硫还原地杆菌
<400> 375
Met Ser Ser Leu Lys Lys Thr Pro Ile Arg Asp Asp Ala Tyr Trp Met
1 5 10 15
Gly Lys Ala Ile Arg Glu Ala Ala Lys Ala Ala Ala Arg Asp Glu Val
20 25 30
Pro Ile Gly Ala Val Ile Val Arg Asp Gly Ala Val Ile Gly Arg Gly
35 40 45
His Asn Leu Arg Glu Gly Ser Asn Asp Pro Ser Ala His Ala Glu Met
50 55 60
Ile Ala Ile Arg Gln Ala Ala Arg Arg Ser Ala Asn Trp Arg Leu Thr
65 70 75 80
Gly Ala Thr Leu Tyr Val Thr Leu Glu Pro Cys Leu Met Cys Met Gly
85 90 95
Ala Ile Ile Leu Ala Arg Leu Glu Arg Val Val Phe Gly Cys Tyr Asp
100 105 110
Pro Lys Gly Gly Ala Ala Gly Ser Leu Tyr Asp Leu Ser Ala Asp Pro
115 120 125
Arg Leu Asn His Gln Val Arg Leu Ser Pro Gly Val Cys Gln Glu Glu
130 135 140
Cys Gly Thr Met Leu Ser Asp Phe Phe Arg Asp Leu Arg Arg Arg Lys
145 150 155 160
Lys Ala Lys Ala Thr Pro Ala Leu Phe Ile Asp Glu Arg Lys Val Pro
165 170 175
Pro Glu Pro
<210> 376
<400> 376
000
<210> 377
<400> 377
000
<210> 378
<400> 378
000
<210> 379
<400> 379
000
<210> 380
<400> 380
000
<210> 381
<400> 381
000
<210> 382
<400> 382
000
<210> 383
<400> 383
000
<210> 384
<211> 24
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 384
Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala
1 5 10 15
Thr Pro Glu Ser Ser Gly Gly Ser
20
<210> 385
<211> 32
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 385
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr
1 5 10 15
Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser
20 25 30
<210> 386
<211> 104
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 386
Gly Gly Ser Gly Gly Ser Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser
1 5 10 15
Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly
20 25 30
Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly
35 40 45
Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly
50 55 60
Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly
65 70 75 80
Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala
85 90 95
Thr Ser Gly Gly Ser Gly Gly Ser
100
<210> 387
<400> 387
000
<210> 388
<400> 388
000
<210> 389
<211> 82
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 389
guuuuagagc uagaaauagc aaguuaaaau aaaggcuagu ccguuaucaa cuugaaaaag 60
uggcaccgag ucggugcuuu uu 82
<210> 390
<400> 390
000
<210> 391
<400> 391
000
<210> 392
<400> 392
000
<210> 393
<400> 393
000
<210> 394
<400> 394
000
<210> 395
<400> 395
000
<210> 396
<400> 396
000
<210> 397
<400> 397
000
<210> 398
<400> 398
000
<210> 399
<400> 399
000
<210> 400
<400> 400
000
<210> 401
<400> 401
000
<210> 402
<400> 402
000
<210> 403
<400> 403
000
<210> 404
<400> 404
000
<210> 405
<400> 405
000
<210> 406
<400> 406
000
<210> 407
<400> 407
000
<210> 408
<400> 408
000
<210> 409
<400> 409
000
<210> 410
<400> 410
000
<210> 411
<400> 411
000
<210> 412
<400> 412
000
<210> 413
<400> 413
000
<210> 414
<400> 414
000
<210> 415
<400> 415
000
<210> 416
<400> 416
000
<210> 417
<211> 345
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 417
Met Glu Val Pro Leu Tyr Asn Ile Phe Gly Asp Asn Tyr Ile Ile Gln
1 5 10 15
Val Ala Thr Glu Ala Glu Asn Ser Thr Ile Tyr Asn Asn Lys Val Glu
20 25 30
Ile Asp Asp Glu Glu Leu Arg Asn Val Leu Asn Leu Ala Tyr Lys Ile
35 40 45
Ala Lys Asn Asn Glu Asp Ala Ala Ala Glu Arg Arg Gly Lys Ala Lys
50 55 60
Lys Lys Lys Gly Glu Glu Gly Glu Thr Thr Thr Ser Asn Ile Ile Leu
65 70 75 80
Pro Leu Ser Gly Asn Asp Lys Asn Pro Trp Thr Glu Thr Leu Lys Cys
85 90 95
Tyr Asn Phe Pro Thr Thr Val Ala Leu Ser Glu Val Phe Lys Asn Phe
100 105 110
Ser Gln Val Lys Glu Cys Glu Glu Val Ser Ala Pro Ser Phe Val Lys
115 120 125
Pro Glu Phe Tyr Glu Phe Gly Arg Ser Pro Gly Met Val Glu Arg Thr
130 135 140
Arg Arg Val Lys Leu Glu Val Glu Pro His Tyr Leu Ile Ile Ala Ala
145 150 155 160
Ala Gly Trp Val Leu Thr Arg Leu Gly Lys Ala Lys Val Ser Glu Gly
165 170 175
Asp Tyr Val Gly Val Asn Val Phe Thr Pro Thr Arg Gly Ile Leu Tyr
180 185 190
Ser Leu Ile Gln Asn Val Asn Gly Ile Val Pro Gly Ile Lys Pro Glu
195 200 205
Thr Ala Phe Gly Leu Trp Ile Ala Arg Lys Val Val Ser Ser Val Thr
210 215 220
Asn Pro Asn Val Ser Val Val Arg Ile Tyr Thr Ile Ser Asp Ala Val
225 230 235 240
Gly Gln Asn Pro Thr Thr Ile Asn Gly Gly Phe Ser Ile Asp Leu Thr
245 250 255
Lys Leu Leu Glu Lys Arg Tyr Leu Leu Ser Glu Arg Leu Glu Ala Ile
260 265 270
Ala Arg Asn Ala Leu Ser Ile Ser Ser Asn Met Arg Glu Arg Tyr Ile
275 280 285
Val Leu Ala Asn Tyr Ile Tyr Glu Tyr Leu Thr Gly Ser Lys Arg Leu
290 295 300
Glu Asp Leu Leu Tyr Phe Ala Asn Arg Asp Leu Ile Met Asn Leu Asn
305 310 315 320
Ser Asp Asp Gly Lys Val Arg Asp Leu Lys Leu Ile Ser Ala Tyr Val
325 330 335
Asn Gly Glu Leu Ile Arg Gly Glu Gly
340 345
<210> 418
<211> 345
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 418
Met Glu Val Pro Leu Tyr Asn Ile Phe Gly Asp Asn Tyr Ile Ile Gln
1 5 10 15
Val Ala Thr Glu Ala Glu Asn Ser Thr Ile Tyr Asn Asn Lys Val Glu
20 25 30
Ile Asp Asp Glu Glu Leu Arg Asn Val Leu Asn Leu Ala Tyr Lys Ile
35 40 45
Ala Lys Asn Asn Glu Asp Ala Ala Ala Glu Arg Arg Gly Lys Ala Lys
50 55 60
Lys Lys Lys Gly Glu Glu Gly Glu Thr Thr Thr Ser Asn Ile Ile Leu
65 70 75 80
Pro Leu Ser Gly Asn Asp Lys Asn Pro Trp Thr Glu Thr Leu Lys Cys
85 90 95
Tyr Asn Phe Pro Thr Thr Val Ala Leu Ser Glu Val Phe Lys Asn Phe
100 105 110
Ser Gln Val Lys Glu Cys Glu Glu Val Ser Ala Pro Ser Phe Val Lys
115 120 125
Pro Glu Phe Tyr Lys Phe Gly Arg Ser Pro Gly Met Val Glu Arg Thr
130 135 140
Arg Arg Val Lys Leu Glu Val Glu Pro His Tyr Leu Ile Met Ala Ala
145 150 155 160
Ala Gly Trp Val Leu Thr Arg Leu Gly Lys Ala Lys Val Ser Glu Gly
165 170 175
Asp Tyr Val Gly Val Asn Val Phe Thr Pro Thr Arg Gly Ile Leu Tyr
180 185 190
Ser Leu Ile Gln Asn Val Asn Gly Ile Val Pro Gly Ile Lys Pro Glu
195 200 205
Thr Ala Phe Gly Leu Trp Ile Ala Arg Lys Val Val Ser Ser Val Thr
210 215 220
Asn Pro Asn Val Ser Val Val Ser Ile Tyr Thr Ile Ser Asp Ala Val
225 230 235 240
Gly Gln Asn Pro Thr Thr Ile Asn Gly Gly Phe Ser Ile Asp Leu Thr
245 250 255
Lys Leu Leu Glu Lys Arg Asp Leu Leu Ser Glu Arg Leu Glu Ala Ile
260 265 270
Ala Arg Asn Ala Leu Ser Ile Ser Ser Asn Met Arg Glu Arg Tyr Ile
275 280 285
Val Leu Ala Asn Tyr Ile Tyr Glu Tyr Leu Thr Gly Ser Lys Arg Leu
290 295 300
Glu Asp Leu Leu Tyr Phe Ala Asn Arg Asp Leu Ile Met Asn Leu Asn
305 310 315 320
Ser Asp Asp Gly Lys Val Arg Asp Leu Lys Leu Ile Ser Ala Tyr Val
325 330 335
Asn Gly Glu Leu Ile Arg Gly Glu Gly
340 345
<210> 419
<211> 1210
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 419
Met Ser Lys Arg His Pro Arg Ile Ser Gly Val Lys Gly Tyr Arg Leu
1 5 10 15
His Ala Gln Arg Leu Glu Tyr Thr Gly Lys Ser Gly Ala Met Arg Thr
20 25 30
Ile Lys Tyr Pro Leu Tyr Ser Ser Pro Ser Gly Gly Arg Thr Val Pro
35 40 45
Arg Glu Ile Val Ser Ala Ile Asn Asp Asp Tyr Val Gly Leu Tyr Gly
50 55 60
Leu Ser Asn Phe Asp Asp Leu Tyr Asn Ala Glu Lys Arg Asn Glu Glu
65 70 75 80
Lys Val Tyr Ser Val Leu Asp Phe Trp Tyr Asp Cys Val Gln Tyr Gly
85 90 95
Ala Val Phe Ser Tyr Thr Ala Pro Gly Leu Leu Lys Asn Val Ala Glu
100 105 110
Val Arg Gly Gly Ser Tyr Glu Leu Thr Lys Thr Leu Lys Gly Ser His
115 120 125
Leu Tyr Asp Glu Leu Gln Ile Asp Lys Val Ile Lys Phe Leu Asn Lys
130 135 140
Lys Glu Ile Ser Arg Ala Asn Gly Ser Leu Asp Lys Leu Lys Lys Asp
145 150 155 160
Ile Ile Asp Cys Phe Lys Ala Glu Tyr Arg Glu Arg His Lys Asp Gln
165 170 175
Cys Asn Lys Leu Ala Asp Asp Ile Lys Asn Ala Lys Lys Asp Ala Gly
180 185 190
Ala Ser Leu Gly Glu Arg Gln Lys Lys Leu Phe Arg Asp Phe Phe Gly
195 200 205
Ile Ser Glu Gln Ser Glu Asn Asp Lys Pro Ser Phe Thr Asn Pro Leu
210 215 220
Asn Leu Thr Cys Cys Leu Leu Pro Phe Asp Thr Val Asn Asn Asn Arg
225 230 235 240
Asn Arg Gly Glu Val Leu Phe Asn Lys Leu Lys Glu Tyr Ala Gln Lys
245 250 255
Leu Asp Lys Asn Glu Gly Ser Leu Glu Met Trp Glu Tyr Ile Gly Ile
260 265 270
Gly Asn Ser Gly Thr Ala Phe Ser Asn Phe Leu Gly Glu Gly Phe Leu
275 280 285
Gly Arg Leu Arg Glu Asn Lys Ile Thr Glu Leu Lys Lys Ala Met Met
290 295 300
Asp Ile Thr Asp Ala Trp Arg Gly Gln Glu Gln Glu Glu Glu Leu Glu
305 310 315 320
Lys Arg Leu Arg Ile Leu Ala Ala Leu Thr Ile Lys Leu Arg Glu Pro
325 330 335
Lys Phe Asp Asn His Trp Gly Gly Tyr Arg Ser Asp Ile Asn Gly Lys
340 345 350
Leu Ser Ser Trp Leu Gln Asn Tyr Ile Asn Gln Thr Val Lys Ile Lys
355 360 365
Glu Asp Leu Lys Gly His Lys Lys Asp Leu Lys Lys Ala Lys Glu Met
370 375 380
Ile Asn Arg Phe Gly Glu Ser Asp Thr Lys Glu Glu Ala Val Val Ser
385 390 395 400
Ser Leu Leu Glu Ser Ile Glu Lys Ile Val Pro Asp Asp Ser Ala Asp
405 410 415
Asp Glu Lys Pro Asp Ile Pro Ala Ile Ala Ile Tyr Arg Arg Phe Leu
420 425 430
Ser Asp Gly Arg Leu Thr Leu Asn Arg Phe Val Gln Arg Glu Asp Val
435 440 445
Gln Glu Ala Leu Ile Lys Glu Arg Leu Glu Ala Glu Lys Lys Lys Lys
450 455 460
Pro Lys Lys Arg Lys Lys Lys Ser Asp Ala Glu Asp Glu Lys Glu Thr
465 470 475 480
Ile Asp Phe Lys Glu Leu Phe Pro His Leu Ala Lys Pro Leu Lys Leu
485 490 495
Val Pro Asn Phe Tyr Gly Asp Ser Lys Arg Glu Leu Tyr Lys Lys Tyr
500 505 510
Lys Asn Ala Ala Ile Tyr Thr Asp Ala Leu Trp Lys Ala Val Glu Lys
515 520 525
Ile Tyr Lys Ser Ala Phe Ser Ser Ser Leu Lys Asn Ser Phe Phe Asp
530 535 540
Thr Asp Phe Asp Lys Asp Phe Phe Ile Lys Arg Leu Gln Lys Ile Phe
545 550 555 560
Ser Val Tyr Arg Arg Phe Asn Thr Asp Lys Trp Lys Pro Ile Val Lys
565 570 575
Asn Ser Phe Ala Pro Tyr Cys Asp Ile Val Ser Leu Ala Glu Asn Glu
580 585 590
Val Leu Tyr Lys Pro Lys Gln Ser Arg Ser Arg Lys Ser Ala Ala Ile
595 600 605
Asp Lys Asn Arg Val Arg Leu Pro Ser Thr Glu Asn Ile Ala Lys Ala
610 615 620
Gly Ile Ala Leu Ala Arg Glu Leu Ser Val Ala Gly Phe Asp Trp Lys
625 630 635 640
Asp Leu Leu Lys Lys Glu Glu His Glu Glu Tyr Ile Asp Leu Ile Glu
645 650 655
Leu His Lys Thr Ala Leu Ala Leu Leu Leu Ala Val Thr Glu Thr Gln
660 665 670
Leu Asp Ile Ser Ala Leu Asp Phe Val Glu Asn Gly Thr Val Lys Asp
675 680 685
Phe Met Lys Thr Arg Asp Gly Asn Leu Val Leu Glu Gly Arg Phe Leu
690 695 700
Glu Met Phe Ser Gln Ser Ile Val Phe Ser Glu Leu Arg Gly Leu Ala
705 710 715 720
Gly Leu Met Ser Arg Lys Glu Phe Ile Thr Arg Ser Ala Ile Gln Thr
725 730 735
Met Asn Gly Lys Gln Ala Glu Leu Leu Tyr Ile Pro His Glu Phe Gln
740 745 750
Ser Ala Lys Ile Thr Thr Pro Lys Glu Met Ser Arg Ala Phe Leu Asp
755 760 765
Leu Ala Pro Ala Glu Phe Ala Thr Ser Leu Glu Pro Glu Ser Leu Ser
770 775 780
Glu Lys Ser Leu Leu Lys Leu Lys Gln Met Arg Tyr Tyr Pro His Tyr
785 790 795 800
Phe Gly Tyr Glu Leu Thr Arg Thr Gly Gln Gly Ile Asp Gly Gly Val
805 810 815
Ala Glu Asn Ala Leu Arg Leu Glu Lys Ser Pro Val Lys Lys Arg Glu
820 825 830
Ile Lys Cys Lys Gln Tyr Lys Thr Leu Gly Arg Gly Gln Asn Lys Ile
835 840 845
Val Leu Tyr Val Arg Ser Ser Tyr Tyr Gln Thr Gln Phe Leu Glu Trp
850 855 860
Phe Leu His Arg Pro Lys Asn Val Gln Thr Asp Val Ala Val Ser Gly
865 870 875 880
Ser Phe Leu Ile Asp Glu Lys Lys Val Lys Thr Arg Trp Asn Tyr Asp
885 890 895
Ala Leu Thr Val Ala Leu Glu Pro Val Ser Gly Ser Glu Arg Val Phe
900 905 910
Val Ser Gln Pro Phe Thr Ile Phe Pro Glu Lys Ser Ala Glu Glu Glu
915 920 925
Gly Gln Arg Tyr Leu Gly Ile Asp Ile Gly Glu Tyr Gly Ile Ala Tyr
930 935 940
Thr Ala Leu Glu Ile Thr Gly Asp Ser Ala Lys Ile Leu Asp Gln Asn
945 950 955 960
Phe Ile Ser Asp Pro Gln Leu Lys Thr Leu Arg Glu Glu Val Lys Gly
965 970 975
Leu Lys Leu Asp Gln Arg Arg Gly Thr Phe Ala Met Pro Ser Thr Lys
980 985 990
Ile Ala Arg Ile Arg Glu Ser Leu Val His Ser Leu Arg Asn Arg Ile
995 1000 1005
His His Leu Ala Leu Lys His Lys Ala Lys Ile Val Tyr Glu Leu
1010 1015 1020
Glu Val Ser Arg Phe Glu Glu Gly Lys Gln Lys Ile Lys Lys Val
1025 1030 1035
Tyr Ala Thr Leu Lys Lys Ala Asp Val Tyr Ser Glu Ile Asp Ala
1040 1045 1050
Asp Lys Asn Leu Gln Thr Thr Val Trp Gly Lys Leu Ala Val Ala
1055 1060 1065
Ser Glu Ile Ser Ala Ser Tyr Thr Ser Gln Phe Cys Gly Ala Cys
1070 1075 1080
Lys Lys Leu Trp Arg Ala Glu Met Gln Val Asp Glu Thr Ile Thr
1085 1090 1095
Thr Gln Glu Leu Ile Gly Thr Val Arg Val Ile Lys Gly Gly Thr
1100 1105 1110
Leu Ile Asp Ala Ile Lys Asp Phe Met Arg Pro Pro Ile Phe Asp
1115 1120 1125
Glu Asn Asp Thr Pro Phe Pro Lys Tyr Arg Asp Phe Cys Asp Lys
1130 1135 1140
His His Ile Ser Lys Lys Met Arg Gly Asn Ser Cys Leu Phe Ile
1145 1150 1155
Cys Pro Phe Cys Arg Ala Asn Ala Asp Ala Asp Ile Gln Ala Ser
1160 1165 1170
Gln Thr Ile Ala Leu Leu Arg Tyr Val Lys Glu Glu Lys Lys Val
1175 1180 1185
Glu Asp Tyr Phe Glu Arg Phe Arg Lys Leu Lys Asn Ile Lys Val
1190 1195 1200
Leu Gly Gln Met Lys Lys Ile
1205 1210
<210> 420
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 420
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Ala Gly Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Asn Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Glu Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 421
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 421
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 422
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 422
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Thr Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Thr Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 423
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 423
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Ser Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 424
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 424
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 425
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 425
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Arg Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 426
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 426
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Leu Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 427
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 427
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 428
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 428
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 429
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 429
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Arg Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 430
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 430
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Ser Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly His His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 431
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 431
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 432
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 432
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly His His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 433
<400> 433
000
<210> 434
<400> 434
000
<210> 435
<400> 435
000
<210> 436
<400> 436
000
<210> 437
<400> 437
000
<210> 438
<400> 438
000
<210> 439
<400> 439
000
<210> 440
<400> 440
000
<210> 441
<400> 441
000
<210> 442
<400> 442
000
<210> 443
<400> 443
000
<210> 444
<400> 444
000
<210> 445
<400> 445
000
<210> 446
<400> 446
000
<210> 447
<400> 447
000
<210> 448
<400> 448
000
<210> 449
<400> 449
000
<210> 450
<400> 450
000
<210> 451
<400> 451
000
<210> 452
<400> 452
000
<210> 453
<400> 453
000
<210> 454
<400> 454
000
<210> 455
<400> 455
000
<210> 456
<400> 456
000
<210> 457
<400> 457
000
<210> 458
<400> 458
000
<210> 459
<400> 459
000
<210> 460
<400> 460
000
<210> 461
<400> 461
000
<210> 462
<400> 462
000
<210> 463
<400> 463
000
<210> 464
<400> 464
000
<210> 465
<400> 465
000
<210> 466
<400> 466
000
<210> 467
<400> 467
000
<210> 468
<400> 468
000
<210> 469
<400> 469
000
<210> 470
<400> 470
000
<210> 471
<400> 471
000
<210> 472
<400> 472
000
<210> 473
<400> 473
000
<210> 474
<400> 474
000
<210> 475
<400> 475
000
<210> 476
<400> 476
000
<210> 477
<400> 477
000
<210> 478
<400> 478
000
<210> 479
<400> 479
000
<210> 480
<400> 480
000
<210> 481
<400> 481
000
<210> 482
<400> 482
000
<210> 483
<400> 483
000
<210> 484
<400> 484
000
<210> 485
<400> 485
000
<210> 486
<400> 486
000
<210> 487
<400> 487
000
<210> 488
<400> 488
000
<210> 489
<400> 489
000
<210> 490
<400> 490
000
<210> 491
<400> 491
000
<210> 492
<400> 492
000
<210> 493
<400> 493
000
<210> 494
<400> 494
000
<210> 495
<400> 495
000
<210> 496
<400> 496
000
<210> 497
<400> 497
000
<210> 498
<400> 498
000
<210> 499
<400> 499
000
<210> 500
<400> 500
000
<210> 501
<400> 501
000
<210> 502
<400> 502
000
<210> 503
<400> 503
000
<210> 504
<400> 504
000
<210> 505
<400> 505
000
<210> 506
<400> 506
000
<210> 507
<400> 507
000
<210> 508
<400> 508
000
<210> 509
<400> 509
000
<210> 510
<400> 510
000
<210> 511
<400> 511
000
<210> 512
<400> 512
000
<210> 513
<400> 513
000
<210> 514
<400> 514
000
<210> 515
<400> 515
000
<210> 516
<400> 516
000
<210> 517
<400> 517
000
<210> 518
<400> 518
000
<210> 519
<400> 519
000
<210> 520
<400> 520
000
<210> 521
<400> 521
000
<210> 522
<400> 522
000
<210> 523
<400> 523
000
<210> 524
<400> 524
000
<210> 525
<400> 525
000
<210> 526
<400> 526
000
<210> 527
<400> 527
000
<210> 528
<400> 528
000
<210> 529
<400> 529
000
<210> 530
<400> 530
000
<210> 531
<400> 531
000
<210> 532
<400> 532
000
<210> 533
<400> 533
000
<210> 534
<400> 534
000
<210> 535
<400> 535
000
<210> 536
<400> 536
000
<210> 537
<400> 537
000
<210> 538
<400> 538
000
<210> 539
<400> 539
000
<210> 540
<400> 540
000
<210> 541
<400> 541
000
<210> 542
<400> 542
000
<210> 543
<400> 543
000
<210> 544
<400> 544
000
<210> 545
<400> 545
000
<210> 546
<400> 546
000
<210> 547
<400> 547
000
<210> 548
<400> 548
000
<210> 549
<400> 549
000
<210> 550
<400> 550
000
<210> 551
<400> 551
000
<210> 552
<400> 552
000
<210> 553
<400> 553
000
<210> 554
<400> 554
000
<210> 555
<400> 555
000
<210> 556
<400> 556
000
<210> 557
<400> 557
000
<210> 558
<400> 558
000
<210> 559
<400> 559
000
<210> 560
<400> 560
000
<210> 561
<400> 561
000
<210> 562
<400> 562
000
<210> 563
<400> 563
000
<210> 564
<400> 564
000
<210> 565
<400> 565
000
<210> 566
<400> 566
000
<210> 567
<400> 567
000
<210> 568
<400> 568
000
<210> 569
<400> 569
000
<210> 570
<400> 570
000
<210> 571
<400> 571
000
<210> 572
<400> 572
000
<210> 573
<400> 573
000
<210> 574
<400> 574
000
<210> 575
<400> 575
000
<210> 576
<400> 576
000
<210> 577
<400> 577
000
<210> 578
<400> 578
000
<210> 579
<400> 579
000
<210> 580
<400> 580
000
<210> 581
<400> 581
000
<210> 582
<400> 582
000
<210> 583
<400> 583
000
<210> 584
<400> 584
000
<210> 585
<400> 585
000
<210> 586
<400> 586
000
<210> 587
<400> 587
000
<210> 588
<400> 588
000
<210> 589
<400> 589
000
<210> 590
<400> 590
000
<210> 591
<400> 591
000
<210> 592
<400> 592
000
<210> 593
<400> 593
000
<210> 594
<400> 594
000
<210> 595
<400> 595
000
<210> 596
<400> 596
000
<210> 597
<400> 597
000
<210> 598
<400> 598
000
<210> 599
<400> 599
000
<210> 600
<400> 600
000
<210> 601
<400> 601
000
<210> 602
<400> 602
000
<210> 603
<400> 603
000
<210> 604
<400> 604
000
<210> 605
<400> 605
000
<210> 606
<400> 606
000
<210> 607
<400> 607
000
<210> 608
<400> 608
000
<210> 609
<400> 609
000
<210> 610
<400> 610
000
<210> 611
<400> 611
000
<210> 612
<400> 612
000
<210> 613
<400> 613
000
<210> 614
<400> 614
000
<210> 615
<400> 615
000
<210> 616
<400> 616
000
<210> 617
<400> 617
000
<210> 618
<400> 618
000
<210> 619
<400> 619
000
<210> 620
<400> 620
000
<210> 621
<400> 621
000
<210> 622
<400> 622
000
<210> 623
<400> 623
000
<210> 624
<400> 624
000
<210> 625
<400> 625
000
<210> 626
<400> 626
000
<210> 627
<400> 627
000
<210> 628
<400> 628
000
<210> 629
<400> 629
000
<210> 630
<400> 630
000
<210> 631
<400> 631
000
<210> 632
<400> 632
000
<210> 633
<400> 633
000
<210> 634
<400> 634
000
<210> 635
<400> 635
000
<210> 636
<400> 636
000
<210> 637
<400> 637
000
<210> 638
<400> 638
000
<210> 639
<400> 639
000
<210> 640
<400> 640
000
<210> 641
<400> 641
000
<210> 642
<400> 642
000
<210> 643
<400> 643
000
<210> 644
<400> 644
000
<210> 645
<400> 645
000
<210> 646
<400> 646
000
<210> 647
<400> 647
000
<210> 648
<400> 648
000
<210> 649
<400> 649
000
<210> 650
<400> 650
000
<210> 651
<400> 651
000
<210> 652
<400> 652
000
<210> 653
<400> 653
000
<210> 654
<400> 654
000
<210> 655
<400> 655
000
<210> 656
<400> 656
000
<210> 657
<400> 657
000
<210> 658
<400> 658
000
<210> 659
<400> 659
000
<210> 660
<400> 660
000
<210> 661
<400> 661
000
<210> 662
<400> 662
000
<210> 663
<400> 663
000
<210> 664
<400> 664
000
<210> 665
<400> 665
000
<210> 666
<400> 666
000
<210> 667
<400> 667
000
<210> 668
<400> 668
000
<210> 669
<400> 669
000
<210> 670
<400> 670
000
<210> 671
<400> 671
000
<210> 672
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 672
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ser Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 673
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 673
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Thr Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 674
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 674
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 675
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 675
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 676
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 676
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 677
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 677
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Leu Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 678
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 678
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Pro Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 679
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 679
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met His Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 680
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 680
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 681
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 681
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 682
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 682
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ser Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 683
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 683
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 684
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 684
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Leu Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 685
<211> 24
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 685
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr
1 5 10 15
Ser Glu Ser Ala Thr Pro Glu Ser
20
<210> 686
<211> 40
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 686
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr
1 5 10 15
Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser
20 25 30
Ser Gly Gly Ser Ser Gly Gly Ser
35 40
<210> 687
<211> 64
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 687
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr
1 5 10 15
Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser
20 25 30
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr
35 40 45
Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser
50 55 60
<210> 688
<211> 92
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 688
Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser
1 5 10 15
Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser
20 25 30
Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu
35 40 45
Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser
50 55 60
Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr
65 70 75 80
Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser
85 90
<210> 689
<400> 689
000
<210> 690
<400> 690
000
<210> 691
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 691
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Leu Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 692
<211> 1767
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 692
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly
385 390 395 400
Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro
405 410 415
Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys
420 425 430
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu
435 440 445
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
450 455 460
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys
465 470 475 480
Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
485 490 495
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp
500 505 510
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys
515 520 525
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu
530 535 540
Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly
545 550 555 560
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu
565 570 575
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
580 585 590
Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg
595 600 605
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly
610 615 620
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe
625 630 635 640
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys
645 650 655
Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp
660 665 670
Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile
675 680 685
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
690 695 700
Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu
705 710 715 720
Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
725 730 735
Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
740 745 750
Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu
755 760 765
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu
770 775 780
Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His
785 790 795 800
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp
805 810 815
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
820 825 830
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser
835 840 845
Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp
850 855 860
Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile
865 870 875 880
Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu
885 890 895
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu
900 905 910
Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu
915 920 925
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn
930 935 940
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile
945 950 955 960
Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
965 970 975
Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys
980 985 990
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val
995 1000 1005
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg
1010 1015 1020
Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
1025 1030 1035
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys
1040 1045 1050
Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
1055 1060 1065
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln
1070 1075 1080
Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys
1085 1090 1095
Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala
1100 1105 1110
Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr
1115 1120 1125
Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys
1130 1135 1140
Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr
1145 1150 1155
Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
1160 1165 1170
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
1175 1180 1185
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr
1190 1195 1200
Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile
1205 1210 1215
Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser
1220 1225 1230
Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser
1235 1240 1245
Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val
1250 1255 1260
Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys
1265 1270 1275
Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg
1280 1285 1290
Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln
1295 1300 1305
Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu
1310 1315 1320
Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile
1325 1330 1335
Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp
1340 1345 1350
Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn
1355 1360 1365
Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr
1370 1375 1380
Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr
1385 1390 1395
Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1400 1405 1410
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1415 1420 1425
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly
1430 1435 1440
Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly
1445 1450 1455
Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
1460 1465 1470
Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val
1475 1480 1485
Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn
1490 1495 1500
Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys
1505 1510 1515
Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val
1520 1525 1530
Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val
1535 1540 1545
Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu
1550 1555 1560
Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val
1565 1570 1575
Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu
1580 1585 1590
Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu
1595 1600 1605
Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe
1610 1615 1620
Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu
1625 1630 1635
Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
1640 1645 1650
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1655 1660 1665
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn
1670 1675 1680
Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile
1685 1690 1695
His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys
1700 1705 1710
Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys
1715 1720 1725
Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu
1730 1735 1740
Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly
1745 1750 1755
Gly Ser Pro Lys Lys Lys Arg Lys Val
1760 1765
<210> 693
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 693
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val His Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Pro Ile Gly Arg His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Ala Ala Leu Leu Ser Tyr Phe Phe Arg Met Arg Arg Gln
340 345 350
Val Phe Lys Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 694
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 694
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Pro Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Arg Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 695
<211> 1767
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 695
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Pro Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Arg Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly
385 390 395 400
Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro
405 410 415
Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys
420 425 430
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu
435 440 445
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
450 455 460
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys
465 470 475 480
Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
485 490 495
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp
500 505 510
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys
515 520 525
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu
530 535 540
Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly
545 550 555 560
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu
565 570 575
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
580 585 590
Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg
595 600 605
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly
610 615 620
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe
625 630 635 640
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys
645 650 655
Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp
660 665 670
Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile
675 680 685
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
690 695 700
Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu
705 710 715 720
Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
725 730 735
Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
740 745 750
Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu
755 760 765
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu
770 775 780
Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His
785 790 795 800
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp
805 810 815
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
820 825 830
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser
835 840 845
Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp
850 855 860
Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile
865 870 875 880
Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu
885 890 895
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu
900 905 910
Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu
915 920 925
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn
930 935 940
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile
945 950 955 960
Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
965 970 975
Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys
980 985 990
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val
995 1000 1005
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg
1010 1015 1020
Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
1025 1030 1035
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys
1040 1045 1050
Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
1055 1060 1065
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln
1070 1075 1080
Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys
1085 1090 1095
Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala
1100 1105 1110
Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr
1115 1120 1125
Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys
1130 1135 1140
Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr
1145 1150 1155
Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
1160 1165 1170
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
1175 1180 1185
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr
1190 1195 1200
Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile
1205 1210 1215
Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser
1220 1225 1230
Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser
1235 1240 1245
Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val
1250 1255 1260
Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys
1265 1270 1275
Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg
1280 1285 1290
Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln
1295 1300 1305
Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu
1310 1315 1320
Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile
1325 1330 1335
Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp
1340 1345 1350
Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn
1355 1360 1365
Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr
1370 1375 1380
Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr
1385 1390 1395
Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1400 1405 1410
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1415 1420 1425
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly
1430 1435 1440
Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly
1445 1450 1455
Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
1460 1465 1470
Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val
1475 1480 1485
Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn
1490 1495 1500
Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys
1505 1510 1515
Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val
1520 1525 1530
Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val
1535 1540 1545
Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu
1550 1555 1560
Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val
1565 1570 1575
Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu
1580 1585 1590
Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu
1595 1600 1605
Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe
1610 1615 1620
Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu
1625 1630 1635
Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
1640 1645 1650
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1655 1660 1665
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn
1670 1675 1680
Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile
1685 1690 1695
His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys
1700 1705 1710
Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys
1715 1720 1725
Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu
1730 1735 1740
Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly
1745 1750 1755
Gly Ser Pro Lys Lys Lys Arg Lys Val
1760 1765
<210> 696
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 696
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ser Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Arg Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 697
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 697
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ser Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Asn Ala Leu Leu Cys Tyr Phe Phe Arg Met Arg Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 698
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 698
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Arg Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 699
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 699
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Asn Ala Leu Leu Cys Tyr Phe Phe Arg Met Arg Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 700
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 700
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 701
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 701
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Leu Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Asn Ala Leu Leu Cys Tyr Phe Phe Arg Met Arg Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 702
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 702
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Leu Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Asn Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 703
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 703
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 704
<211> 1767
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 704
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly
385 390 395 400
Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro
405 410 415
Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys
420 425 430
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu
435 440 445
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
450 455 460
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys
465 470 475 480
Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
485 490 495
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp
500 505 510
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys
515 520 525
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu
530 535 540
Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly
545 550 555 560
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu
565 570 575
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
580 585 590
Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg
595 600 605
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly
610 615 620
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe
625 630 635 640
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys
645 650 655
Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp
660 665 670
Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile
675 680 685
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
690 695 700
Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu
705 710 715 720
Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
725 730 735
Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
740 745 750
Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu
755 760 765
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu
770 775 780
Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His
785 790 795 800
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp
805 810 815
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
820 825 830
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser
835 840 845
Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp
850 855 860
Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile
865 870 875 880
Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu
885 890 895
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu
900 905 910
Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu
915 920 925
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn
930 935 940
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile
945 950 955 960
Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
965 970 975
Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys
980 985 990
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val
995 1000 1005
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg
1010 1015 1020
Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
1025 1030 1035
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys
1040 1045 1050
Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
1055 1060 1065
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln
1070 1075 1080
Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys
1085 1090 1095
Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala
1100 1105 1110
Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr
1115 1120 1125
Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys
1130 1135 1140
Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr
1145 1150 1155
Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
1160 1165 1170
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
1175 1180 1185
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr
1190 1195 1200
Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile
1205 1210 1215
Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser
1220 1225 1230
Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser
1235 1240 1245
Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val
1250 1255 1260
Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys
1265 1270 1275
Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg
1280 1285 1290
Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln
1295 1300 1305
Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu
1310 1315 1320
Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile
1325 1330 1335
Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp
1340 1345 1350
Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn
1355 1360 1365
Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr
1370 1375 1380
Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr
1385 1390 1395
Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1400 1405 1410
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1415 1420 1425
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly
1430 1435 1440
Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly
1445 1450 1455
Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
1460 1465 1470
Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val
1475 1480 1485
Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn
1490 1495 1500
Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys
1505 1510 1515
Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val
1520 1525 1530
Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val
1535 1540 1545
Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu
1550 1555 1560
Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val
1565 1570 1575
Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu
1580 1585 1590
Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu
1595 1600 1605
Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe
1610 1615 1620
Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu
1625 1630 1635
Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
1640 1645 1650
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1655 1660 1665
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn
1670 1675 1680
Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile
1685 1690 1695
His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys
1700 1705 1710
Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys
1715 1720 1725
Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu
1730 1735 1740
Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly
1745 1750 1755
Gly Ser Pro Lys Lys Lys Arg Lys Val
1760 1765
<210> 705
<211> 1767
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 705
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Asn Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly
385 390 395 400
Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro
405 410 415
Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys
420 425 430
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu
435 440 445
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
450 455 460
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys
465 470 475 480
Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
485 490 495
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp
500 505 510
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys
515 520 525
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu
530 535 540
Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly
545 550 555 560
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu
565 570 575
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
580 585 590
Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg
595 600 605
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly
610 615 620
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe
625 630 635 640
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys
645 650 655
Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp
660 665 670
Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile
675 680 685
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
690 695 700
Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu
705 710 715 720
Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
725 730 735
Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
740 745 750
Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu
755 760 765
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu
770 775 780
Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His
785 790 795 800
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp
805 810 815
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
820 825 830
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser
835 840 845
Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp
850 855 860
Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile
865 870 875 880
Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu
885 890 895
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu
900 905 910
Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu
915 920 925
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn
930 935 940
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile
945 950 955 960
Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
965 970 975
Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys
980 985 990
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val
995 1000 1005
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg
1010 1015 1020
Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
1025 1030 1035
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys
1040 1045 1050
Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
1055 1060 1065
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln
1070 1075 1080
Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys
1085 1090 1095
Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala
1100 1105 1110
Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr
1115 1120 1125
Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys
1130 1135 1140
Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr
1145 1150 1155
Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
1160 1165 1170
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
1175 1180 1185
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr
1190 1195 1200
Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile
1205 1210 1215
Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser
1220 1225 1230
Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser
1235 1240 1245
Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val
1250 1255 1260
Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys
1265 1270 1275
Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg
1280 1285 1290
Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln
1295 1300 1305
Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu
1310 1315 1320
Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile
1325 1330 1335
Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp
1340 1345 1350
Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn
1355 1360 1365
Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr
1370 1375 1380
Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr
1385 1390 1395
Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1400 1405 1410
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1415 1420 1425
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly
1430 1435 1440
Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly
1445 1450 1455
Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
1460 1465 1470
Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val
1475 1480 1485
Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn
1490 1495 1500
Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys
1505 1510 1515
Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val
1520 1525 1530
Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val
1535 1540 1545
Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu
1550 1555 1560
Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val
1565 1570 1575
Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu
1580 1585 1590
Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu
1595 1600 1605
Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe
1610 1615 1620
Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu
1625 1630 1635
Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
1640 1645 1650
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1655 1660 1665
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn
1670 1675 1680
Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile
1685 1690 1695
His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys
1700 1705 1710
Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys
1715 1720 1725
Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu
1730 1735 1740
Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly
1745 1750 1755
Gly Ser Pro Lys Lys Lys Arg Lys Val
1760 1765
<210> 706
<211> 1767
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 706
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Leu Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly
385 390 395 400
Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro
405 410 415
Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys
420 425 430
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu
435 440 445
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
450 455 460
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys
465 470 475 480
Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
485 490 495
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp
500 505 510
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys
515 520 525
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu
530 535 540
Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly
545 550 555 560
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu
565 570 575
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
580 585 590
Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg
595 600 605
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly
610 615 620
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe
625 630 635 640
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys
645 650 655
Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp
660 665 670
Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile
675 680 685
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
690 695 700
Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu
705 710 715 720
Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
725 730 735
Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
740 745 750
Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu
755 760 765
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu
770 775 780
Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His
785 790 795 800
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp
805 810 815
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
820 825 830
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser
835 840 845
Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp
850 855 860
Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile
865 870 875 880
Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu
885 890 895
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu
900 905 910
Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu
915 920 925
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn
930 935 940
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile
945 950 955 960
Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
965 970 975
Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys
980 985 990
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val
995 1000 1005
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg
1010 1015 1020
Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
1025 1030 1035
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys
1040 1045 1050
Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
1055 1060 1065
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln
1070 1075 1080
Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys
1085 1090 1095
Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala
1100 1105 1110
Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr
1115 1120 1125
Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys
1130 1135 1140
Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr
1145 1150 1155
Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
1160 1165 1170
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
1175 1180 1185
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr
1190 1195 1200
Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile
1205 1210 1215
Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser
1220 1225 1230
Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser
1235 1240 1245
Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val
1250 1255 1260
Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys
1265 1270 1275
Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg
1280 1285 1290
Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln
1295 1300 1305
Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu
1310 1315 1320
Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile
1325 1330 1335
Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp
1340 1345 1350
Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn
1355 1360 1365
Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr
1370 1375 1380
Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr
1385 1390 1395
Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1400 1405 1410
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1415 1420 1425
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly
1430 1435 1440
Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly
1445 1450 1455
Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
1460 1465 1470
Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val
1475 1480 1485
Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn
1490 1495 1500
Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys
1505 1510 1515
Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val
1520 1525 1530
Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val
1535 1540 1545
Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu
1550 1555 1560
Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val
1565 1570 1575
Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu
1580 1585 1590
Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu
1595 1600 1605
Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe
1610 1615 1620
Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu
1625 1630 1635
Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
1640 1645 1650
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1655 1660 1665
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn
1670 1675 1680
Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile
1685 1690 1695
His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys
1700 1705 1710
Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys
1715 1720 1725
Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu
1730 1735 1740
Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly
1745 1750 1755
Gly Ser Pro Lys Lys Lys Arg Lys Val
1760 1765
<210> 707
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 707
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ser Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Arg Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 708
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 708
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ser Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Asn Ala Leu Leu Cys Tyr Phe Phe Arg Met Arg Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 709
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 709
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Leu Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Asn Ala Leu Leu Cys Tyr Phe Phe Arg Met Arg Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 710
<211> 1773
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 710
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Leu Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Gly Glu Gly
225 230 235 240
Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile
245 250 255
Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile
260 265 270
Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly
275 280 285
Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn
290 295 300
Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro
305 310 315 320
Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu
325 330 335
Cys Asn Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe
340 345 350
Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser
355 360 365
Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr
370 375 380
Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser
385 390 395 400
Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr
405 410 415
Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr
420 425 430
Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp
435 440 445
Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg
450 455 460
Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe
465 470 475 480
Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu
485 490 495
Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile
500 505 510
Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr
515 520 525
Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp
530 535 540
Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly
545 550 555 560
His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp
565 570 575
Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu
580 585 590
Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala
595 600 605
Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro
610 615 620
Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu
625 630 635 640
Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala
645 650 655
Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu
660 665 670
Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys
675 680 685
Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr
690 695 700
Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp
705 710 715 720
Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln
725 730 735
Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly
740 745 750
Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys
755 760 765
Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu
770 775 780
Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp
785 790 795 800
Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile
805 810 815
Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu
820 825 830
Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro
835 840 845
Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu
850 855 860
Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala
865 870 875 880
Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu
885 890 895
Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe
900 905 910
Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met
915 920 925
Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp
930 935 940
Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu
945 950 955 960
Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly
965 970 975
Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu
980 985 990
Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp
995 1000 1005
Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg
1010 1015 1020
Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp
1025 1030 1035
Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp
1040 1045 1050
Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln
1055 1060 1065
Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
1070 1075 1080
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
1085 1090 1095
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser
1100 1105 1110
Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys
1115 1120 1125
Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys
1130 1135 1140
Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala
1145 1150 1155
Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu
1160 1165 1170
Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln
1175 1180 1185
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
1190 1195 1200
Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val
1205 1210 1215
Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp
1220 1225 1230
His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn
1235 1240 1245
Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn
1250 1255 1260
Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg
1265 1270 1275
Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn
1280 1285 1290
Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala
1295 1300 1305
Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
1310 1315 1320
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
1325 1330 1335
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys
1340 1345 1350
Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys
1355 1360 1365
Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu
1370 1375 1380
Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu
1385 1390 1395
Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg
1400 1405 1410
Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala
1415 1420 1425
Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu
1430 1435 1440
Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu
1445 1450 1455
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp
1460 1465 1470
Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile
1475 1480 1485
Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser
1490 1495 1500
Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys
1505 1510 1515
Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val
1520 1525 1530
Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser
1535 1540 1545
Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met
1550 1555 1560
Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1565 1570 1575
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro
1580 1585 1590
Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu
1595 1600 1605
Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro
1610 1615 1620
Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys
1625 1630 1635
Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val
1640 1645 1650
Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser
1655 1660 1665
Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys
1670 1675 1680
Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu
1685 1690 1695
Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly
1700 1705 1710
Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys
1715 1720 1725
Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His
1730 1735 1740
Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln
1745 1750 1755
Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1760 1765 1770
<210> 711
<211> 1775
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 711
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
165 170 175
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
180 185 190
Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr
195 200 205
Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg
210 215 220
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
225 230 235 240
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala
245 250 255
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
260 265 270
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys
275 280 285
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val
290 295 300
Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His
305 310 315 320
Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala
325 330 335
Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln
340 345 350
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly
355 360 365
Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
370 375 380
Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys
385 390 395 400
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
405 410 415
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
420 425 430
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
435 440 445
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
450 455 460
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
465 470 475 480
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
485 490 495
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
500 505 510
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
515 520 525
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
530 535 540
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
545 550 555 560
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
565 570 575
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
580 585 590
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
595 600 605
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
610 615 620
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
625 630 635 640
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
645 650 655
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
660 665 670
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
675 680 685
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
690 695 700
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
705 710 715 720
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
725 730 735
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
740 745 750
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
755 760 765
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
770 775 780
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
785 790 795 800
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
805 810 815
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
820 825 830
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
835 840 845
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
850 855 860
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
865 870 875 880
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
885 890 895
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
900 905 910
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
915 920 925
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
930 935 940
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
945 950 955 960
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
965 970 975
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
980 985 990
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
995 1000 1005
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1010 1015 1020
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1025 1030 1035
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1040 1045 1050
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1055 1060 1065
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1070 1075 1080
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1085 1090 1095
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1100 1105 1110
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1115 1120 1125
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1130 1135 1140
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1145 1150 1155
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1160 1165 1170
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1175 1180 1185
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1190 1195 1200
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1205 1210 1215
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1220 1225 1230
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1235 1240 1245
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1250 1255 1260
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1265 1270 1275
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1280 1285 1290
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1295 1300 1305
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1310 1315 1320
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1325 1330 1335
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1340 1345 1350
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1355 1360 1365
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1370 1375 1380
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1385 1390 1395
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1400 1405 1410
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1415 1420 1425
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1430 1435 1440
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1445 1450 1455
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1460 1465 1470
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1475 1480 1485
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1490 1495 1500
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1505 1510 1515
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1520 1525 1530
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1535 1540 1545
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1550 1555 1560
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1565 1570 1575
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1580 1585 1590
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1595 1600 1605
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1610 1615 1620
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1625 1630 1635
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1640 1645 1650
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1655 1660 1665
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1670 1675 1680
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1685 1690 1695
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1700 1705 1710
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1715 1720 1725
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1730 1735 1740
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1745 1750 1755
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Pro Lys Lys Lys Arg
1760 1765 1770
Lys Val
1775
<210> 712
<400> 712
000
<210> 713
<400> 713
000
<210> 714
<400> 714
000
<210> 715
<400> 715
000
<210> 716
<400> 716
000
<210> 717
<400> 717
000
<210> 718
<400> 718
000
<210> 719
<400> 719
000
<210> 720
<400> 720
000
<210> 721
<400> 721
000
<210> 722
<400> 722
000
<210> 723
<400> 723
000
<210> 724
<400> 724
000
<210> 725
<400> 725
000
<210> 726
<400> 726
000
<210> 727
<400> 727
000
<210> 728
<400> 728
000
<210> 729
<400> 729
000
<210> 730
<400> 730
000
<210> 731
<400> 731
000
<210> 732
<400> 732
000
<210> 733
<400> 733
000
<210> 734
<400> 734
000
<210> 735
<400> 735
000
<210> 736
<400> 736
000
<210> 737
<400> 737
000
<210> 738
<400> 738
000
<210> 739
<400> 739
000
<210> 740
<400> 740
000
<210> 741
<400> 741
000
<210> 742
<400> 742
000
<210> 743
<400> 743
000
<210> 744
<400> 744
000
<210> 745
<400> 745
000
<210> 746
<400> 746
000
<210> 747
<400> 747
000
<210> 748
<400> 748
000
<210> 749
<400> 749
000
<210> 750
<211> 347
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 750
Met Gly Pro Arg Ala Arg Pro Ala Leu Leu Leu Leu Met Leu Leu Gln
1 5 10 15
Thr Ala Val Leu Gln Gly Arg Leu Leu Arg Ser His Ser Leu His Tyr
20 25 30
Leu Phe Met Gly Ala Ser Glu Gln Asp Leu Gly Leu Ser Leu Phe Glu
35 40 45
Ala Leu Gly Tyr Val Asp Asp Gln Leu Phe Val Phe Tyr Asp His Glu
50 55 60
Ser Arg Arg Val Glu Pro Arg Thr Pro Trp Val Ser Ser Arg Ile Ser
65 70 75 80
Ser Gln Met Trp Leu Gln Leu Ser Gln Ser Leu Lys Gly Trp Asp His
85 90 95
Met Phe Thr Val Asp Phe Trp Thr Ile Met Glu Asn His Asn His Ser
100 105 110
Lys Glu Ser His Thr Leu Gln Val Ile Leu Gly Cys Glu Met Gln Glu
115 120 125
Asp Asn Ser Thr Glu Gly Tyr Trp Lys Tyr Gly Tyr Asp Gly Gln Asp
130 135 140
His Leu Glu Phe Cys Pro Asp Thr Leu Asp Trp Arg Ala Ala Glu Pro
145 150 155 160
Arg Ala Trp Pro Thr Lys Leu Glu Trp Glu Arg His Lys Ile Arg Ala
165 170 175
Arg Gln Asn Arg Ala Tyr Leu Glu Arg Asp Cys Pro Ala Gln Leu Gln
180 185 190
Gln Leu Leu Glu Leu Gly Arg Gly Val Leu Asp Gln Gln Val Pro Pro
195 200 205
Leu Val Lys Val Thr His His Val Thr Ser Ser Val Thr Thr Leu Arg
210 215 220
Cys Arg Ala Leu Asn Tyr Tyr Pro Gln Asn Ile Thr Met Lys Trp Leu
225 230 235 240
Lys Asp Lys Gln Pro Met Asp Ala Lys Glu Phe Glu Pro Lys Asp Val
245 250 255
Leu Pro Asn Gly Asp Gly Thr Tyr Gln Gly Trp Ile Thr Leu Ala Val
260 265 270
Pro Pro Gly Glu Glu Gln Arg Tyr Thr Cys Gln Val Glu His Pro Gly
275 280 285
Leu Asp Gln Pro Leu Ile Val Ile Trp Glu Pro Ser Pro Ser Gly Thr
290 295 300
Leu Val Ile Gly Val Ile Ser Gly Ile Ala Val Phe Val Val Ile Leu
305 310 315 320
Phe Ile Gly Ile Leu Phe Ile Ile Leu Arg Lys Arg Gln Gly Ser Arg
325 330 335
Gly Ala Met Gly His Tyr Val Leu Ala Glu Arg
340 345
<210> 751
<400> 751
000
<210> 752
<400> 752
000
<210> 753
<400> 753
000
<210> 754
<400> 754
000
<210> 755
<400> 755
000
<210> 756
<400> 756
000
<210> 757
<400> 757
000
<210> 758
<400> 758
000
<210> 759
<400> 759
000
<210> 760
<400> 760
000
<210> 761
<400> 761
000
<210> 762
<400> 762
000
<210> 763
<400> 763
000
<210> 764
<400> 764
000
<210> 765
<400> 765
000
<210> 766
<400> 766
000
<210> 767
<400> 767
000
<210> 768
<400> 768
000
<210> 769
<400> 769
000
<210> 770
<400> 770
000
<210> 771
<400> 771
000
<210> 772
<400> 772
000
<210> 773
<400> 773
000
<210> 774
<400> 774
000
<210> 775
<400> 775
000
<210> 776
<400> 776
000
<210> 777
<400> 777
000
<210> 778
<400> 778
000
<210> 779
<400> 779
000
<210> 780
<400> 780
000
<210> 781
<400> 781
000
<210> 782
<400> 782
000
<210> 783
<400> 783
000
<210> 784
<400> 784
000
<210> 785
<400> 785
000
<210> 786
<400> 786
000
<210> 787
<400> 787
000
<210> 788
<400> 788
000
<210> 789
<400> 789
000
<210> 790
<400> 790
000
<210> 791
<400> 791
000
<210> 792
<400> 792
000
<210> 793
<400> 793
000
<210> 794
<400> 794
000
<210> 795
<400> 795
000
<210> 796
<400> 796
000
<210> 797
<400> 797
000
<210> 798
<400> 798
000
<210> 799
<400> 799
000
<210> 800
<211> 24
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 800
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr
1 5 10 15
Ser Glu Ser Ala Thr Pro Glu Ser
20
<210> 801
<211> 256
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<220>
<221> MISC_FEATURE
<222> (5)..(120)
<223> 可以不存在
<220>
<221> MISC_FEATURE
<222> (137)..(256)
<223> 可以不存在
<400> 801
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
1 5 10 15
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
20 25 30
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
35 40 45
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
50 55 60
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
65 70 75 80
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
85 90 95
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
100 105 110
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr
115 120 125
Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser
130 135 140
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
145 150 155 160
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
165 170 175
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
180 185 190
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
195 200 205
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
210 215 220
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
225 230 235 240
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser
245 250 255
<210> 802
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 802
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ser Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 803
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 803
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Leu Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 804
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 804
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Leu Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 805
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 805
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Leu Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 806
<400> 806
000
<210> 807
<400> 807
000
<210> 808
<400> 808
000
<210> 809
<400> 809
000
<210> 810
<400> 810
000
<210> 811
<400> 811
000
<210> 812
<400> 812
000
<210> 813
<400> 813
000
<210> 814
<400> 814
000
<210> 815
<400> 815
000
<210> 816
<400> 816
000
<210> 817
<400> 817
000
<210> 818
<400> 818
000
<210> 819
<400> 819
000
<210> 820
<400> 820
000
<210> 821
<400> 821
000
<210> 822
<400> 822
000
<210> 823
<400> 823
000
<210> 824
<400> 824
000
<210> 825
<400> 825
000
<210> 826
<400> 826
000
<210> 827
<400> 827
000
<210> 828
<400> 828
000
<210> 829
<400> 829
000
<210> 830
<400> 830
000
<210> 831
<400> 831
000
<210> 832
<400> 832
000
<210> 833
<400> 833
000
<210> 834
<400> 834
000
<210> 835
<400> 835
000
<210> 836
<400> 836
000
<210> 837
<400> 837
000
<210> 838
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 838
cttgggggcc ccttccccac acta 24
<210> 839
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 839
cttgggggcc ccttccccac act 23
<210> 840
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 840
cttgggggcc ccttccccac ac 22
<210> 841
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 841
cttgggggcc ccttccccac a 21
<210> 842
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 842
cttgggggcc ccttccccac 20
<210> 843
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 843
cttgggggcc ccttcccca 19
<210> 844
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 844
cttgggggcc ccttcccc 18
<210> 845
<211> 17
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 845
cttgggggcc ccttccc 17
<210> 846
<211> 24
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 846
ucaugugggg aaggggcccc caag 24
<210> 847
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 847
caugugggga aggggccccc aag 23
<210> 848
<211> 22
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 848
auguggggaa ggggccccca ag 22
<210> 849
<211> 21
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 849
uguggggaag gggcccccaa g 21
<210> 850
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 850
guggggaagg ggcccccaag 20
<210> 851
<211> 19
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 851
uggggaaggg gcccccaag 19
<210> 852
<211> 18
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 852
ggggaagggg cccccaag 18
<210> 853
<211> 17
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 853
gggaaggggc ccccaag 17
<210> 854
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 854
gggtgctcca cctggtacgt atat 24
<210> 855
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 855
gggtgctcca cctggtacgt ata 23
<210> 856
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 856
gggtgctcca cctggtacgt at 22
<210> 857
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 857
gggtgctcca cctggtacgt a 21
<210> 858
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 858
gggtgctcca cctggtacgt 20
<210> 859
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 859
gggtgctcca cctggtacg 19
<210> 860
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 860
gggtgctcca cctggtac 18
<210> 861
<211> 17
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 861
gggtgctcca cctggta 17
<210> 862
<211> 24
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 862
auauacguac cagguggagc accc 24
<210> 863
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 863
uauacguacc agguggagca ccc 23
<210> 864
<211> 22
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 864
auacguacca gguggagcac cc 22
<210> 865
<211> 21
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 865
uacguaccag guggagcacc c 21
<210> 866
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 866
acguaccagg uggagcaccc 20
<210> 867
<211> 19
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 867
cguaccaggu ggagcaccc 19
<210> 868
<211> 18
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 868
guaccaggug gagcaccc 18
<210> 869
<211> 17
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 869
uaccaggugg agcaccc 17
<210> 870
<211> 21
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 870
gacguaccag guggagcacc c 21
<210> 871
<211> 1047
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 871
atgggcccgc gagccaggcc ggcgcttctc ctcctgatgc ttttgcagac cgcggtcctg 60
caggggcgct tgctgcgttc acactctctg cactacctct tcatgggtgc ctcagagcag 120
gaccttggtc tttccttgtt tgaagctttg ggctacgtgg atgaccagct gttcgtgttc 180
tatgatcatg agagtcgccg tgtggagccc cgaactccat gggtttccag tagaatttca 240
agccagatgt ggctgcagct gagtcagagt ctgaaagggt gggatcacat gttcactgtt 300
gacttctgga ctattatgga aaatcacaac cacagcaagg agtcccacac cctgcaggtc 360
atcctgggct gtgaaatgca agaagacaac agtaccgagg gctactggaa gtacgggtat 420
gatgggcagg accaccttga attctgccct gacacactgg attggagagc agcagaaccc 480
agggcctggc ccaccaagct ggagtgggaa aggcacaaga ttcgggccag gcagaacagg 540
gcctacctgg agagggactg ccctgcacag ctgcagcagt tgctggagct ggggagaggt 600
gttttggacc aacaagtgcc tcctttggtg aaggtgacac atcatgtgac ctcttcagtg 660
accactctac ggtgtcgggc cttgaactac tacccccaga acatcaccat gaagtggctg 720
aaggataagc agccaatgga tgccaaggag ttcgaaccta aagacgtatt gcccaatggg 780
gatgggacct accagggctg gataaccttg gctgtacccc ctggggaaga gcagagatat 840
acgtgccagg tggagcaccc aggcctggat cagcccctca ttgtgatctg ggagccctca 900
ccgtctggca ccctagtcat tggagtcatc agtggaattg ctgtttttgt cgtcatcttg 960
ttcattggaa ttttgttcat aatattaagg aagaggcagg gttcaagagg agccatgggg 1020
cactacgtct tagctgaacg tgagtga 1047

Claims (273)

1.用于使HBG1或HBG2基因的启动子的有义或反义链中的腺苷(A)核碱基脱氨基的方法，所述方法包括使所述启动子与碱基编辑器和与所述碱基编辑器结合的引导RNA接触，其中所述引导RNA(gRNA)包含与所述HBG1和/或HBG2基因的启动子中的靶核酸序列互补的引导序列。
2.权利要求1的方法，其中所述引导序列包含与所述启动子的靶核酸序列100％互补的至少15、16、17、18、19、20、21、22、23、24或25个连续核碱基。
3.权利要求1或2的方法，其中所述碱基编辑器对所述靶核酸序列进行切口。
4.权利要求1-3中任一项的方法，其中所述靶核酸序列包含：
5′-CTTGGGGGCCCCTTCCCCACACTA-3′(SEQ ID NO:838)；
5′-CTTGGGGGCCCCTTCCCCACACT-3′(SEQ ID NO:839)；
5′-CTTGGGGGCCCCTTCCCCACAC-3′(SEQ ID NO:840)；
5′-CTTGGGGGCCCCTTCCCCACA-3′(SEQ ID NO:841)；
5′-CTTGGGGGCCCCTTCCCCAC-3′(SEQ ID NO:842)；
5′-CTTGGGGGCCCCTTCCCCA-3′(SEQ ID NO:843)；
5′-CTTGGGGGCCCCTTCCCC-3′(SEQ ID NO:844)；或
5′-CTTGGGGGCCCCTTCCC-3′(SEQ ID NO:845)。
5.权利要求4的方法，其中所述靶核酸序列进一步包含5′末端处的5′-CCT-3′。
6.权利要求1-4中任一项的方法，其中所述靶核酸序列包含5′-CTTGGGGGCCCCTTCCCCAC-3′(SEQ ID NO:842)。
7.权利要求1-6中任一项的方法，其中所述引导序列包含：
5′-UCAUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:846)；
5′-CAUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:847)；
5′-AUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:848)；
5′-UGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:849)；
5′-GUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:850)；
5′-UGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:851)；
5′-GGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:852)；或
5′-GGGAAGGGGCCCCCAAG-3′(SEQ ID NO:853)。
8.权利要求1-7中任一项的方法，其中所述引导序列包含5′-GUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:850)。
9.权利要求1-8中任一项的方法，其中所述靶核酸序列包含：
5′-CTTGGGGGCCCCTTCCCCACACTA-3′(SEQ ID NO:838)；
5′-CTTGGGGGCCCCTTCCCCACACT-3′(SEQ ID NO:839)；
5′-CTTGGGGGCCCCTTCCCCACAC-3′(SEQ ID NO:840)；
5′-CTTGGGGGCCCCTTCCCCACA-3′(SEQ ID NO:841)；
5′-CTTGGGGGCCCCTTCCCCAC-3′(SEQ ID NO:842)；
5′-CTTGGGGGCCCCTTCCCCA-3′(SEQ ID NO:843)；
5′-CTTGGGGGCCCCTTCCCC-3′(SEQ ID NO:844)；或
5′-CTTGGGGGCCCCTTCCC-3′(SEQ ID NO:845)；并且
其中与SEQ ID NO:845的位置14处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
10.权利要求1-3中任一项的方法，其中所述引导序列包含：
5′-GACAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:254)；
5′-ACAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:255)；
5′-CAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:256)；
5′-AGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:257)；
5′-GAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:258)；或
5′-AUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:259)。
11.权利要求10的方法，其中所述靶核酸序列包含
5′-CCACACTATCTCAATGCAAATATCTGTC-3′(SEQ ID NO:297)；
5′-CCACACTATCTCAATGCAAATATCTGT-3′(SEQ ID NO:298)；
5′-CCACACTATCTCAATGCAAATATCTG-3′(SEQ ID NO:299)；
5′-CCACACTATCTCAATGCAAATATCT-3′(SEQ ID NO:300)；
5′-CCACACTATCTCAATGCAAATATC-3′(SEQ ID NO:301)；或
5′-CCACACTATCTCAATGCAAATAT-3′(SEQ ID NO:302)；并且
其中与SEQ ID NO:302的位置21处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
12.权利要求10或11的方法，其进一步包括使用第二gRNA使所述启动子的有义或反义链中的第二A核碱基脱氨基，所述第二gRNA包含与第二靶核酸序列互补的第二引导序列，其中：
(a)所述第二靶核酸包含SEQ ID NO:845；
(b)所述第二引导序列包含SEQ ID NO:853；或
(c)(a)和(b)两者。
13.权利要求1-3中任一项的方法，其中所述gRNA的引导序列包含：
5′-AUGCAAAUAUCUGUCUGAAACGG-3′(SEQ ID NO:260)。
14.权利要求13的方法，其中所述靶核酸序列包含
5′-CCGTTTCAGACAGATATTTGCAT-3′(SEQ ID NO:303)；
其中与SEQ ID NO:303的位置15、17、18或19的任一个处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
15.权利要求13或14的方法，其进一步包括使用第二gRNA使所述启动子的有义或反义链中的第二A核碱基脱氨基，所述第二gRNA包含与第二靶核酸序列互补的第二引导序列，其中：
(a)所述第二靶核酸包含SEQ ID NO:845或SEQ ID NO:302；
(b)所述第二引导序列包含SEQ ID NO:853或SEQ ID NO:259；或
(c)(a)和(b)两者。
16.权利要求1-3中任一项的方法，其中所述gRNA的引导序列包含核酸序列：
5′-GCAAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:261)；
5′-CAAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:262)；
5′-AAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:263)；
5′-AAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:264)；
5′-AUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:265)；或
5′-UAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:266)。
17.权利要求16的方法，其中所述靶核酸序列包含：
5′-CCAGGGACCGTTTCAGACAGATATTTGC-3′(SEQ ID NO:304)；
5′-CCAGGGACCGTTTCAGACAGATATTTG-3′(SEQ ID NO:305)；
5′-CCAGGGACCGTTTCAGACAGATATTT-3′(SEQ ID NO:306)；
5′-CCAGGGACCGTTTCAGACAGATATT-3′(SEQ ID NO:307)；
5′-CCAGGGACCGTTTCAGACAGATAT-3′(SEQ ID NO:308)；或
5′-CCAGGGACCGTTTCAGACAGATA-3′(SEQ ID NO:309)；
其中与SEQ ID NO:309的位置22处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
18.权利要求16或17的方法，其进一步包括
使用第二gRNA使所述启动子的有义或反义链中的第二A核碱基脱氨基，所述第二gRNA包含与第二靶核酸序列互补的第二引导序列，其中：
(a)所述第二靶核酸包含SEQ ID NO:845、SEQ ID NO:302或SEQ ID NO:303；
(b)所述第二引导序列包含SEQ ID NO:853、SEQ ID NO:259或SEQ ID NO:260；或
(c)(a)和(b)两者。
19.权利要求1-3中任一项的方法，其中所述gRNA的引导序列包含：
5′-AGAUAUUUGCAUUGAGAUAGUGU-3′(SEQ ID NO:267)。
20.权利要求19的方法，其中所述靶核酸序列包含
5′-ACACTATCTCAATGCAAATATCT-3′(SEQ ID NO:310)；
其中与SEQ ID NO:310的位置19处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
21.权利要求19或20的方法，其进一步包括
使用第二gRNA使所述启动子的有义或反义链中的第二A核碱基脱氨基，所述第二gRNA包含与第二靶核酸序列互补的第二引导序列，其中：
(a)所述第二靶核酸包含SEQ ID NO:845、SEQ ID NO:302、SEQ ID NO:303或SEQ IDNO:309；
(b)所述第二引导序列包含SEQ ID NO:853、SEQ ID NO:259、SEQ ID NO:260或SEQ IDNO:266；或
(c)(a)和(b)两者。
22.权利要求1-3中任一项的方法，其中所述gRNA的引导序列包含：
5′-ACAGAUAUUUGCAUUGAGAUAGU-3′(SEQ ID NO:268)。
23.权利要求22的方法，其中所述靶核酸序列包含
5′-ACTATCTCAATGCAAATATCTGT-3′(SEQ ID NO:311)；
其中与SEQ ID NO:311的位置17处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
24.权利要求22或23的方法，其进一步包括进行权利要求1-21中任一项的方法。
25.权利要求1-3中任一项的方法，其中所述gRNA的引导序列包含核酸序列：
5′-GUGGGGAAGGGGCCCCCAAGAGG-3′(SEQ ID NO:269)。
26.权利要求25的方法，其中所述启动子的靶核酸序列包含
5′-CCTCTTGGGGGCCCCTTCCCCAC-3′(SEQ ID NO:312)的核酸位置16和17处的T；
其中与位置16或17的任一个处的T互补的A核碱基的脱氨基导致T至C突变。
27.权利要求25或26的方法，其进一步包括使用第二gRNA使所述启动子的有义或反义链中的第二A核碱基脱氨基，所述第二gRNA包含与第二靶核酸序列互补的第二引导序列，其中：
(a)所述第二靶核酸包含SEQ ID NO:845、SEQ ID NO:302、SEQ ID NO:303、SEQ ID NO:309或SEQ ID NO:311；
(b)所述第二引导序列包含SEQ ID NO:853、SEQ ID NO:259、SEQ ID NO:260或SEQ IDNO:266或SEQ ID NO:268；或
(c)(a)和(b)两者。
28.权利要求1-3中任一项的方法，其中所述gRNA的引导序列包含：
5′-CUUGACCAAUAGCCUUGACAAGG-3′(SEQ ID NO:270)。
29.权利要求28的方法，其中所述靶核酸序列包含
5′-CCTTGTCAAGGCTATTGGTCAAG-3′(SEQ ID NO:313)；
其中与SEQ ID NO:313的位置15、16或19的任一个处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
30.权利要求28或29的方法，其进一步包括使用第二gRNA使所述启动子的有义或反义链中的第二A核碱基脱氨基，所述第二gRNA包含与第二靶核酸序列互补的第二引导序列，其中：
(a)所述第二靶核酸包含SEQ ID NO:845、SEQ ID NO:302、SEQ ID NO:303、SEQ ID NO:309、SEQ ID NO:311或SEQ ID NO:312；
(b)所述第二引导序列包含SEQ ID NO:853、SEQ ID NO:259、SEQ ID NO:260或SEQ IDNO:266、SEQ ID NO:268或SEQ ID NO:269；或
(c)(a)和(b)两者。
31.权利要求1-3中任一项的方法，其中所述gRNA的引导序列包含：
5′-CUUGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:271)；
5′-UUGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:272)；
5′-UGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:273)；
5′-GUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:274)；
5′-UCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:275)；或
5′-CAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:276)。
32.权利要求28的方法，其中所述靶核酸序列包含
5′-CCTTGCCTTGACCAATAGCCTTGACAAG-3′(SEQ ID NO:314)；
5′-CCTTGCCTTGACCAATAGCCTTGACAA-3′(SEQ ID NO:315)；
5′-CCTTGCCTTGACCAATAGCCTTGACA-3′(SEQ ID NO:316)；
5′-CCTTGCCTTGACCAATAGCCTTGAC-3′(SEQ ID NO:317)；
5′-CCTTGCCTTGACCAATAGCCTTGA-3′(SEQ ID NO:318)；或
5′-CCTTGCCTTGACCAATAGCCTTG-3′(SEQ ID NO:319)；
其中与SEQ ID NO:319的位置16、21或22的任一个处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
33.权利要求31或32的方法，其进一步包括使用第二gRNA使所述启动子的有义或反义链中的第二A核碱基脱氨基，所述第二gRNA包含与第二靶核酸序列互补的第二引导序列，其中：
(a)所述第二靶核酸包含SEQ ID NO:845、SEQ ID NO:302、SEQ ID NO:303、SEQ ID NO:309、SEQ ID NO:311、SEQ ID NO:312或SEQ ID NO:313；
(b)所述第二引导序列包含SEQ ID NO:853、SEQ ID NO:259、SEQ ID NO:260或SEQ IDNO:266、SEQ ID NO:268、SEQ ID NO:269或SEQ ID NO:270；或
(c)(a)和(b)两者。
34.权利要求1-3中任一项的方法，其中所述gRNA的引导序列包含：
5′-UUGUCAAGGCUAUUGGUCAAGGC-3′(SEQ ID NO:277)。
35.权利要求34的方法，其中所述靶核酸序列包含
5′-GCCTTGACCAATAGCCTTGACAA-3′(SEQ ID NO:320)；
其中与SEQ ID NO:320的位置17或18的任一个处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
36.权利要求34或35的方法，其进一步包括使用第二gRNA使所述启动子的有义或反义链中的第二A核碱基脱氨基，所述第二gRNA包含与第二靶核酸序列互补的第二引导序列，其中：
(a)所述第二靶核酸包含SEQ ID NO:845、SEQ ID NO:302、SEQ ID NO:303、SEQ ID NO:309、SEQ ID NO:311、SEQ ID NO:312、SEQ ID NO:313或SEQ ID NO:319；
(b)所述第二引导序列包含SEQ ID NO:853、SEQ ID NO:259、SEQ ID NO:260或SEQ IDNO:266、SEQ ID NO:268、SEQ ID NO:269、SEQ ID NO:270或SEQ ID NO:276；或
(c)(a)和(b)两者。
37.权利要求1-3中任一项的方法，其中所述gRNA的引导序列包含：
5′-CUUGUCAAGGCUAUUGGUCAAGG-3′(SEQ ID NO:278)。
38.权利要求37的方法，其中所述靶核酸序列包含
5′-CCTTGACCAATAGCCTTGACAAG-3′(SEQ ID NO:321)；
其中与SEQ ID NO:321的位置16或17的任一个处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
39.权利要求37或38的方法，其进一步包括使用第二gRNA使所述启动子的有义或反义链中的第二A核碱基脱氨基，所述第二gRNA包含与第二靶核酸序列互补的第二引导序列，其中：
(a)所述第二靶核酸包含SEQ ID NO:845、SEQ ID NO:302、SEQ ID NO:303、SEQ ID NO:309、SEQ ID NO:311、SEQ ID NO:312、SEQ ID NO:313、SEQ ID NO:319或SEQ ID NO:320；
(b)所述第二引导序列包含SEQ ID NO:853、SEQ ID NO:259、SEQ ID NO:260或SEQ IDNO:266、SEQ ID NO:268、SEQ ID NO:269、SEQ ID NO:270、SEQ ID NO:276或SEQ ID NO:277；或
(c)(a)和(b)两者。
40.权利要求1-3中任一项的方法，其中所述gRNA的引导序列包含：
5′-UUGACCAAUAGCCUUGACAAGGC-3′(SEQ ID NO:279)。
41.权利要求40的方法，其中所述靶核酸序列包含
5′-GCCTTGTCAAGGCTATTGGTCAA-3′(SEQ ID NO:322)；
其中与SEQ ID NO:322的位置16、17或20的任一个处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
42.权利要求40或41的方法，其进一步包括使用第二gRNA使所述启动子的有义或反义链中的第二A核碱基脱氨基，所述第二gRNA包含与第二靶核酸序列互补的第二引导序列，其中：
(a)所述第二靶核酸包含SEQ ID NO:845、SEQ ID NO:302、SEQ ID NO:303、SEQ ID NO:309、SEQ ID NO:311、SEQ ID NO:312、SEQ ID NO:313、SEQ ID NO:319、SEQ ID NO:320或SEQ ID NO:321；
(b)所述第二引导序列包含SEQ ID NO:853、SEQ ID NO:259、SEQ ID NO:260或SEQ IDNO:266、SEQ ID NO:268、SEQ ID NO:269、SEQ ID NO:270、SEQ ID NO:276、SEQ ID NO:277或SEQ ID NO:278；或
(c)(a)和(b)两者。
43.权利要求1-3中任一项的方法，其中所述gRNA的引导序列包含：
5′-UAGCCUUGACAAGGCAAACUUGA-3′(SEQ ID NO:280)。
44.权利要求43的方法，其中所述靶核酸序列包含
5′-TCAAGTTTGCCTTGTCAAGGCTA-3′(SEQ ID NO:323)；
其中与SEQ ID NO:323的位置15处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
45.权利要求43或44的方法，其进一步包括使用第二gRNA使所述启动子的有义或反义链中的第二A核碱基脱氨基，所述第二gRNA包含与第二靶核酸序列互补的第二引导序列，其中：
(a)所述第二靶核酸包含SEQ ID NO:845、SEQ ID NO:302、SEQ ID NO:303、SEQ ID NO:309、SEQ ID NO:311、SEQ ID NO:312、SEQ ID NO:313、SEQ ID NO:319、SEQ ID NO:320、SEQID NO:321或SEQ ID NO:322；
(b)所述第二引导序列包含SEQ ID NO:853、SEQ ID NO:259、SEQ ID NO:260或SEQ IDNO:266、SEQ ID NO:268、SEQ ID NO:269、SEQ ID NO:270、SEQ ID NO:276、SEQ ID NO:277、SEQ ID NO:278或SEQ ID NO:279；或
(c)(a)和(b)两者。
46.权利要求1-45中任一项的方法，其中使所述启动子中的腺苷核碱基脱氨基导致所述启动子中的T-A碱基对突变为所述启动子中的C-G碱基对。
47.权利要求1-46中任一项的方法，其中使所述启动子中的腺苷核碱基脱氨基导致与遗传性胎儿血红蛋白持续存在症(HPFH)相关的序列。
48.权利要求1-47中任一项的方法，其中使所述启动子中的腺苷核碱基脱氨基引起所述HBG1基因的转录增加。
49.权利要求1-48中任一项的方法，其中使所述启动子中的腺苷核碱基脱氨基引起HBG1蛋白的增加。
50.权利要求1-49中任一项的方法，其中使所述启动子中的腺苷核碱基脱氨基引起所述HBG2基因的转录增加。
51.权利要求1-50中任一项的方法，其中使所述启动子中的腺苷核碱基脱氨基引起HBG2蛋白的增加。
52.权利要求1-51中任一项的方法，其中使所述启动子中的腺苷核碱基脱氨基引起所述HBG1和HBG2基因两者的转录增加。
53.权利要求1-52中任一项的方法，其中使所述启动子中的腺苷核碱基脱氨基引起HBG1和HBG2蛋白的量的增加。
54.权利要求1-53中任一项的方法，其中所述HBG1或HBG2基因的启动子在细胞中。
55.权利要求1-54中任一项的方法，其中所述方法在体外进行。
56.权利要求1-54中任一项的方法，其中所述细胞在受试者中。
57.权利要求56的方法，其中所述方法在体内或离体进行。
58.权利要求56或57的方法，其中使所述启动子中的腺苷核碱基脱氨基对所述受试者赋予遗传性胎儿血红蛋白持续存在症(HPFH)。
59.权利要求56-58中任一项的方法，其中所述受试者患有血液的疾病或病症。
60.权利要求59的方法，其中所述疾病或病症是贫血。
61.权利要求60的方法，其中所述贫血是镰状细胞贫血。
62.权利要求59的方法，其中所述疾病或病症是beta地中海贫血。
63.权利要求59-62中任一项的方法，其中所述疾病或病症是由编码珠蛋白蛋白质的基因或基因的启动子中的突变引起的。
64.权利要求63的方法，其中所述基因是CYGB、HBA1、HBA2、HBB、HBD、HBE1、HBG1、HBG2、HBM、HBQ1、HBZ或MB。
65.用于使HFE基因的有义或反义链中的腺苷(A)核碱基脱氨基的方法，所述方法包括使所述HFE基因与碱基编辑器和与所述碱基编辑器结合的引导RNA接触，其中所述引导RNA包含与所述HFE基因中的靶核酸序列互补的引导序列。
66.权利要求65的方法，其中所述HFE基因包含C至T突变。
67.权利要求66的方法，其中使与所述T互补的腺苷核碱基脱氨基校正所述C至T突变。
68.权利要求66的方法，其中所述HFE基因编码包含Cys至Tyr突变的蛋白质。
69.权利要求68的方法，其中使与所述T互补的腺苷核碱基脱氨基校正所述Cys至Tyr突变。
70.权利要求65-69中任一项的方法，其中所述引导序列包含与所述HFE基因的靶核酸序列100％互补的至少15、16、17、18、19、20、21、22、23、24或25个连续核酸。
71.权利要求65-70中任一项的方法，其中所述碱基编辑器对所述靶序列进行切口。
72.权利要求65-71的方法，其中所述HFE基因中的靶核酸序列包含：
5′-GGGTGCTCCACCTGGTACGTATAT-3′(SEQ ID NO:854)；
5′-GGGTGCTCCACCTGGTACGTATA-3′(SEQ ID NO:855)；
5′-GGGTGCTCCACCTGGTACGTAT-3′(SEQ ID NO:856)；
5′-GGGTGCTCCACCTGGTACGTA-3′(SEQ ID NO:857)；
5′-GGGTGCTCCACCTGGTACGT-3′(SEQ ID NO:858)；
5′-GGGTGCTCCACCTGGTACG-3′(SEQ ID NO:859)；
5′-GGGTGCTCCACCTGGTAC-3′(SEQ ID NO:860)；或
5′-GGGTGCTCCACCTGGTA-3′(SEQ ID NO:861)。
73.权利要求72的方法，其中所述靶核酸序列进一步包含5′末端处的5′-CCT-3′。
74.权利要求65-73中任一项的方法，其中所述HFE基因的靶核酸序列包含5′-GGGTGCTCCACCTGGTACGT-3′(SEQ ID NO:858)。
75.权利要求65-74中任一项的方法，其中所述gRNA的引导序列包含
5′-AUAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:862)；
5′-UAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:863)；
5′-AUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:864)；
5′-UACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:865)；
5′-ACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:866)；
5′-CGUACCAGGUGGAGCACCC-3′(SEQ ID NO:867)；
5′-GUACCAGGUGGAGCACCC-3′(SEQ ID NO:868)；或
5′-UACCAGGUGGAGCACCC-3′(SEQ ID NO:869)。
76.权利要求75的方法，其中所述gRNA的引导序列进一步包含5′末端处的G。
77.权利要求65-76中任一项的方法，其中所述gRNA的引导序列包含核酸序列5′-GACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:870)。
78.权利要求65-77中任一项的方法，其中所述靶核酸序列包含
5′-GGGTGCTCCACCTGGTACGTATAT-3′(SEQ ID NO:854)；
5′-GGGTGCTCCACCTGGTACGTATA-3′(SEQ ID NO:855)；
5′-GGGTGCTCCACCTGGTACGTAT-3′(SEQ ID NO:856)；
5′-GGGTGCTCCACCTGGTACGTA-3′(SEQ ID NO:857)；
5′-GGGTGCTCCACCTGGTACGT-3′(SEQ ID NO:858)；
5′-GGGTGCTCCACCTGGTACG-3′(SEQ ID NO:859)；
5′-GGGTGCTCCACCTGGTAC-3′(SEQ ID NO:860)；或
5′-GGGTGCTCCACCTGGTA-3′(SEQ ID NO:861)；并且
其中与SEQ ID NO:861的位置16处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
79.权利要求65-78中任一项的方法，其中使所述HFE基因中的腺苷核碱基脱氨基导致所述HFE基因中的T-A碱基对突变为所述HFE基因中的C-G碱基对。
80.权利要求65-79中任一项的方法，其中使所述HFE基因中的腺苷核碱基脱氨基导致校正与遗传性血色素沉着症(HHC)相关的序列。
81.权利要求65-80中任一项的方法，其中使所述HFE基因中的腺苷核碱基脱氨基引起从所述HFE基因转录的HFE蛋白的功能增加。
82.权利要求65-81中任一项的方法，其中使所述HFE基因中的腺苷核碱基脱氨基引起HFE稳定性或半衰期的增加。
83.权利要求65-82中任一项的方法，其中所述HFE基因在细胞中。
84.权利要求83的方法，其中所述HFE基因编码包含Cys至Tyr突变的HFE蛋白。
85.权利要求84的方法，其中所述HFE蛋白包含在以下氨基酸序列的残基282处的Cys至Tyr突变(C282Y)：MGPRARPALLLLMLLQTAVLQGRLLRSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDFWTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIWEPSPSGTLVIGVISGIAVFVVILFIGILFIILRKRQGSRGAMGHYVLAER(SEQ ID NO:750)。
86.权利要求83-85中任一项的方法，其中所述细胞是永生化的淋巴样干细胞(lymphoblastoid cell)(LCL)。
87.权利要求65-86中任一项的方法，其中所述方法在体外进行。
88.权利要求65-85中任一项的方法，其中所述细胞在受试者中。
89.权利要求88的方法，其中所述受试者患有铁贮积病症(iron storage disorder)。
90.权利要求89的方法，其中所述铁贮积病症是遗传性血色素沉着症(HHC)。
91.权利要求88-90中任一项的方法，其中所述方法在体内或离体进行。
92.权利要求90的方法，其中使所述HFE基因中的腺苷核碱基脱氨基改善所述受试者中铁贮积病症的一种或多种症状。
93.用于使HBB基因的有义或反义链中的腺苷(A)核碱基脱氨基的方法，所述方法包括使所述HBB基因与碱基编辑器和与所述碱基编辑器结合的引导RNA接触，其中所述引导RNA包含与所述HBB基因中的靶核酸序列互补的引导序列。
94.权利要求93的方法，其中所述HBB基因包含C至T突变。
95.权利要求94的方法，其中使与所述T互补的腺苷核碱基脱氨基校正所述C至T突变。
96.权利要求94的方法，其中所述HBB基因编码包含Glu至Val突变或Glu至Lys突变的蛋白质。
97.权利要求96的方法，其中所述Glu至Val突变或所述Glu至Lys突变在氨基酸序列VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH(SEQ IDNO:340)的氨基酸位置6处。
98.权利要求97的方法，其中使与所述T互补的腺苷核碱基脱氨基校正所述Glu至Val突变或所述Glu至Lys突变。
99.权利要求93-98中任一项的方法，其中所述引导序列包含与所述HBB基因的靶核酸序列100％互补的至少15、16、17、18、19、20、21、22、23、24或25个连续核酸。
100.权利要求93-99中任一项的方法，其中所述碱基编辑器对所述靶序列进行切口。
101.权利要求93-100的方法，其中所述HBB基因中的靶核酸序列包含：
5′-GTGCATCTGACTCCTGTGGAGAA-3′(SEQ ID NO:324)；
5′-GGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:325)；
5′-CCATGGTGCATCTGACTCCTGTGGAGAA-3′(SEQ ID NO:326)；
5′-CCATGGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:327)；
5′-CCATGGTGCATCTGACTCCTGTGGAG-3′(SEQ ID NO:328)；
5′-CCATGGTGCATCTGACTCCTGTGGA-3′(SEQ ID NO:329)；
5′-CCATGGTGCATCTGACTCCTGTGG-3′(SEQ ID NO:330)；
5′-CCATGGTGCATCTGACTCCTGTG-3′(SEQ ID NO:331)；
5′-GCATCTGACTCCTGTGGAGAAGT-3′(SEQ ID NO:332)；
5′-ACCATGGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:333)；或
5′-ACGGCAGACTTCTCCTTAGGAGT-3′(SEQ ID NO:334)。
102.权利要求101的方法，其中所述靶核酸序列包含
5′-GTGCATCTGACTCCTGTGGAGAA-3′(SEQ ID NO:324)，并且
其中与SEQ ID NO:324的位置17处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
103.权利要求101的方法，其中所述靶核酸序列包含
5′-GGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:325)，并且
其中与SEQ ID NO:325的位置18处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
104.权利要求101的方法，其中所述靶核酸序列包含
5′-CCATGGTGCATCTGACTCCTGTGGAGAA-3′(SEQ ID NO:326)；
5′-CCATGGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:327)；
5′-CCATGGTGCATCTGACTCCTGTGGAG-3′(SEQ ID NO:328)；
5′-CCATGGTGCATCTGACTCCTGTGGA-3′(SEQ ID NO:329)；
5′-CCATGGTGCATCTGACTCCTGTGG-3′(SEQ ID NO:330)；或
5′-CCATGGTGCATCTGACTCCTGTG-3′(SEQ ID NO:331)；并且
其中与SEQ ID NO:331的位置22处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
105.权利要求101的方法，其中所述HBB基因中的靶核酸序列包含
5′-GCATCTGACTCCTGTGGAGAAGT-3′(SEQ ID NO:332)，并且
其中与SEQ ID NO:332的位置15处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
106.权利要求101的方法，其中所述HBB基因中的靶核酸序列包含
5′-ACCATGGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:333)，并且
其中与SEQ ID NO:333的位置23处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
107.权利要求101的方法，其中所述HBB基因中的靶核酸序列包含
5′-ACGGCAGACTTCTCCTTAGGAGT-3′(SEQ ID NO:334)，并且
其中与SEQ ID NO:334的位置17处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
108.权利要求93-107中任一项的方法，其中所述gRNA的引导序列包含
5′-UUCUCCACAGGAGUCAGAUGCAC-3′(SEQ ID NO:281)；
5′-UCUCCACAGGAGUCAGAUGCACC-3′(SEQ ID NO:282)；
5′-UUCUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:283)；
5′-UCUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:284)；
5′-CUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:285)；
5′-UCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:286)；
5′-CCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:287)；
5′-CACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:288)；
5′-ACUUCUCCACAGGAGUCAGAUGC-3′(SEQ ID NO:289)；
5′-UCUCCACAGGAGUCAGAUGCACCAUGGU-3′(SEQ ID NO:290)；或
5′-ACUCCUAAGGAGAAGUCUGCCGU-3′(SEQ ID NO:291)。
109.权利要求108的方法，其中所述gRNA的引导序列进一步包含5′末端处的G。
110.权利要求93-109中任一项的方法，其中使所述HBB基因中的腺苷核碱基脱氨基导致所述HBB基因中的T-A碱基对突变为所述HBB基因中的C-G碱基对。
111.权利要求93-110中任一项的方法，其中使所述HBB基因中的腺苷核碱基脱氨基导致校正与镰状细胞病相关的序列。
112.权利要求93-110中任一项的方法，其中使所述HBB基因中的腺苷核碱基脱氨基导致校正与Hb C beta地中海贫血相关的序列。
113.权利要求93-112中任一项的方法，其中使所述HBB基因中的腺苷核碱基脱氨基引起从所述HBB基因转录的beta珠蛋白蛋白质的功能增加。
114.权利要求93-113中任一项的方法，其中使所述HBB基因中的腺苷核碱基脱氨基引起beta珠蛋白稳定性或半衰期的增加。
115.权利要求93-114中任一项的方法，其中所述HBB基因在细胞中。
116.权利要求93-115中任一项的方法，其中所述方法在体外进行。
117.权利要求93-116中任一项的方法，其中所述细胞在受试者中。
118.权利要求117的方法，其中所述受试者患有镰状细胞病。
119.权利要求117的方法，其中所述受试者患有beta地中海贫血。
120.权利要求119的方法，其中所述受试者患有Hb C beta地中海贫血(血红蛋白C病)。
121.权利要求93-120中任一项的方法，其中所述方法在体内或离体进行。
122.权利要求118的方法，其中使所述HBB基因中的腺苷核碱基脱氨基改善所述受试者中镰状细胞病的一种或多种症状。
123.权利要求119或120的方法，其中使所述HBB基因中的腺苷核碱基脱氨基改善beta地中海贫血的一种或多种症状。
124.权利要求94的方法，其中所述HBB基因编码包含Glu至Lys突变的蛋白质。
125.权利要求124的方法，其中所述Glu至Lys突变在氨基酸序列VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH(SEQ ID NO:340)的氨基酸位置26处。
126.权利要求125的方法，其中使与所述T互补的腺苷核碱基脱氨基校正所述Glu至Lys突变。
127.权利要求124-126中任一项的方法，其中所述引导序列包含与所述HBB基因的靶核酸序列100％互补的至少15、16、17、18、19、20、21、22、23、24或25个连续核酸。
128.权利要求124-127中任一项的方法，其中所述碱基编辑器对所述靶序列进行切口。
129.权利要求124-128的方法，其中所述HBB基因中的靶核酸序列包含：
5′-CCTGCCCAGGGCCTTACCACCAA-3′(SEQ ID NO:335)；
5′-ACCTGCCCAGGGCCTTACCACCA-3′(SEQ ID NO:336)；或
5′-CCAACCTGCCCAGGGCCTTACCA-3′(SEQ ID NO:337)。
130.权利要求129的方法，其中所述HBB基因中的靶核酸序列包含
5′-CCTGCCCAGGGCCTTACCACCAA-3′(SEQ ID NO:335)，并且
其中与SEQ ID NO:335的位置15处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
131.权利要求129的方法，其中所述HBB基因中的靶核酸序列包含
5′-ACCTGCCCAGGGCCTTACCACCA-3′(SEQ ID NO:336)，并且
其中与SEQ ID NO:336的位置16处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
132.权利要求129的方法，其中所述HBB基因中的靶核酸序列包含
5′-CCAACCTGCCCAGGGCCTTACCA-3′(SEQ ID NO:337)，并且
其中与位置19处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
133.权利要求124-132中任一项的方法，其中所述gRNA的引导序列包含
5′-UUGGUGGUAAGGCCCUGGGCAGG-3′(SEQ ID NO:292)；
5′-UGGUGGUAAGGCCCUGGGCAGGU-3′(SEQ ID NO:293)；或
5′-UGGUAAGGCCCUGGGCAGGUUGG-3′(SEQ ID NO:294)。
134.权利要求133的方法，其中所述gRNA的引导序列进一步包含5′处的G。
135.权利要求124-134中任一项的方法，其中使所述HBB基因中的腺苷核碱基脱氨基导致所述HBB基因中的T-A碱基对突变为所述HBB基因中的C-G碱基对。
136.权利要求124-136中任一项的方法，其中使所述HBB基因中的腺苷核碱基脱氨基导致校正与Hb C beta地中海贫血相关的序列。
137.权利要求124-136中任一项的方法，其中使所述HBB基因中的腺苷核碱基脱氨基引起从所述HBB基因转录的beta珠蛋白蛋白质的功能增加。
138.权利要求124-137中任一项的方法，其中使所述HBB基因中的腺苷核碱基脱氨基引起beta珠蛋白稳定性或半衰期的增加。
139.权利要求124-138中任一项的方法，其中所述HBB基因在细胞中。
140.权利要求124-139中任一项的方法，其中所述方法在体外进行。
141.权利要求124-140中任一项的方法，其中所述细胞在受试者中。
142.权利要求141的方法，其中所述受试者患有beta地中海贫血。
143.权利要求142的方法，其中所述受试者患有Hb E beta地中海贫血(血红蛋白E病)。
144.权利要求124-143中任一项的方法，其中所述方法在体内或离体进行。
145.权利要求141、142或144的方法，其中使所述HBB基因中的腺苷核碱基脱氨基改善beta地中海贫血的一种或多种症状。
146.用于使F8基因的有义或反义链中的腺苷(A)核碱基脱氨基的方法，所述方法包括使所述F8基因与碱基编辑器和与所述碱基编辑器结合的引导RNA接触，其中所述引导RNA包含与所述F8基因中的靶核酸序列互补的引导序列。
147.权利要求146的方法，其中所述F8基因包含C至T突变。
148.权利要求147的方法，其中使与所述T互补的腺苷核碱基脱氨基校正所述C至T突变。
149.权利要求148的方法，其中所述F8基因编码包含Arg至Cys突变的蛋白质。
150.权利要求149的方法，其中所述Arg至Cys突变在氨基酸序列MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVV VGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY(SEQ ID NO:341)的氨基酸位置612处。
151.权利要求150的方法，其中使与所述T互补的腺苷核碱基脱氨基校正所述Arg至Cys突变。
152.权利要求146-151中任一项的方法，其中所述引导序列包含与所述F8基因的靶核酸序列100％互补的至少15、16、17、18、19、20、21、22、23、24或25个连续核酸。
153.权利要求146-152中任一项的方法，其中所述碱基编辑器对所述靶序列进行切口。
154.权利要求146-153的方法，其中所述F8基因中的靶核酸序列包含：
5′-CCTCACAGAGAATATACAATGCT-3′(SEQ ID NO:338)；或
5′-TCACAGAGAATATACAATGCTTT-3′(SEQ ID NO:339)。
155.权利要求154的方法，其中所述F8基因中的靶核酸序列包含
5′-CCTCACAGAGAATATACAATGCT-3′(SEQ ID NO:338)，并且
其中与SEQ ID NO:388的位置20处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
156.权利要求154的方法，其中所述F8基因中的靶核酸序列包含
5′-TCACAGAGAATATACAATGCTTT-3′(SEQ ID NO:339)，并且
其中与SEQ ID NO:339的位置18处的T互补的A核碱基的脱氨基导致所述靶核酸序列中的T至C突变。
157.权利要求146-156中任一项的方法，其中所述gRNA的引导序列包含
5′-AGCAUUGUAUAUUCUCUGUGAGG-3′(SEQ ID NO:295)；或
5′-AAAGCAUUGUAUAUUCUCUGUGA-3′(SEQ ID NO:296)。
158.权利要求157的方法，其中所述gRNA的引导序列进一步包含5′末端处的G。
159.权利要求146-158中任一项的方法，其中使所述F8基因中的腺苷核碱基脱氨基导致所述F8基因中的T-A碱基对突变为所述F8基因中的C-G碱基对。
160.权利要求146-159中任一项的方法，其中使所述F8基因中的腺苷核碱基脱氨基导致校正与血友病相关的序列。
161.权利要求160的方法，其中所述血友病是血友病A。
162.权利要求146-161中任一项的方法，其中使所述F8基因中的腺苷核碱基脱氨基导致校正与血友病相关的序列。
163.权利要求146-162中任一项的方法，其中使所述F8基因中的腺苷核碱基脱氨基引起从所述F8基因转录的因子VIII蛋白的功能增加。
164.权利要求146-163中任一项的方法，其中使所述F8基因中的腺苷核碱基脱氨基引起因子VIII稳定性或半衰期的增加。
165.权利要求146-164中任一项的方法，其中所述F8基因在细胞中。
166.权利要求146-165中任一项的方法，其中所述方法在体外进行。
167.权利要求146-166中任一项的方法，其中所述细胞在受试者中。
168.权利要求167的方法，其中所述受试者患有血友病。
169.权利要求168的方法，其中所述受试者患有血友病A。
170.权利要求146-169中任一项的方法，其中所述方法在体内或离体进行。
171.权利要求168或169的方法，其中使所述F8基因中的腺苷核碱基脱氨基改善所述受试者中血友病的一种或多种症状。
172.权利要求1-171中任一项的方法，其中所述碱基编辑器包含融合蛋白，所述融合蛋白包含(i)核酸可编程DNA结合蛋白(napDNAbp)和(ii)腺苷脱氨酶。
173.权利要求172的方法，其中所述融合蛋白进一步包含核定位信号(NLS)。
174.权利要求173的方法，其中所述NLS是二分NLS。
175.权利要求173或174的方法，其中所述NLS包含氨基酸序列MKRTADGSEFEPKKKRKV(SEQ ID NO:342)、KRTADGSEFEPKKKRKV(SEQ ID NO:343)或PKKKRKV(SEQ ID NO:4)。
176.权利要求172-175中任一项的方法，其中所述腺苷脱氨酶是大肠杆菌TadA(ecTadA)。
177.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含与SEQ ID NO:1、64-84、420-437、672-684或802-805的任一个至少80％、85％、90％、95％、98％、99％或99.5％相同的氨基酸序列。
178.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含与SEQ ID NO:1的氨基酸序列至少80％、85％、90％、95％、98％、99％或99.5％相同的氨基酸序列。
179.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含SEQ ID NO:1、64-84、420-437、672-684或802-805的任一个的氨基酸序列。
180.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶由SEQ ID NO:1、64-84、420-437、672-684或802-805的任一个的氨基酸序列组成。
181.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含SEQ ID NO:1中H36L、P48S、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、E155V、I156F和K157N突变的一个或多个，或另一脱氨酶中的相应突变。
182.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含SEQ ID NO:1中H36L、P48S、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、E155V、I156F和K157N突变的每一个，或另一脱氨酶中的相应突变。
183.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含SEQ ID NO:1中H36L、P48S、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、E155V、I156F和K157N突变的一个或多个，或另一脱氨酶中的相应突变。
184.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含SEQ ID NO:1中H36L、P48S、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、E155V、I156F和K157N突变的每一个，或另一脱氨酶中的相应突变。
185.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含SEQ ID NO:1中W23L、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、E155V、I156F和K157N突变的一个或多个，或另一脱氨酶中的相应突变。
186.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含SEQ ID NO:1中W23L、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、E155V、I156F和K157N突变的每一个，或另一脱氨酶中的相应突变。
187.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含SEQ ID NO:1中W23L、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、R152P、E155V、I156F和K157N突变的一个或多个，或另一脱氨酶中的相应突变。
188.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含SEQ ID NO:1中W23L、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、A142N、S146C、D147Y、R152P、E155V、I156F和K157N突变的每一个，或另一脱氨酶中的相应突变。
189.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含SEQ ID NO:1中W23R、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、R152P、E155V、I156F和K157N突变的一个或多个，或另一脱氨酶中的相应突变。
190.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含SEQ ID NO:1中W23R、H36L、P48A、R51L、L84F、A106V、D108N、H123Y、S146C、D147Y、R152P、E155V、I156F和K157N突变的每一个，或另一脱氨酶中的相应突变。
191.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含来自SEQ ID NO:1中图7的任一个ABE的一个或多个突变，或另一脱氨酶中的一个或多个相应突变。
192.权利要求172-176中任一项的方法，其中所述腺苷脱氨酶包含来自SEQ ID NO:1中图7的任一个ABE的突变的每一个，或另一脱氨酶中的相应突变。
193.权利要求172-192中任一项的方法，其中所述核酸可编程DNA结合蛋白(napDNAbp)是Cas9结构域、Cpf1结构域、CasX结构域、CasY结构域、C2c1结构域、C2c2结构域或C2c3结构域。
194.权利要求193的方法，其中所述Cas9结构域选自下组：死亡Cas9(dCas9)结构域、Cas9切口酶(nCas9)结构域和核酸酶活性Cas9结构域。
195.权利要求194的方法，其中所述Cas9结构域是Cas9切口酶(nCas9)结构域。
196.权利要求195的方法，其中所述Cas9切口酶结构域包含SEQ ID NO:35中所示的氨基酸序列。
197.权利要求172-196中任一项的方法，其中所述融合蛋白进一步包含所述核酸可编程DNA结合蛋白(napDNAbp)和所述腺苷脱氨酶之间的一个或多个接头。
198.权利要求197的方法，其中所述一个或多个接头包含SEQ ID NO:10、37-40、384-386、685-688或800-801的任一个中所示的氨基酸序列。
199.权利要求198的方法，其中所述接头包含SEQ ID NO:800中所示的氨基酸序列。
200.权利要求172-199中任一项的方法，其中所述融合蛋白进一步包含第二腺苷脱氨酶。
201.权利要求200的方法，其中所述第二腺苷脱氨酶是ecTadA。
202.权利要求201的方法，其中所述第一腺苷脱氨酶和所述第二腺苷脱氨酶是相同的。
203.权利要求201的方法，其中所述第一腺苷脱氨酶和所述第二腺苷脱氨酶是不同的。
204.权利要求200-203中任一项的方法，其中所述第二腺苷脱氨酶包含SEQ ID NO:1的氨基酸序列。
205.权利要求200-203中任一项的方法，其中所述第二腺苷脱氨酶由SEQ ID NO:1的氨基酸序列组成。
206.权利要求200-203中任一项的方法，其中所述第二腺苷脱氨酶包含与SEQ ID NO:1、64-84、420-437、672-684或802-805的任一个至少80％、85％、90％、95％、98％、99％或99.5％相同的氨基酸序列。
207.权利要求200-203中任一项的方法，其中所述第二腺苷脱氨酶包含SEQ ID NO:1、64-84、420-437、672-684或802-805的任一个的氨基酸序列。
208.权利要求200-203中任一项的方法，其中所述第二腺苷脱氨酶由SEQ ID NO:1、64-84、420-437、672-684或802-805的任一个的氨基酸序列组成。
209.权利要求200-203中任一项的方法，其中所述第二腺苷脱氨酶包含与SEQ ID NO:1的氨基酸序列至少80％、85％、90％、95％、98％、99％或99.5％相同的氨基酸序列。
210.权利要求172-209中任一项的方法，其中所述融合蛋白包含以下结构：
[第一腺苷脱氨酶]-[第二腺苷脱氨酶]-[napDNAbp]；
[第二腺苷脱氨酶]-[第一腺苷脱氨酶]-[napDNAbp]；
[第一腺苷脱氨酶]-[第二腺苷脱氨酶]-[napDNAbp]-[NLS]；或
[第二腺苷脱氨酶]-[第一腺苷脱氨酶]-[napDNAbp]-[NLS]；
其中所述napDNAbp是Cas9结构域，并且其中“-”表示任选的接头序列的存在。
211.权利要求210的方法，其中所述Cas9结构域是Cas9切口酶(nCas9)。
212.权利要求200-211中任一项的方法，其中所述第一腺苷脱氨酶和所述第二脱氨酶经由接头融合，所述接头包含氨基酸序列(SGGS)n-SGSETPGTSESATPES-(SGGS)n(SEQ ID NO:801)，其中n是1、2、3、4或5。
213.权利要求200-212中任一项的方法，其中所述第一腺苷脱氨酶和所述第二脱氨酶经由接头融合，所述接头包含氨基酸序列(SGGS)2-SGSETPGTSESATPES-(SGGS)2(SEQ ID NO:800)。
214.权利要求200-213中任一项的方法，其中所述第一腺苷脱氨酶或所述第二腺苷脱氨酶经由接头与所述napDNAbp融合，所述接头包含氨基酸序列(SGGS)n-SGSETPGTSESATPES-(SGGS)n(SEQ ID NO:801)，其中n是1、2、3、4或5。
215.权利要求200-214中任一项的方法，其中所述第一腺苷脱氨酶或所述第二腺苷脱氨酶经由接头与所述napDNAbp融合，所述接头包含氨基酸序列(SGGS)2-SGSETPGTSESATPES-(SGGS)2(SEQ ID NO:800)。
216.权利要求200-215中任一项的方法，其中所述第一腺苷脱氨酶是SEQ ID NO:805的腺苷脱氨酶；并且所述第二腺苷脱氨酶是SEQ ID NO:1的腺苷脱氨酶。
217.权利要求200-215中任一项的方法，其中所述第一腺苷脱氨酶是SEQ ID NO:804的腺苷脱氨酶；并且所述第二腺苷脱氨酶是SEQ ID NO:1的腺苷脱氨酶。
218.权利要求200-215中任一项的方法，其中所述第一腺苷脱氨酶是SEQ ID NO:803的腺苷脱氨酶；并且所述第二腺苷脱氨酶是SEQ ID NO:1的腺苷脱氨酶。
219.权利要求200-215中任一项的方法，其中所述第一腺苷脱氨酶是SEQ ID NO:802的腺苷脱氨酶；并且所述第二腺苷脱氨酶是SEQ ID NO:1的腺苷脱氨酶。
220.权利要求200-215中任一项的方法，其中所述第一腺苷脱氨酶是SEQ ID NO:682的腺苷脱氨酶；并且所述第二腺苷脱氨酶是SEQ ID NO:1的腺苷脱氨酶。
221.权利要求172-220中任一项的方法，其中所述融合蛋白进一步包含(ii)与所述核酸可编程DNA结合蛋白(napDNAbp)结合的引导RNA，其中所述引导RNA(gRNA)包含与所述HBG1和/或HBG2基因的启动子中的靶核酸序列互补的引导序列。
222.权利要求221的方法，其中所述引导RNA的引导序列包含核酸序列
5′-UCAUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:846)；
5′-CAUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:847)；
5′-AUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:848)；
5′-UGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:849).
5′-GUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:850)；
5′-UGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:851)；
5′-GGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:852)；或
5′-GGGAAGGGGCCCCCAAG-3′(SEQ ID NO:853)。
223.权利要求222的方法，其中所述gRNA的引导序列进一步包含5′末端处的G。
224.权利要求222或223的方法，其中所述gRNA的引导序列包含5′-GUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:850)。
225.权利要求172-220中任一项的方法，其中所述融合蛋白进一步包含(ii)与所述核酸可编程DNA结合蛋白(napDNAbp)结合的引导RNA，其中所述引导RNA包含与所述HFE基因中的靶核酸序列互补的引导序列。
226.权利要求225的方法，其中所述gRNA的引导序列包含核酸序列：
5′-AUAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:862)；
5′-UAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:863)；
5′-AUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:864)；
5′-UACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:865)；
5′-ACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:866)；
5′-CGUACCAGGUGGAGCACCC-3′(SEQ ID NO:867)；
5′-GUACCAGGUGGAGCACCC-3′(SEQ ID NO:868)；或
5′-UACCAGGUGGAGCACCC-3′(SEQ ID NO:869)。
227.权利要求226的方法，其中所述gRNA的引导序列进一步包含权利要求226中列出的序列的任一个的5′末端处的G。
228.权利要求225-227中任一项的方法，其中所述gRNA的引导序列包含核酸序列5’-GACGUACCAGGUGGAGCACCC-3’(SEQ ID NO:870)。
229.权利要求172-220中任一项的方法，其中所述融合蛋白进一步包含(ii)与所述核酸可编程DNA结合蛋白(napDNAbp)结合的引导RNA，其中所述引导RNA(gRNA)包含与所述HBG1和/或HBG2基因的启动子中的靶核酸序列互补的引导序列。
230.权利要求229的方法，其中所述引导RNA的引导序列包含核酸序列
5′-GACAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:254)；
5′-ACAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:255)；
5′-CAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:256)；
5′-AGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:257)；
5′-GAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:258)；
5′-AUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:259)；
5′-AUGCAAAUAUCUGUCUGAAACGG-3′(SEQ ID NO:260)；
5′-GCAAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:261)；
5′-CAAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:262)；
5′-AAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:263)；
5′-AAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:264)；
5′-AUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:265)；
5′-UAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:266)；
5′-AGAUAUUUGCAUUGAGAUAGUGU-3′(SEQ ID NO:267)；
5′-ACAGAUAUUUGCAUUGAGAUAGU-3′(SEQ ID NO:268)；
5′-GUGGGGAAGGGGCCCCCAAGAGG-3′(SEQ ID NO:269)；
5′-CUUGACCAAUAGCCUUGACAAGG-3′(SEQ ID NO:270)；
5′-CUUGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:271)；
5′-UUGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:272)；
5′-UGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:273)；
5′-GUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:274)；
5′-UCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:275)；
5′-CAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:276)；
5′-UUGUCAAGGCUAUUGGUCAAGGC-3′(SEQ ID NO:277)；
5′-CUUGUCAAGGCUAUUGGUCAAGG-3′(SEQ ID NO:278)；
5′-UUGACCAAUAGCCUUGACAAGGC-3′(SEQ ID NO:279)；或
5′-UAGCCUUGACAAGGCAAACUUGA-3′(SEQ ID NO:280)。
231.权利要求230的方法，其中所述gRNA的引导序列进一步包含权利要求230中列出的序列的任一个的5′末端处的G。
232.权利要求172-220中任一项的方法，其中所述融合蛋白进一步包含(ii)与所述核酸可编程DNA结合蛋白(napDNAbp)结合的引导RNA，其中所述引导RNA包含与所述HBB基因中的靶核酸序列互补的引导序列。
233.权利要求232的方法，其中所述gRNA的引导序列包含核酸序列：
5′-UUCUCCACAGGAGUCAGAUGCAC-3′(SEQ ID NO:281)；
5′-UCUCCACAGGAGUCAGAUGCACC-3′(SEQ ID NO:282)；
5′-UUCUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:283)；
5′-UCUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:284)；
5′-CUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:285)；
5′-UCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:286)；
5′-CCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:287)；
5′-CACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:288)；
5′-ACUUCUCCACAGGAGUCAGAUGC-3′(SEQ ID NO:289)；
5′-UCUCCACAGGAGUCAGAUGCACCAUGGU-3′(SEQ ID NO:290)；
5′-ACUCCUAAGGAGAAGUCUGCCGU-3′(SEQ ID NO:291)；
5′-UUGGUGGUAAGGCCCUGGGCAGG-3′(SEQ ID NO:292)；
5′-UGGUGGUAAGGCCCUGGGCAGGU-3′(SEQ ID NO:293)；或
5′-UGGUAAGGCCCUGGGCAGGUUGG-3′(SEQ ID NO:294)。
234.权利要求233的方法，其中所述gRNA的引导序列进一步包含权利要求233中列出的序列的任一个的5′末端处的G。
235.权利要求172-220中任一项的方法，其中所述融合蛋白进一步包含(ii)与所述核酸可编程DNA结合蛋白(napDNAbp)结合的引导RNA，其中所述引导RNA包含与所述F8基因中的靶核酸序列互补的引导序列。
236.权利要求235的方法，其中所述gRNA的引导序列包含核酸序列：
5′-AGCAUUGUAUAUUCUCUGUGAGG-3′(SEQ ID NO:295)；或
5′-AAAGCAUUGUAUAUUCUCUGUGA-3′(SEQ ID NO:296)。
237.权利要求236的方法，其中所述gRNA的引导序列进一步包含权利要求236中列出的序列的任一个的5′末端处的G。
238.权利要求1-237中任一项的方法，其中所述碱基编辑器包含SEQ ID NO:707的氨基酸序列或由其组成。
239.权利要求1-237中任一项的方法，其中所述碱基编辑器包含SEQ ID NO:708的氨基酸序列或由其组成。
240.权利要求1-237中任一项的方法，其中所述碱基编辑器包含SEQ ID NO:709的氨基酸序列或由其组成。
241.权利要求1-237中任一项的方法，其中所述碱基编辑器包含SEQ ID NO:710的氨基酸序列或由其组成。
242.权利要求1-237中任一项的方法，其中所述碱基编辑器包含SEQ ID NO:711的氨基酸序列或由其组成。
243.权利要求1-242中任一项的方法，其中所述方法导致小于20％、19％、18％、16％、14％、12％、10％、8％、6％、4％、2％、1％、0.5％、0.2％或0.1％的插入/缺失形成。
244.权利要求1-243中任一项的方法，其中使A核碱基脱氨基的效率是至少5％。
245.权利要求244的方法，其中使A核碱基脱氨基的效率是至少10％、15％、20％、25％、30％、35％、40％、45％、50％、60％、70％、80％、90％、95％或98％。
246.包含核酸构建体的试剂盒，其包含
(a)编码碱基编辑器融合蛋白的核酸序列，所述碱基编辑器融合蛋白包含(i)核酸可编程DNA结合蛋白(napDNAbp)和(ii)能够使DNA中的腺苷脱氨基的腺苷脱氨酶；
和
(b)引导RNA或编码所述引导RNA的表达构建体，其中所述引导RNA包含与以下的有义或反义链中的靶核酸序列互补的引导序列
(i)HBG1和/或HBG2基因的启动子；
(ii)HFE基因；
(iii)HBB基因；或
(iv)F8基因。
247.权利要求246的试剂盒，其中所述靶核酸序列是所述HBG1和/或HBG2基因的启动子中的核酸序列。
248.权利要求247的试剂盒，其中所述靶核酸序列包含核酸序列:
5′-CTTGGGGGCCCCTTCCCCACACTA-3′(SEQ ID NO:838)；
5′-CTTGGGGGCCCCTTCCCCACACT-3′(SEQ ID NO:839)；
5′-CTTGGGGGCCCCTTCCCCACAC-3′(SEQ ID NO:840)；
5′-CTTGGGGGCCCCTTCCCCACA-3′(SEQ ID NO:841)；
5′-CTTGGGGGCCCCTTCCCCAC-3′(SEQ ID NO:842)；
5′-CTTGGGGGCCCCTTCCCCA-3′(SEQ ID NO:843)；
5′-CTTGGGGGCCCCTTCCCC-3′(SEQ ID NO:844)；或
5′-CTTGGGGGCCCCTTCCC-3′(SEQ ID NO:845)。
249.权利要求248的试剂盒，其中所述靶核酸进一步包含权利要求238中列出的序列的任一个的5′末端处的5′-CCT-3′。
250.权利要求247-249中任一项的试剂盒，其中所述启动子中的靶核酸序列包含核酸序列5′-CTTGGGGGCCCCTTCCCCAC-3′(SEQ ID NO:842)。
251.权利要求246的试剂盒，其中所述靶核酸序列是所述HFE基因中的核酸序列。
252.权利要求250的试剂盒，其中所述HFE基因中的靶核酸序列包含核酸序列:
5′-GGGTGCTCCACCTGGTACGTATAT-3′(SEQ ID NO:854)；
5′-GGGTGCTCCACCTGGTACGTATA-3′(SEQ ID NO:855)；
5′-GGGTGCTCCACCTGGTACGTAT-3′(SEQ ID NO:856)；
5′-GGGTGCTCCACCTGGTACGTA-3′(SEQ ID NO:857)；
5′-GGGTGCTCCACCTGGTACGT-3′(SEQ ID NO:858)；
5′-GGGTGCTCCACCTGGTACG-3′(SEQ ID NO:859)；
5′-GGGTGCTCCACCTGGTAC-3′(SEQ ID NO:860)；或
5′-GGGTGCTCCACCTGGTA-3′(SEQ ID NO:861)。
253.权利要求251的试剂盒，其中所述靶核酸进一步包含权利要求241中列出的序列的任一个的5′末端处的5′-CCT-3′。
254.权利要求250-252中任一项的试剂盒，其中所述HFE基因中的靶核酸序列包含核酸序列5′-GGGTGCTCCACCTGGTACGT-3′(SEQ ID NO:858)。
255.权利要求246的试剂盒，其中所述靶核酸序列是所述HBG1和/或HBG2基因的启动子中的核酸序列。
256.权利要求254的试剂盒，其中所述靶核酸序列包含核酸序列:
5′-CCACACTATCTCAATGCAAATATCTGTC-3′(SEQ ID NO:297)；
5′-CCACACTATCTCAATGCAAATATCTGT-3′(SEQ ID NO:298)；
5′-CCACACTATCTCAATGCAAATATCTG-3′(SEQ ID NO:299)；
5′-CCACACTATCTCAATGCAAATATCT-3′(SEQ ID NO:300)；
5′-CCACACTATCTCAATGCAAATATC-3′(SEQ ID NO:301)；
5′-CCACACTATCTCAATGCAAATAT-3′(SEQ ID NO:302)；
5′-CCGTTTCAGACAGATATTTGCAT-3′(SEQ ID NO:303)；
5′-CCAGGGACCGTTTCAGACAGATATTTGC-3′(SEQ ID NO:304)；
5′-CCAGGGACCGTTTCAGACAGATATTTG-3′(SEQ ID NO:305)；
5′-CCAGGGACCGTTTCAGACAGATATTT-3′(SEQ ID NO:306)；
5′-CCAGGGACCGTTTCAGACAGATATT-3′(SEQ ID NO:307)；
5′-CCAGGGACCGTTTCAGACAGATAT-3′(SEQ ID NO:308)；
5′-CCAGGGACCGTTTCAGACAGATA-3′(SEQ ID NO:309)；
5′-ACACTATCTCAATGCAAATATCT-3′(SEQ ID NO:310)；
5′-ACTATCTCAATGCAAATATCTGT-3′(SEQ ID NO:311)；
5′-CCTCTTGGGGGCCCCTTCCCCAC-3′(SEQ ID NO:312)；
5′-CCTTGTCAAGGCTATTGGTCAAG-3′(SEQ ID NO:313)；
5′-CCTTGCCTTGACCAATAGCCTTGACAAG-3′(SEQ ID NO:314)；
5′-CCTTGCCTTGACCAATAGCCTTGACAA-3′(SEQ ID NO:315)；
5′-CCTTGCCTTGACCAATAGCCTTGACA-3′(SEQ ID NO:316)；
5′-CCTTGCCTTGACCAATAGCCTTGAC-3′(SEQ ID NO:317)；
5′-CCTTGCCTTGACCAATAGCCTTGA-3′(SEQ ID NO:318)；
5′-CCTTGCCTTGACCAATAGCCTTG-3′(SEQ ID NO:319)；
5′-GCCTTGACCAATAGCCTTGACAA-3′(SEQ ID NO:320)；
5′-CCTTGACCAATAGCCTTGACAAG-3′(SEQ ID NO:321)；
5′-GCCTTGTCAAGGCTATTGGTCAA-3′(SEQ ID NO:322)；或
5′-TCAAGTTTGCCTTGTCAAGGCTA-3′(SEQ ID NO:323)。
257.权利要求256的试剂盒，其中所述靶核酸序列是所述HBB基因中的核酸序列。
258.权利要求256的试剂盒，其中所述HBB基因中的靶核酸序列包含核酸序列:
5′-GTGCATCTGACTCCTGTGGAGAA-3′(SEQ ID NO:324)；
5′-GGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:325)；
5′-CCATGGTGCATCTGACTCCTGTGGAGAA-3′(SEQ ID NO:326)；
5′-CCATGGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:327)；
5′-CCATGGTGCATCTGACTCCTGTGGAG-3′(SEQ ID NO:328)；
5′-CCATGGTGCATCTGACTCCTGTGGA-3′(SEQ ID NO:329)；
5′-CCATGGTGCATCTGACTCCTGTGG-3′(SEQ ID NO:330)；
5′-CCATGGTGCATCTGACTCCTGTG-3′(SEQ ID NO:331)；
5′-GCATCTGACTCCTGTGGAGAAGT-3′(SEQ ID NO:332)；
5′-ACCATGGTGCATCTGACTCCTGTGGAGA-3′(SEQ ID NO:333)；
5′-ACGGCAGACTTCTCCTTAGGAGT-3′(SEQ ID NO:334)；
5′-CCTGCCCAGGGCCTTACCACCAA-3′(SEQ ID NO:335)；
5′-ACCTGCCCAGGGCCTTACCACCA-3′(SEQ ID NO:336)；或
5′-CCAACCTGCCCAGGGCCTTACCA-3′(SEQ ID NO:337)。
259.权利要求246的试剂盒，其中所述靶核酸序列是所述F8基因中的核酸序列。
260.权利要求258的试剂盒，其中所述F8基因中的靶核酸序列包含核酸序列:
5′-CCTCACAGAGAATATACAATGCT-3′(SEQ ID NO:338)；或
5′-TCACAGAGAATATACAATGCTTT-3′(SEQ ID NO:339)。
261.复合物，其包含(i)本文提供的任何融合蛋白和(ii)引导RNA，其中所述引导RNA包含包括以下核酸序列的引导序列
5′-UCAUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:846)；
5′-CAUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:847)；
5′-AUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:848)；
5′-UGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:849).
5′-GUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:850)；
5′-UGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:851)；
5′-GGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:852)；或
5′-GGGAAGGGGCCCCCAAG-3′(SEQ ID NO:853)。
262.复合物，其包含(i)本文提供的任何融合蛋白和(ii)引导RNA，其中所述引导RNA包含包括以下核酸序列的引导序列
5′-AUAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:862)；
5′-UAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:863)；
5′-AUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:864)；
5′-UACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:865)；
5′-ACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:866)；
5′-CGUACCAGGUGGAGCACCC-3′(SEQ ID NO:867)；
5′-GUACCAGGUGGAGCACCC-3′(SEQ ID NO:868)；或
5′-UACCAGGUGGAGCACCC-3′(SEQ ID NO:869)。
263.复合物，其包含(i)本文提供的任何融合蛋白和(ii)引导RNA，其中所述引导RNA包含包括以下核酸序列的引导序列
5′-GACAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:254)；
5′-ACAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:255)；
5′-CAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:256)；
5′-AGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:257)；
5′-GAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:258)；
5′-AUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:259)；
5′-AUGCAAAUAUCUGUCUGAAACGG-3′(SEQ ID NO:260)；
5′-GCAAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:261)；
5′-CAAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:262)；
5′-AAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:263)；
5′-AAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:264)；
5′-AUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:265)；
5′-UAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:266)；
5′-AGAUAUUUGCAUUGAGAUAGUGU-3′(SEQ ID NO:267)；
5′-ACAGAUAUUUGCAUUGAGAUAGU-3′(SEQ ID NO:268)；
5′-GUGGGGAAGGGGCCCCCAAGAGG-3′(SEQ ID NO:269)；
5′-CUUGACCAAUAGCCUUGACAAGG-3′(SEQ ID NO:270)；
5′-CUUGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:271)；
5′-UUGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:272)；
5′-UGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:273)；
5′-GUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:274)；
5′-UCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:275)；
5′-CAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:276)；
5′-UUGUCAAGGCUAUUGGUCAAGGC-3′(SEQ ID NO:277)；
5′-CUUGUCAAGGCUAUUGGUCAAGG-3′(SEQ ID NO:278)；
5′-UUGACCAAUAGCCUUGACAAGGC-3′(SEQ ID NO:279)；或
5′-UAGCCUUGACAAGGCAAACUUGA-3′(SEQ ID NO:280)。
264.复合物，其包含(i)本文提供的任何融合蛋白和(ii)引导RNA，其中所述引导RNA包含包括以下核酸序列的引导序列
5′-UUCUCCACAGGAGUCAGAUGCAC-3′(SEQ ID NO:281)；
5′-UCUCCACAGGAGUCAGAUGCACC-3′(SEQ ID NO:282)；
5′-UUCUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:283)；
5′-UCUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:284)；
5′-CUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:285)；
5′-UCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:286)；
5′-CCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:287)；
5′-CACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:288)；
5′-ACUUCUCCACAGGAGUCAGAUGC-3′(SEQ ID NO:289)；
5′-UCUCCACAGGAGUCAGAUGCACCAUGGU-3′(SEQ ID NO:290)；
5′-ACUCCUAAGGAGAAGUCUGCCGU-3′(SEQ ID NO:291)；
5′-UUGGUGGUAAGGCCCUGGGCAGG-3′(SEQ ID NO:292)；
5′-UGGUGGUAAGGCCCUGGGCAGGU-3′(SEQ ID NO:293)；或
5′-UGGUAAGGCCCUGGGCAGGUUGG-3′(SEQ ID NO:294)。
265.复合物，其包含(i)本文提供的任何融合蛋白和(ii)引导RNA，其中所述引导RNA包含包括以下核酸序列的引导序列
5′-AGCAUUGUAUAUUCUCUGUGAGG-3′(SEQ ID NO:295)；或
5′-AAAGCAUUGUAUAUUCUCUGUGA-3′(SEQ ID NO:296)。
266.引导RNA(sgRNA)，其包含核酸序列
5′-UCAUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:846)；
5′-CAUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:847)；
5′-AUGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:848)；
5′-UGUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:849).
5′-GUGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:850)；
5′-UGGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:851)；
5′-GGGGAAGGGGCCCCCAAG-3′(SEQ ID NO:852)；
5′-GGGAAGGGGCCCCCAAG-3′(SEQ ID NO:853)；
5′-AUAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:862)；
5′-UAUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:863)；
5′-AUACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:864)；
5′-UACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:865)；
5′-ACGUACCAGGUGGAGCACCC-3′(SEQ ID NO:866)；
5′-CGUACCAGGUGGAGCACCC-3′(SEQ ID NO:867)；
5′-GUACCAGGUGGAGCACCC-3′(SEQ ID NO:868)；
5′-UACCAGGUGGAGCACCC-3′(SEQ ID NO:869)；
5′-GACAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:254)；
5′-ACAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:255)；
5′-CAGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:256)；
5′-AGAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:257)；
5′-GAUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:258)；
5′-AUAUUUGCAUUGAGAUAGUGUGG-3′(SEQ ID NO:259)；
5′-AUGCAAAUAUCUGUCUGAAACGG-3′(SEQ ID NO:260)；
5′-GCAAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:261)；
5′-CAAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:262)；
5′-AAAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:263)；
5′-AAUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:264)；
5′-AUAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:265)；
5′-UAUCUGUCUGAAACGGUCCCUGG-3′(SEQ ID NO:266)；
5′-AGAUAUUUGCAUUGAGAUAGUGU-3′(SEQ ID NO:267)；
5′-ACAGAUAUUUGCAUUGAGAUAGU-3′(SEQ ID NO:268)；
5′-GUGGGGAAGGGGCCCCCAAGAGG-3′(SEQ ID NO:269)；
5′-CUUGACCAAUAGCCUUGACAAGG-3′(SEQ ID NO:270)；
5′-CUUGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:271)；
5′-UUGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:272)；
5′-UGUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:273)；
5′-GUCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:274)；
5′-UCAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:275)；
5′-CAAGGCUAUUGGUCAAGGCAAGG-3′(SEQ ID NO:276)；
5′-UUGUCAAGGCUAUUGGUCAAGGC-3′(SEQ ID NO:277)；
5′-CUUGUCAAGGCUAUUGGUCAAGG-3′(SEQ ID NO:278)；
5′-UUGACCAAUAGCCUUGACAAGGC-3′(SEQ ID NO:279)；
5′-UAGCCUUGACAAGGCAAACUUGA-3′(SEQ ID NO:280)；
5′-UUCUCCACAGGAGUCAGAUGCAC-3′(SEQ ID NO:281)；
5′-UCUCCACAGGAGUCAGAUGCACC-3′(SEQ ID NO:282)；
5′-UUCUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:283)；
5′-UCUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:284)；
5′-CUCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:285)；
5′-UCCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:286)；
5′-CCACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:287)；
5′-CACAGGAGUCAGAUGCACCAUGG-3′(SEQ ID NO:288)；
5′-ACUUCUCCACAGGAGUCAGAUGC-3′(SEQ ID NO:289)；
5′-UCUCCACAGGAGUCAGAUGCACCAUGGU-3′(SEQ ID NO:290)；
5′-ACUCCUAAGGAGAAGUCUGCCGU-3′(SEQ ID NO:291)；
5′-UUGGUGGUAAGGCCCUGGGCAGG-3′(SEQ ID NO:292)；
5′-UGGUGGUAAGGCCCUGGGCAGGU-3′(SEQ ID NO:293)；或
5′-UGGUAAGGCCCUGGGCAGGUUGG-3′(SEQ ID NO:294)；
5′-AGCAUUGUAUAUUCUCUGUGAGG-3′(SEQ ID NO:295)；或
5′-AAAGCAUUGUAUAUUCUCUGUGA-3′(SEQ ID NO:296)。
267.权利要求265的引导RNA，其中所述引导RNA是单一引导RNA(sgRNA)。
268.核酸，其编码权利要求265或266的引导RNA。
269.载体，其包含权利要求267的核酸。
270.权利要求268的载体，其中所述载体包含驱动所述引导RNA表达的异源启动子。
271.药物组合物，其包含权利要求260或264的复合物；权利要求265或266的引导RNA；权利要求267的核酸；或权利要求268或269的载体。
272.权利要求270的药物组合物，其进一步包含药学上可接受的赋形剂。
273.权利要求270或271的药物组合物，其进一步包含阳离子脂质或阳离子聚合物。
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