CN111718418B - 一种增强基因编辑的融合蛋白及其应用 - Google Patents

一种增强基因编辑的融合蛋白及其应用 Download PDF

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CN111718418B
CN111718418B CN201910210107.1A CN201910210107A CN111718418B CN 111718418 B CN111718418 B CN 111718418B CN 201910210107 A CN201910210107 A CN 201910210107A CN 111718418 B CN111718418 B CN 111718418B
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CN111718418A (zh
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李大力
尹树明
张梅
陈曦
张晓辉
王立人
刘明耀
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Shanghai Bangyao Biological Technology Co ltd
East China Normal University
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East China Normal University
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Abstract

本发明涉及一种增强基因编辑的融合蛋白及其应用。具体地，本发明提供一种增强型融合蛋白。相比野生型的基因编辑蛋白，本发明的增强型融合蛋白可显著提高体内或体外的基因编辑效率。

Description

一种增强基因编辑的融合蛋白及其应用
技术领域
本发明涉及生物技术领域，具体地涉及一种增强基因编辑的融合蛋白及其应用。
背景技术
基因编辑技术是通过人为地实现双链DNA的断裂，利用双链DNA断裂的修复机制来实现基因操纵的一门技术。目前已有的基因编辑技术包括ZFN、TALEN和CRISPR/Cas9技术，其中以CRISPR/Cas9技术应用最为广泛。CRISPR/Csa9技术是来源于细菌或古细菌的一种获得性免疫机制，利用一条单链向导RNA(sgRNA)和Csa9蛋白定向的在基因组特定位置产生DNA双链断裂，进而通过内源的非同源末端连接(NHEJ)或同源重组(HDR)修复机制，来实现目的基因的敲除或特定基因或片段的插入。
CRISPR/Cas9技术虽然有着无比强大的功能，但也有其缺点，如：1.脱靶问题；2.PAM的限制导致靶点选择的有限性；3.一些新出来的工具的编辑效率总体偏低(XCas9和SpCas0-NG)，并且目前对于基因编辑工具的优化和改造主要是基于增强工具的精确性和增强靶向范围的改造，还没有针对于基因编辑工具本身来广泛的提高各类基因编辑工具的方法。
因此，本领域迫切需与开发一种新的提高基因编辑效率的方法。
发明内容
本发明的目的是提供一种新的提高基因编辑效率的方法。
本发明第一方面提供了一种融合蛋白，所述融合蛋白的结构如下式I或I’所示：
C-A-L-B (I)
B-L-A-C (I’)
式中，
A为基因编辑蛋白，
B为DNA双链结合结构域，
C为任选的碱基编辑器元件；
L为无或连接肽，
各“-”独立地为连接肽或肽键或非肽键。
在另一优选例中，当融合蛋白的结构如式I’所示时，C为无。
在另一优选例中，所述非肽键包括PEG。
在另一优选例中，所述基因编辑蛋白选自下组：Cas9、Cas9a、Cas12、Cas12a、Cas12b、Cas13、Cas14、或其组合。
在另一优选例中，所述基因编辑蛋白包括野生型或突变型的基因编辑蛋白。
在另一优选例中，所述基因编辑蛋白选自下组：酿脓链球菌(Streptococcuspyogenes)、葡萄球菌(Staphylococcus aureus)、氨基酸球菌属(Acidaminococcus sp)、毛螺科菌(Lachnospiraceae bacterium)、或其组合。
在另一优选例中，所述野生型的基因编辑蛋白的氨基酸序列如SEQ ID NO.:1、14或15所示。
在另一优选例中，所述碱基编辑器元件的氨基酸序列如SEQ ID NO.:2或12所示。
在另一优选例中，所述DNA双链结合结构域为非序列特异性的DNA双链结合结构域。
在另一优选例中，所述DNA双链结合结构域选自下组：HMG-D、Sac7d、或其组合。
在另一优选例中，所述DNA双链结合结构域包括野生型的DNA双链结合结构域和突变型的DNA双链结合结构域。
在另一优选例中，所述DNA双链结合结构域来源于果蝇或古细菌。
在另一优选例中，所述DNA双链结合结构域的氨基酸序列如SEQ ID NO.:10或11所示。
在另一优选例中，所述连接肽的长度为1-100aa，较佳地，15-85aa，更佳地，25-70aa。
在另一优选例中，所述连接肽为具有n个重复的如Gly-Gly-Ser所示的序列，其中n为2-8，优选地n为3-6。
在另一优选例中，所述连接肽的氨基酸序列选自下组：
(1)氨基酸序列如SEQ ID NO.:3-7中任一所示的多肽；
(2)将SEQ ID NO.:3-7中任一所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-8个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(1)所述多肽功能的由SEQ ID NO.:3-7中任一所示氨基酸序列的多肽衍生的多肽。
在另一优选例中，所述碱基编辑器元件包括胞嘧啶脱氨酶、腺嘌呤脱氨酶。
在另一优选例中，所述胞嘧啶脱氨酶包括Apobec1、Apobec3A。
在另一优选例中，所述腺嘌呤脱氨酶包括TadA。
在另一优选例中，所述融合蛋白具有SEQ ID NO.:8、9、13中任一所示的氨基酸序列。
本发明第二方面提供了一种多核苷酸，所述的多核苷酸编码本发明第一方面所述的融合蛋白。
在另一优选例中，所述的多核苷酸在所述突变蛋白或融合蛋白的ORF的侧翼还额外含有选自下组的辅助元件：信号肽、分泌肽、标签序列(如6His)、或其组合。
在另一优选例中，所述的多核苷酸选自下组：DNA序列、RNA序列、或其组合。
本发明第三方面提供了一种载体，所述的载体含有本发明第二方面所述的多核苷酸。
在另一优选例中，所述载体包含一个或多个启动子，所述启动子可操作地与所述核酸序列、增强子、转录终止信号、多腺苷酸化序列、复制起点、选择性标记、核酸限制性位点、和/或同源重组位点连接。
在另一优选例中，所述载体包括质粒、病毒载体。
在另一优选例中，所述的病毒载体选自下组：腺相关病毒(AAV)、腺病毒、慢病毒、逆转录病毒、疱疹病毒、SV40、痘病毒、或其组合。
在另一优选例中，所述载体包括表达载体、穿梭载体、整合载体。
本发明第三方面提供了一种宿主细胞，所述宿主细胞含有本发明第三方面所述的载体，或其基因组中整合有本发明第二方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞为真核细胞，如酵母细胞、植物细胞或哺乳动物细胞(包括人和非人哺乳动物)。
在另一优选例中，所述的宿主细胞为原核细胞，如大肠杆菌。
在另一优选例中，所述酵母细胞选自下组的一种或多种来源的酵母：毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为马克斯克鲁维酵母、和/或乳酸克鲁维酵母。
在另一优选例中，所述宿主细胞选自下组：大肠杆菌、麦胚细胞，昆虫细胞，SF9、Hela、HEK293、CHO、酵母细胞、或其组合。
本发明第五方面提供了一种产生本发明第一方面所述的融合蛋白的方法，包括步骤：
在适合表达的条件下，培养本发明第四方面所述的宿主细胞，从而表达融合蛋白；和/或
分离所述融合蛋白。
本发明第六方面提供了一种基因编辑试剂，所述基因编辑试剂包含本发明第一方面所述的融合蛋白。
在另一优选例中，所述的试剂还包括选自下组的一种或多种试剂：
(a1)gRNA、crRNA、或用于产生所述gRNA或crRNA的载体；
(a2)用于同源定向修复的模板：单链核苷酸序列或质粒载体。
本发明第七方面提供了一种试剂盒，包括本发明第六方面所述的基因编辑试剂。
在另一优选例中，所述试剂盒还包括选自下组的一种或多种试剂：
(a1)gRNA、crRNA、或用于产生所述gRNA或crRNA的载体；
(a2)用于同源定向修复的模板：单链核苷酸序列或质粒载体。
在另一优选例中，所述试剂盒还包括标签或说明书。
本发明第八方面提供了一种本发明第一方面所述的融合蛋白的用途，用于制备试剂或试剂盒，所述试剂或试剂盒用于提高基因编辑效率。
本发明第九方面提供了一种药物组合物，包括：
(a)本发明第一方面所述的融合蛋白、或其编码基因、或其表达载体(vector)；和
(b)药学上可接受的载体(carrier)。
在另一优选例中，所述表达载体包括病毒载体。
在另一优选例中，所述的病毒载体选自下组：腺相关病毒(AAV)、腺病毒、慢病毒、逆转录病毒、疱疹病毒、SV40、痘病毒、或其组合。
在另一优选例中，所述的载体选自下组：慢病毒、腺病毒、腺相关病毒(AAV)、或其组合，较佳地，所述载体为腺相关病毒(AAV)。
在另一优选例中，所述药物组合物的剂型选自下组：冻干制剂、液体制剂、或其组合。
在另一优选例中，所述药物组合物的剂型为注射剂型。
在另一优选例中，所述药物组合物中还包括其他用于基因治疗的药物。
在另一优选例中，所述其他用于基因治疗的药物选自下组：反义核苷酸药物、EDIT-101药物、CTX001、或其组合。
在另一优选例中，所述药物组合物为细胞制剂。
本发明第十方面提供了一种药盒，包括：
(a1)第一容器，以及位于所述第一容器中的本发明第一方面所述的融合蛋白、其编码基因、或其表达载体，或含有本发明第一方面所述的融合蛋白的药物。
在另一优选例中，所述药盒还包括：
(a2)第二容器，以及位于所述第二容器中的其他用于基因治疗的药物，或含有其他用于基因治疗的药物的药物。
在另一优选例中，所述的第一容器和第二容器是相同或不同的容器。
在另一优选例中，所述的第一容器的药物是含权利要求1所述的融合蛋白的单方制剂。
在另一优选例中，所述的第二容器的药物是含其他用于基因治疗的药物的单方制剂。
在另一优选例中，所述药物的剂型选自下组：冻干制剂、液体制剂、或其组合。
在另一优选例中，所述药物的剂型为注射剂型。
本发明第十一方面提供了一种本发明第一方面所述的融合蛋白的用途，用于制备用于基因治疗的药物。
本发明第十二方面提供了一种提高基因编辑效率的方法，包括步骤：
在本发明第一方面所述的融合蛋白或本发明第六方面所述的基因编辑试剂存在下，对细胞进行基因编辑，从而提高基因编辑效率。
在另一优选例中，所述细胞包括人或非人哺乳动物细胞(如灵长类动物或家畜)。
在另一优选例中，所述细胞包括癌细胞或正常细胞。
在另一优选例中，所述细胞选自下组：肾脏细胞、肝脏细胞、神经细胞、心脏细胞、上皮细胞、肌细胞、体细胞、骨髓细胞、内皮细胞、或其组合。
在另一优选例中，所述细胞选自下组：293细胞、A549细胞、SW626细胞、HT-3细胞、PA-1细胞、或其组合。
在另一优选例中，所述细胞包括HEK293T。
在另一优选例中，所述的基因编辑在一体外反应体系中进行。
在另一优选例中，所述的体外反应体系中，所述融合蛋白或基因编辑试剂的含量为100ng－700ng，较佳地，200ng－600ng，更佳地，300ng—500ng。
在另一优选例中，所述方法为非诊断性和非治疗性的。
在另一优选例中，所述细胞为体外的细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了对于两个不同的内源靶点的编辑效率，说明DNA双链结合结构域和Cas9的不同连接方式，以及不同长度的linker对效率的提高都有区别，综合选出HMG-D结构域通过L4长度的linker连接在Cas9的N端最好。即：HMG-D-L4-Cas9，其中，H代表HMG-D；S代表Sac7d；L1-L5代表不同长度的linker；mutH代表突变的HMG-D(V32A和T33A突变，降低了结合活性)；C代表Cas9。
图2显示了HMG-D-L4-Cas9在其他内源性靶点的编辑效率也是可以提高的，效率提高＞20％，较佳地，＞40％，更佳地，＞60％(如80％)，最高可达2倍，其中，H代表HMG-D。
图3显示了双链结合结构域HMG-D可以提高其他来源Cas9蛋白(如：SaCas9)的效率，效率提高了20％以上。
图4显示了显示了双链结合结构域HMG-D可以提高非Cas9蛋白(如：AsCas12a)的效率，效率提高了10-20％。
图5显示了双链结合结构域HMG-D可以提高表观调控工具(如：CRISPR-VPR)的效率，效率可以提高2倍。其中，endCas9即为HMG-D-L4-dCas9。
图6显示了双链结合结构域HMG-D可以提高单碱基编辑工具ABE的效率，其中，H代表HMG-D。
具体实施方式
本发明人经过广泛而深入的研究，意外地获得一种增强型融合蛋白。相比野生型基因编辑蛋白，本发明的增强型融合蛋白可显著提高体内或体外的基因编辑效率，并且本发明还意外的发现，基因编辑蛋白和DNA双链结合结构域、任选的碱基编辑器元件以及任选的连接肽所形成的融合蛋白，可显著提高基因编辑效率(提高≥20％，如80％，甚至可达2倍)，此外，本发明还意外的发现，本发明的融合蛋白可用于基因治疗。在此基础上，本发明人完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
序列同一性(或同源性)通过沿着预定的比较窗(其可以是参考核苷酸序列或蛋白的长度的50％、60％、70％、80％、90％、95％或100％)比较两个对齐的序列，并且确定出现相同的残基的位置的数目来确定。通常地，这表示为百分比。核苷酸序列的序列同一性的测量是本领域技术人员熟知的方法。
如本文所用，术语“EDIT-101药物”属于基因治疗药物，是一类细胞，具体地，EDIT-101是一种利用CRISPR基因编辑技术治疗遗传性视网膜衰退疾病(LCA10疾病)的药物，EDIT-101通过视网膜下注射给药，将基因编辑系统直接输送到感光细胞中而达到治疗效果。
如本文所用，术语“CTX001”属于基因治疗药物，是一类细胞，具体地，CTX001是基于CRISPR基因编辑技术通过剪切β-地中海贫血患者的BCL11A基因实现治疗目的。
野生型的基因编辑蛋白
如本文所用，“野生型的基因编辑蛋白”是指天然存在的、未经过人工改造的基因编辑蛋白，其核苷酸可以通过基因工程技术来获得，如基因组测序、聚合酶链式反应(PCR)等，其氨基酸序列可由核苷酸序列推导而得到。所述野生型的基因编辑蛋白的来源包括(但并不限于)：酿脓链球菌(Streptococcus pyogenes)、葡萄球菌(Staphylococcus aureus)、氨基酸球菌属(Acidaminococcus sp)、毛螺科菌(Lachnospiraceae bacterium)。
在本发明的一个优选例中，所述野生型基因编辑蛋白的氨基酸序列如SEQ IDNO.:1或14或15所示。
在本发明的一个优选例中，所述基因编辑蛋白包括，但并不限于，Cas9、Cas9a、Cas12、Cas12a、Cas12b、Cas13、Cas14。
DNA双链结合结构域
如本文所用，术语“DNA双链结合结构域”为无序列特异性的DNA双链结合结构域。与序列特异性的DNA双链结合结构域相比，本发明的无序列特异性的DNA双链结合结构域不受DNA序列的限制，理论上可以结合任何DNA序列，因此，可以适用于任何位置的DNA的结合。一种优选的DNA双链结合结构域的序列如SEQ ID NO.:10或11所示。
碱基编辑器
本文提供的任何碱基编辑器能够修饰特定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”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(例如突变或脱氨基化)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产生相对于插入/缺失更大比例的意图修饰(例如，点突变或脱氨基化)。
本发明的任何碱基编辑器能够有效地在核酸(例如基因组内的核酸)中产生意图的突变，如点突变，而不产生大量的非意图突变，诸如非意图点突变。
在本发明中，碱基编辑器包括胞嘧啶脱氨酶和腺嘌呤脱氨酶，其他类型的碱基编辑器只要具备本发明的碱基编辑器的功能也在本发明的保护范围内。
在本发明中，将基因编辑蛋白与碱基编辑器融合后的结构称之为ABE或CBE，其中，ABE为基因编辑蛋白与腺嘌呤脱氨酶融合后的结构，CBE为基因编辑蛋白与胞嘧啶脱氨酶融合后的结构。
一种优选的碱基编辑器的序列如SEQ ID NO.:2或12所示。
融合蛋白
如本文所用，“本发明融合蛋白”、或“多肽”均指本发明第二方面所述的融合蛋白。本发明融合蛋白的结构如下式I或I’所示：
C-A-L-B (I)
B-L-A-C (I’)
式中，
A为基因编辑蛋白，
B为DNA双链结合结构域，
C为任选的碱基编辑器元件；
L为无或连接肽，
各“-”独立地为连接肽或肽键或非肽键。
在本发明中，连接肽的长度对融合蛋白的活性有影响，优选的连接肽的长度为1-100aa，较佳地，15-85aa，更佳地，25-70aa。
一种优选的连接肽如SEQ ID NO.:3-7所示。
如本文所用，术语“融合蛋白”还包括具有上述活性的、SEQ ID NO.:8、9、或13所示的变异形式。这些变异形式包括(但并不限于)：1-3个(通常为1-2个，更佳地1个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为3个以内，较佳地为2个以内，更佳地为1个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式的本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
本发明还包括上述融合蛋白的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明融合蛋白的功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)抗原肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与式I的氨基酸序列相比，有至多3个，较佳地至多2个，更佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供本发明融合蛋白的类似物。这些类似物与SEQ ID NO.:8或9或13所示的多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
在本发明中，在式I中，A为基因编辑蛋白，B为HMG-D或Sac7d，C为腺嘌呤脱氨酶或胞嘧啶脱氨酶或无，L为L1或L2或L3或L4或L5或无。
在一优选实施方式中，在式I中，A为基因编辑蛋白，B为HMG-D，C为或腺嘌呤脱氨酶或胞嘧啶脱氨酶或无，L为L4或L5。
在一优选实施方式中，本发明的融合蛋白还可以包括式I中的A、B、C、L元件中的两种或多种。
在一优选实施方式中，在式I中，A为基因编辑蛋白，B为HMG-9，C为无，L为L4。
在一优选实施方式中，在式I中，A为基因编辑蛋白，B为HMG-D，C为腺嘌呤脱氨酶，L为L5。
在一优选实施方式中，在式I中，A为基因编辑蛋白，B为HMG-D，C为胞嘧啶脱氨酶，L为L5。
在一优选实施方式中，本发明的融合蛋白的氨基酸序列如SEQ ID NO.:8、9或13所示。
腺相关病毒
因腺相关病毒(Adeno-associated virus,AAV)较其他病毒载体小，无致病性，可转染正在分裂和未分裂的细胞等特性，基于AAV载体的针对遗传性疾病的基因治疗方法受到了广泛的关注。
腺相关病毒(adeno-associated virus，AAV),也称腺伴随病毒,属于微小病毒科依赖病毒属,是目前发现的一类结构最简单的单链DNA缺陷型病毒，需要辅助病毒(通常为腺病毒)参与复制。它编码两个末端的反向重复序列(ITR)中的cap和rep基因。ITRs对于病毒的复制和包装具有决定性作用。cap基因编码病毒衣壳蛋白，rep基因参与病毒的复制和整合。AAV能感染多种细胞。
重组腺相关病毒载体(rAAV)源于非致病的野生型腺相关病毒，由于其安全性好、宿主细胞范围广(分裂和非分裂细胞)、免疫源性低，在体内表达外源基因时间长等特点，被视为最有前途的基因转移载体之一，在世界范围内的基因治疗和疫苗研究中得到广泛应用。经过10余年的研究，重组腺相关病毒的生物学特性己被深入了解，尤其是其在各种细胞、组织和体内实验中的应用效果方面已经积累了许多资料。在医学研究中，rAAV被用于多种疾病的基因治疗的研究(包括体内、体外实验)；同时作为一种有特点的基因转移载体，还广泛用于基因功能研究、构建疾病模型、制备基因敲除鼠等方面。
在本发明一个优选的实施例中，载体为重组AAV载体。AAV是相对较小的DNA病毒，其可以稳定和位点特异性方式整合到它们所感染的细胞的基因组中。它们能够感染一大系列的细胞而不对细胞生长、形态或分化产生任何影响，并且它们似乎并不涉及人体病理学。AAV基因组己被克隆、测序及表征。AAV在每个末端包含约145个碱基的反向末端重复序列(ITR)区域，其作为病毒的复制起点。该基因组的其余被分成两个带有衣壳化功能的重要区域：包含涉及病毒复制和病毒基因表达的rep基因的基因组左边部分；以及包含编码病毒衣壳蛋白的cap基因的基因组右边部分。
AAV载体可采用本领域的标准方法制备。任何血清型的腺相关病毒均是合适的。用于纯化载体的方法可见于例如美国专利No.6566118、6989264和6995006，它们的公开内容整体以引用方式并入本文。杂合载体的制备在例如PCT申请No.PCT/US2005/027091中有所描述，该申请的公开内容整体以引用方式并入本文。用于体外和体内转运基因的衍生自AAV的载体的使用己有描述(参见例如国际专利申请公布No.WO91/18088和WO93/09239；美国专利No.4,797,368、6,596,535和5,139,941，以及欧洲专利No.0488528，它们均整体以引用方式并入本文)。这些专利公布描述了其中rep和/或cap基因缺失并被所关注的基因替换的各种来源于AAV的构建体，以及这些构建体在体外(进入培养的细胞中)或体内(直接进入生物体)转运所关注的基因的用途。复制缺陷重组AAV可通过将以下质粒共转染进被人类辅助病毒(例如腺病毒)感染的细胞系而制备：所含的所关注核酸序列的侧翼为两个AAV反向末端重复序列(ITR)区域的质粒，和携带AAV衣壳化基因(rep和cap基因)的质粒。然后通过标准技术纯化所产生的AAV重组体。
在一些实施方案中，重组载体被衣壳化到病毒粒子(例如包括但不限于AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15和AAV16的AAV病毒粒子)中。因此，本公开包括含有本文所述的任何载体的重组病毒粒子(因其包含重组多核苷酸而为重组的)。产生这样的粒子的方法是本领域己知的，并在美国专利No.6,596,535中有所描述。
表达载体和宿主细胞
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明融合蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的融合蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明融合蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，编码融合蛋白的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明融合蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞(如大肠杆菌)，或是低等真核细胞，或是高等真核细胞，如酵母细胞、植物细胞或哺乳动物细胞(包括人和非人哺乳动物)。代表性例子有：大肠杆菌、麦胚细胞，昆虫细胞，SF9、Hela、HEK293、CHO、酵母细胞等。在本发明的一个优选实施方式中，选择酵母细胞(如毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为马克斯克鲁维酵母、和/或乳酸克鲁维酵母)为宿主细胞。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
基因治疗
遗传病的基因治疗(Gene Therapy)是指应用基因工程技术将正常基因引入患者细胞内，以纠正缺陷基因而根治疾病。纠正的途径既可以是原位修复有缺陷的基因，也可以是用有功能的正常基因转入细胞基因组的某一部位，以替代缺陷基因来发挥作用。基因是携带生物遗传信息的基本功能单位，是位于染色体上的一段特定序列。将外源的基因导入生物细胞内必须借助一定的技术方法或载体，基因转移的方法分为生物学方法、物理方法和化学方法。腺病毒载体是目前基因治疗最为常用的病毒载体之一。基因治疗主要是治疗那些对人类健康威胁严重的疾病，包括，但并不限于：遗传病(如血友病、囊性纤维病、家庭性高胆固醇血症等)、恶性肿瘤、心血管疾病、感染性疾病(如艾滋病、类风湿等)。基因治疗是将人的正常基因或有治疗作用的基因通过一定方式导入人体靶细胞以纠正基因的缺陷或者发挥治疗作用，从而达到治疗疾病目的的生物医学高技术。基因治疗与常规治疗方法不同：一般意义上疾病的治疗针对的是因基因异常而导致的各种症状，而基因治疗针对的是疾病的根源--异常的基因本身。基因治疗的靶细胞包括，但并不限于，体细胞、骨髓细胞、肝细胞、神经细胞、内皮细胞、肌细胞。
在本发明中，通过基因治疗将靶基因进行高效的基因编辑(包括基因插入、替换等)，从而恢复基因的正常表达或增强基因的表达，从而治疗相关疾病。
本发明的主要优点包括：
(1)本发明首次发现，本发明的融合蛋白可显著提高体内或体外的基因编辑效率。
(2)本发明首次发现，本发明的融合蛋白可显著提高体内或体外的基因编辑效率，提高幅度≥20％，较佳地，＞40％，更佳地，＞60％(如80％)，最高可达2倍。
(3)本发明首次发现体外转录增强型基因编辑工具的mRNA，提高动物模型构建的成功性。
(4)本发明使用包装增强型基因编辑工具的AAV病毒，表达增强型基因编辑工具的蛋白，提高疾病治疗效果。
(5)本发明首次利用通过融合双链DNA结合结构域来提高基因编辑效率。
(6)本发明首次筛选找到了一种高效提高基因编辑效率的双链DNA结合结构域(如HMG-D),以及其非常优异的融合方式。
(7)本发明首次发现，本发明的双链DNA结合结构域可以广泛的提高各种基因编辑工具的基因编辑效率。
(8)本发明的增强型基因编辑工具可以提高动物模型构建的成功率，以及基因治疗效率。
(9)本发明的增强型基因编辑工具还可用于基因治疗。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring HarborLaboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非有特别说明，否则本发明实施例中的试剂和材料均为市售产品。
通用方法
方法1：(适用于实施例1、实施例2和实施例3)
1、24孔板的HEK293T细胞密度长至60-70％，等摩尔量的质粒通过转染试剂PEI转染进细胞，8-10小时换液，细胞培养一段时间后(Cas9系统培养72小时，碱基编辑系统培养120小时)，收细胞，提基因组。
2、设计合适引物扩增靶点左右共150-180bp的序列，进行Hitom建库，深度测序，分析计算编辑效率。
方法2：(适用于实施例4的动物模型构建)
1、体外转录增强型基因编辑工具的mRNA和对应靶点的sgRNA。
2、小鼠胚胎注射mRNA和sgRNA，移植到代孕母鼠，获得F0代小鼠，鉴定基因，统计小鼠基因型突变率，计算模型构建成功率。
方法3：(适用于实施例4的基因治疗)
1、包装增强型基因编辑工具的腺相关病毒(AAV)和治疗基因的sgRNA，或者同源修复模板。
2、通过静脉注射(尾静脉)或局部注射(肌肉等)包装的AAV至疾病动物模型(小鼠或大鼠)，同时注射对照病毒。
3、定期观察，检测治疗组的动物模型和对照组动物模型的表型，评价治疗效率。
实施例1筛选增强型基因编辑工具
分别合成不同种类的双链DNA结合结构域(HMG-D和Sac7d)，通过设计5种不同长度的linker(L1、L2、L3、L4和L5)(表1)，以及融合在Cas9的N端和C端，在两个内源性靶点(VEGF和HBG1/2)上进行比较，根据结果统计，对这一系列优化所得到的增强型的基因编辑工具进行打分(在效果均很好的基础上所做的相对评分)(不好：A；欠好：AA；较好：AAA；好：AAAA；非常好：AAAAA),结果如下：(注：C表示Cas9；L1-L5表示不同linker；H表示HMG-D结构域；S表示sac7d。)
A：S-L1-C,H-L1-C
AA:S-L2-C,S-L3-C,S-L4-C,H-L2-C
AAA:H-L4-C-L4-S,H-L4-C-L5-S
AAAA:H-L3-C,C-L4-S,C-L5-S,C-L4-H,H-L4-C-L4-H,H-L4-C-L5-H,H-L4-H-L4-C,C-L5-H-L5-H
AAAAA:H-L4-C,C-L5-H
综合统计，发现HMG-D的效果非常好，并且L4和L5长度(32个和64个氨基酸)的linker是最优的linker，通过比较N端和C端连接，发现在最优linker条件下，N端和C端连接的效果相都很好，因此本发明人得到增强型的基因编辑工具，即HMG-D通过L4或L5的linker融合在Cas9的N端或C端是非常好的融合方式(图1)。
同理，这种通过双链DNA结合结构域融合来增强基因编辑效率的增强型基因编辑工具，也可以类比到其他类型的双链DNA结合结构域，如应用广泛锌指蛋白(ZFP)、其他转录因子的DNA结合结构域和来源其他物种的HMG-D或Sac7d，等等。本发明同样可以通过这些双链DNA结合结构域来提高基因编辑效率，因此，本发明最重要的是在于发现了双链DNA结合结构域融合基因编辑工具可以提高基因编辑效率，而其中优选双链DNA结合结构域HMG-D。
实施例2提高SpCas9的基因编辑效率
将获得的增强型基因编辑工具(即本发明的融合蛋白，如HMG-D-L4-SpCas9)进一步在更多的内源性靶点上进行效果方面的比较，通过等摩尔比转染293T细胞，发现在所比较的靶点中和SpCas9相比较，本发明的融合蛋白的编辑效率都有大于20％(或60％、或80％)的提高，最高可达2倍(图2)。
实施例3提高其他种属来源的Cas9(如：SaCas9)的基因编辑效率
通过构建HMG-D-L4-SaCas9表达载体，针对内源性靶点，等摩尔比转染293T细胞，发现融合HMG-D后SaCas9编辑效率也有类似的效果的提升。(图3)
实施例4提高其他非Cas9蛋白(如：AsCas12a)的基因编辑效率
通过构建HMG-D-L4-AsCas12a表达载体，针对内源性靶点，等摩尔比转染293T细胞，发现融合HMG-D后AsCas12a编辑效率也有类似的效果的提升。(图4)
实施例5提高转录调控工具(CRISPR/Cas9a)的转录激活效率
通过构建HMG-D-L4-dCas9-VPR表达载体，针对内源性靶点，等摩尔比转染293T细胞，发现融合HMG-D后dCas9-VPR对于内源性基因的转录激活效率也有类似的效果的提升。(图5)
实施例6提高碱基编辑器(base editor)的编辑效率
由于碱基编辑器(base editor)是通过在Cas9的N端融合了胞嘧啶脱氨酶(CBE)和腺嘌呤脱氨酶(ABE)，所以，在碱基编辑器的Cas9的c端通过L5 linker来融合HMG-D，开发出增强型单碱基编辑工具。通过内源性靶点比较，融合HMG-D后碱基编辑器(如ABE)的编辑效率有了极大的提升，如提高了＞1.5－2倍(图6)。
融合了其他类型碱基编辑器的融合蛋白的编辑效率的提升幅度与融合了ABE的融合蛋白的编辑效率类似或相当。
实施例7提高提高动物模型构建的成功率，以及基因治疗效率
通过体外转录增强型基因编辑工具的mRNA，增加了动物模型构建的成功率。同时，通过包装增强型的基因编辑工具的AAV病毒，也可以提高疾病治疗效率。
对比例1与不是DNA结合结构域融合后效果不好
为了进一步验证DNA结合结构域融合后可以提高基因编辑效率，我们把DNA结合结构域改为GFP蛋白(一种非DNA结合结构域)这种无关的蛋白，发现融合了GFP蛋白后不能提高基因编辑效率(图1)。同时，还对HMG-D结构域进行突变，破坏其DNA结合的能力，构建了3个氨基酸突变的HMG-D结构域(mutHMG-D，简称mutH),通过实验比较，mutHMG-D结构域也不能提高基因编辑效率(图1)。
对比例2HMG-D、Sac7d之外的DNA结合结构域效果不好
为了扩宽DNA结合结构域的范围，又测试了一些单链DNA结合结构域(如Rad51)，融合Rad51后同样不能提高基因编辑效率(图1)。因此，结果表明，本发明上述筛选的HMG-D和Sac7d的双链DNA结合结构域是非常有效的。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 华东师范大学
上海邦耀生物科技有限公司
<120> 一种增强基因编辑的融合蛋白及其应用
<130> P2018-2357
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1423
<212> PRT
<213> 酿脓链球菌(Streptococcus pyogenes)
<400> 1
Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp
1 5 10 15
Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg Lys Val
20 25 30
Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu
35 40 45
Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr
50 55 60
Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His
65 70 75 80
Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu
85 90 95
Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr
100 105 110
Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu
115 120 125
Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe
130 135 140
Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn
145 150 155 160
Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His
165 170 175
Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu
180 185 190
Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu
195 200 205
Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe
210 215 220
Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile
225 230 235 240
Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser
245 250 255
Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys
260 265 270
Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr
275 280 285
Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln
290 295 300
Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln
305 310 315 320
Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser
325 330 335
Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr
340 345 350
Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His
355 360 365
Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu
370 375 380
Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly
385 390 395 400
Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys
405 410 415
Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu
420 425 430
Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser
435 440 445
Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg
450 455 460
Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu
465 470 475 480
Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg
485 490 495
Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile
500 505 510
Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln
515 520 525
Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu
530 535 540
Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr
545 550 555 560
Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro
565 570 575
Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe
580 585 590
Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe
595 600 605
Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp
610 615 620
Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile
625 630 635 640
Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu
645 650 655
Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu
660 665 670
Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys
675 680 685
Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys
690 695 700
Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp
705 710 715 720
Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile
725 730 735
His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val
740 745 750
Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly
755 760 765
Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp
770 775 780
Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile
785 790 795 800
Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
805 810 815
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser
820 825 830
Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
835 840 845
Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp
850 855 860
Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile
865 870 875 880
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu
885 890 895
Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu
900 905 910
Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala
915 920 925
Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg
930 935 940
Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu
945 950 955 960
Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
965 970 975
Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val
980 985 990
Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp
995 1000 1005
Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His
1010 1015 1020
Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr
1025 1030 1035 1040
Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1045 1050 1055
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr
1060 1065 1070
Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu
1075 1080 1085
Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr
1090 1095 1100
Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1105 1110 1115 1120
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys
1125 1130 1135
Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1140 1145 1150
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1155 1160 1165
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val
1170 1175 1180
Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys
1185 1190 1195 1200
Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn
1205 1210 1215
Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp
1220 1225 1230
Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly
1235 1240 1245
Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu
1250 1255 1260
Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His
1265 1270 1275 1280
Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1285 1290 1295
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile
1300 1305 1310
Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys
1315 1320 1325
Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln
1330 1335 1340
Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1345 1350 1355 1360
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr
1365 1370 1375
Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1380 1385 1390
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys
1395 1400 1405
Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys
1410 1415 1420
<210> 2
<211> 1792
<212> PRT
<213> 人工序列(artificial sequence)
<400> 2
Met Pro Lys Lys Lys Arg Lys Val Ser Glu Val Glu Phe Ser His Glu
1 5 10 15
Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu
20 25 30
Arg Glu Val Pro Val Gly Ala Val Leu Val His Asn Asn Arg Val Ile
35 40 45
Gly Glu Gly Trp Asn Arg Pro Ile Gly Arg His Asp Pro Thr Ala His
50 55 60
Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr
65 70 75 80
Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Leu Glu Pro Cys Val Met
85 90 95
Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly
100 105 110
Ala Arg Asp Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu
115 120 125
His His Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu
130 135 140
Ala Asp Glu Cys Ala Ala Leu Leu Ser Asp Phe Phe Arg Met Arg Arg
145 150 155 160
Gln Glu Ile Lys Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly
165 170 175
Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu
180 185 190
Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Glu
195 200 205
Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala
210 215 220
Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val
225 230 235 240
Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile Gly Leu
245 250 255
His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly
260 265 270
Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr
275 280 285
Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile
290 295 300
Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala Ala Gly
305 310 315 320
Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg Val Glu
325 330 335
Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Cys Tyr
340 345 350
Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys Ala Gln
355 360 365
Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu
370 375 380
Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser
385 390 395 400
Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr
405 410 415
Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser
420 425 430
Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys
435 440 445
Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala
450 455 460
Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn
465 470 475 480
Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val
485 490 495
Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu
500 505 510
Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu
515 520 525
Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys
530 535 540
Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala
545 550 555 560
Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp
565 570 575
Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val
580 585 590
Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly
595 600 605
Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg
610 615 620
Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu
625 630 635 640
Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys
645 650 655
Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp
660 665 670
Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln
675 680 685
Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu
690 695 700
Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu
705 710 715 720
Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr
725 730 735
Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu
740 745 750
Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly
755 760 765
Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu
770 775 780
Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp
785 790 795 800
Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln
805 810 815
Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe
820 825 830
Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr
835 840 845
Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg
850 855 860
Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn
865 870 875 880
Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu
885 890 895
Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro
900 905 910
Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr
915 920 925
Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser
930 935 940
Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg
945 950 955 960
Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu
965 970 975
Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala
980 985 990
Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp
995 1000 1005
Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu
1010 1015 1020
Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys
1025 1030 1035 1040
Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg
1045 1050 1055
Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly
1060 1065 1070
Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser
1075 1080 1085
Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser
1090 1095 1100
Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1105 1110 1115 1120
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile
1125 1130 1135
Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys
1140 1145 1150
Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg
1155 1160 1165
Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met
1170 1175 1180
Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys
1185 1190 1195 1200
Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu
1205 1210 1215
Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp
1220 1225 1230
Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser
1235 1240 1245
Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp
1250 1255 1260
Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys
1265 1270 1275 1280
Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr
1285 1290 1295
Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser
1300 1305 1310
Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg
1315 1320 1325
Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr
1330 1335 1340
Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1345 1350 1355 1360
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr
1365 1370 1375
Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu
1380 1385 1390
Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu
1395 1400 1405
Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1410 1415 1420
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe
1425 1430 1435 1440
Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1445 1450 1455
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1460 1465 1470
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
1475 1480 1485
Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln
1490 1495 1500
Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp
1505 1510 1515 1520
Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly
1525 1530 1535
Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val
1540 1545 1550
Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly
1555 1560 1565
Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe
1570 1575 1580
Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1585 1590 1595 1600
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met
1605 1610 1615
Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro
1620 1625 1630
Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu
1635 1640 1645
Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1650 1655 1660
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser
1665 1670 1675 1680
Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1685 1690 1695
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1700 1705 1710
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys
1715 1720 1725
Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu
1730 1735 1740
Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu
1745 1750 1755 1760
Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys
1765 1770 1775
Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val
1780 1785 1790
<210> 3
<211> 9
<212> PRT
<213> 人工序列(artificial sequence)
<400> 3
Gly Gly Ser Gly Gly Ser Gly Gly Ser
1 5
<210> 4
<211> 18
<212> PRT
<213> 人工序列(artificial sequence)
<400> 4
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly
1 5 10 15
Gly Ser
<210> 5
<211> 16
<212> PRT
<213> 人工序列(artificial sequence)
<400> 5
Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser
1 5 10 15
<210> 6
<211> 32
<212> PRT
<213> 人工序列(artificial sequence)
<400> 6
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> 7
<211> 48
<212> PRT
<213> 人工序列(artificial sequence)
<400> 7
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 Ser Glu Thr Pro Gly Thr
20 25 30
Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser
35 40 45
<210> 8
<211> 1567
<212> PRT
<213> 人工序列(artificial sequence)
<400> 8
Met Ser Asp Lys Pro Lys Arg Pro Leu Ser Ala Tyr Met Leu Trp Leu
1 5 10 15
Asn Ser Ala Arg Glu Ser Ile Lys Arg Glu Asn Pro Gly Ile Lys Val
20 25 30
Thr Glu Val Ala Lys Arg Gly Gly Glu Leu Trp Arg Ala Met Lys Asp
35 40 45
Lys Ser Glu Trp Glu Ala Lys Ala Ala Lys Ala Lys Asp Asp Tyr Asp
50 55 60
Arg Ala Val Lys Glu Phe Glu Ala Asn Gly Gly Ser Ser Ala Ala Asn
65 70 75 80
Gly Gly Gly Ala Lys Lys Arg Ala Lys Pro Ala Lys Lys Val Ala Lys
85 90 95
Lys Ser Lys Lys Glu Glu Ser Asp Glu Asp Asp Asp Asp Glu Ser Glu
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
Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp
145 150 155 160
Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg Lys Val
165 170 175
Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu
180 185 190
Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr
195 200 205
Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His
210 215 220
Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu
225 230 235 240
Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr
245 250 255
Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu
260 265 270
Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe
275 280 285
Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn
290 295 300
Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His
305 310 315 320
Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu
325 330 335
Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu
340 345 350
Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe
355 360 365
Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile
370 375 380
Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser
385 390 395 400
Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys
405 410 415
Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr
420 425 430
Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln
435 440 445
Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln
450 455 460
Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser
465 470 475 480
Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr
485 490 495
Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His
500 505 510
Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu
515 520 525
Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly
530 535 540
Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys
545 550 555 560
Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu
565 570 575
Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser
580 585 590
Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg
595 600 605
Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu
610 615 620
Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg
625 630 635 640
Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile
645 650 655
Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln
660 665 670
Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu
675 680 685
Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr
690 695 700
Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro
705 710 715 720
Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe
725 730 735
Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe
740 745 750
Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp
755 760 765
Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile
770 775 780
Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu
785 790 795 800
Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu
805 810 815
Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys
820 825 830
Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys
835 840 845
Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp
850 855 860
Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile
865 870 875 880
His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val
885 890 895
Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly
900 905 910
Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp
915 920 925
Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile
930 935 940
Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
945 950 955 960
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser
965 970 975
Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
980 985 990
Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp
995 1000 1005
Gln 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 Leu
1025 1030 1035 1040
Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu
1045 1050 1055
Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala
1060 1065 1070
Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg
1075 1080 1085
Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu
1090 1095 1100
Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
1105 1110 1115 1120
Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val
1125 1130 1135
Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp
1140 1145 1150
Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His
1155 1160 1165
Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr
1170 1175 1180
Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1185 1190 1195 1200
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr
1205 1210 1215
Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu
1220 1225 1230
Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr
1235 1240 1245
Asn 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 Lys
1265 1270 1275 1280
Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1285 1290 1295
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1300 1305 1310
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val
1315 1320 1325
Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys
1330 1335 1340
Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn
1345 1350 1355 1360
Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp
1365 1370 1375
Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly
1380 1385 1390
Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu
1395 1400 1405
Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His
1410 1415 1420
Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1425 1430 1435 1440
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile
1445 1450 1455
Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys
1460 1465 1470
Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln
1475 1480 1485
Ala 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 Thr
1505 1510 1515 1520
Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1525 1530 1535
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys
1540 1545 1550
Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys
1555 1560 1565
<210> 9
<211> 1952
<212> PRT
<213> 人工序列(artificial sequence)
<400> 9
Met Pro Lys Lys Lys Arg Lys Val Ser Glu Val Glu Phe Ser His Glu
1 5 10 15
Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu
20 25 30
Arg Glu Val Pro Val Gly Ala Val Leu Val His Asn Asn Arg Val Ile
35 40 45
Gly Glu Gly Trp Asn Arg Pro Ile Gly Arg His Asp Pro Thr Ala His
50 55 60
Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr
65 70 75 80
Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Leu Glu Pro Cys Val Met
85 90 95
Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly
100 105 110
Ala Arg Asp Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu
115 120 125
His His Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu
130 135 140
Ala Asp Glu Cys Ala Ala Leu Leu Ser Asp Phe Phe Arg Met Arg Arg
145 150 155 160
Gln Glu Ile Lys Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly
165 170 175
Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu
180 185 190
Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Glu
195 200 205
Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala
210 215 220
Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val
225 230 235 240
Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile Gly Leu
245 250 255
His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly
260 265 270
Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr
275 280 285
Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile
290 295 300
Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala Ala Gly
305 310 315 320
Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg Val Glu
325 330 335
Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Cys Tyr
340 345 350
Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys Ala Gln
355 360 365
Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu
370 375 380
Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser
385 390 395 400
Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr
405 410 415
Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser
420 425 430
Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys
435 440 445
Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala
450 455 460
Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn
465 470 475 480
Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val
485 490 495
Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu
500 505 510
Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu
515 520 525
Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys
530 535 540
Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala
545 550 555 560
Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp
565 570 575
Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val
580 585 590
Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly
595 600 605
Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg
610 615 620
Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu
625 630 635 640
Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys
645 650 655
Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp
660 665 670
Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln
675 680 685
Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu
690 695 700
Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu
705 710 715 720
Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr
725 730 735
Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu
740 745 750
Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly
755 760 765
Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu
770 775 780
Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp
785 790 795 800
Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln
805 810 815
Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe
820 825 830
Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr
835 840 845
Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg
850 855 860
Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn
865 870 875 880
Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu
885 890 895
Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro
900 905 910
Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr
915 920 925
Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser
930 935 940
Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg
945 950 955 960
Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu
965 970 975
Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala
980 985 990
Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp
995 1000 1005
Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu
1010 1015 1020
Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys
1025 1030 1035 1040
Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg
1045 1050 1055
Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly
1060 1065 1070
Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser
1075 1080 1085
Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser
1090 1095 1100
Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1105 1110 1115 1120
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile
1125 1130 1135
Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys
1140 1145 1150
Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg
1155 1160 1165
Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met
1170 1175 1180
Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys
1185 1190 1195 1200
Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu
1205 1210 1215
Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp
1220 1225 1230
Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser
1235 1240 1245
Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp
1250 1255 1260
Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys
1265 1270 1275 1280
Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr
1285 1290 1295
Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser
1300 1305 1310
Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg
1315 1320 1325
Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr
1330 1335 1340
Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1345 1350 1355 1360
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr
1365 1370 1375
Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu
1380 1385 1390
Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu
1395 1400 1405
Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1410 1415 1420
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe
1425 1430 1435 1440
Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1445 1450 1455
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1460 1465 1470
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
1475 1480 1485
Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln
1490 1495 1500
Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp
1505 1510 1515 1520
Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly
1525 1530 1535
Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val
1540 1545 1550
Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly
1555 1560 1565
Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe
1570 1575 1580
Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1585 1590 1595 1600
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met
1605 1610 1615
Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro
1620 1625 1630
Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu
1635 1640 1645
Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1650 1655 1660
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser
1665 1670 1675 1680
Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1685 1690 1695
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1700 1705 1710
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys
1715 1720 1725
Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu
1730 1735 1740
Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu
1745 1750 1755 1760
Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys
1765 1770 1775
Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val
1780 1785 1790
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr
1795 1800 1805
Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Ser Glu Thr Pro Gly Thr
1810 1815 1820
Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser
1825 1830 1835 1840
Met Ser Asp Lys Pro Lys Arg Pro Leu Ser Ala Tyr Met Leu Trp Leu
1845 1850 1855
Asn Ser Ala Arg Glu Ser Ile Lys Arg Glu Asn Pro Gly Ile Lys Val
1860 1865 1870
Thr Glu Val Ala Lys Arg Gly Gly Glu Leu Trp Arg Ala Met Lys Asp
1875 1880 1885
Lys Ser Glu Trp Glu Ala Lys Ala Ala Lys Ala Lys Asp Asp Tyr Asp
1890 1895 1900
Arg Ala Val Lys Glu Phe Glu Ala Asn Gly Gly Ser Ser Ala Ala Asn
1905 1910 1915 1920
Gly Gly Gly Ala Lys Lys Arg Ala Lys Pro Ala Lys Lys Val Ala Lys
1925 1930 1935
Lys Ser Lys Lys Glu Glu Ser Asp Glu Asp Asp Asp Asp Glu Ser Glu
1940 1945 1950
<210> 10
<211> 112
<212> PRT
<213> 人工序列(artificial sequence)
<400> 10
Met Ser Asp Lys Pro Lys Arg Pro Leu Ser Ala Tyr Met Leu Trp Leu
1 5 10 15
Asn Ser Ala Arg Glu Ser Ile Lys Arg Glu Asn Pro Gly Ile Lys Val
20 25 30
Thr Glu Val Ala Lys Arg Gly Gly Glu Leu Trp Arg Ala Met Lys Asp
35 40 45
Lys Ser Glu Trp Glu Ala Lys Ala Ala Lys Ala Lys Asp Asp Tyr Asp
50 55 60
Arg Ala Val Lys Glu Phe Glu Ala Asn Gly Gly Ser Ser Ala Ala Asn
65 70 75 80
Gly Gly Gly Ala Lys Lys Arg Ala Lys Pro Ala Lys Lys Val Ala Lys
85 90 95
Lys Ser Lys Lys Glu Glu Ser Asp Glu Asp Asp Asp Asp Glu Ser Glu
100 105 110
<210> 11
<211> 66
<212> PRT
<213> 人工序列(artificial sequence)
<400> 11
Met Val Lys Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp
1 5 10 15
Thr Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Val Ser Phe
20 25 30
Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala Val Ser Glu Lys
35 40 45
Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu
50 55 60
Lys Lys
65
<210> 12
<211> 1667
<212> PRT
<213> 人工序列(artificial sequence)
<400> 12
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu
20 25 30
Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg
35 40 45
Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly
50 55 60
Arg His Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val
65 70 75 80
Glu Val Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro
85 90 95
Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly
100 105 110
Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val
115 120 125
Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg
130 135 140
Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln
145 150 155 160
Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn
165 170 175
Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp
180 185 190
Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro
195 200 205
Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe
210 215 220
Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile
225 230 235 240
Leu Trp Ala Thr Gly Leu Lys Ser Gly Gly Ser Ser Gly Gly Ser Ser
245 250 255
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
260 265 270
Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala
275 280 285
Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys
290 295 300
Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser
305 310 315 320
Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr
325 330 335
Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg
340 345 350
Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met
355 360 365
Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu
370 375 380
Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile
385 390 395 400
Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu
405 410 415
Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile
420 425 430
Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile
435 440 445
Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile
450 455 460
Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn
465 470 475 480
Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys
485 490 495
Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys
500 505 510
Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro
515 520 525
Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu
530 535 540
Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile
545 550 555 560
Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp
565 570 575
Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys
580 585 590
Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln
595 600 605
Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys
610 615 620
Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr
625 630 635 640
Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro
645 650 655
Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn
660 665 670
Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile
675 680 685
Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln
690 695 700
Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys
705 710 715 720
Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly
725 730 735
Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr
740 745 750
Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser
755 760 765
Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys
770 775 780
Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn
785 790 795 800
Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala
805 810 815
Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys
820 825 830
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys
835 840 845
Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg
850 855 860
Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys
865 870 875 880
Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp
885 890 895
Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu
900 905 910
Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
915 920 925
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu
930 935 940
Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe
945 950 955 960
Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His
965 970 975
Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser
980 985 990
Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser
995 1000 1005
Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu
1010 1015 1020
Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1025 1030 1035 1040
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg
1045 1050 1055
Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln
1060 1065 1070
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys
1075 1080 1085
Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
1090 1095 1100
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val
1105 1110 1115 1120
Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr
1125 1130 1135
Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu
1140 1145 1150
Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys
1155 1160 1165
Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly
1170 1175 1180
Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val
1185 1190 1195 1200
Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg
1205 1210 1215
Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys
1220 1225 1230
Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe
1235 1240 1245
Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp
1250 1255 1260
Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1265 1270 1275 1280
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val
1285 1290 1295
Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala
1300 1305 1310
Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile
1315 1320 1325
Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1330 1335 1340
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr
1345 1350 1355 1360
Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1365 1370 1375
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1380 1385 1390
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys
1395 1400 1405
Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val
1410 1415 1420
Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu
1425 1430 1435 1440
Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro
1445 1450 1455
Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu
1460 1465 1470
Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg
1475 1480 1485
Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu
1490 1495 1500
Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1505 1510 1515 1520
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe
1525 1530 1535
Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser
1540 1545 1550
Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val
1555 1560 1565
Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1570 1575 1580
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala
1585 1590 1595 1600
Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1605 1610 1615
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1620 1625 1630
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly
1635 1640 1645
Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys
1650 1655 1660
Arg Lys Val
1665
<210> 13
<211> 1827
<212> PRT
<213> 人工序列(artificial sequence)
<400> 13
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu
20 25 30
Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg
35 40 45
Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly
50 55 60
Arg His Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val
65 70 75 80
Glu Val Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro
85 90 95
Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly
100 105 110
Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val
115 120 125
Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg
130 135 140
Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln
145 150 155 160
Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn
165 170 175
Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp
180 185 190
Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro
195 200 205
Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe
210 215 220
Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile
225 230 235 240
Leu Trp Ala Thr Gly Leu Lys Ser Gly Gly Ser Ser Gly Gly Ser Ser
245 250 255
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
260 265 270
Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala
275 280 285
Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys
290 295 300
Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser
305 310 315 320
Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr
325 330 335
Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg
340 345 350
Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met
355 360 365
Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu
370 375 380
Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile
385 390 395 400
Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu
405 410 415
Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile
420 425 430
Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile
435 440 445
Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile
450 455 460
Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn
465 470 475 480
Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys
485 490 495
Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys
500 505 510
Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro
515 520 525
Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu
530 535 540
Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile
545 550 555 560
Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp
565 570 575
Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys
580 585 590
Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln
595 600 605
Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys
610 615 620
Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr
625 630 635 640
Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro
645 650 655
Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn
660 665 670
Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile
675 680 685
Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln
690 695 700
Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys
705 710 715 720
Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly
725 730 735
Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr
740 745 750
Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser
755 760 765
Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys
770 775 780
Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn
785 790 795 800
Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala
805 810 815
Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys
820 825 830
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys
835 840 845
Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg
850 855 860
Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys
865 870 875 880
Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp
885 890 895
Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu
900 905 910
Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
915 920 925
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu
930 935 940
Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe
945 950 955 960
Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His
965 970 975
Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser
980 985 990
Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser
995 1000 1005
Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu
1010 1015 1020
Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1025 1030 1035 1040
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg
1045 1050 1055
Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln
1060 1065 1070
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys
1075 1080 1085
Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
1090 1095 1100
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val
1105 1110 1115 1120
Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr
1125 1130 1135
Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu
1140 1145 1150
Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys
1155 1160 1165
Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly
1170 1175 1180
Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val
1185 1190 1195 1200
Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg
1205 1210 1215
Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys
1220 1225 1230
Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe
1235 1240 1245
Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp
1250 1255 1260
Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1265 1270 1275 1280
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val
1285 1290 1295
Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala
1300 1305 1310
Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile
1315 1320 1325
Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1330 1335 1340
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr
1345 1350 1355 1360
Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1365 1370 1375
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1380 1385 1390
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys
1395 1400 1405
Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val
1410 1415 1420
Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu
1425 1430 1435 1440
Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro
1445 1450 1455
Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu
1460 1465 1470
Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg
1475 1480 1485
Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu
1490 1495 1500
Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1505 1510 1515 1520
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe
1525 1530 1535
Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser
1540 1545 1550
Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val
1555 1560 1565
Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1570 1575 1580
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala
1585 1590 1595 1600
Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1605 1610 1615
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1620 1625 1630
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly
1635 1640 1645
Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys
1650 1655 1660
Arg Lys Val Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr
1665 1670 1675 1680
Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Ser Glu Thr
1685 1690 1695
Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser
1700 1705 1710
Gly Gly Ser Met Ser Asp Lys Pro Lys Arg Pro Leu Ser Ala Tyr Met
1715 1720 1725
Leu Trp Leu Asn Ser Ala Arg Glu Ser Ile Lys Arg Glu Asn Pro Gly
1730 1735 1740
Ile Lys Val Thr Glu Val Ala Lys Arg Gly Gly Glu Leu Trp Arg Ala
1745 1750 1755 1760
Met Lys Asp Lys Ser Glu Trp Glu Ala Lys Ala Ala Lys Ala Lys Asp
1765 1770 1775
Asp Tyr Asp Arg Ala Val Lys Glu Phe Glu Ala Asn Gly Gly Ser Ser
1780 1785 1790
Ala Ala Asn Gly Gly Gly Ala Lys Lys Arg Ala Lys Pro Ala Lys Lys
1795 1800 1805
Val Ala Lys Lys Ser Lys Lys Glu Glu Ser Asp Glu Asp Asp Asp Asp
1810 1815 1820
Glu Ser Glu
1825
<210> 14
<211> 1057
<212> PRT
<213> 葡萄球菌(Staphylococcus aureus)
<400> 14
Met Glu Ala Ser Met Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly
1 5 10 15
Ile Thr Ser Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val
20 25 30
Ile Asp Ala Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn
35 40 45
Glu Gly Arg Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg
50 55 60
Arg His Arg Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu
65 70 75 80
Leu Thr Asp His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg
85 90 95
Val Lys Gly Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala
100 105 110
Leu Leu His Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val
115 120 125
Glu Glu Asp Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg
130 135 140
Asn Ser Lys Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu
145 150 155 160
Arg Leu Lys Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys
165 170 175
Thr Ser Asp Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys
180 185 190
Ala Tyr His Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu
195 200 205
Leu Glu Thr Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro
210 215 220
Phe Gly Trp Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His
225 230 235 240
Cys Thr Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn
245 250 255
Ala Asp Leu Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr
260 265 270
Arg Asp Glu Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile
275 280 285
Glu Asn Val Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala
290 295 300
Lys Glu Ile Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr
305 310 315 320
Ser Thr Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile
325 330 335
Lys Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu
340 345 350
Asp Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile
355 360 365
Gln Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile
370 375 380
Glu Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser
385 390 395 400
Leu Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp
405 410 415
Asn Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val
420 425 430
Asp Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe
435 440 445
Ile Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val
450 455 460
Ile Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile
465 470 475 480
Glu Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn
485 490 495
Glu Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile
500 505 510
Ile Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile
515 520 525
Lys Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala
530 535 540
Ile Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp
545 550 555 560
His Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys
565 570 575
Val Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro
580 585 590
Phe Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe
595 600 605
Lys Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys
610 615 620
Thr Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser
625 630 635 640
Val Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala
645 650 655
Thr Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn
660 665 670
Leu Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu
675 680 685
Arg Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His
690 695 700
His Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys
705 710 715 720
Glu Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met
725 730 735
Phe Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln
740 745 750
Glu Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys
755 760 765
Asp Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn
770 775 780
Arg Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys
785 790 795 800
Gly Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp
805 810 815
Asn Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu
820 825 830
Met Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met
835 840 845
Glu Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu
850 855 860
Thr Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val
865 870 875 880
Ile Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp
885 890 895
Ile Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser
900 905 910
Leu Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys
915 920 925
Phe Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr
930 935 940
Glu Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile
945 950 955 960
Ser Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile
965 970 975
Lys Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu
980 985 990
Leu Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr
995 1000 1005
Leu Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile
1010 1015 1020
Ala Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly
1025 1030 1035 1040
Asn Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys
1045 1050 1055
Gly
<210> 15
<211> 1307
<212> PRT
<213> 氨基酸球菌属(Acidaminococcus sp)
<400> 15
Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln
20 25 30
Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys
35 40 45
Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln
50 55 60
Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile
65 70 75 80
Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile
85 90 95
Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly
100 105 110
Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile
115 120 125
Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys
130 135 140
Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg
145 150 155 160
Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg
165 170 175
Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg
180 185 190
Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe
195 200 205
Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn
210 215 220
Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val
225 230 235 240
Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp
245 250 255
Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu
260 265 270
Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn
275 280 285
Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro
290 295 300
Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu
305 310 315 320
Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr
325 330 335
Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu
340 345 350
Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His
355 360 365
Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr
370 375 380
Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys
385 390 395 400
Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu
405 410 415
Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser
420 425 430
Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala
435 440 445
Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys
450 455 460
Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu
465 470 475 480
Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe
485 490 495
Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser
500 505 510
Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val
515 520 525
Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp
530 535 540
Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn
545 550 555 560
Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys
565 570 575
Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys
580 585 590
Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys
595 600 605
Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr
610 615 620
Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys
625 630 635 640
Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln
645 650 655
Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala
660 665 670
Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr
675 680 685
Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr
690 695 700
Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His
705 710 715 720
Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu
725 730 735
Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys
740 745 750
Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu
755 760 765
Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln
770 775 780
Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His
785 790 795 800
Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr
805 810 815
Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His
820 825 830
Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn
835 840 845
Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe
850 855 860
Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln
865 870 875 880
Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu
885 890 895
Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg
900 905 910
Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu
915 920 925
Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu
930 935 940
Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val
945 950 955 960
Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile
965 970 975
His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu
980 985 990
Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu
995 1000 1005
Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu Asn
1010 1015 1020
Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly Val Leu
1025 1030 1035 1040
Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala Lys Met Gly
1045 1050 1055
Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro Tyr Thr Ser Lys
1060 1065 1070
Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe Val Trp Lys Thr Ile
1075 1080 1085
Lys Asn His Glu Ser Arg Lys His Phe Leu Glu Gly Phe Asp Phe Leu
1090 1095 1100
His Tyr Asp Val Lys Thr Gly Asp Phe Ile Leu His Phe Lys Met Asn
1105 1110 1115 1120
Arg Asn Leu Ser Phe Gln Arg Gly Leu Pro Gly Phe Met Pro Ala Trp
1125 1130 1135
Asp Ile Val Phe Glu Lys Asn Glu Thr Gln Phe Asp Ala Lys Gly Thr
1140 1145 1150
Pro Phe Ile Ala Gly Lys Arg Ile Val Pro Val Ile Glu Asn His Arg
1155 1160 1165
Phe Thr Gly Arg Tyr Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala
1170 1175 1180
Leu Leu Glu Glu Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu
1185 1190 1195 1200
Pro Lys Leu Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val
1205 1210 1215
Ala Leu Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr
1220 1225 1230
Gly Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys
1235 1240 1245
Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp Ala
1250 1255 1260
Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu Asn His
1265 1270 1275 1280
Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile Ser Asn Gln
1285 1290 1295
Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn
1300 1305

Claims (20)

1.一种融合蛋白，其特征在于，所述融合蛋白的结构如下式I或I’所示：
C-A-L-B (I)
B-L-A-C (I’)
式中，
A为基因编辑蛋白，
B为DNA双链结合结构域，所述DNA双链结合结构域选自下组：HMG-D、Sac7d、或其组合；
C为任选的碱基编辑器元件；
L为无或连接肽，
各“-”独立地为连接肽或肽键或非肽键；
所述基因编辑蛋白选自下组：Cas9、Cas12a、或其组合；所述基因编辑蛋白选自下组：酿脓链球菌(Streptococcus pyogenes)、葡萄球菌(Staphylococcus aureus)、氨基酸球菌属(Acidaminococcus sp)、或其组合；所述连接肽的氨基酸序列为如SEQ ID NO.:4-7中任一所示的多肽。
2.如权利要求1所述的融合蛋白，其特征在于，当融合蛋白的结构如式I’所示时，C为无。
3.如权利要求1所述的融合蛋白，其特征在于，所述DNA双链结合结构域的氨基酸序列如SEQ ID NO.:10或11所示。
4.如权利要求1所述的融合蛋白，其特征在于，所述碱基编辑器元件包括胞嘧啶脱氨酶、腺嘌呤脱氨酶。
5.如权利要求1所述的融合蛋白，其特征在于，所述碱基编辑器元件的氨基酸序列如SEQ ID NO.:2或12所示。
6.如权利要求1所述的融合蛋白，其特征在于，所述DNA双链结合结构域为非序列特异性的DNA双链结合结构域。
7.如权利要求1所述的融合蛋白，其特征在于，所述融合蛋白具有SEQ ID NO.:8、9、13中任一所示的氨基酸序列。
8.一种多核苷酸，其特征在于，所述的多核苷酸编码权利要求1所述的融合蛋白。
9.一种载体，其特征在于，所述的载体含有权利要求8所述的多核苷酸。
10.一种宿主细胞，其特征在于，所述宿主细胞含有权利要求9所述的载体，或其基因组中整合有权利要求8所述的多核苷酸。
11.一种产生权利要求1所述的融合蛋白的方法，其特征在于，包括步骤：
在适合表达的条件下，培养权利要求10所述的宿主细胞，从而表达融合蛋白；和/或
分离所述融合蛋白。
12.一种基因编辑试剂，其特征在于，所述基因编辑试剂包含权利要求1所述的融合蛋白。
13.一种试剂盒，其特征在于，包括权利要求12所述的基因编辑试剂。
14.一种权利要求1所述的融合蛋白的用途，其特征在于，用于制备试剂或试剂盒，所述试剂或试剂盒用于提高基因编辑效率。
15.一种药物组合物，其特征在于，包括：
(a)权利要求1所述的融合蛋白、或其编码基因、或其表达载体；和
(b)药学上可接受的载体。
16.如权利要求15所述的药物组合物，其特征在于，所述药物组合物中还包括其他用于基因治疗的药物。
17.一种药盒，其特征在于，包括：
(a1)第一容器，以及位于所述第一容器中的权利要求1所述的融合蛋白、或其编码基因、或其表达载体，或含有权利要求1所述的融合蛋白的药物。
18.一种权利要求1所述的融合蛋白的用途，其特征在于，用于制备用于基因治疗的药物。
19.一种提高基因编辑效率的方法，其特征在于，包括步骤：
在权利要求1所述的融合蛋白或权利要求12所述的基因编辑试剂存在下，对细胞进行基因编辑，从而提高基因编辑效率，所述细胞为体外的细胞。
20.如权利要求19所述的方法，其特征在于，所述的基因编辑在一体外反应体系中进行。
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