CN112195164A - 工程化的Cas效应蛋白及其使用方法 - Google Patents

工程化的Cas效应蛋白及其使用方法 Download PDF

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CN112195164A
CN112195164A CN202011414384.3A CN202011414384A CN112195164A CN 112195164 A CN112195164 A CN 112195164A CN 202011414384 A CN202011414384 A CN 202011414384A CN 112195164 A CN112195164 A CN 112195164A
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李伟
周琪
陈阳灿
胡艳萍
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Beijing Institute Of Stem Cell And Regenerative Medicine
Institute of Zoology of CAS
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Abstract

本文提供了用于工程化改造酶诸如Cas核酸酶以增加其酶活性的方法。还提供了工程化的Cas效应蛋白，包括工程化的Cas12b、Cas12i和Cas9核酸酶及其衍生物，以及其使用方法。

Description

工程化的Cas效应蛋白及其使用方法
技术领域
本申请总体上涉及生物技术领域。更具体地说，本申请涉及具有提高的活性(例如，基因编辑活性)的工程化Cas效应蛋白的方法和组合物。
背景技术
基因组编辑是在基因组研究和多种应用中的重要且有用的技术。有多个系统可用于基因组编辑，包括成簇规律间隔短回文重复序列(CRISPR)-Cas系统、转录激活子样效应因子核酸酶(TALEN)系统以及锌指核酸酶(ZFN)系统。
CRISPR-Cas系统是一种高效且具有成本效益的基因组编辑技术，可广泛应用于从酵母、植物到斑马鱼和人类的一系列真核生物中(参见综述：Van der Oost 2013, Science339: 768-770，以及Charpentier and Doudna, 2013, Nature 495: 50-51)。CRISPR-Cas系统通过结合Cas效应蛋白和CRISPR RNA(crRNA)在古细菌和细菌中提供适应性免疫。迄今为止，基于该系统的突出的功能上和进化上的模块性，已经对包括六型(I–VI型)两类(第1类和第2类)的CRISPR-Cas系统进行了表征。在第2类CRISPR-Cas系统中，II型Cas9系统和V型-A/B/E/J Cas12a/Cas12b/Cas12e/Cas12j系统已被利用来进行基因组编辑，并为生物医学研究提供了广阔的前景。
但是，当前的CRISPR-Cas系统具有多种局限性，包括有限的基因编辑效率。因此，需要改进方法和系统，用以进行有效的跨多基因座的基因组编辑。
发明内容
为了满足以上和其他需求，本公开提供了用于工程化改造酶（诸如Cas核酸酶）以改善其酶活性的方法、工程化的Cas效应蛋白以及使用该工程化Cas效应蛋白的方法。
在一方面，本申请提供了工程化改造酶的方法，其包括：(a)获得多种工程化的酶，每种工程化的酶包含一个或多个突变，所述突变增加了参比酶的多个柔性区中的柔性区柔性；以及(b)从所述多种工程化的酶中选择一种或多种工程化的酶，其中所述一种或多种工程化的酶与所述参比酶相比具有增加的活性。在一些实施方案中，所述方法还包括确定参比酶中的多个柔性区。
在根据上述方法中任一种的一些实施方案中，所述多个柔性区是基于所述参比酶的氨基酸序列来确定。在一些实施方案中，所述多个柔性区是在不参考所述参比酶或其等同物的三维结构的情况下确定的。在一些实施方案中，所述多个柔性区是使用选自下组的程序来确定的：PredyFlexy、FoldUnfold、PROFbval、Flexserv、FlexPred、DynaMine和Disomine。在一些实施方案中，所述多个柔性区是使用DynaMine来确定的。
在根据上述方法中任一种的一些实施方案中，所述方法包括：(i)计算所述参比酶的每个氨基酸残基的柔性得分，其中较高的柔性得分表示较低的构象柔性；(ii)选择在Xi位置的多个峰氨基酸残基，其中每个峰氨基酸残基的柔性得分低于预定阈值，并且低于在Xi-5至Xi-1以及Xi+1和Xi+5位置的氨基酸残基的柔性得分；以及(iii)将所述多个柔性区定义为氨基酸残基Xi-2至Xi+2。
在根据上述方法中任一种的一些实施方案中，所述多个柔性区位于无规卷曲处。
在根据上述方法中任一种的一些实施方案中，所述一个或多个突变包括在柔性区中插入一个或多个甘氨酸(G)残基。在一些实施方案中，所述一个或多个突变包括在柔性区中插入两个G残基。在根据上述方法中任一种的一些实施方案中，所述一个或多个G残基插入在所述柔性区中的柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、丝氨酸(S)、天冬酰胺(N)、天冬氨酸(D)、组氨酸(H)、蛋氨酸(M)、苏氨酸(T)、谷氨酸(E)、谷氨酰胺(Q)、赖氨酸(K)、精氨酸(R)、丙氨酸(A)和脯氨酸(P)。在一些实施方案中，根据以下优先级选择柔性氨基酸残基：G>S>N>D>H>M>T>E>Q>K>R>A>P。
在根据上述方法中任一种的一些实施方案中，所述一个或多个突变包括用一个或多个G残基替换一个或多个非G残基。在一些实施方案中，所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：亮氨酸(L)、异亮氨酸(I)、缬氨酸(V)、半胱氨酸(C)、酪氨酸(Y)、苯丙氨酸(F)和色氨酸(W)。
在根据上述方法中任一种的一些实施方案中，所述酶是细菌或古细菌酶。在一些实施方案中，所述酶是Cas核酸酶。在一些实施方案中，所述Cas核酸酶选自下组：Cas9、Cas12a、Cas12b、Cas12c、Cas12d、Cas12f、Cas12g、Cas12h、Cms1、Cas12i、Cas12j、Cas12k和CasX。在一些实施方案中，所述多个柔性区在所述参比Cas核酸酶的与DNA和/或RNA相互作用的域中。在一些实施方案中，步骤(b)中描述的活性是位点特异性核酸酶活性。在一些实施方案中，所述活性是真核细胞(例如，哺乳动物细胞)中的基因编辑活性。在一些实施方案中，所述活性是人细胞中的基因编辑活性。在一些实施方案中，在所述细胞中的基因组位点（genomic locus），步骤(b)中的选定的工程化的Cas核酸酶的基因编辑效率比所述参比Cas核酸酶高至少约20%(例如，至少约30%、40%、50%、60%、70%、80%或更多)。在一些实施方案中，在所述细胞中的多个基因组位点，步骤(b)中选择的工程化的Cas核酸酶的平均基因编辑效率比所述参比Cas核酸酶高至少约20% (例如，至少约30%、40%、50%、60%，70%、80%或更多)。在一些实施方案中，所述基因编辑效率是使用如下方法来测量的：T7核酸内切酶1 (T7E1)测定、靶DNA的测序、由分解追踪插入缺失(TIDE)测定或通过扩增子分析进行插入缺失检测(IDAA)测定。
本申请的另一方面提供了使用上述方法中任一种方法来获得的工程化的Cas核酸酶。
在一些实施方案中，本申请提供了工程化的Cas核酸酶，其包含一个或多个突变，所述突变增加了包含参比Cas核酸酶的至少5个连续氨基酸残基的柔性区的柔性，其中所述工程化的Cas核酸酶与参比Cas核酸酶相比具有增加的活性。在一些实施方案中，所述柔性区是基于所述参比Cas核酸酶的氨基酸序列来确定的。在一些实施方案中，所述柔性区是使用选自下组的程序来确定的：PredyFlexy、FoldUnfold、PROFbval、Flexserv、FlexPred、DynaMine和Disomine。在一些实施方案中，所述柔性区是使用DynaMine来确定的，并且其中在所述柔性区中具有最高柔性的氨基酸残基的柔性得分S2 预测不大于约0.8。
在根据上述工程化的Cas核酸酶中任一种的一些实施方案中，所述柔性区具有5个连续的氨基酸残基，其中第3个氨基酸残基具有最低的柔性得分，并且其中较高的柔性得分表示较低的构象柔性。在根据上述工程化的Cas核酸酶中任一种的一些实施方案中，所述柔性区位于无规卷曲处。
在根据上述工程化的Cas核酸酶中任一种的一些实施方案中，所述柔性区在所述参比Cas核酸酶的与DNA和/或RNA相互作用的结构域中。
在根据上述工程化的Cas核酸酶中任一种的一些实施方案中，所述参比Cas核酸酶选自下组：Cas9、Cas12a、Cas12b、Cas12c、Cas12d、Cas12f、Cas12g、Cas12h、Cms1、Cas12i、Cas12j、Cas12k和CasX。
在一些实施方案中，本申请提供了工程化的Cas12b核酸酶，其包含一个或多个突变，所述突变增加了对应于参比Cas12b核酸酶中氨基酸残基835-839的柔性区的柔性，其中所述氨基酸残基编号是基于SEQ ID NO: 1，其中所述工程化的Cas12b核酸酶与所述参比Cas12b核酸酶相比具有增加的活性。在一些实施方案中，所述工程化的Cas12b核酸酶包含与SEQ ID NO: 2具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%中任一个)序列同一性的氨基酸序列。
在一些实施方案中，本申请提供了工程化的Cas12i核酸酶，其包含一个或多个突变，所述突变增加了参比Cas12i核酸酶中的柔性区的柔性，所述柔性区选自对应于以下的区的组：氨基酸残基228-232、氨基酸残基439-443、氨基酸残基478-482、氨基酸残基500-504、氨基酸残基775-779和氨基酸残基925-929，其中所述氨基酸残基编号基于SEQ ID NO:8，其中所述工程化的Cas12i核酸酶与所述参比Cas12i核酸酶相比具有增加的活性。在根据上述工程化的Cas12i核酸酶中任一种的一些实施方案中，所述柔性区对应于氨基酸残基439-443或氨基酸残基925-929，其中氨基酸残基编号基于SEQ ID NO: 8。在一些实施方案中，所述工程化的Cas12i核酸酶包含与选自下组SEQ ID NOs: 14、18和20的氨基酸序列具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、98%、99%或100%中任一个)序列同一性的氨基酸序列。
在一些实施方案中，本申请提供了工程化的Cas9核酸酶，其包含一个或多个突变，所述突变增加了参比Cas9核酸酶中的柔性区的柔性，所述柔性区选自对应于以下的区的组：氨基酸残基39-43、氨基酸残基135-139、氨基酸残基176-180、氨基酸残基274-278、氨基酸残基351-355、氨基酸残基389-393、氨基酸残基521-525、氨基酸残基541-545、氨基酸残基755-759、氨基酸残基774-778、氨基酸残基786-790、氨基酸残基811-815、氨基酸残基848-852、氨基酸残基855-859、氨基酸残基874-878、氨基酸氨基酸残基891-895、氨基酸残基1019-1023和氨基酸残基1036-1040，其中所述氨基酸残基编号基于SEQ ID NO: 25，其中所述工程化的Cas9核酸酶与所述参比Cas9核酸酶相比具有增强的活性。在根据上述工程化的Cas9核酸酶中任一种的一些实施方案中，所述柔性区选自对应于以下的区的组：氨基酸残基135-139、氨基酸残基176-180、氨基酸残基541-545、氨基酸残基755-759和氨基酸残基811-815，其中所述氨基酸残基编号基于SEQ ID NO: 25。在根据上述任一种工程化的Cas9核酸酶的一些实施方案中，所述工程化的核酸酶包含与选自下组SEQ ID NOs: 27、28、33、34、和41的氨基酸序列具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%中任一个)序列同一性的氨基酸序列。
在一些实施方案中，本申请提供了工程化的Cas9核酸酶，其包含一个或多个突变，所述突变增加了参比Cas9核酸酶中的柔性区的柔性，所述柔性区选自对应于以下的区的组：氨基酸残基45-49、氨基酸残基84-88、氨基酸残基116-120、氨基酸残基128-132、氨基酸残基216-220、氨基酸残基318-322、氨基酸残基387-391、氨基酸残基497-501、氨基酸残基583-587、氨基酸残基594-598、氨基酸残基614-618、氨基酸残基696-700和氨基酸残基739-743，其中所述氨基酸残基编号基于SEQ ID NO: 53，其中所述工程化的Cas9核酸酶与所述参比Cas9核酸酶相比具有增强的活性。在一些实施方案中，所述柔性区对应于氨基酸残基45-49或氨基酸残基116-120，其中所述氨基酸残基编号基于SEQ ID NO: 53。在一些实施方案中，所述工程化的Cas9核酸酶包含与选自下组SEQ ID NOs: 54和58-59的氨基酸序列具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%中任一个)序列同一性的氨基酸序列。
在根据上述工程化的Cas核酸酶中任一种的一些实施方案中，所述一个或多个突变包括在柔性区中插入一个或多个G残基。在一些实施方案中，所述一个或多个突变包括在柔性区中插入两个G残基。在一些实施方案中，所述一个或多个G残基被插入到所述柔性区中的柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、丝氨酸(S)、天冬酰胺(N)、天冬氨酸(D)、组氨酸(H)、蛋氨酸(M)、苏氨酸(T)、谷氨酸(E)、谷氨酰胺(Q)、赖氨酸(K)、精氨酸(R)、丙氨酸(A)和脯氨酸(P)，其中所述柔性氨基酸残基是根据以下优先级选择的：G>S>N>D>H>M>T>E>Q>K>R>A>P。
在根据上述工程化的Cas核酸酶中任一种的一些实施方案中，所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：亮氨酸(L)、异亮氨酸(I)、缬氨酸(V)、半胱氨酸(C)、酪氨酸(Y)、苯丙氨酸(F)和色氨酸(W)。
在根据上述工程化的Cas核酸酶中任一种的一些实施方案中，所述工程化的Cas核酸酶包含一个或多个突变，所述突变增加了所述参比Cas核酸酶中两个或更多个柔性区的柔性。
在根据上述工程化的Cas核酸酶中任一种的一些实施方案中，所述Cas核酸酶的增加的活性是位点特异性核酸酶活性。
在根据上述工程化的Cas核酸酶中任一种的一些实施方案中，所述Cas核酸酶的增加的活性是在真核细胞(例如，哺乳动物细胞)中的基因编辑活性。在一些实施方案中，所述Cas核酸酶的增加的活性是人细胞中的基因编辑活性。在一些实施方案中，在所述细胞中的基因组位点，所述工程化的Cas核酸酶的基因编辑效率比所述参比Cas核酸酶高至少约20%(例如，至少约30%、40%、50%、60%、70%、80%或更多)。在一些实施方案中，在所述细胞中的多个基因组位点，所述工程化的Cas核酸酶的平均基因编辑效率比所述参比Cas核酸酶高至少约20%(例如，至少约30%、40%、50%、60%、70%、80%或更多)。在一些实施方案中，所述基因编辑效率是使用如下方法来测量的：T7核酸内切酶1 (T7E1)测定、靶DNA的测序、由分解追踪插入缺失(TIDE)测定或通过扩增子分析进行插入缺失检测(IDAA)测定。
在一些实施方案中，本申请提供了Cas核酸酶，其包含选自下组的氨基酸序列：SEQID NOs. 2、14、18、20、27、28、33、34、41、54和58-59。
在一些实施方案中，本申请提供了工程化的Cas效应蛋白，其包含上述工程化的Cas核酸酶中任一种或其功能衍生物。在一些实施方案中，所述工程化的Cas核酸酶或其功能衍生物具有酶活性。在一些实施方案中，所述效应蛋白能够诱导DNA分子中的双链断裂。在一些实施方案中，所述工程化的Cas效应蛋白能够诱导DNA分子中的单链断裂。在一些实施方案中，所述效应蛋白包含所述工程化的Cas核酸酶的酶失活突变体。
在根据上述工程化Cas效应蛋白中任一种的一些实施方案中，所述工程化Cas效应蛋白还包含与所述工程化的Cas核酸酶或其功能衍生物融合的功能结构域。在一些实施方案中，所述功能结构域选自下组：翻译起始结构域、转录阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域(例如，CBE或ABE结构域)、逆转录酶结构域、报告分子结构域(例如，荧光结构域)和核酸酶结构域。
在根据上述工程化的Cas效应蛋白中任一种的一些实施方案中，所述工程化的Cas效应蛋白包含：含有所述工程化的Cas核酸酶或其功能衍生物的N末端部分的第一多肽和含有所述工程化的Cas核酸酶或其功能衍生物的C末端部分的第二多肽，其中所述第一多肽和所述第二多肽能够在包含指导序列的指导RNA的存在下彼此缔合，以形成与靶核酸特异性结合的成簇规律间隔短回文重复序列(CRISPR)复合物，所述靶核酸包含与所述指导序列互补的靶序列。在一些实施方案中，所述第一多肽和所述第二多肽各自包含二聚化结构域。在一些实施方案中，所述第一二聚结构域和所述第二二聚结构域在诱导剂存在下彼此缔合。在一些实施方案中，所述第一多肽和所述第二多肽不包含二聚化结构域。
本申请的另一方面提供了工程化的CRISPR-Cas系统，其包括：(a)上述工程化的Cas效应蛋白中任一种；(b)包含与靶序列互补的指导序列的指导RNA，或编码所述指导RNA的一种或多种核酸，其中所述工程化的Cas效应蛋白和所述指导RNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合包含所述靶序列的靶核酸并诱导所述靶核酸的修饰。在一些实施方案中，所述指导RNA是包含所述指导序列的crRNA。在一些实施方案中，所述系统包括编码多个crRNA的前体指导RNA阵列（array）。在一些实施方案中，所述指导RNA包含crRNA和tracrRNA。在一些实施方案中，所述指导RNA是单个指导RNA(sgRNA)。在一些实施方案中，所述指导RNA包含crRNA和scoutRNA。在一些实施方案中，其中所述工程化的Cas效应蛋白是主编辑器，所述指导RNA是pegRNA。
在根据上述CRISPR-Cas系统中任一种的一些实施方案中，所述系统包含一种或多种编码所述工程化的Cas效应蛋白的载体。在一些实施方案中，所述一种或多种载体选自下组：逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关的载体和单纯疱疹载体。在一些实施方案中，所述一种或多种载体是腺相关病毒(AAV)载体。在一些实施方案中，AAV载体还编码所述指导RNA(例如，crRNA、sgRNA或前体指导RNA阵列)。
本申请的另一方面提供了工程化的CRISPR-Cas系统，包括：(a) Cas12i效应蛋白，其包含Cas12i核酸酶(例如，Cas12i2)或其功能衍生物；(b) crRNA，其在包含至少四个U残基和与靶序列互补的指导序列的重复序列中用一个非U残基替换一个或多个尿苷(U)残基；其中所述Cas12i效应蛋白和所述crRNA能够形成CRISPR复合物，所述复合物与包含靶序列的靶核酸特异性结合并诱导所述靶核酸的修饰。
本申请的另一方面提供了检测样品中靶核酸的方法，所述方法包括：(a)使样品与上述工程化的CRISPR-Cas系统中任一种和单链且不与所述指导RNA的指导序列杂交的加标签的检测核酸接触；以及(b)测量通过所述工程化的Cas效应蛋白切割所述加标签的检测核酸而产生的可检测信号，从而检测所述靶核酸。
本申请的另一方面提供了修饰包含靶序列的靶核酸的方法，包括使所述靶核酸与上述工程化的CRISPR-Cas系统中任一种接触。在一些实施方案中，所述方法在体外进行。在一些实施方案中，所述靶核酸存在于细胞中。在一些实施方案中，所述细胞是细菌细胞、酵母细胞、哺乳动物细胞、植物细胞或动物细胞。在一些实施方案中，所述方法离体进行。在一些实施方案中，所述方法在体内进行。
在根据上述修饰靶核酸的方法中任一种的一些实施方案中，通过所述工程化的CRISPR-Cas系统切割所述靶核酸或改变所述靶核酸中的靶序列。在一些实施方案中，通过所述工程化的CRISPR-Cas系统改变靶核酸的表达。在一些实施方案中，所述靶核酸是基因组DNA。在一些实施方案中，所述靶序列与疾病或病况相关。
在根据上述修饰靶核酸的方法中任一种的一些实施方案中，所述工程化的CRISPR-Cas系统包括编码多个crRNA的前体指导RNA阵列，其中每个crRNA包含不同的指导序列。
在根据上述修饰靶核酸的方法中任一种的实施方案中，所述方法在约4oC至约67oC(例如，约4oC至约15oC，约15oC至40oC，约4oC至约37oC，或约40oC至约67oC)的温度下进行。
本申请的另一方面提供了治疗与个体细胞中靶核酸相关的疾病或病症的方法，包括采用使用上述工程化的CRISPR-Cas系统的方法中任一种来修饰所述个体的细胞中的靶核酸，从而治疗所述疾病或病症。在一些实施方案中，所述疾病或病症选自下组：癌症、心血管疾病、遗传性疾病、自身免疫疾病、代谢性疾病、神经退行性疾病、眼病、细菌感染和病毒感染。
本申请的另一方面提供了工程化的细胞，其包含经修饰的靶核酸，其中所述靶核酸已使用上述方法之一进行修饰。
在一些实施方案中，本申请提供了工程化的非人类动物，其包含一种或多种上述工程化的细胞。
本申请的另一方面提供了修饰靶核酸中靶序列的方法，包括使所述靶核酸与工程化的CRISPR-Cas系统在约40℃至约67℃的温度下接触，其中所述工程化的CRISPR-Cas系统包括：(a) Cas12i2效应蛋白，其包含Cas12i2核酸酶或其功能衍生物；(b) crRNA，其包含与所述靶序列互补的指导序列；其中所述Cas12i2效应蛋白和所述crRNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合包含所述靶序列的靶核酸并诱导所述靶核酸的修饰。
本申请的另一方面提供了工程化的crRNA，其在包含至少四个U残基的重复序列中用一个非U残基替换一个或多个尿苷(U)残基。在一些实施方案中，所述工程化的crRNA包含约17-25个核苷酸长度的间隔区序列。在一些实施方案中，所述工程化的crRNA包含约20个核苷酸长度的间隔区序列。在一些实施方案中，所述工程化的crRNA包含含有SEQ ID NO:173的核酸序列的重复序列。
还提供了用于上述方法中任一种的组合物、试剂盒和制品。
具体地，本申请还涉及以下各项：
1. 工程化改造酶的方法，包括：
(a)获得多种工程化的酶，每种工程化的酶包含一个或多个突变，所述突变增加了参比酶的多个柔性区中的柔性区柔性；以及
(b)从所述多种工程化的酶中选择一种或多种工程化的酶，其中所述一种或多种工程化的酶与所述参比酶相比具有增加的活性。
2. 如项1所述的方法，其中所述多个柔性区是基于所述参比酶的氨基酸序列来确定的。
3. 如项2所述的方法，其中所述多个柔性区是在不参考所述参比酶或其等同物的三维结构情况下确定的。
4. 如项3所述的方法，其中所述多个柔性区是使用选自下组的程序来确定的：PredyFlexy、FoldUnfold、PROFbval、Flexserv、FlexPred、DynaMine和Disomine。
5. 如项4所述的方法，其中所述多个柔性区是使用DynaMine来确定的。
6. 如项1-5中任一项所述的方法，还包括：
(i)计算所述参比酶的每个氨基酸残基的柔性得分，其中较高的柔性得分表示较低的构象柔性；
(ii)选择在Xi位置的多个峰氨基酸残基，其中每个峰氨基酸残基的柔性得分低于预定阈值，并且低于在Xi-5至Xi-1、Xi+1和Xi+5位置的氨基酸残基的柔性得分；以及
(iii)将所述多个柔性区定义为氨基酸残基Xi-2至Xi+2。
7. 如项1-6中任一项所述的方法，其中所述多个柔性区位于无规卷曲处。
8. 如项1-7中任一项所述的方法，其中所述一个或多个突变包括在柔性区中插入一个或多个甘氨酸(G)残基。
9. 如项8所述的方法，其中所述一个或多个突变包括在柔性区中插入两个G残基。
10. 如项8或9所述的方法，其中所述一个或多个G残基插入在所述柔性区中的柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、丝氨酸(S)、天冬酰胺(N)、天冬氨酸(D)、组氨酸(H)、蛋氨酸(M)、苏氨酸(T)、谷氨酸(E)、谷氨酰胺(Q)、赖氨酸(K)、精氨酸(R)、丙氨酸(A)和脯氨酸(P)。
11. 如项1-10中任一项所述的方法，其中所述一个或多个突变包括用一个或多个G残基替换一个或多个非G残基。
12. 如项11所述的方法，其中所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：亮氨酸(L)、异亮氨酸(I)、缬氨酸(V)、半胱氨酸(C)、酪氨酸(Y)、苯丙氨酸(F)和色氨酸(W)。
13. 如项1-12中任一项所述的方法，其中所述酶是细菌或古细菌酶。
14. 如项13的方法，其中所述酶是Cas核酸酶。
15. 如项14的方法，其中所述Cas核酸酶选自下组：Cas9、Cas12a、Cas12b、Cas12c、Cas12d、Cas12f、Cas12g、Cas12h、Cms1、Cas12i、Cas12j、Cas12k和CasX。
16. 如项14或15所述的方法，其中所述多个柔性区在所述参比Cas核酸酶的与DNA和/或RNA相互作用的结构域中。
17. 如项14-16中任一项所述的方法，其中所述活性是位点特异性核酸酶活性。
18. 如项14-17中任一项所述的方法，其中所述活性是真核细胞中的基因编辑活性。
19. 如项18的方法，其中所述活性是人细胞中的基因编辑活性。
20. 如项18或19所述的方法，其中在所述细胞中的一个基因组位点，步骤(b)中选定的工程化的Cas核酸酶的基因编辑效率比所述参比Cas核酸酶高至少约20%。
21. 如项18-20中任一项所述的方法，其中在所述细胞中的多个基因组位点，步骤(b)中选择的工程化的Cas核酸酶的平均基因编辑效率比所述参比Cas核酸酶高至少约20%。
22. 如项20或21所述的方法，其中所述基因编辑效率是使用如下方法来测量的：T7核酸内切酶1 (T7E1)测定、靶DNA的测序、由分解追踪插入缺失(TIDE)测定或通过扩增子分析进行插入缺失检测(IDAA)测定。
23. 使用项14-22中任一项所述的方法获得的工程化的Cas核酸酶。
24. 工程化的Cas核酸酶，其包含一个或多个突变，所述突变增加了包含参比Cas核酸酶的至少5个连续氨基酸残基的柔性区柔性，其中所述工程化的Cas核酸酶与所述参比Cas核酸酶相比具有提高的活性。
25. 如项24所述的工程化的Cas核酸酶，其中所述柔性区是基于所述参比Cas核酸酶的氨基酸序列来确定的。
26. 如项25所述的工程化的Cas核酸酶，其中所述柔性区是使用选自下组的程序来确定的：PredyFlexy、FoldUnfold、PROFbval、Flexserv、FlexPred、DynaMine和Disomine。
27. 如项26所述的工程化的Cas核酸酶，其中所述柔性区是使用DynaMine来确定的，并且其中在所述柔性区中具有最高柔性的氨基酸残基的柔性得分S2 预测不大于约0.8。
28. 如项24-27中任一项所述的工程化的Cas核酸酶，其中所述柔性区具有5个连续的氨基酸残基，其中第3个氨基酸残基具有最低的柔性得分，并且其中较高的柔性得分表示较低的构象柔性。
29. 如项24-28中任一项所述的工程化的Cas核酸酶，其中所述柔性区位于无规卷曲处。
30. 如项24-29中任一项所述的工程化的Cas核酸酶，其中所述柔性区在所述参比Cas核酸酶的与DNA和/或RNA相互作用的结构域中。
31. 如项24-30中任一项所述的工程化的Cas核酸酶，其中所述参比Cas核酸酶选自下组：Cas9、Cas12a、Cas12b、Cas12c、Cas12d、Cas12f、Cas12g、Cas12h、Cms1、Cas12i、Cas12j、Cas12k和CasX。
32. 工程化的Cas12b核酸酶，其包含一个或多个突变，所述突变增加了对应于参比Cas12b核酸酶中氨基酸残基835至839的柔性区柔性，其中所述氨基酸残基编号基于SEQID NO: 1，其中所述工程化的Cas12b核酸酶与所述参比Cas12b核酸酶相比具有提高的活性。
33. 如项32所述的工程化的Cas12b核酸酶，其包含与SEQ ID NO: 2具有至少约85%序列同一性的氨基酸序列。
34. 工程化的Cas12i核酸酶，其包含一个或多个突变，所述突变增加了参比Cas12i核酸酶中的柔性区的柔性，所述柔性区选自对应于以下的区的组：氨基酸残基228-232、氨基酸残基439-443、氨基酸残基478-482、氨基酸残基500-504、氨基酸残基775-779和氨基酸残基925-929，其中所述氨基酸残基编号基于SEQ ID NO: 8，其中所述工程化的Cas12i核酸酶与所述参比Cas12i核酸酶相比具有增加的活性。
35. 如项34所述的工程化的Cas12i核酸酶，其中所述柔性区对应于氨基酸残基439-443或氨基酸残基925-929，其中所述氨基酸残基编号基于SEQ ID NO: 8。
36. 如项34或35所述的工程化的Cas12i核酸酶，其包含与选自下组SEQ ID NOs:14、18和20的氨基酸序列具有至少约85%序列同一性的氨基酸序列。
37. 工程化的Cas9核酸酶，其包含一个或多个突变，所述突变增加了参比Cas9核酸酶中的柔性区的柔性，所述柔性区选自对应于以下的区的组：氨基酸残基39-43，氨基酸残基135-139，氨基酸残基176-180、氨基酸残基274-278、氨基酸残基351-355、氨基酸残基389-393、氨基酸残基521-525、氨基酸残基541-545、氨基酸残基755-759、氨基酸残基774-778、氨基酸残基786-790、氨基酸残基811-815、氨基酸残基848-852、氨基酸残基855-859、氨基酸残基874-878、氨基酸残基891-895、氨基酸残基1019-1023和氨基酸残基1036-1040，其中所述氨基酸残基编号基于SEQ ID NO: 25，其中所述工程化的Cas9核酸酶与所述参比Cas9核酸酶相比具有增强的活性。
38. 如项37所述的工程化的Cas9核酸酶，其中所述柔性区选自对应于以下的区的组：氨基酸残基135-139、氨基酸残基176-180、氨基酸残基541-545、氨基酸残基755-759和氨基酸残基811-815，其中所述氨基酸残基编号基于SEQ ID NO: 25。
39. 如项37或38所述的工程化的Cas9核酸酶，其包含与选自下组SEQ ID NOs:27、28、33、34、和41的氨基酸序列具有至少约85%序列同一性的氨基酸序列。
40. 工程化的Cas9核酸酶，其包含一个或多个突变，所述突变增加了参比Cas9核酸酶中的柔性区的柔性，所述柔性区选自对应于以下的区的组：氨基酸残基45-49、氨基酸残基84-88、氨基酸残基116-120、氨基酸残基128-132、氨基酸残基216-220、氨基酸残基318-322、氨基酸残基387-391、氨基酸残基497-501、氨基酸残基583-587、氨基酸残基594-598、氨基酸残基614-618、氨基酸残基696-700和氨基酸残基739-743，其中所述氨基酸残基编号基于SEQ ID NO: 53，其中所述工程化的Cas9核酸酶与所述参比Cas9核酸酶相比具有增加的活性。
41. 如项40所述的工程化的Cas9核酸酶，其中所述柔性区对应于氨基酸残基45-49或氨基酸残基116-120，其中所述氨基酸残基编号基于SEQ ID NO: 53。
42. 如项40或41所述的工程化的Cas9核酸酶，其包含与选自下组SEQ ID NOs: 54和58-59的氨基酸序列具有至少约85%序列同一性的氨基酸序列。
43. 如项24-42中任一项所述的工程化的Cas核酸酶，其中所述一个或多个突变包括在所述柔性区中插入一个或多个G残基。
44. 如项43所述的工程化的Cas核酸酶，其中所述一个或多个突变包括在柔性区中插入两个G残基。
45. 如项43或44所述的工程化的Cas核酸酶，其中所述一个或多个G残基被插入到所述柔性区中的柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、丝氨酸(S)、天冬酰胺(N)、天冬氨酸(D)、组氨酸(H)、蛋氨酸(M)、苏氨酸(T)、谷氨酸(E)、谷氨酰胺(Q)、赖氨酸(K)、精氨酸(R)、丙氨酸(A)和脯氨酸(P)。
46. 如项24-45中任一项所述的工程化的Cas核酸酶，其中所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：亮氨酸(L)、异亮氨酸(I)、缬氨酸(V)、半胱氨酸(C)、酪氨酸(Y)、苯丙氨酸(F)和色氨酸(W)。
47. 如项24-46中任一项所述的工程化的Cas核酸酶，其包含一个或多个突变，其增加了所述参比Cas核酸酶中两个或更多个柔性区的柔性。
48. 如项24-47中任一项所述的工程化的Cas核酸酶，其中所述活性是位点特异性核酸酶活性。
49. 如项24-48中任一项所述的工程化的Cas核酸酶，其中所述活性是在真核细胞中的基因编辑活性。
50. 如项49所述的工程化的Cas核酸酶，其中所述活性是在人细胞中的基因编辑活性。
51. 如项49或50所述的工程化的Cas核酸酶，其中在所述细胞中的一个基因组位点，所述工程化的Cas核酸酶的基因编辑效率比所述参比Cas核酸酶高至少约20%。
52. 如项49-51中任一项所述的工程化的Cas核酸酶，其中在所述细胞中的多个基因组位点，所述工程化的Cas核酸酶的平均基因编辑效率比所述参比Cas核酸酶高至少约20%。
53. 如项49-52中任一项所述的工程化的Cas核酸酶，其中所述基因编辑效率是使用如下方法来测量的：T7核酸内切酶1 (T7E1)测定、靶DNA的测序、由分解追踪插入缺失(TIDE)测定或通过扩增子分析进行插入缺失检测(IDAA)测定。
54. 工程化的Cas核酸酶，其包含选自下组SEQ ID NOs: 2、14、18、20、27、28、33、34、41、54和58-59的氨基酸序列。
55. 工程化的Cas效应蛋白，其包含项23-54中任一项所述的工程化的Cas核酸酶或其功能衍生物。
56. 如项55所述的工程化的Cas效应蛋白，其中所述工程化的Cas核酸酶或其功能衍生物具有酶活性。
57. 如项56所述的工程化的Cas效应蛋白，其中所述效应蛋白能够诱导DNA分子中的双链断裂。
58. 如项56所述的工程化的Cas效应蛋白，其中所述效应蛋白能够诱导DNA分子中的单链断裂。
59. 如项55所述的工程化的Cas效应蛋白，其中所述效应蛋白包含所述工程化的Cas核酸酶的酶失活突变体。
60. 如项55-59中任一项所述的工程化的Cas效应蛋白，其还包含与所述工程化的Cas核酸酶或其功能衍生物融合的功能结构域。
61. 如项60所述的工程化的Cas效应蛋白，其中所述功能结构域选自下组：翻译起始结构域、转录阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域、逆转录酶结构域、报告分子结构域和核酸酶结构域。
62. 如项55-61中任一项所述的工程化的Cas效应蛋白，其包含：含有所述工程化的Cas核酸酶或其功能衍生物的N末端部分的第一多肽和含有所述工程化的Cas核酸酶或其功能衍生物的C末端部分的第二多肽，其中所述第一多肽和所述第二多肽能够在包含指导序列的指导RNA的存在下彼此缔合，以形成与靶核酸特异性结合的成簇规律间隔短回文重复序列(CRISPR)复合物，所述靶核酸包含与所述指导序列互补的靶序列。
63. 如项62所述的工程化的Cas效应蛋白，其中所述第一多肽和所述第二多肽各自包含二聚化结构域。
64. 如项63所述的工程化的Cas效应蛋白，其中所述第一二聚化结构域和所述第二二聚化结构域在诱导剂存在下彼此缔合。
65. 如项62所述的工程化的Cas效应蛋白，其中所述第一多肽和所述第二多肽不包含二聚化结构域。
66. 工程化的CRISPR-Cas系统，包括：
(a)项55-65中任一项所述的工程化的Cas效应蛋白；以及
(b)包含与靶序列互补的指导序列的指导RNA，或编码所述指导RNA的一种或多种核酸，
其中所述工程化的Cas效应蛋白和所述指导RNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合包含所述靶序列的靶核酸并诱导所述靶核酸的修饰。
67. 如项66所述的工程化的CRISPR-Cas系统，其中所述指导RNA是包含所述指导序列的crRNA。
68. 如项66或67所述的工程化的CRISPR-Cas系统，其包括编码多个crRNA的前体指导RNA阵列。
69. 如项66所述的工程化的CRISPR-Cas系统，其中所述指导RNA包含crRNA和tracrRNA。
70. 如项69所述的工程化的CRISPR-Cas系统，其中所述指导RNA是单个指导RNA(sgRNA)。
71. 如项66-70中任一项所述的工程化的CRISPR-Cas系统，其包含一种或多种编码所述工程化的Cas效应蛋白的载体。
72. 如项71所述的工程化的CRISPR-Cas系统，其中所述一种或多种载体是腺相关病毒(AAV)载体。
73. 如项72所述的工程化的CRISPR-Cas系统，其中所述AAV载体还编码所述指导RNA。
74. 检测样品中靶核酸的方法，包括：
(a)使样品与项66-68中任一项所述的工程化的CRISPR-Cas系统和单链且不与所述指导RNA的指导序列杂交的加标签的检测核酸接触；以及
(b)测量通过所述工程化的Cas效应蛋白切割所述加标签的检测核酸而产生的可检测信号，从而检测所述靶核酸。
75. 修饰包含靶序列的靶核酸的方法，包括使所述靶核酸与项66-73中任一项所述的工程化的CRISPR-Cas系统接触。
76. 如项75所述的方法，其中所述方法在体外进行。
77. 如项75所述的方法，其中所述靶核酸存在于细胞中。
78. 如项77所述的方法，其中所述细胞是细菌细胞、酵母细胞、哺乳动物细胞、植物细胞或动物细胞。
79. 如项77或78所述的方法，其中所述方法离体进行。
80. 如项77或78所述的方法，其中所述方法在体内进行。
81. 如项75-80中任一项所述的方法，其中通过所述工程化的CRISPR-Cas系统切割所述靶核酸或改变所述靶核酸中的靶序列。
82. 如项75-80中任一项所述的方法，其中通过所述工程化的CRISPR-Cas系统改变所述靶核酸的表达。
83. 如项75-82中任一项所述的方法，其中所述靶核酸是基因组DNA。
84. 如项75-83中任一项所述的方法，其中所述靶序列与疾病或病症相关。
85. 如项75-84中任一项所述的方法，其中所述工程化的CRISPR-Cas系统包括编码多个crRNA的前体指导RNA阵列，其中每个crRNA均包含不同的指导序列。
86. 如项75-85中任一项所述的方法，其中所述方法在约4℃至约67℃的温度下进行。
87. 如项66-73中任一项所述的工程化的CRISPR-Cas系统在治疗与个体的细胞中靶核酸相关的疾病或病症的药物中的用途。
88. 如项87所述的用途，其中所述疾病或病症选自下组：癌症、心血管疾病、遗传性疾病、自身免疫性疾病、代谢性疾病、神经退行性疾病、眼病、细菌感染和病毒感染。
89. 工程化的细胞，其包含经修饰的靶核酸，其中使用如项75-86中任一项所述的方法修饰过所述靶核酸。
90. 工程化的非人类动物，其包含一种或多种如项89所述的工程化的细胞。
应理解为清楚起见，在单独实施方案的上下文中描述的本公开的某些特征，也可以在单个实施方案中组合提供。相反，为简洁起见，在单个实施方案的上下文中描述的本公开的各种特征，也可以单独地或以任何合适的子组合来提供。涉及特定方法步骤、试剂或条件或组合物组分的实施方案的所有组合被本公开明确地涵盖，并且在本文中公开，如同各自和每个组合均被单独地和明确地公开。
附图说明
图1A显示了用于工程化具有提高的活性的Cas蛋白的示例性设计管线。在此示例中，使用DynaMine软件识别了候选柔性区，并引入了甘氨酸替代(单下划线)或插入(双下划线)以生成Cas蛋白变体。然后将变体与eGFP一起克隆到表达载体中，并用变体Cas + GFP质粒和sgRNA或crRNA质粒转染细胞，以测试所述变体的编辑效率。
图1B显示了根据获得的Cas蛋白S2序参数得分谱确定峰氨基酸。
图2显示了BhCas12b4的柔性(S2)得分谱。
图3显示了在人细胞中使用示出的sgRNA由野生型BhCas12b4产生的插入缺失%（相比于enBhCas12b4）。图3中最后一个图显示了对于所有测试的位点产生的插入缺失%。
图4显示了Cas12i2的柔性(S2)得分谱。
图5显示了通过计算确定的Cas12i2的二级结构区域。
图6显示了使用四个不同crRNA，Cas12i2变体与WT Cas12i相比的基因编辑效率(插入缺失%)。
图7显示了与单独的WT Cas12i或变体2.2和6.1相比，具有变体2.2+6.1的组合突变的Cas12i2变体(命名为enCas12i2)的基因编辑效率(插入缺失%)。enCas12i2 (2.2+6.1)显示出提高的编辑效率。
图8A显示了与SpCas9和BhCas12b-v4相比，enCas12i2在人细胞中的整体基因组编辑效率。对于enCas12i2在46个位点、对于SpCas9在18个位点且对于BhCas12bv4在23个位点分析了编辑效率(插入缺失%)。
图8B显示了在人293T细胞中enCas12i2在包括NTTA、NTTC、NTTG和NTTT以及ATTN、CTTN、GTTN和TTTN的不同原间隔区相邻基序(PAM)位点的平均编辑效率。
图9显示了enCas12i2在体内对前体crRNA的加工。与使用单个crRNA相比，使用靶向3个位点的前体crRNA，enCas12i2具有相当的基因组编辑活性。
图10显示了Cas12i2和enCas12i2对DNA质粒的体外切割。
图11A显示了通过野生型Cas12i2和工程化的Cas12i2变体检测含有XBP靶序列的dsDNA。
图11B显示了证明在核酸检测实验中野生型Cas12i2可切割经由rU连接的荧光报告分子的结果。
图12显示了GeoCas9的柔性(S2)得分谱。
图13显示了由计算确定的GeoCas9的二级结构区域。
图14显示了靶向深度测序（Targeted deep sequencing）测定的结果，以确定通过工程化的GeoCas9变体产生插入缺失的效率。箭头指示了具有显著提高的编辑效率的工程化的GeoCas9变体。
图15显示了SaCas9的选定柔性区和相应结构域的位置。
图16显示了T7酶切割测定的结果，以测试SaCas9变体的编辑效率。工程化的SaCas9变体1.1、3.1、3.2(以方框轮廓表示)显示出显著提高的基因编辑效率。
图17A显示了野生型Cas12i1和Cas12i2的体外dsDNA切割。
图17B显示了用于测试Cas12i1或Cas12i2的切割的crRNA序列。
图18显示了在人类基因组靶位点使用crRNA，由野生型Cas12i1和Cas12i2产生的插入缺失百分比。使用T7核酸内切酶1(T7E1)测定来测量基因编辑效率。我们发现，Cas12i2能够在更广泛的靶标中产生插入缺失，这表明它是工程学的有希望的候选者。
图19显示了与使用Cas12i1的野生型crRNA序列相比，所示出的crRNA突变体的编辑效率。
图20显示了证明Cas12i2在12℃-67℃的较宽温度范围内具有酶活性(即能够切割dsDNA)的结果。
图21显示了使用crRNA阵列(前体crRNA)在人细胞中的多个基因组靶位点的Cas12i2编辑。
图22显示了通过使用在crRNA的1-19个碱基之一处具有单个碱基错配的crRNA，测试Cas12i2产生插入缺失的能力来定义Cas12i2的种子序列的结果。
图23显示了对于Cas12i2的20bp最佳间隔区长度的鉴定。测试了Cas12i2使用17-25 bp间隔区长度范围，采用两个不同crRNA (CCR5-1和CCR5-2)产生插入缺失的能力。
具体实施方式
本申请提供了通过引入氨基酸突变来工程化改造酶的方法，所述氨基酸突变增强了所述酶的柔性区中的柔性，这导致体外和体内酶活性的增加。本文所述的方法适用于多种Cas核酸酶，包括Cas12b、Cas12i和Cas9。值得注意的是，本文描述的方法不依赖于所述Cas核酸酶的三维结构。例如，本文所述的方法已经成功地应用于Cas12i，以产生在广泛的基因座位范围具有改善的基因组编辑效率的多种工程化的Cas12i蛋白。还提供了工程化的Cas效应蛋白(例如，Cas12b、Cas12i和Cas9)及其使用方法。
定义
除非另有定义，本文中使用的所有技术和科学术语具有与本公开所属领域的普通技术人员通常所理解的相同含义。
如本文所用，“效应蛋白”是指具有活性如位点特异性结合活性、单链DNA切割活性、双链DNA切割活性、单链RNA切割活性或转录调节活性的蛋白。
如本文所用，“指导RNA”和“gRNA”在本文中可互换使用，是指能够与Cas效应蛋白和靶核酸(例如，双链DNA)形成复合物的RNA。指导RNA可以包含单个RNA分子或通过两个或更多个RNA分子，其通过在两个或更多个RNA分子中的互补区域杂交而彼此结合。当与双RNA指导的Cas核酸酶如Cas9结合使用时，指导RNA包含crRNA和tracrRNA。当与单个RNA指导的Cas核酸酶如Cas12i结合使用时，指导RNA不包含tracrRNA或另外的反式激活RNA。本文还考虑了可以被加工成多个crRNA的前体指导RNA阵列，并且对于某些CRISPR/Cas系统，加工后的crRNA进一步与tracrRNA或另一种反式激活RNA(例如scoutRNA)结合以指导Cas效应蛋白。“crRNA”或“CRISPR RNA”包含与靶核酸(例如，双链DNA)的靶序列具有足够互补性的指导序列，其指导CRISPR复合物与靶核酸的序列特异性结合。“tracrRNA”或“反式激活CRISPRRNA”与crRNA部分互补并与之碱基配对，并可在crRNA的成熟中发挥作用。“单一指导RNA”或“sgRNA”是在单个分子中具有相互融合的crRNA和tracrRNA的工程化的指导RNA。
术语“核酸”、“多核苷酸”和“核苷酸序列”可互换使用，是指任何长度的核苷酸的聚合形式，包括脱氧核糖核苷酸、核糖核苷酸、其组合及其类似物。“寡核苷酸”和“低聚核苷酸”可互换使用，是指具有不超过约50个核苷酸的短多核苷酸。
如本文所用，“互补性”是指核酸通过传统的沃森-克里克(Watson-Crick)碱基配对与另一核酸形成氢键的能力。互补性百分比表示可与第二种核酸形成氢键(即，沃森-克里克碱基配对)的核酸分子中的残基百分比(例如，10分之5、6、7、8、9、10，分别互补约50%、60%、70%、80%、90%和100%)。“完全互补”是指核酸序列的所有连续残基与第二核酸序列中相同数目的连续残基形成氢键。如本文所用，“基本上互补”是指在约40、50、60、70、80、100、150、200、250个或更多个核苷酸的区域上，互补程度为至少约70%、75%、80%、85%、90%、95%、97%、98%、99%或100中任一个，或指在严格条件下杂交的两种核酸。
如本文所用，用于杂交的“严格条件”是指与靶序列具有互补性的核酸主要与靶序列杂交，而基本上不与非靶序列杂交的条件。严格条件通常是序列依赖性的，并且取决于许多因素而变化。通常，序列越长，序列与其靶序列特异性杂交的温度越高。严格条件的非限制性例子详细描述于Tijssen (1993), Laboratory Techniques In Biochemistry AndMolecular Biology- Hybridization With Nucleic Acid Probes，第一部分第二章"Overview of principles of hybridization and the strategy of nucleic acidprobe assay,” Elsevier, N,Y。
“杂交”是指一种或多种多核苷酸反应形成复合物的反应，所述复合物通过核苷酸残基的碱基之间的氢键而稳定化。氢键可通过沃森克里克碱基配对、霍格斯坦(Hoogstein)结合或以任何其他序列特异性方式发生。能够与给定序列杂交的序列称为所述给定序列的“互补体”。
针对核酸序列的“序列同一性百分比(%)”定义为，在通过允许空缺(gaps)来比对序列(如有必要)以实现最大的序列同一性百分比后，候选序列中与特定核酸序列中的核苷酸相同的核苷酸百分比。针对肽、多肽或蛋白质序列的“序列同一性百分比(%)”，是在通过允许空缺来比对序列(如有必要)以实现最大的序列同源性百分比后，候选序列中与特定肽或氨基酸序列中的氨基酸残基相同替换的氨基酸残基的百分比。为了确定氨基酸序列同一性百分比的目的，比对可以以本领域技术范围内的各种方式来实现，例如，使用诸如BLAST、BLAST-2、ALIGN或MEGALIGNTM (DNASTAR)软件之类的公众可获得的计算机软件。本领域技术人员可以确定用于测量比对的合适参数，包括在所比较序列的全长上实现最大比对所需的任何算法。
术语“多肽”和“肽”在本文可互换使用，是指任何长度的氨基酸的聚合物。所述聚合物可以是直链或支链的，它可以包含经修饰的氨基酸，并且可以被非氨基酸中断。蛋白质可以具有一个或多个多肽。该术语还涵盖已经过修饰的氨基酸聚合物；例如，二硫键的形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作（诸如与标记组分的缀合）。
如本文所用，“变体”解释为分别不同于参比多核苷酸或多肽但保留必要特性的多核苷酸或多肽。多核苷酸的典型变体与另一参比多核苷酸的核酸序列不同。变体核酸序列的变化可以改变或可以不改变参比多核苷酸编码的多肽的氨基酸序列。核苷酸变化可导致参比序列编码的多肽中的氨基酸替换、添加、缺失、融合和截短，如下所述。多肽的典型变体与另一参比多肽在氨基酸序列上不同。通常，差异是有限的，使得参比多肽和变体的序列总体上非常相似，并且在许多区域是相同的。变体和参比多肽的氨基酸序列可以通过一个或多个替换、添加、缺失的任何组合而不同。替换或插入的氨基酸残基可以是或可以不是遗传密码编码的氨基酸残基。多核苷酸或多肽的变体可以是天然存在的（诸如等位基因变体），或者可以是未知天然存在的变体。多核苷酸和多肽的非天然存在的变体可以通过诱变技术，通过直接合成，以及通过本领域技术人员已知的其他重组方法来制备。
如本文所用，术语“野生型”具有本领域技术人员通常理解的含义，意指当它存在于大自然中时，将其与突变体或变体区分开的、典型形式的生物体、菌株、基因或特征。它可以与自然界中的资源隔离开来，并没有被刻意修饰。
如本文所用，术语“非天然存在”或“工程化的”可互换使用，是指人工参与。当这些术语用于描述核酸分子或多肽时，是指所述核酸分子或多肽至少基本上不含其天然缔合的或天然存在的至少一种其他组分。
如本文所用，术语“直系同源物(orthologue/ ortholog)”具有本领域普通技术人员通常理解的含义。作为进一步的指导，本文所指的蛋白质的“直系同源物”是指属于不同物种的蛋白质，其执行与作为其直系同源物的蛋白质相同或相似的功能。
如本文所用，术语“同一性”用于表示两个多肽之间或两个核酸之间的序列匹配。当两个比较序列中的一个位置被相同的碱基或氨基酸单体亚基占据时(例如，两个DNA分子的每个中的一个位置都被腺嘌呤占据，或者两个多肽的每个中的一个位置被赖氨酸占据)，那么在那个位置每个分子均相同。这两个序列之间的“同一性百分比”是两个序列共有的匹配位置数除以要比较的位置数 x 100的函数。例如，如果两个序列的10个位置中有6个匹配，则这两个序列具有60%的同一性。例如，DNA序列CTGACT和CAGGTT具有50%的同一性(总共6个位置中有3个匹配)。通常，当两个序列进行比对以产生最大的同一性时，进行这种比较。这种比对可以通过例如Needleman et al. (1970) J. Mol. Biol. 48: 443-453中的方法来实现，所述方法可方便地通过计算机程序如比对(Align)程序(DNAstar，Inc.)来进行。也可以采用PAM 120权重残基表，使用E. Meyers和W. Miller的算法(Comput. ApplBiosci., 4: 11-17 (1988))集成到ALIGN程序(2.0版)中。空缺长度罚分12和空缺罚分4，用于确定两个氨基酸序列之间的同一性百分比。此外，可以使用集成到GCG软件包(可从www.gcg.com获得)的GAP程序中的Needleman和Wunsch(J MoI Biol. 48: 444-453(1970))算法，采用Blossum 62矩阵或PAM250矩阵，空缺权重为16、14、12、10、8、6或4，长度权重为1、2、3、4、5或6，以确定两个氨基酸序列之间的同一性百分比。
本文所用的“细胞”应理解为不仅指特定的单个细胞，而且指该细胞的后代或潜在后代。因为由于突变或环境影响，可能在后代中发生某些修饰，所以此类后代可能事实上与亲本细胞不同，但仍包括在本文术语的范围内。
如本文所用，术语“转导”和“转染”包括本领域已知的使用感染剂(如病毒)或其他方式将DNA引入细胞中以表达目的蛋白质或分子的方法。除了病毒或类似病毒的试剂外，还有基于化学的转染方法，如使用磷酸钙、树状聚合物，脂质体或阳离子聚合物(例如DEAE-葡聚糖或聚乙烯亚胺)的转染方法；非化学方法，如电穿孔、细胞挤压(cell squeezing)、声致穿孔(sonoporation)、光学转染、穿刺转染(impalefection)、原生质体融合、质粒递送或转座子；基于颗粒的方法，如使用基因枪、磁转染或磁体辅助转染、颗粒轰击；以及杂交方法（诸如核转染）。
如本文所用，术语“转染的”、“转化的”或“转导的”是指将外源核酸转移或引入宿主细胞的过程。“转染的”、“转化的”或“转导的”细胞是已经用外源核酸转染、转化或转导的细胞。
术语“体内”是指从其中获得细胞的该生物体内。“离体”或“体外”是指从其中获得细胞的该生物体外。
如本文所用，“处理(treatment/treating)”是用于获得有益的或期望的结果(包括临床结果)的方法。为了本发明的目的，有益的或期望的临床结果包括但不限于以下的一种或多种：减轻由疾病引起的一种或多种症状，减轻疾病的程度，稳定疾病(例如预防或延缓疾病的恶化)，预防或延缓疾病的扩散(例如转移)，预防或延缓疾病的复发，降低疾病的复发率，延缓或减慢疾病的进展，改善疾病状态，提供疾病的(部分或全部)缓解，减少治疗该疾病所需的一种或多种其他药物的剂量，延缓疾病的进展，提高生活质量，和/或延长生存期。“处理”还包括减少癌症的病理后果。本发明的方法考虑了这些处理的方面中的任何一个或多个。
如本文所用，术语“有效量”是指足以处理特定病症、病况或疾病(如改善、缓解、减轻和/或延迟其一种或多种症状)的化合物或组合物的量。如本领域中所理解的，“有效量”可以以一次或多次给药，即，可能需要单次给药或多此给药来达到期望的处理终点。
“受试者”、“个体”或“患者”在本文中可互换使用，以达到治疗目的，是指任何归类为哺乳动物的动物，包括人类、家畜和农场动物，以及动物园、体育馆或宠物动物如狗、马、猫、牛等。在一些实施方案中，所述个体是人类个体。
应理解，本文所述的本发明的实施方案包括“由...组成”和/或“基本上由...组成”的实施方案。
在本文中对“约”值或参数的提及包括(并描述了)针对该值或参数本身的变化。例如，提及“大约X”的描述，包括对“X”的描述。
如本文所用，对“不”值或参数的提及通常意指并描述了“除…外”值或参数。例如，所述方法不用于处理X型癌症，意味着所述方法用于处理除X型以外的癌症。
如本文所用，术语“大约X-Y”具有与“大约X至大约Y”相同的含义。
如本文和所附权利要求书中所使用的，单数形式“一个/一种(a/an)”和“所述”包括复数对象，除非上下文另外明确指出。还应注意，权利要求可以被撰写为排除任何可选的要素。因此此陈述旨在作为与权利要求要素的叙述结合使用诸如“只”、“仅”等排他性术语的先行基础，或使用“否”的限制。
如本文所用，术语“和/或”在词语诸如“A和/或B”中，旨在既包括A和B；A或B；A(单独)；以及B(单独)。同样地，如本文所用，术语“和/或”在词语诸如“A、B和/或C”中，旨在包括以下每个实施方案：A、B和C；A、B或C；A或C；A或B；B或C；A和C；A和B；B和C；A(单独)；B(单独)；以及C(单独)。
工程化改造酶的方法
本申请提供了工程化改造酶的方法，其包括：(a)获得多种工程化的酶，每种工程化的酶包含一个或多个突变，所述突变增加了参比酶的多个柔性区中的柔性区柔性；以及(b)从所述多种工程化的酶中选择一种或多种工程化的酶，其中所述一种或多种工程化的酶与所述参比酶相比具有增加的活性。在一些实施方案中，所述方法还包括确定所述参比酶中的多个柔性区。在一些实施方案中，所述参比酶或其等同物的三维结构无法获得。在一些实施方案中，所述酶是细菌或古细菌的酶。在一些实施方案中，在真核细胞诸如哺乳动物细胞(例如人细胞)中测量活性。
在一些实施方案中，提供了工程化Cas核酸酶的方法，其包括：(a)确定参比Cas核酸酶中的多个柔性区(例如，基于参比Cas核酸酶的一级序列)；(b)获得多种工程化的Cas核酸酶，其各自包含一个或多个突变，所述突变增加了所述参比Cas核酸酶的多个柔性区中的柔性区柔性；以及(c)从多种工程化的Cas核酸酶中选择一种或多种工程化的Cas核酸酶，其中所述一种或多种工程化的Cas核酸酶与参比Cas核酸酶相比具有提高的活性。在一些实施方案中，使用选自下组的程序确定多个柔性区：PredyFlexy、FoldUnfold、PROFbval、Flexserv、FlexPred、DynaMine和Disomine。在一些实施方案中，所述Cas核酸酶选自下组：Cas9、Cas12a、Cas12b、Cas12c、Cas12d、Cas12f、Cas12g、Cas12h、Cms1、Cas12i、Cas12j、Cas12k和CasX。在一些实施方案中，所述多个柔性区位于无规卷曲处。在一些实施方案中，所述多个柔性区在参比Cas核酸酶的与DNA和/或RNA相互作用的结构域中。在一些实施方案中，所述柔性区的长度为至少约5个(例如5个)氨基酸。在一些实施方案中，所述一个或多个突变包括在柔性区中插入一个或多个(例如2个)甘氨酸(G)残基。在一些实施方案中，所述一个或多个G残基插入在柔性区中的柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、丝氨酸(S)、天冬酰胺( N)、天冬氨酸(D)、组氨酸(H)、蛋氨酸(M)、苏氨酸(T)、谷氨酸(E)、谷氨酰胺(Q)、赖氨酸(K)、精氨酸(R)、丙氨酸(A)和脯氨酸(P)。在一些实施方案中，根据以下优先级选择所述柔性氨基酸残基：G>S>N>D>H>M>T>E>Q>K>R>A>P。在一些实施方案中，所述一个或多个突变包括用一个或多个G残基替换一个或多个非G残基。在一些实施方案中，所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：亮氨酸(L)、异亮氨酸(I)、缬氨酸(V)、半胱氨酸(C)、酪氨酸(Y)、苯丙氨酸(F)和色氨酸(W)。在一些实施方案中，所述活性是位点特异性核酸酶活性。在一些实施方案中，所述活性是真核细胞(例如人细胞)中的基因编辑活性。在一些实施方案中，所述基因编辑效率是使用如下方法来测量的：T7核酸内切酶1 (T7E1)测定、靶DNA的测序、由分解追踪插入缺失(TIDE)测定或通过扩增子分析进行插入缺失检测(IDAA)测定。
在一些实施方案中，提供了工程化Cas核酸酶的方法，其包括：(a)计算参比Cas核酸酶的每个氨基酸残基的柔性得分(例如，基于所述参比Cas核酸酶的一级序列)，其中较高的柔性得分表示较低的构象柔性；(b)选择在Xi位置的多个峰氨基酸残基，其中每个峰氨基酸残基的柔性得分低于预定阈值，并且低于在Xi-5至Xi-1、Xi+1和Xi+5位置的氨基酸残基的柔性得分；(c)选择多个柔性区（作为氨基酸残基Xi-2至Xi+2）；(d)获得多种工程化的Cas核酸酶，每种工程化的Cas核酸酶包含一个或多个突变，所述突变增加了所述参比Cas核酸酶的多个柔性区中柔性区柔性，其中所述一个或多个突变包括在柔性区中插入一个或多个(例如，2个)G残基，和/或用一个或多个G残基替换一个或多个非G残基；以及(e)从所述多种工程化的Cas核酸酶中选择一种或多种工程化的Cas核酸酶，其中所述一种或多种工程化的Cas核酸酶与所述参比Cas核酸酶相比具有增加的活性。在一些实施方案中，所述多个柔性区是使用选自下组的程序来确定的：PredyFlexy、FoldUnfold、PROFbval、Flexserv、FlexPred、DynaMine和Disomine。在一些实施方案中，所述Cas核酸酶选自下组：Cas9、Cas12a、Cas12b、Cas12c、Cas12d、Cas12f、Cas12g、Cas12h、Cms1、Cas12i、Cas12j、Cas12k和CasX。在一些实施方案中，所述多个柔性区位于无规卷曲处。在一些实施方案中，所述多个柔性区在所述参比Cas核酸酶的与DNA和/或RNA相互作用的结构域中。在一些实施方案中，所述一个或多个G残基插入在柔性区中的柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、丝氨酸(S)、天冬酰胺( N)、天冬氨酸(D)、组氨酸(H)、蛋氨酸(M)、苏氨酸(T)、谷氨酸(E)、谷氨酰胺(Q)、赖氨酸(K)、精氨酸(R)、丙氨酸(A)和脯氨酸(P)。在一些实施方案中，根据以下优先级选择所述柔性氨基酸残基：G>S>N>D>H>M>T>E>Q>K>R>A>P。在一些实施方案中，所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：亮氨酸(L)、异亮氨酸(I)、缬氨酸。(V)、半胱氨酸(C)、酪氨酸(Y)、苯丙氨酸(F)和色氨酸(W)。在一些实施方案中，所述活性是位点特异性核酸酶活性。在一些实施方案中，所述活性是真核细胞(例如人细胞)中的基因编辑活性。在一些实施方案中，所述基因编辑效率是使用如下方法来测量的：T7核酸内切酶1 (T7E1)测定、靶DNA的测序、由分解追踪插入缺失(TIDE)测定或通过扩增子分析进行插入缺失检测(IDAA)测定。
还提供了使用本文所述任何一种方法获得的工程化的酶(例如，工程化的Cas核酸酶)以及包含本文所述多种工程化的酶的文库。
确定多个柔性区
所述参比酶中的多个柔性区可以使用本领域中已知的任何方法来确定。在一些实施方案中，仅基于所述参比酶的氨基酸序列确定多个柔性区。在一些实施方案中，基于所述参比酶的结构信息确定多个柔性区，包括例如二级结构、晶体结构、NMR结构等。在一些实施方案中，在不参考所述参比酶的结构信息(例如三维结构)的情况下确定多个柔性区。在一些实施方案中，所述参比酶或其等同物的三维结构无法获得。
在本文提供的方法的一些实施方案中，基于所述参比酶的氨基酸序列确定多个柔性区。在一些实施方案中，所述多个柔性区是使用选自下组的程序来确定的：PredyFlexy、FoldUnfold、PROFbval、Flexserv、FlexPred、DynaMine和Disomine。例如，Yu等人已经描述了基于氨基酸序列来确定柔性区的方法(Engineering proteins for thermostabilitythrough rigidifying flexible sites. Biotechnology Advances Volume 32, Issue2, March–April 2014, Pages 308-315)。
在一些实施方案中，基于溶液中蛋白质的NMR化学位移数据确定多个柔性区。在一些实施方案中，使用DynaMine确定多个柔性区(Cilia et al. The DynaMine webserver:predicting protein dynamics from sequence. Nucleic Acids Res. 2014 Jul 1; 42(Web Server issue): W264–W270)。DynaMine利用溶液中蛋白质的NMR化学位移数据来获得对氨基酸序列与骨架动力学之间关系的定量理解，并预测柔性区。从这些数据开发出来的DynaMine预测因子仅基于序列信息就可以预测针对骨架动力学的蛋白质的残基水平潜力，这与3D结构信息的方法相反。这种方法揭示了大量可用的缺乏针对动力学分析的结构信息的蛋白质序列。在一些实施方案中，柔性谱是S2得分谱，其中较低的S2得分表示较高的柔性。
在一些实施方案中，使用分子动力学模拟(例如，模拟的均方根波动)来确定多个柔性区。在一些实施例中，基于B因子(例如，晶体学)数据确定多个柔性区。在一些实施方案中，使用Tarun等人描述的PredyFlexy确定多个柔性区(In silico prediction ofprotein flexibility with local structure approach. Biochimie (165), October2019, Pages 150-155)。
在一些实施方案中，使用每个残基的预期平均接触数作为给定区域是折叠还是未折叠的指标来确定多个柔性区，诸如使用FoldUnfold (Oxana et al. FoldUnfold: webserver for the prediction of disordered regions in protein chain,Bioinformatics, Volume 22, Issue 23, 1 December 2006, Pages 2948–2949)。
在一些实施方案中，通过从氨基酸序列计算归一化的B值来确定多个柔性区，诸如使用PROFbval (Schlessinger et al. PROFbval: predict flexible and rigidresidues in proteins. Bioinformatics, Volume 22, Issue 7, 1 April 2006, Pages891–893)。
在一些实施方案中，使用多重粗粒度方法、结构数据库和原子模型的组合来确定多个柔性区，诸如使用Flexserv (Camps et al. FlexServ: an integrated tool forthe analysis of protein flexibility. Bioinformatics 2009 Jul 1;25(13):1709-10)。
在一些实施方案中，使用经训练的监督模式识别方法、支持向量机(SVM) 来确定多个柔性区，诸如使用FlexPred Web服务器(Kuznetsov1 et al. FlexPred: a web-server for predicting residue positions involved in conformational switchesin proteins. Bioinformation. 2008; 3(3): 134–136)。
在一些实施方案中，基于使用循环神经网络计算的蛋白质紊乱来确定多个柔性区，诸如使用DisoMine (Orlando et al. Prediction of disordered regions inproteins with recurrent Neural Networks and protein dynamics. bioRxiv2020.05.25.115253; doi: https://doi.org/10.1101/2020.05.25.115253)。
在一些实施方案中，确定多个柔性区包括：(i)计算所述参比酶的每个氨基酸残基的柔性得分，其中较高的柔性得分表示较低的构象柔性；(ii)选择在Xi位置的多个峰氨基酸残基，其中每个峰氨基酸残基的柔性得分低于预定阈值，并且低于在Xi-5至Xi-1、Xi+1和Xi+5位置的氨基酸残基的柔性得分；以及(iii)将所述多个柔性区定义为氨基酸残基Xi-2至Xi+2。在一些实施方案中，使用DynaMine确定柔性区，并且在所述柔性区中具有最高柔性的氨基酸残基的柔性得分S2 预测不大于约0.8 (例如，不大于约0.75或0.7)。
在一些实施方案中，柔性区具有5个连续的氨基酸残基，其中第3个氨基酸残基具有最低的柔性得分，并且其中较高的柔性得分表示较低的构象柔性。
在一些实施方案中，柔性区位于蛋白质的无规卷曲处或接头区中。在一些实施方案中，基于可用的结构数据确定无规卷曲处或接头区。在一些实施方案中，基于与具有已知结构的直系同源物的同源性来确定无规卷曲处或接头区。在一些实施方案中，基于氨基酸序列确定无规卷曲处或接头区。在一些实施方案中，没有3D结构数据可用于参比蛋白质或参比蛋白质的直系同源物。在一些实施方案中，柔性区不位于无规卷曲处或接头区中。在一些实施方案中，柔性区位于距无规卷曲处或接头区约10个氨基酸之内，诸如约9、8、7、6、5、4、3、2或1个氨基酸中的任何一个。在一些实施方案中，柔性区比无规卷曲处或接头区更长。在一些实施方案中，柔性区比无规卷曲处或接头区更短。在一些实施方案中，柔性区的至少一部分位于α螺旋或β链中。
在一些实施方案中，柔性区位于所述参比酶的功能结构域中。在一些实施方案中，柔性区位于所述参比酶的催化位点附近。在一些实施方案中，柔性区不位于所述参比酶的功能结构域中。在一些实施方案中，所述参比酶是参比核酸酶。在一些实施方案中，柔性区在与DNA和/或RNA相互作用的参比核酸酶(例如，Cas核酸酶)的结构域中。在一些实施方案中，柔性区不位于与DNA和/或RNA相互作用的参比核酸酶的结构域中。在一些实施方案中，所述参比酶是Cas核酸酶，其选自下组：Cas9、Cas12a、Cas12b、Cas12c、Cas12d、Cas12f、Cas12g、Cas12h、Cms1、Cas12i、Cas12j、Cas12k和CasX。在第三节的“其他工程化的Cas效应蛋白”小节中，讨论了可以使用本文所述方法进行工程化的其他Cas核酸酶。在一些实施方案中，所述参比酶是Argonaut蛋白。
在一些实施方案中，所述工程化的酶(例如，Cas核酸酶)包含一个或多个突变，所述突变增加了两个或更多个(例如，2、3、4、5、6个)柔性区柔性。在一些实施方案中，不同柔性区中的一个或多个突变在增加所述工程化的酶相对于所述参比酶的活性中具有协同作用。
产生具有能增加柔性的突变的变体
在一些实施方案中，所述方法包括：在包含参比Cas核酸酶的至少5个连续氨基酸残基的柔性区中产生一个或多个G残基的插入和/或疏水氨基酸残基的G替换，其中所述工程化的Cas核酸酶与所述参比Cas核酸酶相比具有增强的活性。在一些实施方案中，柔性区具有5个连续的氨基酸残基，其中第3个氨基酸残基具有最低的柔性得分，并且其中较高的柔性得分表示较低的构象柔性。在一些实施方案中，所述一个或多个突变包括在柔性区中插入一个或多个(例如2个)G残基。在一些实施方案中，所述一个或多个G残基插入在柔性区中柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、丝氨酸(S)、天冬酰胺( N)、天冬氨酸(D)、组氨酸(H)、蛋氨酸(M)、苏氨酸(T)、谷氨酸(E)、谷氨酰胺(Q)、赖氨酸(K)、精氨酸(R)、丙氨酸(A)和脯氨酸(P)。在一些实施方案中，根据以下优先级选择所述柔性氨基酸残基：G>S>N>D>H>M>T>E>Q>K>R>A>P。
在一些实施方案中，所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：L、I、V、C、Y、F和W。在一些实施方案中，所述工程化的Cas核酸酶包含一个或多个增加两个或多个柔性区的柔性的突变。在一些实施方案中，所述参比Cas核酸酶选自下组：Cas9、Cas12a、Cas12b、Cas12c、Cas12d、Cas12f、Cas12g、Cas12h、Cms1、Cas12i、Cas12j、Cas12k和CasX。在一些实施方案中，所述方法包括：产生一个或多个G或S残基的插入和/或在柔性区中用G或S残基替换疏水氨基酸残基。在一些实施方案中，所述方法包括：产生用更具柔性的氨基酸残基替换疏水残基(例如，L、I、V、C、Y、F或W)或具更少柔性的氨基酸残基。在一些实施方案中，根据以下优先级选择所述更具柔性的氨基酸残基：G>S>N>D>H>M>T>E>Q>K>R>A>P。
选择具有增强活性的工程化的酶
在一些实施方案中，所述方法包括从多种工程化的酶中选择一种或多种工程化的酶，其中所述一种或多种工程化的酶与所述参比酶相比具有增加的活性，其中所述活性是位点特异性核酸酶活性。在一些实施方案中，所述活性是真核细胞中的基因编辑活性。在一些实施方案中，所述活性是人细胞中的基因编辑活性。
在一些实施方案中，所述增加的活性是位点特异性核酸酶活性。在一些实施方案中，所述位点特异性核酸酶活性是在体外确定的。在一些实施方案中，所述位点特异性核酸酶活性是在细胞中确定的。可以使用本领域中已知的方法来评估位点特异性核酸酶的活性，所述方法包括例如，如本文提供的实施例中所述的基于琼脂糖凝胶电泳的体外切割测定。在一些实施方案中，工程化的酶(例如工程化的Cas核酸酶)变体具有与所选择的参比酶相比增加至少约20%、30%、40%、60%、70%、80%、90%、1.5倍、2倍、3倍、4倍、5倍或更多中任一个的位点特异性核酸酶活性。
在一些实施方案中，所述活性是细胞如原核细胞或真核细胞中的基因编辑活性。可以使用本领域中已知的方法来确定细胞中工程化的酶的基因编辑效率，包括例如T7核酸内切酶1 (T7E1)测定、靶DNA测序(包括例如Sanger序列和二代测序)、由分解追踪插入缺失(TIDE)测定或通过扩增子分析进行插入缺失检测(IDAA)测定。参见，例如，Sentmanat MFet al., “A survey of validation strategies for CRISPR-Cas9 editing,”Scientific Reports, 2018, 8，文章编号888，其通过整体引用而并入本文。在一些实施方案中，例如，如本文实施例中所述，使用靶向的二代测序(NGS)来测量细胞中工程化的酶的基因编辑效率。
在一些实施方案中，所述活性是在真核细胞诸如植物细胞或哺乳动物细胞中的基因编辑活性。在一些实施方案中，在真核细胞中工程化的酶(例如工程化的Cas核酸酶)变体具有与所选择的参比酶相比增加至少约20%、30%、40%、60%、70%、80%、90%、1.5倍、2倍、3倍、4倍、5倍或更多中任一个的位点特异性核酸酶活性。
在一些实施方案中，所述活性是在人细胞例如293T细胞中的基因编辑活性。在一些实施方案中，在人细胞中工程化的酶(例如工程化的Cas核酸酶)变体具有与所选择的参比酶相比增加至少约20%、30%、40%、60%、70%、80%、90%、1.5倍、2倍、3倍、4倍、5倍或更多中任一个的位点特异性核酸酶活性。
在一些实施方案中，所述方法还包括将来自多种工程化的酶的一种或多种工程化的酶的突变进行组合的步骤，其中所述一种或多种工程化的酶与参比酶相比具有增加的活性。在一些实施方案中，所述组合的突变包含在一个或多个(例如2、3、4或更多个)不同柔性区中的突变。
工程化的Cas效应蛋白
本申请提供了工程化的Cas效应蛋白，其具有改善的活性，例如靶标结合、双链切割活性、切口酶活性和/或基因编辑活性。在一些实施方案中，使用第II部分中描述的工程化方法中任一种获得所述工程化的Cas核酸酶。在一些实施方案中，提供了工程化的Cas效应蛋白(例如，Cas核酸酶、Cas切口酶、Cas融合效应蛋白或分割型(split) Cas效应蛋白)，其包含本文所述工程化的Cas核酸酶或其功能衍生物中的任一种。
在一些实施方案中，提供了工程化的Cas核酸酶，其包含一个或多个突变，所述突变增加了包含参比Cas核酸酶的至少5个连续氨基酸残基的柔性区的柔性，其中所述工程化的Cas核酸酶与所述参比Cas酶相比具有提高的活性。在一些实施方案中，基于所述参比Cas核酸酶的氨基酸序列例如使用选自下组的的程序来确定所述柔性区：PredyFlexy、FoldUnfold、PROFbval、Flexserv、FlexPred、DynaMine和Disomine。在一些实施方案中，使用DynaMine确定所述柔性区，并且其中在所述柔性区中具有最高柔性的氨基酸残基的柔性得分S2 预测不大于约0.8 (例如，不大于约0.75或0.7)。在一些实施方案中，所述柔性区具有5个连续的氨基酸残基，其中第3个氨基酸残基具有最低的柔性得分，并且其中较高的柔性得分表示较低的构象柔性。在一些实施方案中，所述柔性区位于无规卷曲处。在一些实施方案中，所述柔性区在参比Cas核酸酶的与DNA和/或RNA相互作用的结构域中。在一些实施方案中，所述工程化的Cas核酸酶包含一个或多个增加两个或多个柔性区的柔性的突变。在一些实施方案中，所述参比Cas核酸酶选自下组：Cas9、Cas12a、Cas12b、Cas12c、Cas12d、Cas12f、Cas12g、Cas12h、Cms1、Cas12i、Cas12j、Cas12k和CasX。
在一些实施方案中，提供了工程化的Cas核酸酶，其包括在包含参比Cas核酸酶的至少5个连续氨基酸残基的柔性区中插入一个或多个G残基和/或替换疏水氨基酸残基，其中所述工程化的Cas核酸酶与所述参比Cas核酸酶相比具有增强的活性。在一些实施方案中，所述柔性区具有5个连续的氨基酸残基，其中第3个氨基酸残基具有最低的柔性得分，并且其中较高的柔性得分表示较低的构象柔性。在一些实施方案中，所述一个或多个突变包括在所述柔性区中插入一个或多个(例如2个) G残基。在一些实施方案中，所述一个或多个G残基插入在所述柔性区中柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、S、N、D、H、M、T、E、Q、K、R、A和P。在一些实施方案中，根据以下优先级选择所述柔性氨基酸残基：G>S>N>D>H>M>T>E>Q>K>R>A>P。在一些实施方案中，所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：L、I、V、C、Y、F和W。在一些实施方案中，所述工程化的Cas核酸酶包含一个或多个增加两个或多个柔性区的柔性的突变。在一些实施方案中，所述参比Cas核酸酶选自下组：Cas9、Cas12a、Cas12b、Cas12c、Cas12d、Cas12f、Cas12g、Cas12h、Cms1、Cas12i、Cas12j、Cas12k和CasX。
在一些实施方案中，提供了工程化的Cas效应蛋白，其包含工程化的Cas核酸酶或其功能衍生物，其中所述工程化的Cas核酸酶包含一个或多个突变，所述突变增加了包含参比Cas核酸酶的至少5个连续氨基酸残基的柔性区的柔性，其中所述工程化的Cas核酸酶与所述参比Cas核酸酶相比具有增加的活性。在一些实施方案中，使用DynaMine确定所述柔性区，并且其中在所述柔性区中具有最高柔性的氨基酸残基的柔性得分S2 pred不大于约0.8(例如，不大于约0.75或0.7)。在一些实施方案中，所述柔性区具有5个连续的氨基酸残基，其中第3个氨基酸残基具有最低的柔性得分，并且其中较高的柔性得分表示较低的构象柔性。在一些实施方案中，所述工程化的Cas核酸酶包括在所述柔性区中插入一个或多个G残基和/或替换疏水氨基酸残基。在一些实施方案中，所述一个或多个突变包括在所述柔性区中插入一个或多个(例如2个) G残基。在一些实施方案中，所述一个或多个G残基插入在柔性区中柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、S、N、D、H、M、T、E、Q、K、R、A和P。在一些实施方案中，根据以下优先级选择所述柔性氨基酸残基：G>S>N>D>H>M>T>E>Q>K>R>A>P。在一些实施方案中，所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：L、I、V、C、Y、F和W。在一些实施方案中，所述工程化的Cas核酸酶包含一种或多种增加两个或多个柔性区的柔性的突变。在一些实施方案中，所述参比Cas核酸酶选自下组：Cas9、Cas12a、Cas12b、Cas12c、Cas12d、Cas12f、Cas12g、Cas12h、Cms1、Cas12i、Cas12j、Cas12k和CasX。在一些实施方案中，所述工程化的Cas核酸酶的功能衍生物是具有酶活性Cas核酸酶、Cas切口酶、失去酶活的Cas、融合效应蛋白(例如，转录激活因子、转录阻遏物、碱基编辑器或引物编辑器)或分割型Cas效应蛋白(例如，可诱导的分割型Cas效应蛋白或自诱导的分割型Cas效应蛋白)。
还提供了工程化的CRISPR-Cas系统，其包含本文所述工程化的Cas效应蛋白(例如，工程化的Cas核酸酶) 和指导RNA(包括前体指导RNA阵列、crRNA、单个指导RNA，或crRNA和tracrRNA) 中的任一种。在一些实施方案中，所述工程化的CRISPR-Cas系统包含一种或多种编码所述工程化的Cas效应蛋白和/或所述指导RNA的核酸分子。在一些实施方案中，所述工程化的CRISPR-Cas系统包含一种或多种编码所述工程化的Cas效应蛋白和/或所述指导RNA的载体。在一些实施方案中，提供了编码所述工程化的Cas效应蛋白的载体。在一些实施方案中，所述载体还包含指导RNA。在一些实施方案中，所述载体选自下组：逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关的载体和单纯疱疹载体。在一些实施方案中，所述载体是腺相关病毒(AAV)载体。
本文所述的工程化的Cas核酸酶具有突变，所述突变增加了参比Cas核酸酶(如天然存在的野生型Cas核酸酶)中一个或多个(例如1、2、3个或更多个)柔性区的构象柔性。与所述参比Cas核酸酶相比，可以组合两个或更多个柔性区中的突变以提供所述工程化的Cas核酸酶中活性的协同增加。如在第II部分中所述，可以使用本领域中已知的方法，例如基于以上在第II部分“工程化改造酶的方法”中描述的一种或多种方法，确定参比Cas核酸酶中的柔性区。在一些实施方案中，参比Cas的柔性区是基于所述参比Cas核酸酶的氨基酸序列确定的。在一些实施方案中，参比Cas核酸酶的柔性区是使用选自下组的程序来确定的：PredyFlexy、FoldUnfold、PROFbval、Flexserv、FlexPred、DynaMine和Disomine。
所述柔性区可包含所述参比Cas核酸酶的5个或更多个(例如5、6、7，8、9、10、11、12、13、14、15或更多个)连续氨基酸残基。在一些实施方案中，所述柔性区具有所述参比Cas核酸酶的5个连续氨基酸残基。可通过首先选择所述参比Cas核酸酶的位置X处的峰氨基酸残基来定义所述柔性区，其中所述峰氨基酸残基具有低于预定阈值(例如，如DynaMine所确定的S2 pred为0.8或更小)的柔性得分，并且其中所述峰氨基酸残基的柔性得分低于X-5至X-1位氨基酸残基(即，位于所述峰氨基酸残基的N末端侧翼的5个氨基酸残基)以及X+1至X+5位氨基酸残基(即，位于所述峰氨基酸残基的C末端侧翼的5个氨基酸残基)的柔性得分，其中较高的柔性得分表示较低的构象柔性。在一些实施方案中，所述柔性区具有5个连续的氨基酸残基，其中第3个氨基酸残基具有最低的柔性得分，并且其中较高的柔性得分表示较低的构象柔性。
在一些实施方案中，使用DynaMine确定参比Cas核酸酶的柔性区。在一些实施方案中，在所述柔性区的柔性区中具有最高柔性的氨基酸残基(即，峰氨基酸残基)位于如DynaMine所确定的上下文相关的柔性区中。在一些实施方案中，在所述柔性区的柔性区中具有最高柔性的氨基酸残基(即，峰氨基酸残基)位于如DynaMine所确定的柔性区中。在一些实施方案中，基于DynaMine中的计算，在柔性区中具有最高柔性的氨基酸残基(即，峰氨基酸残基)的柔性得分S2 pred不大于约0.8，例如不大于约0.79、0.78、0.77、0.76、0.75、0.74、0.73、0.72、0.71、0.7或更小值中的任一个。在一些实施方案中，柔性区的氨基酸残基在上下文相关的柔性区中和/或如DynaMine所确定的柔性区中。在一些实施方案中，基于DynaMine中的计算，柔性区中的每个氨基酸残基的柔性得分S2 pred不大于约0.8，例如，不大于约0.79、0.78、0.77、0.76、0.75、0.74、0.73、0.72， 0.71，0.7或更小值中的任一个。
在一些实施方案中，参比Cas核酸酶的柔性区位于无规卷曲处。在一些实施方案中，参比Cas核酸酶的柔性区不位于α螺旋中。在一些实施方案中，参比Cas核酸酶的柔性区不位于β链中。在一些实施方案中，参比Cas核酸酶的柔性区不位于无规卷曲处。在一些实施方案中，参比Cas核酸酶的柔性区的至少一部分位于α螺旋或β链中。
在一些实施方案中，参比Cas核酸酶的柔性区在与DNA相互作用的参比Cas核酸酶的结构域中。在一些实施方案中，参比Cas核酸酶的柔性区在与RNA(例如指导RNA，如crRNA和/或tracrRNA)相互作用的参比Cas核酸酶的结构域中。在一些实施方案中，参比Cas核酸酶的柔性区在参比Cas核酸酶的不与DNA或RNA相互作用的结构域中。在一些实施方案中，参比Cas核酸酶的柔性区位于参比Cas核酸酶的功能结构域之间。
可以使用能增加所述柔性区柔性的任何突变。已经基于实验数据(例如晶体结构、NMR和其他蛋白质动力学研究)表征了20个天然存在的氨基酸残基的柔性。柔性氨基酸残基包括G、T、R、S、N、Q、D、P、E、K、A、M；刚性氨基酸残基包括W、Y、F、C、I、V、H、L。在一些实施方案中，氨基酸的柔性取决于其相邻氨基酸的身份。参见例如Smith DK et al., “Improvedamino acid flexibility parameters,” Protein Sci., 2003, 12(5): 1060-1072。
在一些实施方案中，所述柔性区的一个或多个突变包括与柔性构象相关的一个或多个(例如1、2、3个或更多个)氨基酸残基的插入。所述插入可发生在柔性区的任何位置。在一些实施方案中，所述插入是在柔性区中峰氨基酸的N末端或C末端。在一些实施方案中，所述插入是在柔性氨基酸残基的N末端或C末端，例如具有如下优先级：G>S>N>D>H>M>T>E>Q>K>R>A>P。在一些实施方案中，其中在柔性区中有不止一个优选的柔性氨基酸(例如两个G)，所述插入是在更接近于峰氨基酸残基的优选柔性氨基酸残基的N末端或C末端进行的。在一些实施方案中，其中在柔性区中存在两个优选的柔性氨基酸残基(例如，两个G)，并且它们与峰氨基酸残基具有相同的距离，所述插入是在这样的优选柔性氨基酸残基N末端或C末端进行的，即其具有的相邻氨基酸残基在柔性方面比其他优选的柔性氨基酸残基具有的相邻氨基酸残基有更高的优先级(即，G>S>N>D>H>M>T>E>Q>K>R>A>P)。在一些实施方案中，其中在柔性区中存在两个优选的柔性氨基酸残基(例如，两个G)，它们与峰氨基酸残基具有相同的距离，并且它们的相邻氨基酸残基在柔性方面具有相同的优先级，所述插入是在这样的优选柔性氨基酸残基的N末端或C末端进行的，即其更靠近所述参比Cas核酸酶的N末端。甘氨酸(G)被广泛接受为柔性氨基酸残基。在一些实施方案中，所述柔性区的一个或多个突变包括在所述柔性区中插入一个或多个(例如1、2、3个或更多个) G。在一些实施方案中，所述一个或多个突变包括在所述柔性区中插入两个G残基。在一些实施方案中，所述一个或多个G残基插入在所述柔性区中柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、S、N、D、H、M、T、E、Q、K、R、A和P。在一些实施方案中，根据以下优先级选择所述柔性氨基酸残基：G>S>N>D>H>M>T>E>Q>K>R>A>P。也可以考虑插入其他柔性氨基酸残基或柔性氨基酸残基的基团，例如S、T、GS、SG、GGS等。
在一些实施方案中，所述柔性区的一个或多个突变包括将一个或多个与具有较少柔性的构象相关的氨基酸残基(例如，刚性氨基酸残基)替换为一个或多个具有较多柔性的构象的氨基酸残基(例如，柔性氨基酸残基)。在一些实施方案中，所述柔性区的一个或多个突变包括用柔性氨基酸残基替换柔性区中的疏水氨基酸残基。在一些实施方案中，所述疏水氨基酸残基选自下组：L、I、V、C、Y、F和W。在一些实施方案中，所述柔性区的一个或多个突变包括用柔性氨基酸残基替换柔性区中的两个或多个(例如，2、3或4个)疏水氨基酸残基。在一些实施方案中，所述柔性氨基酸残基为G、S、T、N、D、H、K或R。在一些实施方案中，所述柔性氨基酸残基为G、S或T。在一些实施方案中，所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：L、I、V、C、Y、F和W。如果所述突变不会对所述工程化的Cas核酸酶的折叠和/或活性产生不利影响，则也考虑了本文所述的柔性区中的插入和替换的组合。
在一些实施方案中，所述工程化的Cas核酸酶是1类或2类CRISPR-Cas系统的Cas效应蛋白。在一些实施方案中，所述工程化的Cas核酸酶是I-A型、I-B型、I-C型、I-D型、I-E型、I-F型、II-A型、II-B型、II-C型、III-A型、III-B型、IV-A型、IV-B型、V-A型、V-B型、V-F型、V-U3型、V-U4型、V-U2型、V-U1型、V-C型、V-D型、V-E型、V-U5型、V-G型、V-H型、V-I型、V-K型和VI型CRISPR-Cas系统的Cas效应蛋白。参见，例如，Makarova KS and Koonin EV,“Annotation and Classification of CRISPR-Cas Systems”, Methods Mol. Biol.,2015, 1311:47-75; Markarova KS et al., “An updated evolutionaryclassification of CRISPR-Cas systems,” Nat. Rev. Microbiol. 2015, 13: 722-736; Yan WX et al., “Functionally diverse type V CRISPR-Cas systems,”Science, 2019, 363 (6422): 88-91; 以及 Pinilla-Redondo R. et al., “Type IVCRISPR-Cas systems are highly diverse and involved in competition betweenplasmids,” Nucleic Acids Research, 2020, 48(4): 2000-2012，上述文献通过整体引用而并入本文。在一些实施方案中，所述工程化的Cas核酸酶选自下组：Cas9、Cas12a、Cas12b、Cas12c、Cas12d、Cas12f、Cas12g、Cas12h、Cms1、Cas12i、Cas12j、Cas12k和CasX。在一些实施方案中，所述工程化的Cas核酸酶是Cas9。在一些实施方案中，所述工程化的Cas核酸酶是Cas12i。在一些实施方案中，所述工程化的Cas核酸酶是Cas12b。
所述工程化的Cas核酸酶与所述参比Cas核酸酶相比具有增加的活性。在一些实施方案中，所述活性是靶DNA结合活性。在一些实施方案中，所述活性是位点特异性核酸酶活性。在一些实施方案中，所述活性是双链DNA切割活性。在一些实施方案中，所述活性是单链DNA切割活性，包括例如位点特异性DNA切割活性或非特异性DNA切割活性。在一些实施方案中，所述活性是单链RNA切割活性，例如位点特异性RNA切割活性或非特异性RNA切割活性。在一些实施方案中，所述活性是在体外测量的。在一些实施方案中，所述活性是在细胞如细菌细胞、植物细胞或真核细胞中测量的。在一些实施方案中，所述活性是在哺乳动物细胞如啮齿动物细胞或人细胞中测量的。在一些实施方案中，所述活性是在人细胞诸如293T细胞中测量的。在一些实施方案中，所述工程化的Cas核酸酶具有与参比Cas核酸酶相比增加至少约20%、30%、40%、60%、70%、80%、90%、1.5倍、2倍、3倍、4倍、5倍或更多中任一个的位点特异性核酸酶活性。所述工程化的Cas核酸酶的位点特异性核酸酶活性可以使用本领域中已知的方法来测量，包括例如，凝胶迁移测定。
在一些实施方案中，所述活性是细胞中的基因编辑活性。在一些实施方案中，所述细胞是细菌细胞、植物细胞或真核细胞。在一些实施方案中，所述细胞是哺乳动物细胞如啮齿动物细胞或人细胞。在一些实施方案中，所述细胞是293T细胞。在一些实施方案中，所述活性是在细胞中靶基因组位点的插入缺失形成活性，例如通过所述工程化的Cas核酸酶对靶核酸进行位点特异性切割和通过非同源末端连接(NHEJ)机制进行DNA修复。在一些实施方案中，所述活性是在细胞中靶基因组位点插入外源核酸序列，例如通过所述工程化的Cas核酸酶对靶核酸进行位点特异性切割和通过同源重组(HR)机制进行DNA修复。在一些实施方案中，所述工程化的Cas核酸酶与参比Cas核酸酶相比在细胞(例如人细胞如293T细胞)的基因组位点处增加至少约20%、30%、40%、60%、70%、80%、90%、1.5倍、2倍、3倍、4倍、5倍或更多中任一个的基因编辑(例如，插入缺失形成)活性。在一些实施方案中，所述工程化的Cas核酸酶与参比Cas核酸酶相比在细胞(例如人细胞如293T细胞)的多个(例如2、3、4、5、6、7、8、9、10个或更多个)基因组位点处增加至少约20%、30%、40%、60%、70%、80%、90%、1.5倍、2倍、3倍、4倍、5倍或更多中任一个的基因编辑(例如，插入缺失形成)活性。在一些实施方案中，所述工程化的Cas核酸酶与所述参比Cas核酸酶相比，能够编辑更多数目的基因组位点。在一些实施方案中，所述工程化的Cas核酸酶的共有PAM序列与所述参比Cas核酸酶相同。
可使用本领域已知的方法确定细胞中工程化的Cas核酸酶的基因编辑效率，包括例如T7核酸内切酶1 (T7E1)测定、靶DNA的测序(包括例如，Sanger序列，以及二代测序)、由分解追踪插入缺失(TIDE)测定或通过扩增子分析进行插入缺失检测(IDAA)测定。参见例如Sentmanat MF et al., “A survey of validation strategies for CRISPR-Cas9editing,” Scientific Reports, 2018, 8，文章编号888，其通过整体引用而并入本文。在一些实施方案中，例如，如本文实施例中所述，使用靶向的二代测序(NGS)来测量细胞中所述工程化的Cas核酸酶的基因编辑效率。用于确定所述工程化的Cas核酸酶的基因编辑效率的示例性基因组位点包括但不限于CCR5、AAVS、CD34、RNF2和EMX1。
本申请还提供了基于本文所述工程化的Cas核酸酶中任一种的工程化的Cas效应蛋白。在一些实施方案中，所述工程化的Cas效应蛋白包含所述工程化的Cas核酸酶的功能衍生物，如以下“功能衍生物”部分中所述的功能衍生物中任一种。
在一些实施方案中，所述工程化的Cas效应蛋白具有位点特异性核酸酶活性。在一些实施方案中，所述工程化的Cas效应蛋白可诱导靶DNA分子中的双链断裂。在一些实施方案中，所述工程化的Cas效应蛋白包含具有酶活性的工程化的Cas核酸酶。在一些实施方案中，所述工程化的Cas效应蛋白是Cas切口酶，其可诱导靶DNA分子中的单链断裂。在一些实施方案中，所述工程化的Cas效应蛋白包含所述工程化的Cas核酸酶的切口酶突变体。
在一些实施方案中，所述工程化的Cas效应蛋白包含所述工程化的Cas核酸酶的酶失活(即，失去酶活的)突变体。在一些实施方案中，所述工程化的Cas效应蛋白还包含与所述工程化的Cas核酸酶或其功能衍生物融合的一个或多个功能结构域。在一些实施方案中，所述工程化的Cas效应蛋白包含与所述工程化的Cas核酸酶的无酶活性突变体融合的功能结构域。在一些实施方案中，所述功能结构域选自下组：翻译起始结构域、转录阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域(例如，CBE或ABE结构域)、逆转录酶结构域、报告分子结构域(例如，荧光结构域)和核酸酶结构域。
在一些实施方案中，所述工程化的Cas效应蛋白包含基于所述工程化的Cas核酸酶或其功能衍生物的分割型Cas多肽。在一些实施方案中，所述工程化的Cas效应蛋白包含：含有所述工程化的Cas核酸酶或其功能衍生物的N末端部分的第一多肽和含有所述工程化的Cas核酸酶或其功能衍生物的C末端部分的第二多肽，其中所述第一多肽和所述第二多肽能够在包含指导序列的指导RNA存在下彼此缔合以形成CRISPR复合物，所述CRISPR复合物特异性结合包含与所述指导序列互补的靶序列的靶核酸。在一些实施方案中，所述分割型Cas效应蛋白是可诱导的，例如，每个分割型Cas多肽包含能够在诱导剂(例如雷帕霉素)存在下彼此缔合的二聚结构域。在一些实施方案中，所述分割型Cas效应蛋白是自诱导的，例如，所述分割型Cas多肽不包含二聚化结构域，并且它们能够在指导RNA存在下彼此缔合。
工程化的Cas12b效应蛋白
本申请提供了工程化的Cas12b效应蛋白(例如，Cas12b核酸酶、Cas12b切口酶、Cas12b融合效应蛋白、分割型Cas12b效应蛋白)，其具有改善的活性(例如，靶标结合、双链切割活性、切口酶活性和/或基因编辑活性)。
在一些实施方案中，提供了工程化的Cas12b核酸酶，其包含一个或多个增加对应于氨基酸残基835-839的柔性区柔性的突变，其中所述氨基酸残基的编号基于SEQ ID NO:1，其中所述工程化的Cas12b核酸酶与参比Cas12b核酸酶相比具有增强的活性。在一些实施方案中，所述参比Cas12b核酸酶是BhCas12b (例如，BhCas12bv4)。在一些实施方案中，所述一个或多个突变包括在柔性区中插入一个或多个(例如2个) G残基。在一些实施方案中，所述一个或多个G残基插入在所述柔性区中的柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、S、N、D、H、M、T、E、Q、K、R、A和P。在一些实施方案中，根据以下优先级选择所述柔性氨基酸残基：G>S>N>D>H>M>T>E>Q>K>R>A>P。在一些实施方案中，所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：L、I、V、C、Y、F和W。在一些实施方案中，所述工程化的Cas12b核酸酶包含SEQ ID NO: 83的氨基酸序列。在一些实施方案中，所述工程化的Cas12b核酸酶包含与SEQ ID NO: 2的序列具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中的任一个)序列同一性的氨基酸序列。
在一些实施方案中，提供了工程化的Cas12b核酸酶，其包含氨基酸序列SEQ IDNO: 2。
SEQ ID NO: 1：外村尚芽孢杆菌(Bacillus hisashii) Cas12b版本4 (BhCas12b-v4)氨基酸序列
MATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYGERSRFENSRLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCRVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSM
SEQ ID NO: 2：enBhCas12bv4 (BhCas12b-v4 1.1)氨基酸序列
MATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPGGERSRFENSRLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCRVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSM
V-B型CRISPR-Cas12b (也称为C2c1)系统已被鉴定为双RNA(即crRNA和tracrRNA)指导的DNA核酸内切酶系统，其具有与Cas9和Cas12a不同的特征(Shmakov, S. et al.Mol. Cell 60, 385–397 (2015))。首先，据报道在用crRNA/tracrRNA双螺旋重组时，Cas12b在体外产生远离原间隔区相邻基序(PAM)位点的粘性末端。第二，尽管Cas12b的RuvC结构域与Cas9和Cas12a的相似，但其推定的Nuc结构域与Cas9的HNH结构域和Cas12a的Nuc结构域没有序列或结构上的相似性。此外，Cas12b蛋白比最广泛使用的SpCas9和Cas12a更小(例如，AacCas12b：1,129个氨基酸(aa)；SpCas9：1,369 aa；AsCas12a：1,353 aa；LbCas12a：1,228aa)，这使得在基因疗法中Cas12b适合于腺相关病毒(AAV)介导的体内递送。与小尺寸的Cas9蛋白诸如SaCas9和CjCas9相比，Cas12b识别更简单的PAM序列(例如AacCas12b：5'-TTN-3' (SEQ ID NO: 3)；与SaCas9：5'-NNGRRT-3' (SEQ ID NO: 4)、CjCas9：5'-NNNNRYAC-3' (SEQ ID NO: 5))，其显著增加了基因组中Cas12b的靶向范围。另外，Cas12b具有最小的脱靶效应，因此可以作为治疗和临床应用上的更安全的选择。
来自多种生物体的Cas12b (C2c1)核酸酶可以用作参比Cas12b核酸酶，以提供本申请的工程化的Cas12b效应蛋白。示例性的Cas12b核酸酶已经描述于例如，Shmakov, S.et al.Mol. Cell 60, 385–397 (2015)；Shmakov, S. et al.Nat. Rev. Microbiol. 15,169–182 (2017)；WO2016205764和WO2020/087631，以上文献通过整体引用而并入本文。
在一些实施方案中，所述工程化的Cas12b效应蛋白基于选自下组的参比Cas12b蛋白(例如，Cas12b核酸酶)：嗜酸脂环酸芽孢杆菌(Alicyclobacillus acidiphilus)的Cas12b蛋白(AaCas12b)，kakegawensis脂环酸芽孢杆菌(Alicyclobacillus kakegawensis)的Cas12b (AkCas12b)，大孢脂环酸芽孢杆菌(Alicyclobacillus macrosporangiidus)的Cas12b (AmCas12b)，外村尚芽孢杆菌(Bacillus hisashii)的Cas12b (BhCas12b)，芽孢杆菌(Bacillus)的BsCas12b，芽孢杆菌的Bs3Cas12b，非常脱硫弧菌(Desulfovibrio inopinatus)的Cas12b (DiCas12b)，沉积物莱斯氏菌(Laceyella sediminis)的Cas12b (LsCas12b)，螺旋体菌(Spirochaetes bacterium)的Cas12b(SbCas12b)，热生肿块芽孢杆菌(Tuberibacillus calidus)的Cas12b (TcCas12b)及其功能衍生物。天然存在的Cas12b蛋白的序列是已知的，例如在UniProtKB IDs：T0D7A2、A0A6I3SPI6和AOA6I7FUC4中，其内容通过整体引用而并入本文。
在一些实施方案中，所述参比Cas12b蛋白是来自外村尚芽孢杆菌的Cas12b核酸酶(BhCas12b)或其功能衍生物。在一些实施方案中，所述工程化的Cas12b效应蛋白基于包含与SEQ ID NO: 6的氨基酸序列具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%或99%中任一种)序列同一性的氨基酸序列的参比Cas12b蛋白。在一些实施方案中，所述工程化的Cas12b效应蛋白基于包含SEQ ID NO: 6的氨基酸序列的参比Cas12b核酸酶。BhCas12b中的示例性柔性区和增加所述柔性区柔性的突变在图2-3中示出。
在一些实施方案中，所述参比Cas12b蛋白是来自嗜酸脂环酸芽孢杆菌的Cas12b核酸酶(AaCas12b)或其功能衍生物。在一些实施方案中，所述工程化的Cas12b效应蛋白基于包含与SEQ ID NO: 7的氨基酸序列具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%或99%中任一种)序列同一性的氨基酸序列的参比Cas12b蛋白。在一些实施方案中，所述工程化的Cas12b效应蛋白基于包含SEQ ID NO: 7的氨基酸序列的参比Cas12b核酸酶。
注意，与参比Cas12b蛋白具有某种序列同一性(例如，至少约60%、70%、80%、85%、90%、95%、98%或更多中任一个)的直系同源物或其片段可以用作设计本申请的工程化的Cas12b效应蛋白的基础。技术人员可以基于目的和应用来确定适用于本申请的Cas12b直系同源物或其片段的序列同一性的百分比。用于确定序列同一性值的方法可参见：Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press,New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W.,ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994;Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987;以及Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M StocktonPress, New York, 1991。WO2020/087631中描述了多种Cas12b直系同源物，该申请通过整体引用而并入本文。
天然存在的Cas12b核酸酶具有多种结构域。在一些实施方案中，参比Cas12b核酸酶从N末端到C末端包含：第一WED结构域(WED-1；也称为OBD-1结构域)，第一REC结构域(REC1)，第二WED结构域(WED-II；也称为OBD-II结构域)，第一RuvC结构域(RuvC-I)，桥螺旋(BH)结构域，第二RuvC结构域(RuvC-II)，第一Nuc结构域(Nuc-I；也称为UK-I结构域)，第三RuvC结构域(RuvC-III)和第二Nuc结构域(Nuc-II；也称为UK-II结构域)。可以使用本领域中的已知方法，如基于参比Cas12b核酸酶的晶体结构(例如，对于AaCas12b的PDB ID Nos：5U30、5U31、5U33、5U34和5WQE)和/或与参比Cas12b核酸酶中已知功能结构域的序列同源性来确定结构域边界。在一些实施方案中，AaCas12b具有以下结构域：WEB-I结构域(氨基酸残基1-14)，REC1结构域(氨基酸残基15-386)，WED-II结构域(氨基酸残基387-518)，RuvC-I结构域(氨基酸残基519-628)，BH结构域(氨基酸残基629-658)，REC2结构域(氨基酸残基659-784)，RuvC-II结构域(氨基酸残基785-900)，Nuc-I结构域(氨基酸残基901-974)，RuvC-III结构域(氨基酸残基975-993)，以及Nuc-II结构域(氨基酸残基994-1129)，其中所述氨基酸编号基于SEQ ID NO: 7。
酸土脂环酸芽孢杆菌(Alicyclobacillus acidoterrestris) Cas12b与sgRNA结合为二元复合物并且与靶DNA结合为三元复合物的晶体结构已描述于Yang H., et al.Cell 167:1814-1828(2016) and Liu L. et al. Mol. Cell 65:310-322 (2017)。简言之，晶体结构显示了2个不连续的REC(识别区，残基15-386、658- 784)和NUC(核酸酶，残基1-14、387-658和785-1129)叶(lobe，由多个结构域组成）。crRNA (或单个指导RNA，sgRNA)在两个叶之间的中央通道中结合。PAM识别具有序列特异性，并且主要通过与REC1(螺旋-1)和WED-II (OBD-II)结构域的相互作用而发生。sgRNA-靶DNA异源双链主要以不依赖序列的方式结合至REC叶。
在一些实施方案中，所述工程化的Cas12b核酸酶是基于天然存在的Cas12b核酸酶的功能变体。在一些实施方案中，所述功能变体具有一个或多个突变，如氨基酸替换、插入和缺失。举例来说，与野生型天然存在的Cas12b核酸酶相比，所述功能变体可以包含1、2、3、4、5、6、7、8、9、10个或更多个氨基酸替换中的任一个。在一些实施方案中，所述一个或多个替换是保守替换。在一些实施方案中，所述功能变体具有天然存在的Cas12b核酸酶的所有结构域。在一些实施方案中，所述功能变体不具有天然存在的Cas12b核酸酶的一个或多个结构域。
还提供了基于本文所述工程化的Cas12b核酸酶中任一种的工程化的Cas12b效应蛋白。在一些实施方案中，所述工程化的Cas12b效应蛋白具有酶活性。在一些实施方案中，所述工程化的Cas12b效应蛋白是切割靶双螺旋核酸(例如，双螺旋DNA)的两条链的核酸酶。在一些实施方案中，所述工程化的Cas12b效应蛋白是切口酶，即切割靶双螺旋核酸(例如，双螺旋DNA)的单链。在一些实施方案中，所述工程化的Cas12b效应蛋白包含所述工程化的Cas12b核酸酶的酶失活突变体。Cas12b核酸酶活性位点中一个或多个氨基酸残基的突变会导致失去酶活的Cas12b。例如，在人细胞中AaCas12b的R785A、R911A或D977A突变体没有核酸酶活性。参见，例如，Teng F. et al., Cell Discovery, 4，文章编号：63(2018)，其通过整体引用而并入本文。在一些实施方案中，所述工程化的Cas12b效应蛋白包含工程化的Cas12b，其具有对应于AaCas12b的R785A突变的突变。在一些实施方案中，所述工程化的Cas12b效应蛋白包含工程化的Cas12b，其具有一个或多个对应于AaCas12b的R785A、R911A或D977A的突变。在一些实施方案中，所述工程化的Cas12b效应蛋白包括工程化的Cas12b，其具有对应于AaCas12b的R911A突变的突变。在一些实施方案中，所述工程化的Cas12b效应蛋白包含工程化的Cas12b，其具有对应于AaCas12b的D977A突变的突变。
在一些实施方案中，提供了工程化的Cas12b切口酶。在一些实施方案中，提供了工程化的Cas12b融合效应蛋白，其包含与功能结构域融合的工程化的Cas12b核酸酶或其功能衍生物(例如，工程化的Cas12b核酸酶的酶失活突变体)，所述功能结构域诸如为翻译起始结构域、转录阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域(例如CBE或ABE结构域)、逆转录酶结构域、报告分子结构域(例如荧光结构域)和核酸酶结构域。在一些实施方案中，提供了工程化的分割型Cas12b效应蛋白。分割型Cas12b效应蛋白已经在例如PCT/CN2020/111057中描述，其通过整体引用而并入本文。
还提供了工程化的CRISPR-Cas12b系统，其包含本文所述工程化的Cas12b效应蛋白中的任一种，以及包含与靶序列互补的指导序列的指导RNA，或一种或多种编码所述指导RNA的核酸，其中所述工程化的Cas12b效应蛋白和所述指导RNA能够形成CRISPR复合物，其与包含靶序列的靶核酸特异性结合并诱导所述靶核酸的修饰。在一些实施方案中，所述指导RNA包含crRNA和tracrRNA。在一些实施方案中，所述指导RNA是sgRNA。在一些实施方案中，所述指导RNA是可以被加工成crRNA和tracrRNA的前体分子。在一些实施方案中，所述指导RNA是编码多个crRNA的前体RNA阵列，并且其中每个加工的crRNA与tracrRNA缔合。在一些实施方案中，所述工程化的Cas12b效应蛋白和/或所述指导RNA由一种或多种载体如AAV载体编码。在一些实施方案中，所述工程化的CRISPR-Cas12b系统是核糖核蛋白(RNP)复合物，其包含结合至所述指导RNA的所述工程化的Cas12b效应蛋白。
工程化的Cas12i效应蛋白
本申请提供了工程化的Cas12i效应蛋白，其具有改善的活性(例如靶标结合、双链切割活性、切口酶活性和/或基因编辑活性)。
在一些实施方案中，提供了工程化的Cas12i核酸酶，其包含一个或多个突变，所述突变增加了参比Cas12i核酸酶中柔性区的柔性，所述柔性区选自对应于以下的区的组：氨基酸残基228-232、氨基酸残基439-443、氨基酸残基478-482、氨基酸残基500-504、氨基酸残基775-779和氨基酸残基925-929，其中所述氨基酸残基编号基于SEQ ID NO: 8，其中所述工程化的Cas12i核酸酶与参比Cas12i核酸酶相比具有增加的活性。在一些实施方案中，所述柔性区对应于氨基酸残基439-443或氨基酸残基925-929，其中所述氨基酸残基编号基于SEQ ID NO: 8。在一些实施方案中，所述参比Cas12i核酸酶是Cas12i2。在一些实施方案中，所述一个或多个突变包括在所述柔性区中插入一个或多个(例如2个) G残基。在一些实施方案中，所述一个或多个G残基插入在所述柔性区中柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、S、N、D、H、M、T、E、Q、K、R、A和P。在一些实施方案中，根据以下优先级选择所述柔性氨基酸残基：G>S>N>D>H>M>T>E>Q>K> R>A>P。在一些实施方案中，所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：L、I、V、C、Y、F和W。在一些实施方案中，所述工程化的Cas12i核酸酶包含一个或多个选自下组SEQ ID NOs: 96-105的氨基酸序列。在一些实施方案中，所述工程化的Cas12i核酸酶包含一种或多种选自下组SEQ ID NOs: 99和104-105的氨基酸序列。在一些实施方案中，所述工程化的Cas12i核酸酶包含与SEQ ID NO: 8具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中的任一个)序列同一性的氨基酸序列。
在一些实施方案中，提供了包含SEQ ID NO: 99的氨基酸序列的工程化的Cas12i2核酸酶。在一些实施方案中，所述工程化的Cas12i2核酸酶包含与SEQ ID NO: 14具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中的任一个)序列同一性的氨基酸序列。在一些实施方案中，提供了包含SEQ ID NO: 14的氨基酸序列的工程化的Cas12i核酸酶。
在一些实施方案中，提供了包含SEQ ID NO: 104的氨基酸序列的工程化的Cas12i2核酸酶。在一些实施方案中，所述工程化的Cas12i2核酸酶包含与SEQ ID NO: 19具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中的任一个)序列同一性的氨基酸序列。在一些实施方案中，提供了包含SEQ ID NO: 18的氨基酸序列的工程化的Cas12i核酸酶。
在一些实施方案中，提供了包含SEQ ID NO: 105的氨基酸序列的工程化的Cas12i2核酸酶。在一些实施方案中，所述工程化的Cas12i2核酸酶包含与SEQ ID NO: 18具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中的任一个)序列同一性的氨基酸序列。在一些实施方案中，提供了包含SEQ ID NO: 19的氨基酸序列的工程化的Cas12i核酸酶。
在一些实施方案中，提供了工程化的Cas12i2核酸酶，其包含SEQ ID NO: 99的氨基酸序列和SEQ ID NO: 104的氨基酸序列。在一些实施方案中，所述工程化的Cas12i2核酸酶包含与SEQ ID NO: 20具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中的任一个)序列同一性的氨基酸序列。在一些实施方案中，提供了包含SEQ ID NO: 20的氨基酸序列的工程化的Cas12i核酸酶。
SEQ ID NO: 8：野生Cas12i2氨基酸序列
MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKLSQNLRAKNIGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS
SEQ ID NO: 14：Cas12i2-2.2氨基酸序列
MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGGGNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKLSQNLRAKNIGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS
SEQ ID NO: 18：Cas12i2-6.1氨基酸序列
MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKLSQNLRAKNGGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS
SEQ ID NO: 19：Cas12i2-6.2氨基酸序列
MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKLSQNLRAKNIGGGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS
SEQ ID NO: 20：enCas12i2 (Cas12i2-2.2+6.1)氨基酸序列
MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGGGNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKLSQNLRAKNGGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS
V-I型CRISPR-Cas12i已被鉴定为RNA-指导的DNA核酸内切酶系统。与诸如Cas12b或Cas9的CRISPR-Cas系统不同，基于Cas12i的CRISPR系统不需要tracrRNA序列。在一些实施方案中，所述RNA指导序列包括crRNA。通常，本文所述的crRNA包括直接重复序列和间隔区序列。在某些实施方案中，所述crRNA包括与指导序列或间隔区序列连接的直接重复序列、基本由或由其组成。在一些实施方案中，所述crRNA包括直接重复序列、间隔区序列和直接重复序列(DR-间隔区-DR)，其是其他CRISPR系统中的前体crRNA (pre- crRNA)构型的典型特征。在一些实施方案中，所述crRNA包括截短的直接重复序列和间隔区序列，其是经加工的或成熟的crRNA的典型特征。在一些实施方案中，所述CRISPR-Cas效应蛋白与RNA指导序列形成复合物，并且所述间隔区序列将复合物引导至与靶核酸的序列特异性结合，所述靶核酸与间隔区序列互补。
在一些实施方案中，本申请的工程化的Cas12i是核酸内切酶，其结合靶序列的特定位点并在指导RNA的指导下切割，并且具有DNA和RNA内切核酸酶活性。在一些实施方案中，所述Cas12i能够通过加工前体crRNA阵列来进行自主的crRNA生物发生。自主的前体crRNA处理可促进Cas12i的递送，从而实现双切口应用，因为可以由单个crRNA转录物靶向两个单独的基因组位点。然后，Cas12i蛋白将CRISPR阵列加工成两个同源的crRNA，从而形成配对的切口复合物。V-I型(Cas12i)效应蛋白的复用（Multiplexing）是利用所述效应蛋白的前体crRNA处理能力完成的，其中可以针对具有不同序列的多个靶标在单个RNA指导序列上编程。这样，可以同时操纵多个基因或DNA靶标以用于治疗应用。在一些实施方案中，所述指导RNA包含由CRISPR阵列表达的前体crRNA，所述CRISPR阵列由与未加工的DR序列交错的靶序列组成，通过所述效应蛋白的内在前体crRNA加工而重复以使得能够同时靶向一个、两个或多个位点。
在一些实施方案中，VI型CRISPR-Cas效应蛋白能够识别原间隔区相邻基序(PAM)，并且所述靶核酸包括PAM或由其组成，PAM包括核酸序列5'-TTN-3' (SEQ ID NO: 21)、5'-TTH-3' (SEQ ID NO: 22)、5'-TTY-3' (SEQ ID NO: 23)或5'-TTC-3' (SEQ ID NO: 24)，或者由其组成。
来自多种生物体的Cas12i核酸酶可以用作所述参比Cas12i核酸酶，以提供本申请的工程化的Cas12i效应蛋白。示例性Cas12i核酸酶已经描述于例如WO2019/201331A1和US2020/0063126A1中，其通过整体引用而并入本文。在一些实施方案中，所述参比Cas12i蛋白具有酶活性。在一些实施方案中，所述参比Cas12i是核酸酶，即切割靶双螺旋核酸(例如，双螺旋DNA)的两条链。在一些实施方案中，所述参比Cas12i是切口酶，即切割靶双螺旋核酸(例如，双螺旋DNA)的单链。在一些实施方案中，所述参比Cas12i蛋白是酶失活的。在一些实施方案中，所述参比Cas12i核酸酶是Cas12i1 (例如，SEQ ID NO: 9)、Cas12i2 (例如，SEQID NO: 8)或Cas12i-Phi (例如，SEQ ID NO: 10)。在一些实施方案中，所述参比Cas12i核酸酶包含选自下组SEQ ID NOs: 8-10的氨基酸序列。与Cas12i或其功能衍生物具有一定序列同一性(例如至少约60%、70%、80%、85%、90%、95%、98%或更多中任一个)的直系同源物可以用作设计本申请的工程化的Cas12i效应蛋白的基础。
在一些实施方案中，所述工程化的Cas12i蛋白基于天然存在的Cas12i蛋白的功能变体。在一些实施方案中，所述功能变体具有一个或多个突变，如氨基酸替换、插入和缺失。举例来说，与野生型天然存在的Cas12i蛋白相比，所述功能变体可包含1、2、3、4、5、6、7、8、9、10个或更多个氨基酸替换中的任一个。在一些实施方案中，所述一个或多个替换是保守替换。在一些实施方案中，所述功能变体具有天然存在的Cas12i蛋白的所有结构域。在一些实施方案中，所述功能变体不具有天然存在的Cas12i蛋白的一个或多个结构域。
还提供了基于本文所述工程化的Cas12i核酸酶中任一种的工程化的Cas12i效应蛋白。在一些实施方案中，所述工程化的Cas12i效应蛋白具有酶活性。在一些实施方案中，所述工程化的Cas12i效应蛋白是切割靶双螺旋核酸(例如，双螺旋DNA)的两条链的核酸酶。在一些实施方案中，所述工程化的Cas12i效应蛋白是切口酶，即切割靶双螺旋核酸(例如，双螺旋DNA)的单链。在一些实施方案中，所述工程化的Cas12i效应蛋白包含所述工程化的Cas12i核酸酶的酶失活突变体。Cas12i核酸酶活性位点中一个或多个氨基酸残基的突变会导致失去酶活的Cas12i。在一些实施方案中，本文提供的工程化的Cas12i酶可以被修饰以具有减少的核酸酶活性，例如，核酸酶与野生型Cas12i酶相比失活至少50%、至少60%、至少70%、至少80%、至少90%、至少95%，至少97%或100%。所述核酸酶活性可以通过几种方法来降低，例如，将突变引入所述核酸酶或Cas12i酶的PAM相互作用结构域中。在一些实施方案中，鉴定了核酸酶活性的催化残基，并且这些氨基酸残基可以被不同的氨基酸残基(例如，甘氨酸或丙氨酸)替换以降低所述核酸酶活性。Cas12i1的此类突变的实例包括D647A、E894A或D948A。Cas12i2的此类突变的实例包括D599A、E833A或D886A。
在一些实施方案中，提供了工程化的Cas12i切口酶。在一些实施方案中，提供了工程化的Cas12i融合效应蛋白，其包含与功能结构域融合的工程化的Cas12i核酸酶或其功能衍生物(例如，所述工程化的Cas12i核酸酶的酶失活突变体)，所述功能结构域诸如为翻译起始结构域、转录阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域(例如CBE或ABE结构域)、逆转录酶结构域、报告分子结构域(例如荧光结构域)和核酸酶结构域。在一些实施方案中，提供了工程化的Cas12i碱基编辑器，其包含与胞嘧啶脱氨酶结构域或腺苷脱氨酶结构域融合的本文所述工程化的Cas12i核酸酶(例如，enCas12i2或SEQ IDNO: 20)中任一种的催化失活变体。在一些实施方案中，提供了工程化的Cas12i主编辑器，其包含与逆转录酶结构域融合的本文所述工程化的Cas12i核酸酶(例如，enCas12i2或SEQID NO: 20)中任一种的催化失活变体。在一些实施方案中，提供了工程化的分割型Cas12i效应蛋白。
还提供了工程化的CRISPR-Cas12i系统，其包含本文所述工程化的Cas12i效应蛋白中任一种，以及包含与靶序列互补的指导序列的指导RNA，或编码所述指导RNA的一种或多种核酸，其中所述工程化的Cas12i效应蛋白和所述指导RNA能够形成CRISPR复合物，其与包含靶序列的靶核酸特异性结合并诱导所述靶核酸的修饰。在一些实施方案中，所述指导RNA包含crRNA。在一些实施方案中，不需要tracrRNA。在一些实施方案中，所述指导RNA包含从CRISPR阵列表达的前体crRNA，所述CRISPR阵列由与未加工的DR序列交织的靶序列组成，通过所述效应蛋白的内在前体crRNA加工而重复，以使得能够同时靶向一个、两个或多个位点。在一些实施方案中，所述指导RNA是编码多个crRNA的前体RNA阵列。在一些实施方案中，所述工程化的Cas12i效应蛋白和/或所述指导RNA由一种或多种载体诸如AAV载体编码。在一些实施方案中，所述工程化的CRISPR-Cas12i系统是核糖核蛋白(RNP)复合物，其包含结合至所述指导RNA的工程化的Cas12i效应蛋白。
工程化的Cas9效应蛋白
本申请提供了具有改善的活性(例如靶标结合、双链切割活性、切口酶活性和/或基因编辑活性)的工程化的Cas9效应蛋白。
在一些实施方案中，提供了工程化的Cas9核酸酶，其包含一个或多个突变，所述突变增加了参比Cas9核酸酶中柔性区的柔性，所述柔性区选自对应于以下的区的组：氨基酸残基39-43、氨基酸残基135-139、氨基酸残基176-180、氨基酸残基274-278、氨基酸残基351-355、氨基酸残基389-393、氨基酸残基521-525、氨基酸残基541-545、氨基酸氨基酸残基755-759、氨基酸残基774-778、氨基酸残基786-790、氨基酸残基811-815、氨基酸残基848-852、氨基酸残基855-859、氨基酸残基874-878、氨基酸残基891-895、氨基酸残基1019-1023和氨基酸残基1036-1040，其中所述氨基酸残基编号基于SEQ ID NO: 25，并且其中所述工程化的Cas9核酸酶与所述参比Cas9核酸酶相比具有增强的活性。在一些实施方案中，所述参比Cas9核酸酶是GeoCas9。在一些实施方案中，所述参比Cas9核酸酶中的柔性区选自对应于以下的区的组：氨基酸残基135-139、氨基酸残基176-180、氨基酸残基541-545、氨基酸残基755-759和氨基酸残基811-815，其中所述氨基酸残基编号基于SEQ ID NO: 25。在一个实施方案中，所述一个或多个突变包括在所述柔性区中插入一个或多个(例如2个) G残基。在一些实施方案中，所述一个或多个G残基插入在所述柔性区中柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、S、N、D、H、M、T、E、Q、K、R、A和P。在一些实施方案中，根据以下优先级选择所述柔性氨基酸残基：G>S>N>D>H>M>T>E>Q>K>R>A>P。在一些实施方案中，所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：L、I、V、C、Y、F和W。在一些实施方案中，所述工程化的Cas9核酸酶包含一个或多个选自下组SEQ ID NOs: 138-175的氨基酸序列。在一些实施方案中，所述工程化的Cas9核酸酶包含一种或多种选自下组SEQ ID NOs：139、140、145-146、和153的氨基酸序列。在一些实施方案中，所述工程化的Cas9核酸酶包含与SEQ ID NO: 25具有至少约85%(例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中任一个)序列同一性的氨基酸序列。
在一些实施方案中，提供了包含SEQ ID NO: 139氨基酸序列的工程化的GeoCas9核酸酶。在一些实施方案中，所述工程化的Cas9核酸酶包含与SEQ ID NO: 27具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中任一个)序列同一性的氨基酸序列。在一些实施方案中，提供了包含SEQ ID NO: 27氨基酸序列的工程化的Cas9核酸酶。
在一些实施方案中，提供了包含SEQ ID NO: 140氨基酸序列的工程化的GeoCas9核酸酶。在一些实施方案中，所述工程化的Cas9核酸酶包含与SEQ ID NO: 29具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中任一个)序列同一性的氨基酸序列。在一些实施方案中，提供了包含SEQ ID NO: 28氨基酸序列的工程化的Cas9核酸酶。
在一些实施方案中，提供了包含SEQ ID NO: 145氨基酸序列的工程化的GeoCas9核酸酶。在一些实施方案中，所述工程化的Cas9核酸酶包含与SEQ ID NO: 33具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中任一个)序列同一性的氨基酸序列。在一些实施方案中，提供了包含SEQ ID NO: 33氨基酸序列的工程化的Cas9核酸酶。
在一些实施方案中，提供了包含SEQ ID NO: 146氨基酸序列的工程化的GeoCas9核酸酶。在一些实施方案中，所述工程化的Cas9核酸酶包含与SEQ ID NO: 34具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中任一个)序列同一性的氨基酸序列。在一些实施方案中，提供了包含SEQ ID NO: 34氨基酸序列的工程化的Cas9核酸酶。
在一些实施方案中，提供了包含SEQ ID NO: 153氨基酸序列的工程化的GeoCas9核酸酶。在一些实施方案中，所述工程化的Cas9核酸酶包含与SEQ ID NO: 40具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中任一个)序列同一性的氨基酸序列。在一些实施方案中，提供了包含SEQ ID NO: 41氨基酸序列的工程化的Cas9核酸酶。
SEQ ID NO: 25：野生GeoCas9氨基酸序列
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAAIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETVRPLQSTRD
SEQ ID NO: 27：GeoCas9-2.1氨基酸序列
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKRRGFKSNRKSERGGSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAAIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETVRPLQSTRD
SEQ ID NO: 28：GeoCas9-3.1氨基酸序列
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGGGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAAIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETVRPLQSTRD
SEQ ID NO: 33：GeoCas9-8.1氨基酸序列
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGGGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAAIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETVRPLQSTRD
SEQ ID NO: 34：GeoCas9-9.1氨基酸序列
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAAIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHGPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETVRPLQSTRD
SEQ ID NO: 41：GeoCas9-12.1氨基酸序列
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAAIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRMPKRSGTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETVRPLQSTRD在一些实施方案中，提供了工程化的Cas9核酸酶，其包含一个或多个突变，所述突变增加了参比Cas9核酸酶中柔性区的柔性，所述柔性区选自对应于以下的区的组：氨基酸残基45-49、氨基酸残基84-88、氨基酸残基116-120、氨基酸残基128-132、氨基酸残基216-220、氨基酸残基318-322、氨基酸残基387-391、氨基酸残基497-501、氨基酸残基583-587、氨基酸残基594-598、氨基酸残基614-618、氨基酸残基696-700和氨基酸残基739-743，其中所述氨基酸残基编号基于SEQ ID NO: 53，并且其中所述工程化的Cas9核酸酶与参比Cas9核酸酶相比具有增加的活性。在一些实施方案中，所述参比Cas9核酸酶中的柔性区对应于氨基酸残基45-49或氨基酸残基116-120，其中所述氨基酸残基编号基于SEQ ID NO: 53。在一些实施方案中，所述参比Cas9核酸酶是SaCas9。在一些实施方案中，所述一个或多个突变包括在所述柔性区中插入一个或多个(例如2个) G残基。在一些实施方案中，所述一个或多个G残基插入在所述柔性区中柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、S、N、D、H、M、T、E、Q、K、R、A和P。在一些实施方案中，根据以下优先级选择所述柔性氨基酸残基：G>S>N>D>H>M>T>E>Q>K>R>A>P。在一些实施方案中，所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：L、I、V、C、Y、F和W。在一些实施方案中，所述工程化的Cas9核酸酶包含一个或多个选自下组SEQ ID NOs: 199-217的氨基酸序列。在一些实施方案中，所述工程化的Cas9核酸酶包含一个或多个选自下组SEQ ID NOs：199、203和204的氨基酸序列。在一些实施方案中，所述工程化的Cas9核酸酶包含与SEQ ID NO: 53具有至少约85% (例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中任一个)序列同一性的氨基酸序列。
在一些实施方案中，提供了包含SEQ ID NO: 199氨基酸序列的工程化的SaCas9核酸酶。在一些实施方案中，所述工程化的Cas9核酸酶包含与SEQ ID NO: 54具有至少约85%(例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中任一个)序列同一性的氨基酸序列。在一些实施方案中，提供了包含SEQ ID NO: 54氨基酸序列的工程化的Cas9核酸酶。
在一些实施方案中，提供了包含SEQ ID NO: 203氨基酸序列的工程化的SaCas9核酸酶。在一些实施方案中，所述工程化的Cas9核酸酶包含与SEQ ID NO: 58具有至少约85%(例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中任一个)序列同一性的氨基酸序列。在一些实施方案中，提供了包含SEQ ID NO: 58氨基酸序列的工程化的Cas9核酸酶。
在一些实施方案中，提供了包含SEQ ID NO: 204氨基酸序列的工程化的SaCas9核酸酶。在一些实施方案中，所述工程化的Cas9核酸酶包含与SEQ ID NO: 59具有至少约85%(例如，至少约90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多中任一个)序列同一性的氨基酸序列。在一些实施方案中，提供了包含SEQ ID NO: 59氨基酸序列的工程化的Cas9核酸酶。
SEQ ID NO: 53：野生SaCas9氨基酸序列
MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGGS
SEQ ID NO: 54：SaCas9-1.1氨基酸序列
MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGGGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGGS
SEQ ID NO: 58：SaCas9-3.1氨基酸序列
MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGGHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGGS
SEQ ID NO: 59：SaCas9-3.2氨基酸序列
MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGGGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGGS
II型CRISPR-Cas9系统是双RNA-指导的(即crRNA和tracrRNA) DNA核酸内切酶系统。与反式激活crRNA (tracrRNA)碱基配对的成熟crRNA形成一个双RNA结构，所述RNA结构指导CRISPR相关蛋白Cas9在靶DNA中引入双链(ds)断裂。在与crRNA指导序列互补的位点，Cas9HNH核酸酶结构域切割互补链，而Cas9 RuvC-样结构域切割非互补链。当将双-tracrRNA:crRNA工程化为单个RNA嵌合体时，它还指导序列特异性Cas9 dsDNA切割。靶向DNA的RNA(在本文中也称为“crRNA”；或“指导RNA”；或“gRNA”)包括：i)第一片段，其包含与靶DNA中靶序列互补的核苷酸序列；ii)第二片段，其与定点（site-directed）多肽相互作用；iii)转录终止子。
来自多种生物体的Cas9核酸酶可以用作所述参比Cas9核酸酶，以提供本申请的工程化的Cas9效应蛋白。示例性Cas9蛋白已描述于例如，US8697359、US10266850和US20170145425，其通过整体引用而并入本文。
在一些实施方案中，所述工程化的Cas9效应蛋白基于这样的参比Cas9蛋白(例如，Cas9核酸酶)，其选自来自以下细菌的Cas9蛋白的：肺炎链球菌(Streptococcus pneumoniae)(Csn1)、化脓性链球菌(Streptococcus pyogenes)(SpCas9)或嗜热链球菌(Streptococcus thermophiles)(StCas9)、金黄色葡萄球菌(Staphylococcus aureus)(SaCas9)，脑膜炎奈瑟氏球菌(Neisseria meningitides)(Nm2Cas9)，空肠弯曲菌(Campylobacter jejuni)(CjCas9)，嗜热脂肪地芽孢杆菌(Geobacillus stearothermophilus)(GeoCas9)和密螺旋体(Treponema denticola)(TdCas9)，并且可能包括源自这些生物体的突变Cas9。在一些实施方案中，所述参比Cas9蛋白对于某些应用可以具有期望的性质，诸如靶向嗜热菌。例如，与化脓性链球菌Cas9 (SpCas9)的45°C相比，GeoCas9在高达70°C的温度下具有活性。在一些实施方案中，所述参比Cas9核酸酶包含选自下组SEQ ID NOs: 25、53或72-73的氨基酸序列。与Cas9或其功能衍生物具有一定序列同一性(例如至少约60%、70%、80%、85%、90%、95%、98%或更多中任一个)的直系同源物可以用作设计本申请的工程化的Cas9效应蛋白的基础。
天然存在的Cas9核酸酶具有多种结构域。在一些实施方案中，参比Cas9核酸酶包含具有中央HNH内切核酸酶结构域和分割型RuvC/RNaseH结构域的结构域架构。在一些实施方案中，参比Cas9核酸酶包含具有保守架构的4个关键基序。基序1、2和4是类似RuvC的基序，而基序3是HNH基序。可以使用本领域中的已知方法，诸如基于参比Cas9核酸酶的晶体结构(例如，PDB ID Nos: 5CZZ、4OGC、5X2G、6JOO)来确定结构域边界。
还提供了基于本文所述工程化的Cas9核酸酶中任一种的工程化的Cas9效应蛋白。在一些实施方案中，所述工程化的Cas9效应蛋白具有酶活性。在一些实施方案中，所述工程化的Cas9效应蛋白是切割靶双螺旋核酸(例如，双螺旋DNA)的两条链的核酸酶。在一些实施方案中，所述工程化的Cas9效应蛋白是切口酶，即切割靶双螺旋核酸(例如，双螺旋DNA)的单链。例如，来自化脓性链球菌的Cas9的RuvC I催化结构域中的天冬氨酸至丙氨酸替换(D10A)将Cas9从切割两条链的核酸酶转变成切口酶(切割一条单链)。使Cas9成为切口酶的突变的其他实例包括但不限于：H840A、N854A和N863A。在一些实施方案中，所述工程化的Cas9效应蛋白包含所述工程化的Cas9核酸酶的酶失活突变体。Cas9核酸酶活性位点中一个或多个氨基酸残基的突变可导致Cas9酶失活。例如，Cas9的两个或更多个催化结构域(RuvCI、RuvC II和RuvC III)可以被突变，以产生基本上缺乏所有DNA切割活性的突变的Cas9。在一些实施方案中，将D10A突变与H840A、N854A或N863A突变中一种或多种组合，以产生基本上缺乏所有DNA切割活性的Cas9酶。在一些实施方案中，相对于其非突变形式当突变酶的DNA切割活性小于约25%、10%、5%、1%、0.1%、0.01%或更低时，认为CRISPR酶基本上缺乏所有DNA切割活性。其他突变可能是有用的；如果Cas9或其他CRISPR酶来自化脓性链球菌以外的物种，则可以对相应氨基酸进行突变以实现相似的作用。
在一些实施方案中，提供了工程化的Cas9切口酶。在一些实施方案中，提供了工程化的Cas9融合效应蛋白，其包含与功能结构域融合的工程化的Cas9核酸酶或其功能衍生物(例如，工程化的Cas9核酸酶的酶失活突变体)，所述功能结构域诸如为翻译起始结构域、转录阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域(例如CBE或ABE结构域)、逆转录酶结构域、报告分子结构域(例如荧光结构域)或核酸酶结构域。在一些实施方案中，提供了工程化的分割型Cas9效应蛋白。分割型Cas9效应蛋白已描述于例如WO2016/112242，其通过整体引用而并入本文。
还提供了工程化的CRISPR-Cas9系统，其包含本文所述工程化的Cas9效应蛋白中任一种，以及包含与靶序列互补的指导序列的指导RNA，或一种或多种编码该指导RNA的核酸，其中所述工程化的Cas9效应蛋白和所述指导RNA能够形成CRISPR复合物，其与包含靶序列的靶核酸特异性结合并诱导所述靶核酸的修饰。在一些实施方案中，所述指导RNA包含crRNA和tracrRNA。在一些实施方案中，所述指导RNA是sgRNA。在一些实施方案中，所述指导RNA是可以被加工成crRNA和tracrRNA的前体分子。在一些实施方案中，所述指导RNA是编码多个crRNA的前体RNA阵列，并且其中每个经加工的crRNA与tracrRNA相关。在一些实施方案中，所述工程化的Cas9效应蛋白和/或所述指导RNA由一种或多种载体如AAV载体编码。在一些实施方案中，所述工程化的CRISPR-Cas9系统是核糖核蛋白(RNP)复合物，其包含结合至所述指导RNA的所述工程化的Cas9效应蛋白。
其他工程化的Cas效应蛋白
本申请提供了工程化的Cas效应蛋白(例如，Cas核酸酶、Cas切口酶、Cas融合效应蛋白、分割型Cas效应蛋白)，其具有改善的活性(例如，靶标结合、双链切割活性、切口酶活性和/或基因编辑活性)。用于工程化改造的合适参比Cas蛋白例如，可以包括：Casl、CaslB、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Csnl、Csxl2、CaslO、Csyl、Csy2、Csy3、Csel、Cse2、Cscl、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmrl、Cmr3、Cmr4、Cmr5、Cmr6、Csbl、Csb2、Csb3、Csxl7、Csxl4、CsxlO、Csxl6、CsaX、Csx3、Csxl、Csxl5、Csfl、Csf2、Csf3、Csf4、Cpfl (也称为Casl2a)、CasX (Cas12e)、Cas12c、Cas12d、Cas12g、Cas12k、Cas12i、cas12f、Cas12j、Cas12w、Cas12v、CasX、CasY、CasZ，或其等同物或修改版本。有关示例性Cas参比蛋白的描述，请参见综述文章：Zhang F (2019). Development of CRISPR-Cas systems forgenome editing and beyond. Quarterly Reviews of Biophysics 52, e6, 1-31；Li Y& Peng N. Endogenous CRISPR-Cas system-based genome editing andantimicrobials: review and prospects. Frontiers in microbiology 10 (2019):2471。
在一些实施方案中，所述参比Cas核酸酶是Cas12a、Cas12b (如“工程化的Cas12b效应蛋白”小节中描述的参比蛋白中任一种)、Cas9 (如“工程化的Cas9效应蛋白”小节中描述的参比蛋白中任一种)、Cas12i (如“工程化的Cas12i效应蛋白”小节中描述的参比蛋白中任一种)、Cas12f、Cas12j或CasX(Cas12e)。
在一些实施方案中，所述参比蛋白是V-A型CRISPR-Cas系统的Cas12a蛋白(先前称为Cpf1)。V-A型系统不需要tracrRNA，可以简化指导RNA的设计。来自多种生物体的Cas12a(Cpf1)核酸酶可用作参比Cas12a核酸酶，以提供本申请的工程化的Cas12a效应蛋白。示例性的Cas12a核酸酶已描述于例如US10648020；US10669540；US9790490；US20180282713和WO2018188571，其通过整体引用而并入本文。
在一些实施方案中，所述工程化的Cas12a效应蛋白基于参比Cas12a蛋白(例如，Cas12a核酸酶)，其选自来自以下物种的Cas12a蛋白：普雷沃氏菌(Prevotella)和弗朗西斯菌(Francisella)，如新弗朗西斯菌(Francisella novicida)(FnCas12a或FnCpf1)，或氨基酸球菌(Acidaminococcus)(AsCas12a或AsCpf1)或毛螺菌科(Lachnospiraceae)细菌(LbCas12a或LbCpf1p)。在一些实施方案中，所述参比Cas12a核酸酶包含选自下组SEQ IDNOs: 74-75的氨基酸序列。与Cas12a或其功能衍生物具有一定序列同一性(例如至少约60%、70%、80%、85%、90%、95%、98%或更多中任一个)的直系同源物，可以用作设计本申请中工程化的Cas12a效应蛋白的基础。
氨基酸球菌Cas12a-crRNA-靶DNA复合物的晶体结构已描述于Yamano T,Nishimasu H, Zetsche B, et al. Crystal structure of Cpf1 in complex withguide RNA and target DNA. Cell. 2016; 165: 949–962，可用PDB代码5B43获取。也可获取LbCas12a (LbCpf1)的晶体结构(例如，PDB代码5XUU、5XH6和5XUT)。
在一些实施方案中，所述工程化的Cas12a效应蛋白包含一种或多种减少或消除核酸酶活性的突变。例如，FnCpf1 RuvC结构域中合适的突变氨基酸位置包括但不限于：D917A、E1006A、E1028A、D1227A、D1255A、N1257A、D917A、E1006A、E1028A、D1227A、D1255A和N1257A。在一些实施方案中，基本降低核酸酶活性的点突变包括在推定的第二核酸酶结构域中的突变，如N580A、N584A、T587A、W609A、D610A、K613A、E614A、D616A、K624A、D625A、K627A和Y629A。在另一个实例中，AsCpf1p RuvC结构域中的突变是D908A、E993A和D1263A，其中D908A、E993A和D1263A突变使AsCpf1效应蛋白的DNA切割活性完全失活。在一些实施方案中，LbCpf1p RuvC结构域中的突变包括但不限于832、947或1180。在一些实施方案中，LbCpf1 RuvC结构域中的突变是LbD832A、E925A、D947A或D1180A，其中LbD832A、E925A、D947A或D1180A突变使Cas12a蛋白的DNA切割活性完全失活。本文考虑了在其他工程化的Cas12a中与针对FnCpf1和LbCpf1描述的那些位置相对应的位置处的突变。
在一些实施方案中，所述参比蛋白是CasX(也称为Cas12e)蛋白。来自多种生物体的CasX核酸酶可以用作所述参比CasX核酸酶，以提供本申请的工程化的CasX效应蛋白。在一些实施方案中，本发明方法和/或组合物的工程化的CasX蛋白是(或衍生自)天然存在的(野生型)蛋白。示例性CasX核酸酶已描述于例如US10570415、WO2018/202800和WO2019/084148，其通过整体引用而并入本文。
在一些实施方案中，所述工程化的CasX效应蛋白基于参比CasX蛋白(例如，CasX核酸酶)，其选自来自δ-变形菌(Deltaproteobacter) (DpbCasX或CasX1)和浮霉菌(Plantomycetes) (PlmCasX或CasX2)的CasX蛋白。在一些实施方案中，所述参比CasX核酸酶包含选自下组SEQ ID NOs: 76-77的氨基酸序列。与CasX或其功能衍生物具有一定序列同一性(例如至少约60%、70%、80%、85%、90%、95%、98%或更多中任一个)的直系同源物可以用作设计本申请工程化的CasX效应蛋白的基础。
在一些实施方案中，与先前鉴定的CRISPR-Cas核酸内切酶相比，CasX蛋白短，因此使用该蛋白作为替代物提供的优点是编码该蛋白的核苷酸序列相对短。例如，这在需要编码所述工程化的CasX蛋白的核酸的情况下是有用的，例如在采用病毒载体(例如AAV载体)以递送至细胞如真核细胞(例如，哺乳动物细胞、人细胞、小鼠细胞，体外、离体、体内)用于研究和/或临床应用的情况。还应注意的是，在低温(例如10-17℃)收集的环境样品中存在带有天然存在的CasX CRISPR位点的细菌。因此，预期CasX能够在低温(例如10-14°C、10-17°C、10-20°C)下良好发挥作用。
对DpbCasX-sgRNA-DNA复合物(PDB代码6NY2、6NY1和6NY3)的冷冻EM结构进行分析后，发现了非靶链结合(NTSB)和靶链装载(TSL)结构域(Liu-J-J, OrlovaN, OakesBL, etal. CasX enzymes comprise a distinct family of RNA-guided genome editors.Nature. 2019; 566:218–223)。TSL结构域的位置类似于Cas12a (PDB 5B43)中所谓的“Nuc”结构域的位置。这些结构域在CasX和Cas12a酶中执行类似的功能：在非靶DNA链被RuvC结构域切割后，它们弯曲sgRNA-DNA双螺旋。这种构象变化允许靶DNA链被RuvC结构域切割。因此，与Cas9使用不同结构域HNH和RuvC来切割每条DNA链相比，Cas12e和Cas12a都依赖单个核酸酶结构域进行双链DNA切割。在Cas12e和Cas12a中，较大的结构变化改变了DNA链对于RuvC核酸酶的可及性，并以此方式弥补了第二个核酸酶结构域的缺乏。
Cas12a和Cas12X均生成带有粘性末端的产品。相反，Cas9蛋白主要产生平末端。值得注意的是，刘俊杰(Jun-Jie Liu)等人发现DpbCasX产生的粘性末端大约10个核苷酸长，其比Cas12a蛋白通常产生的3–5nt突出端更长。Cas12a和CasX产生的5′-突出端可用于通过直接DNA连接在体内或体外将DNA片段插入基因组。
与Cas12a相反，CasX酶除了crRNA外还需要tracrRNA来进行DNA靶标识别。
在一些实施方案中，所述参比Cas核酸酶是Cas12f。示例性Cas12f核酸酶已公开于例如WO2020088450中。在一些实施方案中，所述参比Cas核酸酶是Cas12i。示例性Cas12i核酸酶已公开于例如WO2020098772中。
在一些实施方案中，所述参比Cas核酸酶是Cas12j (例如，SEQ ID NO: 78)。
变体
本申请提供了工程化的Cas效应蛋白，其包含本文所述工程化的Cas核酸酶的功能变体。在一些实施方案中，当与相应的工程化的Cas核酸酶的氨基酸序列相比时，功能变体的氨基酸序列具有至少一个氨基酸残基的不同(例如，具有缺失、插入、替换和/或融合)。在一些实施方案中，所述功能变体具有一个或多个突变，如氨基酸替换、插入和/或缺失。举例来说，与工程化的Cas核酸酶相比，功能变体可包含1、2、3、4、5、6、7、8、9、10个或更多个氨基酸中任一个替换。在一些实施方案中，所述一个或多个替换是保守替换。在一些实施方案中，所述功能变体具有工程化的Cas核酸酶的所有结构域。在一些实施方案中，所述功能变体不具有工程化的Cas核酸酶的一个或多个结构域。
对于本文所述Cas变体蛋白(例如，切口酶Cas蛋白、失活或催化失活的Cas(dCas)、融合Cas)中任一种，所述Cas变体可包括具有上述相同参数(例如，存在的结构域、同一性百分比等)的Cas蛋白序列。
催化活性
在一些实施方案中，所述功能变体与其工程化的Cas核酸酶的非突变形式相比，具有不同的催化活性。在一些实施方案中，所述突变(例如，氨基酸替换、插入和/或缺失)在Cas效应蛋白的催化结构域(例如，RuvC结构域)中。在一些实施方案中，所述变体包含多个催化结构域中的突变。切割双链靶核酸的一条链而不切割另一条链的Cas效应蛋白，在本文中被称为“切口酶”(例如“切口酶Cas”)。在本文中，基本上不具有核酸酶活性的Cas蛋白称为失活Cas蛋白(“dCas”)(声明：在融合Cas效应蛋白的情况下，异源多肽(融合伴侣)可以提供核酸酶活性，将在下面进行详细说明)。在一些实施方案中，相对于其非突变形式而言，当突变酶的DNA切割活性小于约25%、10%、5%、1%、0.1%、0.01%或更低时，认为Cas效应蛋白基本上缺乏所有的DNA切割活性。
Cas功能变体的示例性突变描述于上述Cas12b、Cas12i和Cas9小节，以及WO2016205764、WO2020/087631、WO2019/201331A1、US2020/0063126A1、US8697359、US10266850、US20170145425、US10648020、US10669540、US9790490、US20180282713、WO2018188571、US10570415、WO2018/202800和WO2019/084148中，其通过整体引用而并入本文。
分割型Cas效应蛋白
本申请还提供了基于本文所述工程化的Cas效应蛋白中任一种的分割型Cas效应蛋白。分割型Cas效应蛋白对于递送可能是有利的。在一些实施方案中，所述工程化的Cas效应蛋白被分割成酶的两个部分，可以将它们重构在一起以提供基本起作用的Cas效应蛋白。Cas效应蛋白(例如，Cas12和Cas9蛋白)的分割形式已描述于例如WO2016/112242、WO2016/205749和PCT/CN 2020 /111057，其通过整体引用而并入本文。
在一些实施方案中，提供了分割型Cas效应蛋白，其包含第一多肽和第二多肽，所述第一多肽包含本文所述工程化的Cas核酸酶中任一种或其功能衍生物的N末端部分，所述第二多肽包含所述工程化的Cas核酸酶或其功能衍生物的C末端部分，其中所述第一多肽和第二多肽能够在包含指导序列的指导RNA存在下彼此缔合，以形成与靶核酸特异性结合的CRISPR复合物，所述靶核酸包含与所述指导序列互补的靶序列。在一些实施方案中，所述第一多肽和第二多肽各自包含二聚化结构域。在一些实施方案中，所述第一二聚化结构域和第二二聚化结构域在诱导剂(例如雷帕霉素)存在下彼此缔合。在一些实施方案中，所述第一多肽和第二多肽不包含二聚化结构域。在一些实施方案中，所述分割型Cas效应蛋白是自诱导的。
可以以不影响催化结构域的方式进行分割。Cas效应蛋白可以用作核酸酶(包括切口酶)或可以是灭活的酶，其本质上是具有很少或没有催化活性(例如由于其催化结构域中的突变)的RNA指导的DNA结合蛋白。
在一些实施方案中，Cas蛋白的核酸酶叶和α-螺旋叶表达为分开的多肽。尽管所述叶不自行相互作用，但RNA指导序列将它们募集到一个复合物中，所述复合物概括了全长Cas酶的活性并催化位点特异性的DNA切割。在一些实施方案中，经修饰的RNA指导序列可以用于通过防止二聚化而消除分割型酶的活性，从而允许诱导型二聚化系统的发展。所述分割型酶描述于例如，Wright, Addison V., et al. “Rational design of a split-Cas9enzyme complex,” Proc. Nat'l. Acad. Sci., 112.10 (2015): 2984-2989，其通过整体引用并入本文。
本文所述分割型Cas效应蛋白部分可设计为通过将参比工程化的Cas效应蛋白(例如全长工程化的Cas12b、Cas12i、Cas9、Cas12a或CasX效应蛋白或其功能变体)在一个分割位置处分开(即，分割)成两半，所述位置是所述参比Cas效应蛋白的N端部分与C端部分分开的点。在一些实施方案中，所述N末端部分包含氨基酸残基1至X，而所述C末端部分包含氨基酸残基X+1至所述参比Cas效应蛋白的C末端。在该实例中，编号是连续的，但这并非必需的，因为还考虑了氨基酸(或编码它们的核苷酸)可以从分割的末端和/或突变(例如，插入、删除和替换)中任一个在所述多肽链的内部区域修剪而来，条件是重构的Cas效应蛋白保留了足够的DNA结合活性(如果需要)、DNA切口酶或切割活性，例如与所述参比Cas效应蛋白相比，具有至少40%、50%、60%、70%、80%、90%或95%活性。
可以通过计算机(in silico)设计分割点并将其克隆到构建体中。在此过程中，可以将突变引入分割型Cas效应蛋白，并且可以去除非功能结构域。在一些实施方案中，所述分割型Cas效应蛋白的两个部分或片段(即，N末端和C末端片段)可以形成完整的Cas效应蛋白，其包含例如完整Cas效应蛋白序列的至少70%、至少80%、至少90%、至少95%或至少99%。
所述分割型Cas效应蛋白可各自包含一个或多个二聚化结构域。在一些实施方案中，所述第一多肽包含与第一分割型Cas效应蛋白部分融合的第一二聚结构域，并且所述第二多肽包含与第二分割型Cas效应蛋白部分融合的第二二聚结构域。可通过肽接头(例如，柔性肽接头如GS接头)或化学键将所述二聚化结构域融合至所述分割型Cas效应蛋白部分。在一些实施方案中，所述二聚化结构域与所述分割型Cas效应蛋白部分的N末端融合。在一些实施方案中，所述二聚化结构域与所述分割型Cas效应蛋白部分的C末端融合。
在一些实施方案中，所述分割型Cas效应蛋白不包含任何二聚化结构域。
在一些实施方案中，所述二聚化结构域促进两个分割型Cas效应蛋白部分的缔合。在一些实施方案中，所述分割型Cas效应蛋白部分被诱导剂诱导而缔合或二聚化为功能性Cas效应蛋白。在一些实施方案中，所述分割型Cas效应蛋白包含可诱导的二聚化结构域。在一些实施方案中，所述二聚化结构域不是可诱导的二聚化结构域，即，所述二聚化结构域在诱导剂不存在下进行二聚化。
诱导剂可以是除指导RNA(例如sgRNA)以外的诱导能量源或诱导分子。所述诱导剂通过诱导的二聚化结构域的二聚化将两个分割型Cas效应蛋白部分重构为功能性Cas效应蛋白。在一些实施方案中，所述诱导剂通过可诱导的二聚化结构域的诱导缔合的作用将两个分割型Cas效应蛋白部分聚集在一起。在一些实施方案中，在没有诱导剂的情况下，两个分割型Cas效应蛋白部分不彼此缔合以重构为功能性Cas效应蛋白。在一些实施方案中，在没有诱导剂的情况下，两个分开的Cas效应蛋白部分可以在指导RNA(例如sgRNA)存在下彼此缔合以重构为功能性Cas效应蛋白。
本申请的诱导剂可以是热、超声、电磁能或化合物。在一些实施方案中，所述诱导剂是抗生素、小分子、激素、激素衍生物、类固醇或类固醇衍生物。在一些实施方案中，所述诱导剂是脱落酸(ABA)、多西霉素(DOX)、异丙基苯甲酸(cumate)、雷帕霉素、4-羟基他莫昔芬(4OHT)、雌激素或蜕皮激素。在一些实施方案中，所述分割型Cas效应系统是选自下组的诱导剂控制的系统：基于抗生素的诱导系统、基于电磁能的诱导系统、基于小分子的诱导系统、基于核受体的诱导系统和基于激素的诱导系统。在一些实施方案中，所述分割型Cas效应系统是选自下组的诱导剂控制的系统：四环素(Tet)/DOX诱导系统、光诱导系统、ABA诱导系统、异丙基苯甲酸(cumate)阻遏物/操纵子系统、4OHT/雌激素诱导系统、基于蜕皮激素的诱导系统和FKBP12/FRAP (FKBP12-雷帕霉素复合物)诱导系统。这样的诱导剂也讨论于本文和PCT/US2013/051418，其通过整体引用而并入本文。FRB/FKBP/雷帕霉素系统已描述于Paulmurugan and Gambhir, Cancer Res, August 15, 2005 65; 7413，以及Crabtree et al., Chemistry & Biology 13, 99-107, Jan 2006，其通过整体引用而并入本文。
在一些实施方案中，成对的分割型Cas效应蛋白是分开的并且是无活性的，直到诱导了所述二聚化结构域的二聚化(例如，FRB和FKBP)，该二聚化导致功能性Cas效应蛋白核酸酶的再组装。在一些实施方案中，包含诱导型二聚体(例如FRB)的第一半部分的第一分割型Cas效应蛋白被分开递送，和/或处在与包含诱导型二聚体(例如FKBP)的第二半部分的第二分割型Cas效应蛋白分开的位置。
可用于本文所述的诱导剂控制的分割型Cas效应系统中的其他示例性的基于FKBP的诱导系统包括但不限于：在FK506存在下与钙调神经磷酸酶(CNA)进行二聚化的FKBP；在FKCsA存在下与CyP-Fas进行二聚化的FKBP；在雷帕霉素存在下与FRB进行二聚化的FKBP；在香豆霉素存在下与GryB进行二聚化的GyrB；在赤霉素存在下与GID1进行二聚化的GAI；或在HaXS存在下与HaloTag进行二聚化的Snap-tag。
也考虑了FKBP家族本身内的替代方案。例如，在FK1012存在下FKBP进行均二聚(即，一个FKBP与另一种FKBP进行二聚化)。
在一些实施方案中，所述二聚化结构域是FKBP，而诱导剂是FK1012。在一些实施方案中，所述二聚化结构域是GryB，而诱导剂是香豆霉素。在一些实施方案中，所述二聚化结构域是ABA，而诱导剂是赤霉素。
在一些实施方案中，可以在不存在诱导剂的情况下自动诱导(即，自动激活或自诱导)所述分割型Cas效应蛋白部分以缔合/二聚化为功能性Cas效应蛋白。不受任何理论或假设的束缚，可以通过与指导RNA如sgRNA结合来介导所述分割型Cas效应蛋白部分的自动诱导。在一些实施方案中，所述第一多肽和第二多肽不包含二聚化结构域。在一些实施方案中，所述第一多肽和第二多肽包含二聚化结构域。
在一些实施方案中，本文所述分割型Cas效应系统(包括诱导剂控制的和自动诱导的系统)的重构的Cas效应蛋白，相对于参比Cas效应蛋白编辑效率，具有至少70% (如至少约70%、75%、80%、85%、90%、95%、98%、99%或更多效率或100%效率中任一种)的编辑效率。
在一些实施方案中，本文所述诱导剂控制的分割型Cas效应系统的重构的Cas效应蛋白，在没有诱导剂存在下(即由于自动诱导)，相对于参比Cas效应蛋白编辑效率，其具有不超过50%(如不超过约50%、45%、40%、35%、30%、25%、20%、15%、10%、5%或更低效率，或0%效率中任一个)的编辑效率。
融合Cas效应蛋白
本申请还提供了工程化的Cas效应蛋白，其包含另外的蛋白结构域和/或组分，如接头、核定位/输出序列、功能结构域和/或报告蛋白。
在一些实施方案中，所述工程化的Cas效应蛋白是包含一个或多个异源蛋白结构域(例如，约或大于约1、2、3、4、5、6、7、8、9、10个或更多个结构域)以及所述工程化的Cas核酸酶的核酸靶向结构域或其功能衍生物的蛋白复合物。在一些实施方案中，所述工程化的Cas效应蛋白是包含与所述工程化的Cas核酸酶融合的一个或多个异源蛋白结构域(例如约或多于约1、2、3、4、5、6、7、8、9、10个或更多个结构域)的融合蛋白。
在一些实施方案中，本申请的工程化的Cas效应蛋白可包含(例如，通过融合蛋白，如通过一个或多个肽接头，例如GS肽接头等) 一个或多个功能结构域或缔合(例如，通过多种蛋白的共表达)于它。在一些实施方案中，所述一个或多个功能结构域是酶结构域。这些功能结构域可以具有多种活性，例如DNA和/或RNA甲基化酶活性、脱甲基酶活性、转录激活活性、转录抑制活性、转录释放因子活性、组蛋白修饰活性、RNA切割活性、DNA切割活性、核酸结合活性和开关活性(例如，光诱导的)。在一些实施方案中，所述一个或多个功能结构域是转录激活结构域(即，反式激活结构域)或阻遏物结构域。在一些实施方案中，所述一个或多个功能结构域是组蛋白修饰结构域。在一些实施方案中，所述一个或多个功能结构域是转座酶结构域、HR(同源重组)机构结构域、重组酶结构域和/或整合酶结构域。在一些实施方案中，所述功能结构域是Krüppel相关盒(KRAB)、VP64、VP16、Fok1、P65、HSF1、MyoD1、生物素-APEX、APOBEC1、AID、PmCDA1、Tad1和M-MLV逆转录酶。在一些实施方案中，所述功能结构域选自下组：翻译起始结构域、转录阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域(例如，CBE或ABE结构域)、逆转录酶结构域、报告分子结构域(例如，荧光结构域)和核酸酶结构域。
在一些实施方案中，所述工程化的Cas效应蛋白中一个或多个功能结构域的定位允许功能结构域的正确空间定向以影响具有所赋予功能作用的靶标。例如，如果所述功能结构域是转录激活子(例如，VP16、VP64或p65)，则将所述转录激活子放置在使其能够影响靶标转录的空间取向上。同样地，定位转录阻遏物以影响靶标的转录，并且将核酸酶(例如，Fok1)定位以切割或部分切割所述靶标。在一些实施方案中，所述功能结构域位于所述工程化的Cas效应蛋白的N末端。在一些实施方案中，所述功能结构域位于工程化的Cas效应蛋白的C末端。在一些实施方案中，所述工程化的Cas效应蛋白在N末端包含第一功能结构域，并在C末端包含第二功能结构域。在一些实施方案中，所述工程化Cas效应蛋白包含与一个或多个功能结构域融合的本文所述工程化的Cas核酸酶中任一种的催化失活突变体。
在一些实施方案中，所述工程化的Cas效应蛋白是转录激活子。在一些实施方案中，所述工程化的Cas效应蛋白包含与反式激活结构域融合的本文所述工程化的Cas核酸酶中任一种的酶失活变体。在一些实施方案中，所述反式激活域选自下组：VP64、p65、HSF1、VP16、MyoD1、HSF1、RTA、SET7/9及其组合。在一些实施方案中，所述反式激活结构域包含VP64、p65和HSF1。在一些实施方案中，所述工程化的Cas效应蛋白包含两个分割型Cas效应多肽，每个都与反式激活结构域融合。
在一些实施方案中，所述工程化的Cas效应蛋白是转录阻遏物。在一些实施方案中，所述工程化的Cas效应蛋白包含与转录阻遏结构域融合的本文所述工程化的Cas核酸酶中任一种的酶失活变体。在一些实施方案中，所述转录阻遏物结构域选自下组：Krüppel相关盒(KRAB)、EnR、NuE、NcoR、SID、SID4X及其组合。在一些实施方案中，所述工程化的Cas效应蛋白包含两个分割型Cas效应多肽，每个都与转录阻遏结构域融合。
在一些实施方案中，所述工程化的Cas效应蛋白是碱基编辑器，如胞嘧啶编辑器或腺苷编辑器。在一些实施方案中，所述工程化的Cas效应蛋白包含与核碱基编辑结构域融合的本文所述任何工程化的Cas核酸酶中任一种的酶失活变体，所述核碱基编辑结构域诸如为胞嘧啶碱基编辑(CBE)结构域或腺苷碱基编辑(ABE)域。在一些实施方案中，所述核碱基编辑结构域是DNA编辑结构域。在一些实施方案中，所述核碱基编辑结构域具有脱氨酶活性。在一些实施方案中，所述核碱基编辑结构域是胞嘧啶脱氨酶结构域。在一些实施方案中，所述核碱基编辑结构域是腺苷脱氨酶结构域。基于Cas核酸酶的示例性碱基编辑器描述于例如，WO2018/165629A1和WO2019/226953A1，其通过整体引用而并入本文。示例性CBE结构域包括但不限于：活化诱导的胞苷脱氨酶或AID (例如hAID)、载脂蛋白B mRNA编辑复合物或APOBEC(例如大鼠APOBEC1、hAPOBEC3 A/B/C/D/E/F/G)和PmCDA1。示例性ABE结构域包括但不限于：TadA、ABE8及其变体(参见例如Gaudelli et al., 2017, Nature 551: 464-471; and Richter et al., 2020, Nature Biotechnology 38: 883-891)。在一些实施方案中，所述功能性结构域是APOBEC1结构域，例如包含SEQ ID NO: 218氨基酸序列的大鼠APOBEC1结构域。在一些实施方案中，所述功能性结构域是TadA结构域，例如包含SEQ IDNO: 219氨基酸序列的大肠杆菌(E. coli) TadA结构域。在一些实施方案中，所述工程化的Cas效应蛋白还包含一个或多个核定位序列。
在一些实施方案中，所述工程化的Cas效应蛋白是主编辑器。基于Cas9的主编辑器描述于例如，A. Anzalone et al., Nature, 2019, 576 (7785): 149-157，其通过整体引用而并入本文。在一些实施方案中，所述工程化的Cas效应蛋白包含与逆转录酶结构域融合的本文所述工程化的Cas核酸酶中任一种的切口酶变体。在一些实施方案中，所述功能结构域是逆转录酶结构域。在一些实施方案中，所述逆转录酶结构域是M-MLV逆转录酶或其变体，例如具有D200N、T306K、W313F、T330P和L603W的一个或多个突变的M-MLV逆转录酶。在一些实施方案中，所述逆转录酶结构域包含SEQ ID NO: 220或221的氨基酸序列。在一些实施方案中，提供了包含所述主编辑器的工程化CRISPR/Cas系统。在一些实施方案中，所述工程化的CRISPR/Cas系统还包含第二Cas切口酶，例如基于与主编辑器的相同的工程化的Cas核酸酶。在一些实施方案中，所述工程化的CRISPR/Cas系统包含主编辑器指导RNA (pegRNA)，其包含引物结合位点和逆转录酶(RT)模板序列。
在一些实施方案中，本申请提供了具有与分割型Cas效应蛋白部分之一或两者缔合(即，结合或融合)的一个或多个(例如，1、2、3、4、5、6个或更多个)功能结构域的分割型Cas效应系统。所述功能结构域可以作为所第一和/或第二分割型Cas效应蛋白的一部分提供，作为该构建体内的融合物。所述功能结构域通常通过肽接头(诸如GS接头)与分割型Cas效应蛋白中其他部分融合(例如，分割型Cas效应蛋白部分)。这些功能结构域可用于基于催化失活Cas效应蛋白重新改换所述分割型Cas效应系统的功能。
在一些实施方案中，所述工程化的Cas效应蛋白包含一个或多个核定位序列(NLS)和/或一个或多个核输出序列(NES)。示例性的NLS序列包括例如PKKKRKVPG(SEQ ID NO:79)和ASPKKKRKV(SEQ ID NO: 80)。NLS和/或NES可以可操作地连接至所述工程化的Cas效应蛋白的N末端和/或C末端或所述工程化的Cas效应蛋白中的多肽链。
在一些实施方案中，所述工程化的Cas效应蛋白可以编码另外的组分，例如报告蛋白。在一些实施方案中，所述工程化的Cas效应蛋白包含荧光蛋白，例如GFP。这样的系统可以允许对基因组位点进行成像(例如，参见“Dynamic Imaging of Genomic Loci inLiving Human Cells by an Optimized CRISPR/Cas System” Chen B et al. Cell2013)。在一些实施方案中，所述工程化的Cas效应蛋白是可以用于对基因组位点成像的可诱导的分割型Cas效应系统。
工程化的CRISPR-Cas系统
还提供了工程化的CRISPR-Cas系统，其包括：(a)本文所述工程化的Cas效应蛋白中任一种(例如，工程化的Cas核酸酶、切口酶、分割型Cas、转录阻遏物、转录激活子、碱基编辑器或主编辑器)；以及(b)包含与靶序列互补的指导序列的指导RNA，或编码所述指导RNA的一种或多种核酸，其中所述工程化的Cas效应蛋白和所述指导RNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合于包含所述靶序列的靶核酸并诱导所述靶核酸的修饰。在一些实施方案中，所述工程化的CRISPR-Cas系统包含编码所述工程化的Cas效应蛋白和/或所述指导RNA的一种或多种核酸。在一些实施方案中，所述工程化的CRISPR-Cas系统包含前体指导RNA阵列，其可以例如通过所述工程化的Cas效应蛋白加工成多个crRNA。在一些实施方案中，所述工程化的CRISPR-Cas系统包含一种或多种编码所述工程化的Cas效应蛋白和/或所述指导RNA的载体。在一些实施方案中，所述工程化的CRISPR-Cas系统包含核糖核蛋白(RNP)复合物，其包含结合至所述指导RNA的所述工程化的Cas效应蛋白。
本申请的工程化的CRISPR-Cas系统可包含任何合适的指导RNA。指导RNA(gRNA)可以包含能够与目标靶核酸(诸如细胞中的目标基因组位点)中的靶序列杂交的指导序列。在一些实施方案中，所述gRNA包含含有所述指导序列的CRISPR RNA(crRNA)序列。在一些实施方案中，所述gRNA包含反式激活CRISPR RNA (tracrRNA)序列。在一些实施方案中，指导RNA是单指导RNA(sgRNA)。在一些实施方案中，所述sgRNA包含tracrRNA和crRNA。
在一些实施方案中，本文提供的CRISPR-Cas系统不需要tracrRNA序列(例如，CRISPR-Cas12i或CRISPR-Cas12a系统)。在一些实施方案中，所述指导RNA包含crRNA。通常，本文所述的crRNA包括直接重复序列和间隔区序列。在某些实施方案中，所述crRNA包括与指导序列或间隔区序列连接的直接重复序列、基本由或由它组成。在一些实施方案中，所述crRNA包括直接重复序列、间隔区序列和直接重复序列(DR-间隔区序列-DR)，其是前体crRNA (pre-crRNA)构型的典型特征。在一些实施方案中，所述crRNA包括截短的直接重复序列和间隔区序列，其是经加工的或成熟的crRNA的典型特征。在一些实施方案中，所述CRISPR-Cas效应蛋白与RNA指导序列形成复合物，并且所述间隔区序列将所述复合物引导至与靶核酸进行序列特异性结合，所述靶核酸与间隔区序列互补。
一些V型Cas酶，如Cas12c/d酶需要scoutRNA。参见Harrington et al., 2020,Mol. Cell., 79: 1-9。在一些实施方案中，所述指导RNA包含crRNA和scoutRNA。
在一些实施方案中，所述指导RNA是包含指导序列的crRNA。在一些实施方案中，所述工程化的CRISPR-Cas系统包含编码多个crRNA的前体指导RNA阵列。在一些实施方案中，所述Cas效应蛋白切割所述前体指导RNA阵列以产生多个crRNA。在一些实施方案中，所述工程化的CRISPR-Cas系统包含编码多个crRNA的前体指导RNA阵列，其中每个crRNA包含不同的指导序列。在一些实施方案中，由所述前体指导RNA阵列编码的crRNA与tracrRNA或scoutRNA缔合。
所述指导序列可以具有合适的长度。在一些实施方案中，所述指导序列在约18至约35个核苷酸之间，包括例如18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或35个核苷酸。所述指导序列可以与靶核酸的靶序列具有至少约70%、75%、80%、85%、90%、95%、98%、99%或100%的互补性。
构建体和载体
本文还提供了编码本文所述工程化的Cas效应蛋白中任一种的构建体、载体和表达系统。在一些实施方案中，所述构建体、载体或表达系统还包含一种或多种gRNA(例如，sgRNA)或crRNA阵列。
“载体”是包含分离的核酸并且可以用于将所述分离的核酸递送至细胞内部的物质组合物。许多载体是本领域已知的，包括但不限于：线性多核苷酸、与离子或两亲性化合物缔合的多核苷酸、质粒和病毒。通常，合适的载体包含在至少一种生物中起作用的复制起点、启动子序列、方便的限制性核酸内切酶位点和一种或多种选择性标记物。术语“载体”也应被解释为包括非质粒和非病毒化合物，其促进核酸转移到细胞中，诸如例如，聚赖氨酸化合物、脂质体等。
在一些实施方案中，所述载体是病毒载体。病毒载体的实例包括但不限于：腺病毒载体、腺相关病毒载体、慢病毒载体、逆转录病毒载体、牛痘载体、单纯疱疹病毒载体及其衍生物。在一些实施方案中，所述载体是噬菌体载体。病毒载体技术是本领域众所周知的，并且例如描述于Sambrook等人(2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York)，以及其他病毒学和分子生物学手册。
已经开发了许多基于病毒的系统，用于将基因转移到哺乳动物细胞中。例如，逆转录病毒为基因递送系统提供了便利的平台。可以使用本领域已知的技术将异源核酸插入载体，并包装在逆转录病毒颗粒中。然后可以分离重组病毒，并在体外或离体递送至所述工程化的哺乳动物细胞。许多逆转录病毒系统是本领域已知的。在一些实施方案中，使用腺病毒载体。许多腺病毒载体是本领域已知的。在一些实施方案中，使用慢病毒载体。在一些实施方案中，使用自灭活的慢病毒载体。
在某些实施方案中，所述载体是腺相关病毒(AAV)载体，例如AAV2、AAV8或AAV9，其可以按包含至少1×105个颗粒(也称为颗粒单位，pu)的单次给药施用腺病毒或腺相关病毒。在一些实施方案中，所述给药量是至少约1×106个颗粒，至少约1×107个颗粒，至少约1×108个颗粒，或至少约1×109个颗粒的腺相关病毒。递送方法和给药量描述于例如WO2016205764和美国专利No.8,454,972，其通过整体引用而并入本文。
在一些实施方案中，所述载体是重组腺相关病毒(rAAV)载体。例如，在一些实施方案中，可以使用经修饰的AAV载体递送。经修饰的AAV载体可以基于几种衣壳类型中的一种或多种，包括AAV1、AV2、AAV5、AAV6、AAV8、AAV8.2、AAV9、AAV rh10，经修饰的AAV载体(例如经修饰的AAV2，经修饰的AAV3，经修饰的AAV6)和假型AAV(例如AAV2/8，AAV2/5和AAV2/6)。可用于产生rAAV颗粒的示例性AAV载体和技术是本领域已知的(参见例如Aponte-Ubilluset al. (2018) Appl. Microbiol. Biotechnol. 102(3): 1045-54；Zhong et al.(2012) J. Genet. Syndr. Gene Ther. S1: 008；West et al. (1987) Virology 160:38-47 (1987)；Tratschin et al. (1985) Mol. Cell. Biol. 5: 3251-60；美国专利Nos.4,797,368和5,173,414；国际公开第WO2015/054653号和第WO93/24641号，其各自通过引用并入本文)。
用于递送Cas9和其他Cas蛋白的任何已知的AAV载体都可以用于递送本申请的工程化的Cas系统。
将载体引入哺乳动物细胞的方法是本领域已知的。可以通过物理、化学或生物学方法将载体转移到宿主细胞中。
用于将载体引入宿主细胞的物理方法包括：磷酸钙沉淀、脂质转染、粒子轰击、显微注射、电穿孔等。产生包含载体和/或外源核酸的细胞的方法是本领域众所周知的。参见，例如Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York。在一些实施方案中，通过电穿孔将所述载体引入所述细胞。
用于将异源核酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体已成为将基因插入哺乳动物例如人细胞的最广泛使用的方法。
用于将载体引入宿主细胞的化学方法包括胶体分散系统如大分子复合物、纳米胶囊、微球、珠子和基于脂质的系统，所述基于脂质的系统包括水包油乳液、胶束、混合胶束和脂质体。用作体外递送载体的示例性胶体系统是脂质体(例如，人工膜囊泡)。在一些实施方案中，所述工程化的CRISPR-Cas系统以RNP的形式在纳米颗粒中递送。
在一些实施方案中，编码所述CRISPR-Cas系统或其组分的载体或表达系统包含一种或多种可选择或可检测的标记物，该标记物提供了分离或有效选择含有和/或已被CRISPR-Cas系统(例如在早期和大规模)修饰的细胞的手段。
报告基因可用于鉴定潜在转染的细胞和评估调节序列的功能。通常，报告基因是在受体生物体或组织中不存在或不表达的基因，其编码的多肽的表达是通过某些易于检测的性质(例如酶活性)来证明。在将DNA引入受体细胞后的合适时间测定报告基因的表达。合适的报告基因可以包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌的碱性磷酸酶的基因或绿色荧光蛋白基因(例如Ui-Tei et al. FEBS Letters 479: 79-82 (2000))。
确认宿主细胞中异源核酸的存在的其他方法包括例如本领域技术人员众所周知的分子生物学测定，诸如Southern和Northern印迹、RT-PCR和PCR；生化测定，例如通过免疫学方法(如ELISA和Western印迹)检测特定肽的存在或不存在。
在一些实施方案中，编码所述工程化的Cas效应蛋白和/或所述指导RNA的核酸序 列与启动子可操作地连接。在一些实施方案中，所述启动子是相对于使用所述工程化的 CRISPR-Cas系统进行工程化改造的细胞的内源启动子。例如，可以使用本领域已知的任何 方法将编码所述工程化的Cas效应蛋白的核酸敲入工程化的哺乳动物细胞的基因组中位于 内源性启动子下游处。在一些实施方案中，所述内源启动子是丰富蛋白(如β-肌动蛋白)的 启动子。在一些实施方案中，所述内源启动子是可诱导的启动子，例如，可通过工程化的哺 乳动物细胞的内源激活信号来诱导。在一些实施方案中，其中所述工程化的哺乳动物细胞 是T细胞，所述启动子是T细胞活化依赖性启动子(诸如IL-2启动子、NFAT启动子或NF
Figure 6558DEST_PATH_IMAGE001
B启动 子)。
在一些实施方案中，所述启动子是相对于使用所述工程化的CRISPR-Cas系统进行工程化改造的细胞的异源启动子。已经探索了多种启动子以在哺乳动物细胞中表达基因，并且本领域中已知的任何启动子都可以用于本申请中。启动子可大致分为组成型启动子或受调节的启动子，例如诱导型启动子。
在一些实施方案中，编码所述工程化的Cas效应蛋白和/或所述指导RNA的核酸序列与组成型启动子可操作地连接。组成型启动子允许异源基因(也称为转基因)在宿主细胞中组成型表达。本文考虑的示例性组成型启动子包括但不限于：巨细胞病毒(CMV)启动子、人延伸因子-1α (hEF1α)、泛素C启动子(UbiC)、磷酸甘油激酶启动子(PGK)、猿猴病毒40早期启动子(SV40)以及鸡β-肌动蛋白启动子与CMV早期增强子(CAG)耦联。在一些实施方案中，所述启动子是CAG启动子，其包含巨细胞病毒(CMV)早期增强子元件、启动子、鸡β-肌动蛋白基因的第一外显子和第一内含子，以及兔β-珠蛋白基因的剪接受体。
在一些实施方案中，编码所述工程化的CRISPR-Cas蛋白和/或所述指导RNA的核酸序列可操作地连接至诱导型启动子。诱导型启动子属于受调控的启动子类型。所述诱导型启动子可以通过一种或多种条件如物理条件、微环境或宿主细胞的生理状态、诱导物(即，诱导剂)或其组合来诱导。在一些实施方案中，所述诱导条件选自下组：诱导剂、辐照(如电离辐射、光)、温度(如热)、氧化还原状态、肿瘤环境和待通过工程化的CRISPR-Cas系统进行工程化改造的细胞的活化状态。在一些实施方案中，所述启动子可被小分子诱导剂诸如化合物诱导。在一些实施方案中，所述小分子选自下组：强力霉素、四环素、醇、金属或类固醇。化学诱导的启动子已被最广泛地研究。这样的启动子包括其转录活性受小分子化学物质例如强力霉素、四环素、醇、类固醇、金属和其他化合物的存在或不存在调节的启动子。具有逆四环素受控反式激活因子(rtTA)和四环素响应元件启动子(TRE)的强力霉素诱导系统是目前最成熟的系统。WO9429442描述了通过四环素响应性启动子严格控制真核细胞中基因表达。WO9601313公开了四环素调节的转录调节剂。另外，诸如Tet-on系统之类的Tet技术已描述于例如TetSystems.com网站。在本申请中，任何已知的化学调节的启动子均可用于驱动编码所述工程化的CRISPR-Cas蛋白和/或所述指导RNA的表达。
在一些实施方案中，编码所述工程化的Cas效应蛋白(例如，enCas12i2)的核酸序列是经过密码子优化的。
在一些实施方案中，提供了表达构建体，其包含经密码子优化的序列，编码所述工程化的Cas效应蛋白的该序列连接到BPK2104-ccdB载体上。在一些实施方案中，所述表达构建体编码与所述工程化的Cas效应蛋白的C末端可操作连接的标签(例如10xHis标签)。
在一些实施方案中，每种工程化的分割型Cas构建体编码荧光蛋白诸如GFP或RFP。所述报告蛋白可以用于评估所述工程化的Cas蛋白的共定位和/或二聚化，例如使用显微镜。可以使用编码自切割肽诸如T2A、P2A、E2A或F2A肽的序列，将编码工程化的Cas效应蛋白的核酸序列与编码另外的组分的核酸序列融合。
在一些实施方案中，提供了用于哺乳动物细胞(例如人细胞)的表达构建体，所述构建体包含编码所述工程化的Cas效应蛋白的核酸序列。在一些实施方案中，所述表达构建体包含经密码子优化的序列，所述序列编码插入pCAG-2A-eGFP载体中的所述工程化的Cas效应蛋白，从而使Cas蛋白可操作地连接至eGFP。在一些实施方案中，提供第二载体用于在哺乳动物细胞(例如人细胞)中表达指导RNA(例如sgRNA，crRNA或前体crRNA阵列)。在一些实施方案中，编码所述指导RNA的序列在pUC19-U6-i2-cr RNA载体主链中表达。示例性的双载体表达系统如图1所示。
使用方法
本申请的一个方面提供了使用本文所述工程化的Cas效应蛋白或CRISPR-Cas系统中任一种在体外、离体或体内检测靶核酸或修饰核酸的方法，以及使用所述工程化的Cas效应蛋白或CRISPR-Cas系统进行处理或诊断的方法。还提供了本文所述工程化的Cas效应蛋白或CRISPR-Cas系统用于检测或修饰细胞中的核酸，以及用于处理或诊断受试者的疾病或病况的用途；以及包含所述工程化的Cas效应蛋白中任一种或所述工程化的CRISPR-Cas系统的一种或多种组分的组合物在制备用于检测或修饰细胞中的核酸以及用于处理或诊断受试者的疾病或病况的药物中的用途。
使用Cas12i的方法
本申请的一个方面提供了使用Cas12i (包括野生型或工程化的Cas12i效应蛋白)在哺乳动物细胞(例如人细胞)中切割靶核酸和进行基因组编辑的方法。
在一些实施方案中，本申请提供了修饰靶核酸中的靶序列的方法，其包括使所述靶核酸与工程化的CRISPR-Cas系统在约40℃至约67℃的温度下接触，其中所述工程化的CRISPR-Cas该系统包括：(a) Cas12i效应蛋白，其包含Cas12i核酸酶或其功能衍生物；(b)crRNA，其包含与所述靶序列互补的指导序列，其中所述Cas12i效应蛋白和所述crRNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合包含所述靶序列的靶核酸并诱导所述靶核酸的修饰。在一些实施方案中，所述Cas12i效应蛋白是Cas12i2核酸酶或其功能衍生物。在一些实施方案中，所述方法在升高的温度下进行，例如在约40、42、44、46、48、50、52、54、56、58、60、62、64、66或67oC中任一个温度下。在一些实施方案中，所述方法在约40oC至50oC，50oC至60oC，45oC至55oC，55oC至65oC，40oC至60oC，或50oC至67oC的温度下进行。在一些实施方案中，所述Cas12i效应蛋白具有非特异性单链RNA切割活性。
在一些实施方案中，使用本文所述的Cas12i效应蛋白(例如，工程化的Cas12i2蛋白)的方法在4oC至约40oC的温度下进行，如在约4-10、10-20、20-30、30-40、15-37、4-20或20-40 oC的温度下。
本申请还提供了工程化的crRNA，其改善了Cas12i核酸酶(例如，Cas12i1核酸酶)的基因编辑效力。在一些实施方案中，相比包含对应于所述Cas12i2核酸酶的内源性重复序列(例如SEQ ID NO: 171)的crRNA，所述工程化的crRNA使人细胞中的Cas12i2核酸酶的基因编辑活性增加至少约20%(例如，至少约30%、40%、50%、60%、70%、80%或更多)。
在一些实施方案中，提供了工程化的crRNA，其在包含至少四个U残基的重复序列中用非U残基替换一个或多个尿苷(U)残基。在一些实施方案中，提供了编码多种本文所述工程化的crRNA的工程化的前体指导RNA阵列。
在一些实施方案中，所述工程化的crRNA包含约17-25个(例如17、18、19、20、21、22、23、24或25个中任一个)核苷酸长的间隔区序列。在一些实施方案中，所述工程化的crRNA包含约20个核苷酸长的间隔区序列。
在一些实施方案中，所述工程化的crRNA包含含有SEQ ID NO: 173的核酸序列的重复序列。
在一些实施方案中，提供了工程化的CRISPR-Cas系统，包括：(a) Cas12i效应蛋白，其包含Cas12i1核酸酶或其功能衍生物；以及(b) crRNA，其在包含至少四个U残基和与靶序列互补的指导序列的重复序列中用非U残基替换一个或多个尿苷(U)残基；其中所述Cas12i效应蛋白和所述crRNA能够形成CRISPR复合物，所述CRISPR复合物与包含所述靶序列的靶核酸特异性结合并诱导所述靶核酸的修饰。在一些实施方案中，提供了使用所述工程化的CRISPR-Cas系统修饰靶核酸中的靶序列的方法。
本文还考虑了根据该IV部分“使用方法”中描述的方法中任一种，使用本文所述Cas12i (包括野生型或工程化的Cas12i效应蛋白)和/或所述工程化的crRNA修饰哺乳动物细胞中靶核酸的方法、处理方法、检测方法等。
修饰的方法
在一些实施方案中，本申请提供了修饰包含靶序列的靶核酸的方法，所述方法包括使所述靶核酸与本文所述工程化的CRISPR-Cas系统中任一种接触。在一些实施方案中，所述方法在体外进行。在一些实施方案中，所述靶核酸存在于细胞中。在一些实施方案中，所述细胞是细菌细胞、酵母细胞、哺乳动物细胞、植物细胞或动物细胞。在一些实施方案中，所述方法离体进行。在一些实施方案中，所述方法在体内进行。
在一些实施方案中，通过所述工程化的CRISPR-Cas系统切割所述靶核酸或改变所述靶核酸中的靶序列。在一些实施方案中，通过所述工程化的CRISPR-Cas系统改变所述靶核酸的表达。在一些实施方案中，所述靶核酸是基因组DNA。在一些实施方案中，所述靶序列与疾病或病况相关。在一些实施方案中，所述工程化的CRISPR-Cas系统包含编码多个crRNA的前体指导RNA阵列，其中每个crRNA包含不同的指导序列。在一些实施方案中，所述方法在约4oC至约67oC的温度下进行，如约12oC至67oC，4oC至25oC，25oC至约37oC，约37oC至约45oC，约45oC至约50oC，约50oC至约60oC，或约40oC至约67oC中任一种。在一些实施方案中，所述方法在低温(如约4oC至约12oC)下进行。在一些实施方案中，所述方法在高温(如约40oC至约67oC)下进行。
在一些实施方案中，本申请提供了处理与个体细胞中的靶核酸相关的疾病或病况的方法，其包括使用本文所述方法中任一种来修饰所述个体细胞中的所述靶核酸，从而处理所述疾病或病况。在一些实施方案中，所述疾病或病况选自下组：癌症、心血管疾病、遗传性疾病、自身免疫疾病、代谢性疾病、神经退行性疾病、眼病、细菌感染和病毒感染。
本文所述工程化的CRISPR-Cas系统可以用多种方式修饰细胞中的靶核酸，这取决于所述CRISPR-Cas系统中工程化的Cas效应蛋白的类型。在一些实施方案中，所述方法诱导靶核酸中的位点特异性切割。在一些实施方案中，所述方法在细胞如细菌细胞、植物细胞或动物细胞(例如哺乳动物细胞)中切割基因组DNA。在一些实施方案中，所述方法通过切割细胞中的基因组DNA来杀死细胞。在一些实施方案中，所述方法在细胞中切割病毒核酸。
在一些实施方案中，所述方法改变(如增加或减少)细胞中所述靶核酸的表达水平。在一些实施方案中，所述方法例如基于融合至反式激活结构域的无酶活性的Cas蛋白，使用工程化的Cas效应蛋白来提高细胞中所述靶核酸的表达水平。在一些实施方案中，所述方法例如基于融合至转录阻遏结构域的无酶活性的Cas蛋白，使用工程化的Cas效应蛋白来降低细胞中所述靶核酸的表达水平。在一些实施方案中，所述方法例如基于融合至表观遗传修饰结构域的无酶活性的Cas蛋白，使用工程化的Cas效应蛋白将表观遗传修饰引入细胞中的所述靶核酸。本文所述工程化的Cas系统可用于将其他修饰引入所述靶核酸，这取决于所述工程化的Cas效应蛋白所包含的功能结构域。
在一些实施方案中，所述方法改变细胞中所述靶核酸中的靶序列。在一些实施方案中，所述方法将突变引入细胞中的所述靶核酸。在一些实施方案中，所述方法使用一种或多种内源性DNA修复途径，如非同源末端连接(NHEJ)或同源定向重组(HDR)，在细胞中修复靶DNA中诱导的双链断裂，作为由CRISPR复合物进行序列特异性切割的结果。示例性的突变包括但不限于：插入、缺失、替换和移码。在一些实施方案中，所述方法在靶位点处插入供体DNA。在一些实施方案中，供体DNA的插入导致将选择性标记物或报告蛋白引入所述细胞。在一些实施方案中，供体DNA的插入导致基因的敲入。在一些实施方案中，供体DNA的插入导致敲除突变。在一些实施方案中，供体DNA的插入导致替换突变诸如单核苷酸替换。在一些实施方案中，所述方法诱导细胞的表型变化。
在一些实施方案中，所述工程化的CRISPR-Cas系统用于基因电路（geneticcircuit）的一部分，或用于将基因电路插入细胞的基因组DNA中。本文所述的诱导剂控制的工程化的分割型Cas效应蛋白尤其可用作基因电路的组分。基因电路可用于基因疗法。设计和使用基因电路的方法和技术是本领域已知的。可以进一步参考例如Brophy, JenniferAN, and Christopher A. Voigt. "Principles of genetic circuit design." Nature methods 11.5 (2014): 508。
本文所述工程化的CRISPR-Cas系统可用于修饰多种靶核酸。在一些实施方案中，所述靶核酸在细胞中。在一些实施方案中，所述靶核酸是基因组DNA。在一些实施方案中，所述靶核酸是染色体外DNA。在一些实施方案中，所述靶核酸对于细胞是外源的。在一些实施方案中，所述靶核酸是病毒核酸诸如病毒DNA。在一些实施方案中，所述靶核酸是细胞中的质粒。在一些实施方案中，所述靶核酸是经水平转移（horizontally transferred）的质粒。在一些实施方案中，所述靶核酸是RNA。
在一些实施方案中，所述靶核酸是经分离的核酸诸如经分离的DNA。在一些实施方案中，所述靶核酸存在于无细胞的环境中。在一些实施方案中，所述靶核酸是经分离的载体诸如质粒。在一些实施方案中，所述靶核酸是经分离的线性DNA片段。
本文描述的方法适用于任何合适的细胞类型。在一些实施方案中，所述细胞是细菌、酵母细胞、真菌细胞、藻类细胞、植物细胞或动物细胞(例如，哺乳动物细胞，如人细胞)。在一些实施方案中，所述细胞是自然来源的诸如由组织活检分离出的细胞。在一些实施方案中，所述细胞是从体外培养的细胞系分离的细胞。在一些实施方案中，所述细胞来自原代细胞系。在一些实施方案中，所述细胞来自永生化细胞系。在一些实施方案中，所述细胞是基因工程化的细胞。
在一些实施方案中，所述细胞是选自下组的生物的动物细胞：牛、绵羊、山羊、马、猪、鹿、鸡、鸭、鹅、兔和鱼。
在一些实施方案中，所述细胞是选自下组的生物的植物细胞：玉米、小麦、大麦、燕麦、水稻、大豆、油棕、红花、芝麻、烟草、亚麻、棉花、向日葵、珍珠小米、粟米、高粱、油菜、大麻、蔬菜作物、饲料作物、工业作物、木本作物和生物质作物。
在一些实施方案中，所述细胞是哺乳动物细胞。在一些实施方案中，所述细胞是人细胞。在一些实施方案中，所述人细胞是人胚胎肾293T (HEK293T或293T)细胞或HeLa细胞。在一些实施方案中，所述细胞是人胚肾(HEK293T)细胞。在一些实施方案中，所述哺乳动物细胞选自下组：免疫细胞、肝细胞、肿瘤细胞、干细胞、合子、肌肉细胞和皮肤细胞。
在一些实施方案中，所述细胞是选自下组的免疫细胞：细胞毒性T细胞、辅助T细 胞、天然杀伤(NK)T细胞、iNK-T细胞、NK-T样细胞、
Figure 923698DEST_PATH_IMAGE002
T细胞、肿瘤浸润性T细胞和树突状细胞 (DC)激活的T细胞。在一些实施方案中，所述方法产生经修饰的免疫细胞，诸如CAR-T细胞或 TCR-T细胞。
在一些实施方案中，所述细胞是胚胎干(ES)细胞、诱导性多能干(iPS)细胞、配子的祖细胞、配子、合子或胚胎中的细胞。
本文所述的方法可以用于在体内、离体或体外修饰靶细胞，并且可以用改变所述细胞的方式进行，以使得一旦被修饰，所述经修饰的细胞的后代或细胞系就保留经改变的表型。所述经修饰的细胞和后代可以是多细胞生物的一部分，诸如具有离体或体内应用(如基因组编辑和基因疗法)的植物或动物。
在一些实施方案中，所述方法离体进行。在一些实施方案中，将所述工程化的CRISPR-Cas系统引入细胞后，所述经修饰的细胞(例如，哺乳动物细胞)离体繁殖。在一些实施方案中，将所述经修饰的细胞培养以繁殖至少约1天、2天、3天、4天、5天、6天、7天、10天、12天或14天中任一个。在一些实施方案中，将所述经修饰的细胞培养不超过约1天、2天、3天、4天、5天、6天、7天、10天、12天或14天中任一个。在一些实施方案中，进一步评价或筛选所述经修饰的细胞以选择具有一种或多种所需表型或特性的细胞。
在一些实施方案中，所述靶序列是与疾病或病况相关的序列。示例性的疾病或病况包括但不限于：癌症、心血管疾病、遗传性疾病、自身免疫性疾病、代谢性疾病、神经退行性疾病、眼病、细菌感染和病毒感染。在一些实施方案中，所述疾病或病况是遗传疾病。在一些实施方案中，所述疾病或病况是单基因疾病或病况。在一些实施方案中，所述疾病或病况是多基因疾病或病况。
在一些实施方案中，与野生型序列相比，所述靶序列具有突变。在一些实施方案中，所述靶序列具有与疾病或病况相关的单核苷酸多态性(SNP)。
在一些实施方案中，插入所述靶核酸中的供体DNA编码选自下组的生物产物：报告蛋白、抗原特异性受体、治疗性蛋白、抗生素抗性蛋白、RNAi分子、细胞因子、激酶、抗原、抗原特异性受体、细胞因子受体和自杀多肽。在一些实施方案中，所述供体DNA编码治疗性蛋白。在一些实施方案中，所述供体DNA编码可用于基因疗法的治疗性蛋白。在一些实施方案中，所述供体DNA编码治疗性抗体。在一些实施方案中，所述供体DNA编码工程化的受体，诸如嵌合抗原受体(CAR)或工程化的TCR。在一些实施方案中，所述供体DNA编码治疗性RNA，诸如小RNA(例如，siRNA、shRNA或miRNA)或长的非编码RNA (lincRNA)。
本文所述的方法可以用于在两个或更多个(例如2、3、4、5、6、8、10个或更多)不同的靶位点处进行多重基因编辑或调节。在一些实施方案中，所述方法检测或修饰多个靶核酸或靶核酸序列。在一些实施方案中，所述方法包括使靶核酸与包含多个(例如2、3、4、5、6、8、10个或更多) crRNA序列的指导RNA接触，其中每个crRNA均包含不同的靶序列。
还提供了包含经修饰的靶核酸的工程化的细胞，所述细胞使用本文所述方法中任一种来产生。所述工程化的细胞可以用于细胞疗法。可以使用本文所述用于细胞疗法的方法将自体或同种异体细胞用于制备工程化的细胞。
本文所述的方法还可用于生成细胞(例如哺乳动物细胞)的等基因系以研究遗传变体。
还提供了包含本文所述工程化的细胞的工程化非人类动物。在一些实施方案中，所述工程化的非人类动物是经过基因组编辑的非人类动物。所述工程化的非人类动物可以用作疾病模型。
产生非人类基因组编辑或转基因动物的技术是本领域众所周知的，包括但不限于：原核显微注射、病毒感染、对胚胎干细胞和诱导性多能干(iPS)细胞的转化。可以使用的详细方法包括但不限于Sundberg和Ichiki描述的方法(2006, Genetically EngineeredMice Handbook, CRC Press)和Gibson描述的方法(2004, A Primer Of Genome Science2nd ed. Sunderland, Mass.: Sinauer)。
所述工程化的动物可以是任何合适的物种，包括但不限于：牛、马、羊、犬、鹿、猫科动物，山羊、猪、灵长类，以及了解较少的哺乳动物诸如大象、鹿、斑马或骆驼。
处理方法
进一步提供了使用根据本文所述的修饰细胞中的靶核酸的方法中的任一种的处理方法，以及使用根据本文所述的检测靶核酸的方法中任一种的诊断方法。
在一些实施方案中，本申请提供了处理与个体细胞中的靶核酸相关的疾病或病况的方法，其包括使所述靶核酸与本文所述工程化的CRISPR-Cas系统中任一种接触，其中所述指导RNA的指导序列与所述靶核酸的靶序列互补，其中所述工程化的Cas效应蛋白和所述指导RNA彼此缔合以结合至所述靶核酸以修饰所述靶核酸，从而使所述疾病或病况得到处理。在一些实施方案中，将突变(例如，敲除或敲入突变)引入所述靶核酸。在一些实施方案中，所述靶核酸的表达被增强。在一些实施方案中，所述靶核酸的表达被抑制。
在一些实施方案中，本申请提供了处理个体的疾病或病况的方法，其包括向所述个体施用有效量的本文所述工程化的CRISPR-Cas系统中任一种以及编码治疗剂的供体DNA，其中所述指导RNA的指导序列与所述个体的靶核酸的靶序列互补，其中所述工程化的Cas效应蛋白和所述指导RNA彼此结合以结合至所述靶核酸并将供体DNA插入所述靶序列中，从而使所述疾病或病况得到处理。
在一些实施方案中，本申请提供了处理个体的疾病或病况的方法，其包括向所述个体施用有效量的包含经修饰的靶核酸的工程化的细胞，其中所述工程化的细胞是通过使所述细胞与本文所述工程化的CRISPR-Cas系统中任一种接触而制备的，其中所述指导RNA的指导序列与所述靶核酸的靶序列互补，其中所述工程化的Cas效应蛋白和所述指导RNA相互缔合以结合至靶核酸以修饰所述靶核酸。在一些实施方案中，所述工程化的细胞是免疫细胞。
在一些实施方案中，所述个体是人。在一些实施方案中，所述个体是动物，例如模型动物诸如啮齿动物、宠物或农场动物。在一些实施方案中，所述个体是哺乳动物。
在一些实施方案中，所述疾病或病况选自下组：癌症、心血管疾病、遗传性疾病、自身免疫性疾病、代谢性疾病、神经退行性疾病、眼病、细菌感染和病毒感染。在一些实施方案中，所述靶核酸是PCSK9。在一些实施方案中，所述疾病或病况是心血管疾病。在一些实施方案中，所述疾病或病况是冠状动脉疾病。在一些实施方案中，所述方法降低个体的胆固醇水平。在一些实施方案中，所述方法处理个体的糖尿病。
检测方法
本申请还提供了使用具有改善的活性的所述工程化的Cas效应蛋白或CRISPR-Cas系统中任一种来检测靶核酸的方法。使用Cas效应蛋白作为检测试剂可利用以下发现：一旦通过检测到靶DNA而被激活，V型CRISPR/Cas蛋白(例如，Cas 12蛋白，如Cas12a、Cas12b、Cas12c、Cas12d、Cas12e(CasX)和Cas12i)可混杂地切割非靶向单链DNA(ssDNA)。例如使用Cas蛋白作为检测试剂的方法描述于US10253365和WO2020/056924，其通过整体引用而并入本文。
在一些实施方案中，一旦V型CRISPR/Cas效应蛋白(例如，Cas12蛋白，如Cas12a、Cas12b、Cas12c、Cas12d、Cas12e(CasX)或Cas12i)被指导RNA激活（其在样品中包含与指导RNA杂交的靶DNA(即样品中包含靶DNA)时发生），所述Cas效应蛋白成为一种可混杂地切割单链核酸(例如，非靶ssDNA或RNA，即指导RNA的指导序列不与之杂交的单链核酸)的核酸酶。因此，当样品中存在靶DNA(双链或单链)时(例如，在某些情况下超过阈值量)，结果是样品中单链核酸的切割，这可使用任何方便的检测方法进行检测(例如，使用加标签的单链检测核酸如DNA或RNA)。Cas12i可以切割ssDNA和ssRNA。
在一些实施方案中，提供了检测样品中的靶DNA(例如，双链或单链)的方法，其包括：(a)使所述样品与以下项接触：(i)本文所述工程化的V型CRISPR/Cas效应蛋白(例如，Cas12蛋白，如Cas12a、Cas12b、Cas12c、Cas12d、Cas12e (CasX)或Cas12i)中任一种；(ii)指导RNA，其包含与所述靶DNA杂交的指导序列；和(iii)检测核酸，其为单链的(即“单链检测核酸”)并且不与所述指导RNA的指导序列杂交；以及(b)测量通过所述工程化的V型CRISPR/Cas效应蛋白切割所述单链检测核酸而产生的可检测信号。在某些情况下，所述单链检测核酸包括发射荧光的染料对(例如，发射荧光的染料对是荧光共振能量转移(FRET)对、猝灭剂/荧光对)。在某些情况下，所述靶DNA是病毒DNA (例如，乳头状病毒、肝DNA病毒、疱疹病毒、腺病毒、痘病毒、细小病毒等)。在一些实施方案中，所述单链检测核酸是DNA。在一些实施方案中，所述单链检测核酸是RNA。在一些实施方案中，所述工程化的Cas效应蛋白是工程化的Cas12i核酸酶，如enCas12i2。
本公开的用于检测样品中的靶DNA(单链或双链)的方法，可以以高灵敏度检测靶DNA。在一些情况下，本公开的方法可以用于检测存在于包含多个DNA(包括所述靶DNA和多个非靶DNA)的样品中的靶DNA，其中所述靶DNA以每107个非靶DNA中的一个或更多个拷贝存在(例如，每106个非靶DNA中的一个或更多个拷贝，每105个非靶DNA中的一个或更多个拷贝，每104个非靶DNA中的一个或更多个拷贝，每103个非靶DNA中的一个或更多个拷贝，每102个非靶DNA中的一个或更多个拷贝，每50个非靶DNA中的一个或更多个拷贝，每20个非靶DNA中的一个或更多个拷贝，每10个非靶DNA中的一个或更多个拷贝或每5个非靶DNA中的一个或更多个拷贝)。
在一些实施方案中，与所述参比Cas核酸酶相比，本文所述工程化的Cas效应蛋白可以以更高的灵敏度检测靶DNA。在一些实施方案中，与所述参比Cas核酸酶相比，所述工程化的Cas效应蛋白可以以10%、15%、20%、25%、30%、40%、50%、60%、70%、80%、90%或更高的灵敏度检测靶DNA。
递送方法
在一些实施方案中，本文所述工程化的CRISPR-Cas系统或其组分、其核酸分子、或编码或提供其组分的核酸分子可通过多种递送系统诸如质粒或病毒递送至宿主细胞(例如，上面“构建体和载体”小节中描述的载体中的任一种)。在一些实施方案或方法中，所述工程化的CRISPR-Cas系统可以通过其他方法递送，如由所述工程化的Cas效应蛋白及其一个或多个同源RNA指导序列组成的核糖核蛋白复合物的核转染或电穿孔。
在一些实施方案中，通过纳米颗粒或外泌体来递送。
在一些实施方案中，成对的Cas切口酶复合物可以使用纳米颗粒或其他直接蛋白递送方法直接递送，使得包含两个成对的crRNA元件的复合物被共同递送。此外，蛋白可以通过病毒载体或直接地递送至细胞，然后直接递送包含针对双切口的两个成对间隔区的CRISPR阵列。在某些情况下，对于直接RNA递送，可以将RNA缀合于至少一个糖部分诸如N-乙酰基半乳糖胺(GalNAc)(特别是三触角GalNAc)。
试剂盒和制品
还提供了包含本文所述工程化的Cas效应蛋白或工程化的CRISPR-Cas系统中任一种的一种或多种组分的组合物、试剂盒、单位药剂和制品。
在一些实施方案中，提供了试剂盒，其包含：一个或多个AAV载体，其编码本文所述工程化Cas效应蛋白或工程化CRISPR-Cas系统中任一种。在一些实施方案中，所述试剂盒还包含一种或多种指导RNA。在一些实施方案中，所述试剂盒还包含供体DNA。在一些实施方案中，所述试剂盒还包含细胞诸如人细胞。
所述试剂盒可包含一种或多种额外组分，诸如容器、试剂、培养基、细胞因子、缓冲液、抗体等，以允许工程化的细胞的繁殖。所述试剂盒还可包含用于施用组合物的装置。
所述试剂盒还可包含使用本文所述工程化的CRISPR-Cas系统的说明书，诸如检测或修饰靶核酸的方法。在一些实施方案中，所述试剂盒包含用于处理或诊断疾病或病况的说明书。与所述试剂盒组分使用有关的说明书通常包括针对该刻意处理的给药量、给药时间表和给药途径的信息。所述容器可以是单位剂量、散装包装(例如多剂量包装)或亚单位剂量。例如，可以提供这样的试剂盒（其包含足够剂量的本文所公开的组合物）以在延长的时期内提供对个体的有效处理。试剂盒还可以包括多个单位剂量的组合物和使用说明书，其包装数量足以在药房(例如医院药房和复方药房)中存储和使用。
本发明的试剂盒处于合适的包装中。合适的包装包括但不限于：小瓶、瓶子、广口瓶、软包装(例如密封的聚酯薄膜或塑料袋)等。试剂盒可以任选地提供额外的组分，如缓冲液和解释性信息。因此，本申请还提供了一种制品，其包括小瓶(如密封的小瓶)、瓶子、广口瓶、柔性包装等。
所述制品可以包括容器以及在容器上或与容器粘合的标签或包装插页。合适的容器包括例如瓶子、小瓶、注射器等。所述容器可以由多种材料(诸如玻璃或塑料)形成。通常，所述容器容纳有效处理本文所述的疾病或病症的组合物，并且可以具有无菌进入口(例如，容器可以是静脉注射溶液袋或具有可通过皮下注射针刺穿的塞子的小瓶)。所述标签或包装插页表明该组合物用于处理个体中的特定病况。所述标签或包装插页将进一步包括用于将组合物向个体施用的说明书。
包装插页是指通常包含在治疗产品的商业包装中的说明，所述说明包含关于使用此类治疗产品的适应症、用法、给药量、施用、禁忌症和/或警告的信息。
此外，所述制品还可包括第二容器，其包含药学上可接受的缓冲液，如抑菌注射用水(BWFI)、磷酸盐缓冲盐水、林格氏溶液和葡萄糖溶液。从商业和用户的角度来看，它还可以包括其他材料，包括其他缓冲液、稀释剂、过滤器、针头和注射器。
实施例
以下实施例旨在仅作为本发明的示例，因此不应视为以任何方式限制本发明。通过举例而非限制的方式提供以下实施例和详细描述。
实施例1：工程化改造具有改善的效率的酶的管线
本实施例提供了设计具有增强的构象转化动力学的Cas酶的策略，其导致Cas内切核酸酶具有更好的催化效率。本文提供的示例性方法允许工程化改造具有改善的活性(如靶标结合、双链切割活性、切口酶活性和/或基因编辑活性)的Cas蛋白。
我们认为，在人体细胞中Cas构象转变的条件可能不是最佳的，而增强的构象转变动力学可能会导致Cas酶具有更好的催化效率。为了设计具有增强的构象转变动力学的Cas蛋白，我们设计了一种提高Cas酶中柔性区柔性的策略。图1显示了用于设计SaCas9变体的管线，作为该设计工作流程的示例。
增强型Cas酶设计管线（pipeline）
首先，我们使用DynaMine分子动力学预测器确定了Cas酶的柔性区(Cilia et al. TheDynaMine webserver: predicting protein dynamics from sequence. Nucleic AcidsRes. 2014 Jul 1; 42(Web Server issue): W264–W270)。从分子动力学的预测，我们获得了Cas蛋白的S2序参数得分谱。S2序参数的范围可以是0-1，其中1对应于刚性键向量，0对应于完全的柔性。
基于从DynaMine获得的S2序参数得分谱，相比在任一侧(氨基酸峰之前或之后) 5个相邻氨基酸的得分，我们将峰氨基酸(峰aa，氨基酸峰)定义为得分最低的氨基酸。基于此定义，我们确定了峰氨基酸。我们选择在任一侧(峰aa之前或之后)包含一个峰氨基酸及其相邻2个氨基酸的区域作为候选柔性区用于工程化改造。
接下来，我们在所述候选柔性区中工程化改造了具有增强的柔性的Cas变体。在本实施例中，我们使用以下策略来工程化改造具有增强柔性的候选柔性区：
(a)甘氨酸替代
如果所述候选柔性区中存在高度疏水的氨基酸(aa；L、I、V、C、Y、F、W)，则所述疏水残基被甘氨酸(G)替换。
(b)甘氨酸插入
在所述候选柔性区内，我们在柔性区的最柔性aa前面插入了两个G。根据以下优先级顺序选择最柔性的aa：G>S>N>D>H>M>T>E>Q>K>R>A>P。
如果在所述柔性区中有多个均等的最柔性aa，我们将最柔性aa定义为：更接近所述峰aa的氨基酸(aa)。
如果所述区域中有多个均等的最柔性aa，并且两个最柔性aa与峰aa的距离相同，则我们将最柔性aa定义为：相邻aa有优先度的aa。
如在以下“方法”部分中所述进行克隆和纯化得到Cas变体，并在人细胞中测量所述变体的切割效率。
方法
质粒构建
BhCas12bv4、Cas12i2和GeoCas9的编码序列经过密码子优化(人类)并合成。通过基于PCR的定点诱变产生Cas蛋白的变体。为了原核表达，使用T4连接酶将Cas12i2及其变体的编码序列连接到BPK2104-ccdB表达载体中，用XmaI和SpeI消化该载体。该融合构建体在蛋白的C末端包含融合的10xHis标签。Cas效应蛋白通过pCAG-2A-eGFP载体在人293T细胞中表达。将编码Cas蛋白的DNA插入XmaI和NheI之间。通过将含有靶序列的退火寡核苷酸连接到BasI消化的pUC19-U6-i2-crRNA骨架中，构建了在293T中表达BhCas12 bv4、GeoCas9和Cas12i2的sgRNA或crRNA的载体。
蛋白纯化
将原核表达质粒转化到大肠杆菌(E. coli) BL21菌株(λDE3)中，并将转化体接种在含氯霉素(CmR)的固体LB上。拣选3-5个克隆在含CmR的15ml液体LB中于37℃下培养过夜。然后将2ml培养物转移到300ml液体LB中进行培养，直到OD600达到1.2，然后在16°C下用IPTG诱导16 h。细胞沉淀用裂解缓冲液重悬，然后进行超声处理。离心后保留上清液，以备将来纯化。通过使用Ni柱的一步纯化法来获得目标蛋白。将目标蛋白用0.22μm过滤器灭菌，然后以等分试样存储。通过以BSA为标准的Bradford蛋白测定法确定浓度。
体外RNA转录
使用HISRIBETM T7快速高产RNA合成试剂盒(NEB)将含有T7启动子的PCR扩增的dsDNA用作体外转录模板，以产生crRNA。使用Oligo Clean＆Concentrator试剂盒(ZYMOTMResearch)纯化转录的crRNA，并在NANODROPTM 2000 (Thermo Fisher Scientific)上进行定量。
细胞培养、转染以及荧光激活细胞分选法(FACS)
将HEK293T细胞在含1%青霉素-链霉素(Gibco)和10%胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24-细胞培养皿(Corning)中16小时，直到融合度达到70%。通过使用Lipofectamine 3000 (Invitrogen)，将600ng编码Cas蛋白的质粒和3000ng编码crRNA的质粒转染到每个24-细胞培养皿中。转染68h后，用胰蛋白酶-EDTA (0.05%) (Gibco)消化荧光激活细胞分选(FACS) 的HEK293T细胞。使用具有GFP信号的MoFlo XDP (Beckman Coulter)进行细胞分选。
核酸内切酶I (T7EI)测定和靶向深度测序分析以进行基因组修饰
将FACS分选的GFP-阳性293FT细胞用缓冲液L裂解，并在55℃下孵育3小时，然后在95℃下孵育10分钟。使用相应的引物对在不同基因组位点中包含靶位点的dsDNA片段进行PCR扩增。对于T7E1测定，使用200~400ng PCR产物，添加ddH2O至最终体积为10μL。然后将混合物进行重退火程序以形成异源双链dsDNA。然后，在37°C下用1/10体积的NEBUFFERTM 2.1和0.2μL T7EI (NEB)处理混合物50分钟。通过~3%琼脂糖凝胶电泳分析消化的产物。基于先前的方法计算插入缺失。将通过T7E1测定鉴定出的含有突变的PCR产物克隆到TA克隆载体中，然后将其转化到感受态大肠杆菌中。过夜培养后，随机拣选菌落并送去做Sanger测序。对于靶向的深度测序，直接使用细胞裂解液作为模板，通过条形码(barcoded) PCR直接扩增靶位点。纯化PCR产物并归集到几个文库中以进行高通量测序。通过计算包含插入或缺失的读取(reads)的比率，采用CRISPResso2分析插入缺失(%)。数量少于完整读取的0.05%的读取被废弃。
实施例2：工程化的BhCas12b的设计和表征
本实施例描述了使用实施例1中所述的设计管线来设计和表征在人细胞中具有改善的基因编辑活性的外村尚芽孢杆菌(Bacillus hisashii) Cas12bv4 (BhCas12bv4)的工程化的变体。
如上文实施例1中所述，使用DynaMine以计算机方式确定了Cas12bv4的柔性区。如以上实施例1中所述，鉴定用于工程化改造的峰aa和候选柔性区。图2显示了BhCas12bv4的柔性(S2得分)谱，所选峰aa用圆圈表示。下表1显示了柔性区序列(SEQ ID NO: 81)和工程化的变体的Y＞G替换(SEQ ID NO: 82)。氨基酸位置基于SEQ ID NO: 1。BhCas12bv4没有已解析的晶体结构，但是高度同源的BthCas12b (>98%同源性)具有可用的晶体结构，因此根据BthCas122b的结构和同源性确定了如表1所示的BhCas12bv4的接头。
表1：enBhCas12b4的候选柔性区和经修饰的序列。
Figure 765752DEST_PATH_IMAGE003
按照实施例1中所述将所得的BhCas12bv4变体(enBhCas12bv4 1.1，如SEQ ID NO: 2所示)进行克隆和纯化。然后，我们测试了enBhCas12bv4 1.1在人293T细胞中对于11个基因组位点的编辑效率，与野生型BhCas12bv4的效率相比，发现所述工程化的变体的编辑效率得到了显著提高(图3)。在野生型BhCas12bv4的编辑效率较低的基因组位点(诸如RNF2-5、CCR5-8和CCR5-1)，观察到编辑效率的最显著提高(图3)。
实施例3：工程化的Cas12i2的设计和表征
本实施例描述了使用实施例1所述的设计管线来设计和表征在人细胞中具有改善的基因编辑活性的Cas12i2工程化的变体。
Cas12i2没有已知的3D结构，也没有已解析其结构的等同物。因此，我们寻求测试我们的不需要解析结构的方法是否可用于工程化改造具有改善的活性(如靶标结合、双链切割活性、切口酶活性和/或基因编辑活性)的Cas12i2变体。
如上文实施例1中所述，使用DynaMine以计算机方式确定了Cas12i2的柔性区。Cas12i2的柔性(S2得分)谱如图4所示。基于S2得分谱，我们选择S2得分小于0.71的峰作为柔性区。尽管没有已解析的Cas12i2或紧密相关等同物的结构信息，但我们发现我们选择的柔性区与通过计算二级结构预测确定的Cas12i2接头区相关，如图5所示(Buchan DWA,Jones DT (2019). The PSIPRED Protein Analysis Workbench: 20 years on. NucleicAcids Research)。
以下表2中显示了工程化的柔性变体区域的选择的柔性区氨基酸序列和经修饰的氨基酸序列。氨基酸位置基于SEQ ID NO: 8。
表2：工程化的Cas12i2的选择的候选柔性区和经修饰的序列。
Figure 160961DEST_PATH_IMAGE004
基因组编辑的测定
为了分析所述工程化的Cas12i2变异体在人细胞中的活性，在人基因组中选择了四个靶位点(CCR5-3、CCR5-2、RNF2-7和CCR5-8)。在每个位点确定了与野生型Cas12i2相比，Cas12i2变体的编辑效率(插入缺失%)。在测试的变体中，Cas12i2-2.2 (SEQ ID NO: 14)和Cas12i2-6.1 (SEQ ID NO: 18)显著提高了基因编辑效率。值得注意的是，变体6.1显著提高了在所有四个靶位点的编辑效率(图6)。
基于提高的Cas12i2变体2.2和6.1的编辑效率，然后我们测试了通过结合变体2.2和6.1突变是否可以进一步改善在5个靶位点(CCR5-3、CCR5-2、AAVS1-1、CCR5-15、RNF2-7)的编辑效率。组合变体2.2+6.1进一步提高了Cas12i2的编辑效率(图7)。在本文中，我们将组合变体Cas12i2-2.2+6.1称为“enCas12i2” (SEQ ID NO: 20)。
接下来，我们测试了与SpCas9和BhCas12b-v4相比，enCas12i2在人细胞中的总体基因组编辑效率。在46个位点对于enCas12i2， 在18个位点对于Sp Cas9，以及在23个位点对于BhCas12bv4分析了编辑效率(插入缺失%)(图8A)。我们证明了enCas12i2可以有效识别原间隔区相邻基序(PAM)位点NTTA、NTTC、NTTG和NTTT，以及ATTN、CTTN、GTTN和TTTN (图8B)。最后，我们证明了enCas12i2保留了加工前体crRNA的能力(图9)。前体crRNA形式的基因编辑效率等同于单个crRNA形式的基因编辑效率。
体外切割的测定
我们还比较了enCas12i2与野生型Cas12i2的体外质粒切割活性。体外切割测定的详细描述可参见实施例6。简而言之，将靶质粒与由Cas12i2或enCas12i2加crRNA形成的不同浓度的RNP一起孵育。反应在37℃下进行10分钟。如图10所示，相比野生型Cas12i2蛋白，纯化的enCas12i2蛋白在37℃下显示提高的dsDNA切割活性。
核酸检测的测定
最后，我们在核酸检测的测定中使用了野生型和工程化的Cas12i2。简而言之，将60 nMCas12i2或其工程化的变体与带有1X NEBuffer 2 的108 nM crRNA、40 nM激活子、RNA酶抑制剂、200 nM合成FQ-ssDNA-5T检测剂(5'-6-FAM-TTTTT-BHQ1-3')混合于单个20μl反应系统中。反应在Applied Biosystems 7500实时PCR系统(Thermo Fisher)中于37℃进行15个循环，每分钟测量FAM通道。导出ΔRn值，并通过SigmaPlot软件进行分析。反式XBP激活子的序列显示于SEQ ID NO: 230，而XBP1靶位点显示于SEQ ID NO: 231。
如图11A所示，野生型Cas12i2和工程化的Cas12i2变体(2.2、6.1和2.2+6.1)两者 均能够检测含有XBP靶位点的双链DNA (即，反式XBP激活子)。
Figure 975334DEST_PATH_IMAGE005
Rn值对应于检测剂核酸切 割的水平。所述工程化的Cas12i2变体显示出具有比野生型Cas12i2更高的检测活性。
此外，在核酸检测实验中，我们使用含有RNA核苷酸的检测核酸，发现野生型Cas12i2可以切割含有rU (5'-6-FAM-UUUUUU-BHQ1-3')的基于RNA的荧光报告分子(图11B)。
总之，这些结果表明，可以通过我们的工程化改造具有改善活性的Cas蛋白的方法对Cas12i2进行有效的修饰。所述方法不依赖于Cas12i2的三维结构信息。
实施例4：工程化GeoCas9的设计和表征
本实施例描述了使用实施例1所述的设计管线来设计和表征在人细胞中具有改善的基因编辑活性的GeoCas9工程化的变体。
为了证明我们的方法广泛适用于Cas12蛋白以外的Cas蛋白，我们接下来生成和测试了工程化的GeoCas9的变体。
如上文实施例1中所述，使用DynaMine以计算机方式确定了GeoCas9的柔性区。GeoCas9的柔性(S2得分)谱如图12所示。基于S2得分谱，我们从18个氨基酸峰中选择了柔性区，其中S2得分低于0.71。我们选择的柔性区与通过计算二级结构预测确定的GeoCas9接头区相缔合，如图13所示(Buchan DWA, Jones DT (2019). The PSIPRED Protein AnalysisWorkbench: 20 years on. Nucleic Acids Research)。
工程化的柔性变体区域的所选择的柔性区氨基酸序列和经修饰的氨基酸序列如表3所示。所述氨基酸位置基于SEQ ID NO: 25。
表3：工程化的GeoCas9的所选择的候选柔性区和经修饰的序列。
Figure 610845DEST_PATH_IMAGE006
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Figure 76779DEST_PATH_IMAGE008
靶向深度测序的测定
为了筛选工程化的GeoCas9变体以改善基因编辑活性，我们进行了靶向深度测序，以确定由GeoCas9产生的插入缺失的效率。首先通过FACS分选GFP-阳性的293T细胞来分离表达工程化的GeoCas9变体的细胞。将所述细胞在缓冲液L中裂解，并在55℃下孵育3小时，然后在95℃下孵育10分钟。直接使用细胞裂解液作为模板，通过条形码(barcoded) PCR直接扩增包含CD34-2的靶位点。纯化PCR产物并归集到几个文库中以进行高通量测序。通过计算包含插入或缺失的读取(reads)的比率，采用CRISPResso2分析插入缺失(%)。数量少于完整读取的0.05%的读取被废弃。靶向测序结果见图14。箭头指示了其编辑效率得到了显著提高的工程化的GeoCas9。提高效率的突变体有：2.1 3.1 8.1 9.1 12.1。
方法
将HEK293T细胞在含有1%青霉素-链霉素(Gibco)和10%胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24-孔板(Corning)中16小时，直到融合度达到70%。通过使用Lipofectamine 3000 (Invitrogen)将600ng编码Cas蛋白的质粒和3000ng编码crRNA的质粒转染到每个24-孔板中。转染68小时后，用胰蛋白酶-EDTA (0.05%) (Gibco)消化荧光激活细胞分选(FACS) HEK293T细胞。使用带有GFP信号的MoFlo XDP (Beckman Coulter)进行细胞分选。
将FACS分选的GFP-阳性293FT细胞用缓冲液L裂解，并在55℃下孵育3小时，然后在95℃下孵育10分钟。使用相应的引物对在不同基因组位点中包含靶位点的dsDNA片段进行PCR扩增(下表4)。对于T7E1测定，使用200~400ng PCR产物，添加ddH2O至最终体积为10μL。然后将混合物进行重退火程序以形成异源双链dsDNA。然后，在37°C下用1/10体积的NEBUFFERTM 2.1和0.2μL T7EI(NEB)处理混合物50分钟。通过~3%琼脂糖凝胶电泳分析经消化的产物。根据先前的方法计算插入缺失(Cong等，2013)。将通过T7E1测定鉴定出的含有突变的PCR产物克隆到TA克隆载体中，其随后被转化到感受态大肠杆菌中。过夜培养后，随机拣选菌落并送去做Sanger测序。
表4：使用的引物。
Figure 807975DEST_PATH_IMAGE009
实施例5：工程化的SaCas9的设计和表征
本实施例描述了使用实施例1中所述的设计管线来设计和表征在人细胞中具有改善的基因编辑活性的SaCas9工程化的变体。
如上文实施例1中所述，使用DynaMine以计算机方式确定了SaCas9的柔性区。SaCas9的柔性(S2得分)谱如图1所示。基于S2得分谱，我们从13个氨基酸峰中选择了柔性区，其中S2得分低于0.71 (图1中圈出的峰)。
所选择的柔性区和SaCas9的相应结构域的位置如图15所示。工程化的柔性变体区域的所选择的柔性区的氨基酸序列和经修饰的氨基酸序列如表5所示。氨基酸位置基于SEQID NO: 53。
表5：工程化的SaCas9的选择的柔性区氨基酸序列和经修饰的氨基酸序列。
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Figure 555668DEST_PATH_IMAGE011
Figure 292680DEST_PATH_IMAGE012
为了筛选工程化的SaCas9变体以改善基因编辑活性，我们根据上述针对GeoCas9的相同方法进行了T7酶切割测定(图16)。工程化的SaSa9变体1.1(SEQ ID NO: 54)、3.1(SEQ IDNO: 58)和3.2(SEQ ID NO: 59)(由图16中的方框标示)显示出显著改善的基因编辑效率。
实施例6：哺乳动物细胞中Cas12i的基因组编辑
本实例描述了野生型Cas12i1和Cas12i2的表征。
在这里，我们显示Cas12i1和Cas12i2可以在体外切割dsDNA。Cas12i1和Cas12i2在体外生成经切割的dsDNA产物，其通过凝胶电泳解析(图17A)。体外切割测定中使用的cRNA示于图17B。
我们测试了Cas12i1和Cas12i2是否能够在人细胞中进行基因组编辑。我们设计了多个crRNA分别靶向AAVs1 (2个crRNA)、CCR5 (7个crRNA)、CD34 (2个crRNA)和RNF2 (10个crRNA)，并测试了Cas12i1和Cas12i2使用每个crRNA生成插入缺失的能力。我们发现，Cas12i2能够在更广泛的靶标中产生插入缺失，这表明它是用于工程化改造的有前景的候选者(图18)。
基于Cas12i1的野生型crRNA序列，我们设计了三种突变的crRNA序列：
Figure 816065DEST_PATH_IMAGE013
我们发现，使用带有t3c点突变的crRNA，Cas12i1能够生成插入缺失，其效率明显高于使用野生型crRNA序列(图19)。
基于我们发现的Cas12i2能够在广泛的靶标中生成插入缺失，我们接下来试图进一步表征Cas12i2。值得注意的是，我们发现Cas12i2在从12°C-67°C的较宽温度范围内具有酶活性(即能够切割dsDNA)(图20)。这些数据表明，Cas12i2有潜力用于多种生活在不同温度条件下或具有不同体温的生物。
另外，我们发现野生型Cas12i2能够使用crRNA阵列在人细胞中编辑多个基因组靶位点(图21)。
我们接下来确定了野生型Cas12i2的种子序列。CRISPR-Cas核糖核蛋白效应子的靶标结合是通过Cas蛋白部分识别双链PAM基序开始的，然后通过CRISPR RNA部分的指导部使靶标去稳、局部解链和询问(interrogation)。后者过程取决于种子序列，即必须与CRISPR RNA指导序列严格互补的靶部。种子外部的靶标与CRISPR RNA指导序列之间的错配对靶标结合的影响很小，因此导致CRISPR-Cas效应蛋白的脱靶活性。在此我们定义该种子序列。
为了定义Cas12i2的种子序列，我们测试了Cas12i2生成在crRNA的碱基1-19之一处带有单碱基错配的AAVs1-1和RNF2-1 crRNA插入缺失的能力。我们发现Cas12i2的种子序列在1-10bp之内(图22)。
最后，我们确定了Cas12i2的最佳间隔区长度。我们使用间隔区长度从17 bp到25bp的片段测试了Cas12i2用两个不同的crRNA (CCR5-1和CCR5-2)生成插入缺失的能力。我们发现，Cas12i2基因编辑的最佳间隔区长度为20 bp(图23)。
方法
Cas12i表达
对于原核表达，使用T4连接酶将Cas12i2及其变体的编码序列连接到BPK2104-ccdB表达载体中，该载体用XmaI和SpeI消化。
体外切割测定
通过PCR创建用于体外切割测定的模板，并通过DNA Clean＆Concentrator试剂盒(ZYMOTM Research)进行纯化。在单次体外切割测定中，使用100nM模板，以及1mM Cas12i蛋白和2mM crRNA。该反应在1x NEBUFFERTM 3.1 (对于Cas12i1)和1x NEBUFFERTM 2 (对于Cas12i2)中于37℃下进行60分钟。为了确定Cas12i2的热稳定性，将酶切混合物在其酶切缓冲液中的大范围温度(4℃-67℃)下孵育1小时。为了进行质粒切割测定，将质粒与由Cas12i2或enCas12i2加crRNA形成的不同浓度RNP孵育。在1x NEBUFFERTM 2中于37℃反应10分钟。在37℃与蛋白酶(Takara)孵育20分钟后，加入RNA酶混合物(Thermo FisherScientific)终止反应，以在37℃消化crRNA持续20分钟。通过琼脂糖凝胶电泳和溴化乙锭染色来解析所述反应。
序列表
<110> 中国科学院动物研究所
北京干细胞与再生医学研究院
<120> 工程化的Cas效应蛋白及其使用方法
<130> PD01215
<141> 2020-12-07
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85 90 95
Glu Leu Tyr Glu Glu Leu Val Pro Ser Ser Val Glu Lys Lys Gly Glu
100 105 110
Ala Asn Gln Leu Ser Asn Lys Phe Leu Tyr Pro Leu Val Asp Pro Asn
115 120 125
Ser Gln Ser Gly Lys Gly Thr Ala Ser Ser Gly Arg Lys Pro Arg Trp
130 135 140
Tyr Asn Leu Lys Ile Ala Gly Asp Pro Ser Trp Glu Glu Glu Lys Lys
145 150 155 160
Lys Trp Glu Glu Asp Lys Lys Lys Asp Pro Leu Ala Lys Ile Leu Gly
165 170 175
Lys Leu Ala Glu Tyr Gly Leu Ile Pro Leu Phe Ile Pro Tyr Thr Asp
180 185 190
Ser Asn Glu Pro Ile Val Lys Glu Ile Lys Trp Met Glu Lys Ser Arg
195 200 205
Asn Gln Ser Val Arg Arg Leu Asp Lys Asp Met Phe Ile Gln Ala Leu
210 215 220
Glu Arg Phe Leu Ser Trp Glu Ser Trp Asn Leu Lys Val Lys Glu Glu
225 230 235 240
Tyr Glu Lys Val Glu Lys Glu Tyr Lys Thr Leu Glu Glu Arg Ile Lys
245 250 255
Glu Asp Ile Gln Ala Leu Lys Ala Leu Glu Gln Tyr Glu Lys Glu Arg
260 265 270
Gln Glu Gln Leu Leu Arg Asp Thr Leu Asn Thr Asn Glu Tyr Arg Leu
275 280 285
Ser Lys Arg Gly Leu Arg Gly Trp Arg Glu Ile Ile Gln Lys Trp Leu
290 295 300
Lys Met Asp Glu Asn Glu Pro Ser Glu Lys Tyr Leu Glu Val Phe Lys
305 310 315 320
Asp Tyr Gln Arg Lys His Pro Arg Glu Ala Gly Asp Tyr Ser Val Tyr
325 330 335
Glu Phe Leu Ser Lys Lys Glu Asn His Phe Ile Trp Arg Asn His Pro
340 345 350
Glu Tyr Pro Tyr Leu Tyr Ala Thr Phe Cys Glu Ile Asp Lys Lys Lys
355 360 365
Lys Asp Ala Lys Gln Gln Ala Thr Phe Thr Leu Ala Asp Pro Ile Asn
370 375 380
His Pro Leu Trp Val Arg Phe Glu Glu Arg Ser Gly Ser Asn Leu Asn
385 390 395 400
Lys Tyr Arg Ile Leu Thr Glu Gln Leu His Thr Glu Lys Leu Lys Lys
405 410 415
Lys Leu Thr Val Gln Leu Asp Arg Leu Ile Tyr Pro Thr Glu Ser Gly
420 425 430
Gly Trp Glu Glu Lys Gly Lys Val Asp Ile Val Leu Leu Pro Ser Arg
435 440 445
Gln Phe Tyr Asn Gln Ile Phe Leu Asp Ile Glu Glu Lys Gly Lys His
450 455 460
Ala Phe Thr Tyr Lys Asp Glu Ser Ile Lys Phe Pro Leu Lys Gly Thr
465 470 475 480
Leu Gly Gly Ala Arg Val Gln Phe Asp Arg Asp His Leu Arg Arg Tyr
485 490 495
Pro His Lys Val Glu Ser Gly Asn Val Gly Arg Ile Tyr Phe Asn Met
500 505 510
Thr Val Asn Ile Glu Pro Thr Glu Ser Pro Val Ser Lys Ser Leu Lys
515 520 525
Ile His Arg Asp Asp Phe Pro Lys Val Val Asn Phe Lys Pro Lys Glu
530 535 540
Leu Thr Glu Trp Ile Lys Asp Ser Lys Gly Lys Lys Leu Lys Ser Gly
545 550 555 560
Ile Glu Ser Leu Glu Ile Gly Leu Arg Val Met Ser Ile Asp Leu Gly
565 570 575
Gln Arg Gln Ala Ala Ala Ala Ser Ile Phe Glu Val Val Asp Gln Lys
580 585 590
Pro Asp Ile Glu Gly Lys Leu Phe Phe Pro Ile Lys Gly Thr Glu Leu
595 600 605
Tyr Ala Val His Arg Ala Ser Phe Asn Ile Lys Leu Pro Gly Glu Thr
610 615 620
Leu Val Lys Ser Arg Glu Val Leu Arg Lys Ala Arg Glu Asp Asn Leu
625 630 635 640
Lys Leu Met Asn Gln Lys Leu Asn Phe Leu Arg Asn Val Leu His Phe
645 650 655
Gln Gln Phe Glu Asp Ile Thr Glu Arg Glu Lys Arg Val Thr Lys Trp
660 665 670
Ile Ser Arg Gln Glu Asn Ser Asp Val Pro Leu Val Tyr Gln Asp Glu
675 680 685
Leu Ile Gln Ile Arg Glu Leu Met Tyr Lys Pro Tyr Lys Asp Trp Val
690 695 700
Ala Phe Leu Lys Gln Leu His Lys Arg Leu Glu Val Glu Ile Gly Lys
705 710 715 720
Glu Val Lys His Trp Arg Lys Ser Leu Ser Asp Gly Arg Lys Gly Leu
725 730 735
Tyr Gly Ile Ser Leu Lys Asn Ile Asp Glu Ile Asp Arg Thr Arg Lys
740 745 750
Phe Leu Leu Arg Trp Ser Leu Arg Pro Thr Glu Pro Gly Glu Val Arg
755 760 765
Arg Leu Glu Pro Gly Gln Arg Phe Ala Ile Asp Gln Leu Asn His Leu
770 775 780
Asn Ala Leu Lys Glu Asp Arg Leu Lys Lys Met Ala Asn Thr Ile Ile
785 790 795 800
Met His Ala Leu Gly Tyr Cys Tyr Asp Val Arg Lys Lys Lys Trp Gln
805 810 815
Ala Lys Asn Pro Ala Cys Gln Ile Ile Leu Phe Glu Asp Leu Ser Asn
820 825 830
Tyr Asn Pro Gly Gly Glu Arg Ser Arg Phe Glu Asn Ser Arg Leu Met
835 840 845
Lys Trp Ser Arg Arg Glu Ile Pro Arg Gln Val Ala Leu Gln Gly Glu
850 855 860
Ile Tyr Gly Leu Gln Val Gly Glu Val Gly Ala Gln Phe Ser Ser Arg
865 870 875 880
Phe His Ala Lys Thr Gly Ser Pro Gly Ile Arg Cys Arg Val Val Thr
885 890 895
Lys Glu Lys Leu Gln Asp Asn Arg Phe Phe Lys Asn Leu Gln Arg Glu
900 905 910
Gly Arg Leu Thr Leu Asp Lys Ile Ala Val Leu Lys Glu Gly Asp Leu
915 920 925
Tyr Pro Asp Lys Gly Gly Glu Lys Phe Ile Ser Leu Ser Lys Asp Arg
930 935 940
Lys Cys Val Thr Thr His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln
945 950 955 960
Lys Arg Phe Trp Thr Arg Thr His Gly Phe Tyr Lys Val Tyr Cys Lys
965 970 975
Ala Tyr Gln Val Asp Gly Gln Thr Val Tyr Ile Pro Glu Ser Lys Asp
980 985 990
Gln Lys Gln Lys Ile Ile Glu Glu Phe Gly Glu Gly Tyr Phe Ile Leu
995 1000 1005
Lys Asp Gly Val Tyr Glu Trp Val Asn Ala Gly Lys Leu Lys Ile Lys
1010 1015 1020
Lys Gly Ser Ser Lys Gln Ser Ser Ser Glu Leu Val Asp Ser Asp Ile
1025 1030 1035 1040
Leu Lys Asp Ser Phe Asp Leu Ala Ser Glu Leu Lys Gly Glu Lys Leu
1045 1050 1055
Met Leu Tyr Arg Asp Pro Ser Gly Asn Val Phe Pro Ser Asp Lys Trp
1060 1065 1070
Met Ala Ala Gly Val Phe Phe Gly Lys Leu Glu Arg Ile Leu Ile Ser
1075 1080 1085
Lys Leu Thr Asn Gln Tyr Ser Ile Ser Thr Ile Glu Asp Asp Ser Ser
1090 1095 1100
Lys Gln Ser Met
1105
<210> 3
<211> 3
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 3
Thr Thr Asn
1
<210> 4
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 4
Asn Asn Gly Arg Arg Thr
1 5
<210> 5
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 5
Asn Asn Asn Asn Arg Tyr Ala Cys
1 5
<210> 6
<211> 1108
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 6
Met Ala Thr Arg Ser Phe Ile Leu Lys Ile Glu Pro Asn Glu Glu Val
1 5 10 15
Lys Lys Gly Leu Trp Lys Thr His Glu Val Leu Asn His Gly Ile Ala
20 25 30
Tyr Tyr Met Asn Ile Leu Lys Leu Ile Arg Gln Glu Ala Ile Tyr Glu
35 40 45
His His Glu Gln Asp Pro Lys Asn Pro Lys Lys Val Ser Lys Ala Glu
50 55 60
Ile Gln Ala Glu Leu Trp Asp Phe Val Leu Lys Met Gln Lys Cys Asn
65 70 75 80
Ser Phe Thr His Glu Val Asp Lys Asp Glu Val Phe Asn Ile Leu Arg
85 90 95
Glu Leu Tyr Glu Glu Leu Val Pro Ser Ser Val Glu Lys Lys Gly Glu
100 105 110
Ala Asn Gln Leu Ser Asn Lys Phe Leu Tyr Pro Leu Val Asp Pro Asn
115 120 125
Ser Gln Ser Gly Lys Gly Thr Ala Ser Ser Gly Arg Lys Pro Arg Trp
130 135 140
Tyr Asn Leu Lys Ile Ala Gly Asp Pro Ser Trp Glu Glu Glu Lys Lys
145 150 155 160
Lys Trp Glu Glu Asp Lys Lys Lys Asp Pro Leu Ala Lys Ile Leu Gly
165 170 175
Lys Leu Ala Glu Tyr Gly Leu Ile Pro Leu Phe Ile Pro Tyr Thr Asp
180 185 190
Ser Asn Glu Pro Ile Val Lys Glu Ile Lys Trp Met Glu Lys Ser Arg
195 200 205
Asn Gln Ser Val Arg Arg Leu Asp Lys Asp Met Phe Ile Gln Ala Leu
210 215 220
Glu Arg Phe Leu Ser Trp Glu Ser Trp Asn Leu Lys Val Lys Glu Glu
225 230 235 240
Tyr Glu Lys Val Glu Lys Glu Tyr Lys Thr Leu Glu Glu Arg Ile Lys
245 250 255
Glu Asp Ile Gln Ala Leu Lys Ala Leu Glu Gln Tyr Glu Lys Glu Arg
260 265 270
Gln Glu Gln Leu Leu Arg Asp Thr Leu Asn Thr Asn Glu Tyr Arg Leu
275 280 285
Ser Lys Arg Gly Leu Arg Gly Trp Arg Glu Ile Ile Gln Lys Trp Leu
290 295 300
Lys Met Asp Glu Asn Glu Pro Ser Glu Lys Tyr Leu Glu Val Phe Lys
305 310 315 320
Asp Tyr Gln Arg Lys His Pro Arg Glu Ala Gly Asp Tyr Ser Val Tyr
325 330 335
Glu Phe Leu Ser Lys Lys Glu Asn His Phe Ile Trp Arg Asn His Pro
340 345 350
Glu Tyr Pro Tyr Leu Tyr Ala Thr Phe Cys Glu Ile Asp Lys Lys Lys
355 360 365
Lys Asp Ala Lys Gln Gln Ala Thr Phe Thr Leu Ala Asp Pro Ile Asn
370 375 380
His Pro Leu Trp Val Arg Phe Glu Glu Arg Ser Gly Ser Asn Leu Asn
385 390 395 400
Lys Tyr Arg Ile Leu Thr Glu Gln Leu His Thr Glu Lys Leu Lys Lys
405 410 415
Lys Leu Thr Val Gln Leu Asp Arg Leu Ile Tyr Pro Thr Glu Ser Gly
420 425 430
Gly Trp Glu Glu Lys Gly Lys Val Asp Ile Val Leu Leu Pro Ser Arg
435 440 445
Gln Phe Tyr Asn Gln Ile Phe Leu Asp Ile Glu Glu Lys Gly Lys His
450 455 460
Ala Phe Thr Tyr Lys Asp Glu Ser Ile Lys Phe Pro Leu Lys Gly Thr
465 470 475 480
Leu Gly Gly Ala Arg Val Gln Phe Asp Arg Asp His Leu Arg Arg Tyr
485 490 495
Pro His Lys Val Glu Ser Gly Asn Val Gly Arg Ile Tyr Phe Asn Met
500 505 510
Thr Val Asn Ile Glu Pro Thr Glu Ser Pro Val Ser Lys Ser Leu Lys
515 520 525
Ile His Arg Asp Asp Phe Pro Lys Val Val Asn Phe Lys Pro Lys Glu
530 535 540
Leu Thr Glu Trp Ile Lys Asp Ser Lys Gly Lys Lys Leu Lys Ser Gly
545 550 555 560
Ile Glu Ser Leu Glu Ile Gly Leu Arg Val Met Ser Ile Asp Leu Gly
565 570 575
Gln Arg Gln Ala Ala Ala Ala Ser Ile Phe Glu Val Val Asp Gln Lys
580 585 590
Pro Asp Ile Glu Gly Lys Leu Phe Phe Pro Ile Lys Gly Thr Glu Leu
595 600 605
Tyr Ala Val His Arg Ala Ser Phe Asn Ile Lys Leu Pro Gly Glu Thr
610 615 620
Leu Val Lys Ser Arg Glu Val Leu Arg Lys Ala Arg Glu Asp Asn Leu
625 630 635 640
Lys Leu Met Asn Gln Lys Leu Asn Phe Leu Arg Asn Val Leu His Phe
645 650 655
Gln Gln Phe Glu Asp Ile Thr Glu Arg Glu Lys Arg Val Thr Lys Trp
660 665 670
Ile Ser Arg Gln Glu Asn Ser Asp Val Pro Leu Val Tyr Gln Asp Glu
675 680 685
Leu Ile Gln Ile Arg Glu Leu Met Tyr Lys Pro Tyr Lys Asp Trp Val
690 695 700
Ala Phe Leu Lys Gln Leu His Lys Arg Leu Glu Val Glu Ile Gly Lys
705 710 715 720
Glu Val Lys His Trp Arg Lys Ser Leu Ser Asp Gly Arg Lys Gly Leu
725 730 735
Tyr Gly Ile Ser Leu Lys Asn Ile Asp Glu Ile Asp Arg Thr Arg Lys
740 745 750
Phe Leu Leu Arg Trp Ser Leu Arg Pro Thr Glu Pro Gly Glu Val Arg
755 760 765
Arg Leu Glu Pro Gly Gln Arg Phe Ala Ile Asp Gln Leu Asn His Leu
770 775 780
Asn Ala Leu Lys Glu Asp Arg Leu Lys Lys Met Ala Asn Thr Ile Ile
785 790 795 800
Met His Ala Leu Gly Tyr Cys Tyr Asp Val Arg Lys Lys Lys Trp Gln
805 810 815
Ala Lys Asn Pro Ala Cys Gln Ile Ile Leu Phe Glu Asp Leu Ser Asn
820 825 830
Tyr Asn Pro Tyr Gly Glu Arg Ser Arg Phe Glu Asn Ser Arg Leu Met
835 840 845
Lys Trp Ser Arg Arg Glu Ile Pro Arg Gln Val Ala Leu Gln Gly Glu
850 855 860
Ile Tyr Gly Leu Gln Val Gly Glu Val Gly Ala Gln Phe Ser Ser Arg
865 870 875 880
Phe His Ala Lys Thr Gly Ser Pro Gly Ile Arg Cys Arg Val Val Thr
885 890 895
Lys Glu Lys Leu Gln Asp Asn Arg Phe Phe Lys Asn Leu Gln Arg Glu
900 905 910
Gly Arg Leu Thr Leu Asp Lys Ile Ala Val Leu Lys Glu Gly Asp Leu
915 920 925
Tyr Pro Asp Lys Gly Gly Glu Lys Phe Ile Ser Leu Ser Lys Asp Arg
930 935 940
Lys Cys Val Thr Thr His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln
945 950 955 960
Lys Arg Phe Trp Thr Arg Thr His Gly Phe Tyr Lys Val Tyr Cys Lys
965 970 975
Ala Tyr Gln Val Asp Gly Gln Thr Val Tyr Ile Pro Glu Ser Lys Asp
980 985 990
Gln Lys Gln Lys Ile Ile Glu Glu Phe Gly Glu Gly Tyr Phe Ile Leu
995 1000 1005
Lys Asp Gly Val Tyr Glu Trp Val Asn Ala Gly Lys Leu Lys Ile Lys
1010 1015 1020
Lys Gly Ser Ser Lys Gln Ser Ser Ser Glu Leu Val Asp Ser Asp Ile
1025 1030 1035 1040
Leu Lys Asp Ser Phe Asp Leu Ala Ser Glu Leu Lys Gly Glu Lys Leu
1045 1050 1055
Met Leu Tyr Arg Asp Pro Ser Gly Asn Val Phe Pro Ser Asp Lys Trp
1060 1065 1070
Met Ala Ala Gly Val Phe Phe Gly Lys Leu Glu Arg Ile Leu Ile Ser
1075 1080 1085
Lys Leu Thr Asn Gln Tyr Ser Ile Ser Thr Ile Glu Asp Asp Ser Ser
1090 1095 1100
Lys Gln Ser Met
1105
<210> 7
<211> 1129
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 7
Met Ala Val Lys Ser Met Lys Val Lys Leu Arg Leu Asp Asn Met Pro
1 5 10 15
Glu Ile Arg Ala Gly Leu Trp Lys Leu His Thr Glu Val Asn Ala Gly
20 25 30
Val Arg Tyr Tyr Thr Glu Trp Leu Ser Leu Leu Arg Gln Glu Asn Leu
35 40 45
Tyr Arg Arg Ser Pro Asn Gly Asp Gly Glu Gln Glu Cys Tyr Lys Thr
50 55 60
Ala Glu Glu Cys Lys Ala Glu Leu Leu Glu Arg Leu Arg Ala Arg Gln
65 70 75 80
Val Glu Asn Gly His Cys Gly Pro Ala Gly Ser Asp Asp Glu Leu Leu
85 90 95
Gln Leu Ala Arg Gln Leu Tyr Glu Leu Leu Val Pro Gln Ala Ile Gly
100 105 110
Ala Lys Gly Asp Ala Gln Gln Ile Ala Arg Lys Phe Leu Ser Pro Leu
115 120 125
Ala Asp Lys Asp Ala Val Gly Gly Leu Gly Ile Ala Lys Ala Gly Asn
130 135 140
Lys Pro Arg Trp Val Arg Met Arg Glu Ala Gly Glu Pro Gly Trp Glu
145 150 155 160
Glu Glu Lys Ala Lys Ala Glu Ala Arg Lys Ser Thr Asp Arg Thr Ala
165 170 175
Asp Val Leu Arg Ala Leu Ala Asp Phe Gly Leu Lys Pro Leu Met Arg
180 185 190
Val Tyr Thr Asp Ser Asp Met Ser Ser Val Gln Trp Lys Pro Leu Arg
195 200 205
Lys Gly Gln Ala Val Arg Thr Trp Asp Arg Asp Met Phe Gln Gln Ala
210 215 220
Ile Glu Arg Met Met Ser Trp Glu Ser Trp Asn Gln Arg Val Gly Glu
225 230 235 240
Ala Tyr Ala Lys Leu Val Glu Gln Lys Ser Arg Phe Glu Gln Lys Asn
245 250 255
Phe Val Gly Gln Glu His Leu Val Gln Leu Val Asn Gln Leu Gln Gln
260 265 270
Asp Met Lys Glu Ala Ser His Gly Leu Glu Ser Lys Glu Gln Thr Ala
275 280 285
His Tyr Leu Thr Gly Arg Ala Leu Arg Gly Ser Asp Lys Val Phe Glu
290 295 300
Lys Trp Glu Lys Leu Asp Pro Asp Ala Pro Phe Asp Leu Tyr Asp Thr
305 310 315 320
Glu Ile Lys Asn Val Gln Arg Arg Asn Thr Arg Arg Phe Gly Ser His
325 330 335
Asp Leu Phe Ala Lys Leu Ala Glu Pro Lys Tyr Gln Ala Leu Trp Arg
340 345 350
Glu Asp Ala Ser Phe Leu Thr Arg Tyr Ala Val Tyr Asn Ser Ile Val
355 360 365
Arg Lys Leu Asn His Ala Lys Met Phe Ala Thr Phe Thr Leu Pro Asp
370 375 380
Ala Thr Ala His Pro Ile Trp Thr Arg Phe Asp Lys Leu Gly Gly Asn
385 390 395 400
Leu His Gln Tyr Thr Phe Leu Phe Asn Glu Phe Gly Glu Gly Arg His
405 410 415
Ala Ile Arg Phe Gln Lys Leu Leu Thr Val Glu Asp Gly Val Ala Lys
420 425 430
Glu Val Asp Asp Val Thr Val Pro Ile Ser Met Ser Ala Gln Leu Asp
435 440 445
Asp Leu Leu Pro Arg Asp Pro His Glu Leu Val Ala Leu Tyr Phe Gln
450 455 460
Asp Tyr Gly Ala Glu Gln His Leu Ala Gly Glu Phe Gly Gly Ala Lys
465 470 475 480
Ile Gln Tyr Arg Arg Asp Gln Leu Asn His Leu His Ala Arg Arg Gly
485 490 495
Ala Arg Asp Val Tyr Leu Asn Leu Ser Val Arg Val Gln Ser Gln Ser
500 505 510
Glu Ala Arg Gly Glu Arg Arg Pro Pro Tyr Ala Ala Val Phe Arg Leu
515 520 525
Val Gly Asp Asn His Arg Ala Phe Val His Phe Asp Lys Leu Ser Asp
530 535 540
Tyr Leu Ala Glu His Pro Asp Asp Gly Lys Leu Gly Ser Glu Gly Leu
545 550 555 560
Leu Ser Gly Leu Arg Val Met Ser Val Asp Leu Gly Leu Arg Thr Ser
565 570 575
Ala Ser Ile Ser Val Phe Arg Val Ala Arg Lys Asp Glu Leu Lys Pro
580 585 590
Asn Ser Glu Gly Arg Val Pro Phe Cys Phe Pro Ile Glu Gly Asn Glu
595 600 605
Asn Leu Val Ala Val His Glu Arg Ser Gln Leu Leu Lys Leu Pro Gly
610 615 620
Glu Thr Glu Ser Lys Asp Leu Arg Ala Ile Arg Glu Glu Arg Gln Arg
625 630 635 640
Thr Leu Arg Gln Leu Arg Thr Gln Leu Ala Tyr Leu Arg Leu Leu Val
645 650 655
Arg Cys Gly Ser Glu Asp Val Gly Arg Arg Glu Arg Ser Trp Ala Lys
660 665 670
Leu Ile Glu Gln Pro Met Asp Ala Asn Gln Met Thr Pro Asp Trp Arg
675 680 685
Glu Ala Phe Glu Asp Glu Leu Gln Lys Leu Lys Ser Leu Tyr Gly Ile
690 695 700
Cys Gly Asp Arg Glu Trp Thr Glu Ala Val Tyr Glu Ser Val Arg Arg
705 710 715 720
Val Trp Arg His Met Gly Lys Gln Val Arg Asp Trp Arg Lys Asp Val
725 730 735
Arg Ser Gly Glu Arg Pro Lys Ile Arg Gly Tyr Gln Lys Asp Val Val
740 745 750
Gly Gly Asn Ser Ile Glu Gln Ile Glu Tyr Leu Glu Arg Gln Tyr Lys
755 760 765
Phe Leu Lys Ser Trp Ser Phe Phe Gly Lys Val Ser Gly Gln Val Ile
770 775 780
Arg Ala Glu Lys Gly Ser Arg Phe Ala Ile Thr Leu Arg Glu His Ile
785 790 795 800
Asp His Ala Lys Glu Asp Arg Leu Lys Lys Leu Ala Asp Arg Ile Ile
805 810 815
Met Glu Ala Leu Gly Tyr Val Tyr Ala Leu Asp Asp Glu Arg Gly Lys
820 825 830
Gly Lys Trp Val Ala Lys Tyr Pro Pro Cys Gln Leu Ile Leu Leu Glu
835 840 845
Glu Leu Ser Glu Tyr Gln Phe Asn Asn Asp Arg Pro Pro Ser Glu Asn
850 855 860
Asn Gln Leu Met Gln Trp Ser His Arg Gly Val Phe Gln Glu Leu Leu
865 870 875 880
Asn Gln Ala Gln Val His Asp Leu Leu Val Gly Thr Met Tyr Ala Ala
885 890 895
Phe Ser Ser Arg Phe Asp Ala Arg Thr Gly Ala Pro Gly Ile Arg Cys
900 905 910
Arg Arg Val Pro Ala Arg Cys Ala Arg Glu Gln Asn Pro Glu Pro Phe
915 920 925
Pro Trp Trp Leu Asn Lys Phe Val Ala Glu His Lys Leu Asp Gly Cys
930 935 940
Pro Leu Arg Ala Asp Asp Leu Ile Pro Thr Gly Glu Gly Glu Phe Phe
945 950 955 960
Val Ser Pro Phe Ser Ala Glu Glu Gly Asp Phe His Gln Ile His Ala
965 970 975
Asp Leu Asn Ala Ala Gln Asn Leu Gln Arg Arg Leu Trp Ser Asp Phe
980 985 990
Asp Ile Ser Gln Ile Arg Leu Arg Cys Asp Trp Gly Glu Val Asp Gly
995 1000 1005
Glu Pro Val Leu Ile Pro Arg Thr Thr Gly Lys Arg Thr Ala Asp Ser
1010 1015 1020
Tyr Gly Asn Lys Val Phe Tyr Thr Lys Thr Gly Val Thr Tyr Tyr Glu
1025 1030 1035 1040
Arg Glu Arg Gly Lys Lys Arg Arg Lys Val Phe Ala Gln Glu Glu Leu
1045 1050 1055
Ser Glu Glu Glu Ala Glu Leu Leu Val Glu Ala Asp Glu Ala Arg Glu
1060 1065 1070
Lys Ser Val Val Leu Met Arg Asp Pro Ser Gly Ile Ile Asn Arg Gly
1075 1080 1085
Asp Trp Thr Arg Gln Lys Glu Phe Trp Ser Met Val Asn Gln Arg Ile
1090 1095 1100
Glu Gly Tyr Leu Val Lys Gln Ile Arg Ser Arg Val Arg Leu Gln Glu
1105 1110 1115 1120
Ser Ala Cys Glu Asn Thr Gly Asp Ile
1125
<210> 8
<211> 1054
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 8
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg
500 505 510
Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
515 520 525
Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg
530 535 540
Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys
545 550 555 560
Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile
565 570 575
Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly
580 585 590
Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr
595 600 605
Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly
610 615 620
Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu
625 630 635 640
Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr
645 650 655
Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu
660 665 670
Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu
675 680 685
Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr
690 695 700
Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly
705 710 715 720
Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe
725 730 735
Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser
740 745 750
Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe
755 760 765
Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp Glu Gln
770 775 780
Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys Leu Glu
785 790 795 800
Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile Ala Asn
805 810 815
Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile Arg Gly
820 825 830
Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys Ala Asn
835 840 845
Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys Ile Arg
850 855 860
Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly Ser Leu
865 870 875 880
Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp Lys Ala
885 890 895
Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly Asp Trp
900 905 910
Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile Gly Thr
915 920 925
Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His Tyr Glu
930 935 940
Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp Arg Lys
945 950 955 960
Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu Lys Leu
965 970 975
Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly Arg Ile
980 985 990
Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile Val Phe
995 1000 1005
Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala Ala Ala
1010 1015 1020
Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser Asp Glu
1025 1030 1035 1040
Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050
<210> 9
<211> 1093
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 9
Met Ser Asn Lys Glu Lys Asn Ala Ser Glu Thr Arg Lys Ala Tyr Thr
1 5 10 15
Thr Lys Met Ile Pro Arg Ser His Asp Arg Met Lys Leu Leu Gly Asn
20 25 30
Phe Met Asp Tyr Leu Met Asp Gly Thr Pro Ile Phe Phe Glu Leu Trp
35 40 45
Asn Gln Phe Gly Gly Gly Ile Asp Arg Asp Ile Ile Ser Gly Thr Ala
50 55 60
Asn Lys Asp Lys Ile Ser Asp Asp Leu Leu Leu Ala Val Asn Trp Phe
65 70 75 80
Lys Val Met Pro Ile Asn Ser Lys Pro Gln Gly Val Ser Pro Ser Asn
85 90 95
Leu Ala Asn Leu Phe Gln Gln Tyr Ser Gly Ser Glu Pro Asp Ile Gln
100 105 110
Ala Gln Glu Tyr Phe Ala Ser Asn Phe Asp Thr Glu Lys His Gln Trp
115 120 125
Lys Asp Met Arg Val Glu Tyr Glu Arg Leu Leu Ala Glu Leu Gln Leu
130 135 140
Ser Arg Ser Asp Met His His Asp Leu Lys Leu Met Tyr Lys Glu Lys
145 150 155 160
Cys Ile Gly Leu Ser Leu Ser Thr Ala His Tyr Ile Thr Ser Val Met
165 170 175
Phe Gly Thr Gly Ala Lys Asn Asn Arg Gln Thr Lys His Gln Phe Tyr
180 185 190
Ser Lys Val Ile Gln Leu Leu Glu Glu Ser Thr Gln Ile Asn Ser Val
195 200 205
Glu Gln Leu Ala Ser Ile Ile Leu Lys Ala Gly Asp Cys Asp Ser Tyr
210 215 220
Arg Lys Leu Arg Ile Arg Cys Ser Arg Lys Gly Ala Thr Pro Ser Ile
225 230 235 240
Leu Lys Ile Val Gln Asp Tyr Glu Leu Gly Thr Asn His Asp Asp Glu
245 250 255
Val Asn Val Pro Ser Leu Ile Ala Asn Leu Lys Glu Lys Leu Gly Arg
260 265 270
Phe Glu Tyr Glu Cys Glu Trp Lys Cys Met Glu Lys Ile Lys Ala Phe
275 280 285
Leu Ala Ser Lys Val Gly Pro Tyr Tyr Leu Gly Ser Tyr Ser Ala Met
290 295 300
Leu Glu Asn Ala Leu Ser Pro Ile Lys Gly Met Thr Thr Lys Asn Cys
305 310 315 320
Lys Phe Val Leu Lys Gln Ile Asp Ala Lys Asn Asp Ile Lys Tyr Glu
325 330 335
Asn Glu Pro Phe Gly Lys Ile Val Glu Gly Phe Phe Asp Ser Pro Tyr
340 345 350
Phe Glu Ser Asp Thr Asn Val Lys Trp Val Leu His Pro His His Ile
355 360 365
Gly Glu Ser Asn Ile Lys Thr Leu Trp Glu Asp Leu Asn Ala Ile His
370 375 380
Ser Lys Tyr Glu Glu Asp Ile Ala Ser Leu Ser Glu Asp Lys Lys Glu
385 390 395 400
Lys Arg Ile Lys Val Tyr Gln Gly Asp Val Cys Gln Thr Ile Asn Thr
405 410 415
Tyr Cys Glu Glu Val Gly Lys Glu Ala Lys Thr Pro Leu Val Gln Leu
420 425 430
Leu Arg Tyr Leu Tyr Ser Arg Lys Asp Asp Ile Ala Val Asp Lys Ile
435 440 445
Ile Asp Gly Ile Thr Phe Leu Ser Lys Lys His Lys Val Glu Lys Gln
450 455 460
Lys Ile Asn Pro Val Ile Gln Lys Tyr Pro Ser Phe Asn Phe Gly Asn
465 470 475 480
Asn Ser Lys Leu Leu Gly Lys Ile Ile Ser Pro Lys Asp Lys Leu Lys
485 490 495
His Asn Leu Lys Cys Asn Arg Asn Gln Val Asp Asn Tyr Ile Trp Ile
500 505 510
Glu Ile Lys Val Leu Asn Thr Lys Thr Met Arg Trp Glu Lys His His
515 520 525
Tyr Ala Leu Ser Ser Thr Arg Phe Leu Glu Glu Val Tyr Tyr Pro Ala
530 535 540
Thr Ser Glu Asn Pro Pro Asp Ala Leu Ala Ala Arg Phe Arg Thr Lys
545 550 555 560
Thr Asn Gly Tyr Glu Gly Lys Pro Ala Leu Ser Ala Glu Gln Ile Glu
565 570 575
Gln Ile Arg Ser Ala Pro Val Gly Leu Arg Lys Val Lys Lys Arg Gln
580 585 590
Met Arg Leu Glu Ala Ala Arg Gln Gln Asn Leu Leu Pro Arg Tyr Thr
595 600 605
Trp Gly Lys Asp Phe Asn Ile Asn Ile Cys Lys Arg Gly Asn Asn Phe
610 615 620
Glu Val Thr Leu Ala Thr Lys Val Lys Lys Lys Lys Glu Lys Asn Tyr
625 630 635 640
Lys Val Val Leu Gly Tyr Asp Ala Asn Ile Val Arg Lys Asn Thr Tyr
645 650 655
Ala Ala Ile Glu Ala His Ala Asn Gly Asp Gly Val Ile Asp Tyr Asn
660 665 670
Asp Leu Pro Val Lys Pro Ile Glu Ser Gly Phe Val Thr Val Glu Ser
675 680 685
Gln Val Arg Asp Lys Ser Tyr Asp Gln Leu Ser Tyr Asn Gly Val Lys
690 695 700
Leu Leu Tyr Cys Lys Pro His Val Glu Ser Arg Arg Ser Phe Leu Glu
705 710 715 720
Lys Tyr Arg Asn Gly Thr Met Lys Asp Asn Arg Gly Asn Asn Ile Gln
725 730 735
Ile Asp Phe Met Lys Asp Phe Glu Ala Ile Ala Asp Asp Glu Thr Ser
740 745 750
Leu Tyr Tyr Phe Asn Met Lys Tyr Cys Lys Leu Leu Gln Ser Ser Ile
755 760 765
Arg Asn His Ser Ser Gln Ala Lys Glu Tyr Arg Glu Glu Ile Phe Glu
770 775 780
Leu Leu Arg Asp Gly Lys Leu Ser Val Leu Lys Leu Ser Ser Leu Ser
785 790 795 800
Asn Leu Ser Phe Val Met Phe Lys Val Ala Lys Ser Leu Ile Gly Thr
805 810 815
Tyr Phe Gly His Leu Leu Lys Lys Pro Lys Asn Ser Lys Ser Asp Val
820 825 830
Lys Ala Pro Pro Ile Thr Asp Glu Asp Lys Gln Lys Ala Asp Pro Glu
835 840 845
Met Phe Ala Leu Arg Leu Ala Leu Glu Glu Lys Arg Leu Asn Lys Val
850 855 860
Lys Ser Lys Lys Glu Val Ile Ala Asn Lys Ile Val Ala Lys Ala Leu
865 870 875 880
Glu Leu Arg Asp Lys Tyr Gly Pro Val Leu Ile Lys Gly Glu Asn Ile
885 890 895
Ser Asp Thr Thr Lys Lys Gly Lys Lys Ser Ser Thr Asn Ser Phe Leu
900 905 910
Met Asp Trp Leu Ala Arg Gly Val Ala Asn Lys Val Lys Glu Met Val
915 920 925
Met Met His Gln Gly Leu Glu Phe Val Glu Val Asn Pro Asn Phe Thr
930 935 940
Ser His Gln Asp Pro Phe Val His Lys Asn Pro Glu Asn Thr Phe Arg
945 950 955 960
Ala Arg Tyr Ser Arg Cys Thr Pro Ser Glu Leu Thr Glu Lys Asn Arg
965 970 975
Lys Glu Ile Leu Ser Phe Leu Ser Asp Lys Pro Ser Lys Arg Pro Thr
980 985 990
Asn Ala Tyr Tyr Asn Glu Gly Ala Met Ala Phe Leu Ala Thr Tyr Gly
995 1000 1005
Leu Lys Lys Asn Asp Val Leu Gly Val Ser Leu Glu Lys Phe Lys Gln
1010 1015 1020
Ile Met Ala Asn Ile Leu His Gln Arg Ser Glu Asp Gln Leu Leu Phe
1025 1030 1035 1040
Pro Ser Arg Gly Gly Met Phe Tyr Leu Ala Thr Tyr Lys Leu Asp Ala
1045 1050 1055
Asp Ala Thr Ser Val Asn Trp Asn Gly Lys Gln Phe Trp Val Cys Asn
1060 1065 1070
Ala Asp Leu Val Ala Ala Tyr Asn Val Gly Leu Val Asp Ile Gln Lys
1075 1080 1085
Asp Phe Lys Lys Lys
1090
<210> 10
<211> 1046
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 10
Met Val Ser Asp Ser Thr Ile Arg Pro Tyr Thr Ser Lys Leu Ala Pro
1 5 10 15
Asn Asp Pro Lys Leu Lys Met Leu Asn Asp Thr Phe Asn Trp Leu Asp
20 25 30
His Ala Tyr Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe Gly Ala
35 40 45
Ile Glu His Glu Thr Ala Gln Glu Leu Ile Gly Glu Lys Ser Lys Phe
50 55 60
Asp Ala Asp Leu Leu Cys Ala Ile Met Trp Phe Arg Leu Glu Glu Lys
65 70 75 80
Ser Asp Asn Pro Gly Pro Leu Gln Thr Val Glu Gln Arg Met Arg Leu
85 90 95
Phe Gln Lys Tyr Ser Gly His Glu Pro Ser Ser Phe Thr Gln Glu Tyr
100 105 110
Ile Lys Gly Asn Ile Asp Ser Glu Lys Tyr Glu Trp Val Asp Cys Arg
115 120 125
Leu Lys Phe Ile Asp Leu Ala Arg Asn Ile Asn Thr Thr Gln Glu Ser
130 135 140
Leu Lys Ile Asp Ala Cys Thr Leu Phe Met Asn Lys Leu Ile Pro Val
145 150 155 160
Ser Lys Asp Asp Glu Phe Asn Ala Tyr Gly Leu Ile Ser Gln Leu Phe
165 170 175
Gly Thr Gly Lys Lys Glu Asp Arg Ser Ile Lys Ala Ala Met Leu Glu
180 185 190
Glu Ile Ser Asn Ile Leu Ala Asp Lys Lys Pro Asp Thr Trp Glu Glu
195 200 205
Tyr Gln Asp Leu Ile Lys Arg Thr Phe Asp Val Ser Asn Tyr Lys Glu
210 215 220
Leu Lys Glu Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Ser Ser Leu
225 230 235 240
Val Ile Asp Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro Asn Leu
245 250 255
Ile Lys Asn Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys Lys Lys
260 265 270
Thr Val Phe Leu Leu Pro Asn Arg Met Lys Leu Arg Glu Phe Ile Ala
275 280 285
Ser Gln Ile Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val Leu Asn
290 295 300
Arg Ser Met Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile Leu Tyr
305 310 315 320
Thr Asn Glu Lys Glu Glu Arg Asn Asn Glu Ile Gln Glu Leu Leu Lys
325 330 335
Lys Asp Ile Leu Leu Ala Ala Ser Ile Leu Gly Asp Phe Arg Arg Gly
340 345 350
Glu Phe Asn Arg Ser Val Val Ser Lys Asn His Leu Gly Ala Arg Leu
355 360 365
Asn Glu Leu Phe Glu Ile Trp Gln Asp Leu Thr Met Asp Asp Gly Ile
370 375 380
Arg Lys Tyr Val Asp Leu Cys Lys Asp Lys Phe Ser Arg Arg Pro Val
385 390 395 400
Lys Ala Leu Leu Gln Tyr Ile Tyr Pro His Phe Asp Lys Ile Thr Ala
405 410 415
Lys Gln Phe Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu Thr Asn
420 425 430
Asn Arg Lys Lys Ile His Pro Thr Val Thr Gly Pro Thr Val Cys Asn
435 440 445
Trp Gly Pro Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro Asn Gln
450 455 460
Met Val Lys Gly Arg Pro Ala Gly Ser His Gly Met Ile Trp Val Thr
465 470 475 480
Met Lys Val Ile Asp Asn Gly Arg Trp Val Ser His His Leu Pro Phe
485 490 495
His Asn Ser Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu Gly Leu
500 505 510
Pro Thr Lys Asn Gln Pro Arg Thr Lys Gln Leu Gly Thr Gln Val Gly
515 520 525
Ser Ile Ile Ser Ala Thr Lys Leu Ala Ala Leu Lys Ser Gln Glu Glu
530 535 540
Gln Asp Arg Arg Asn Asp Arg Lys Asn Arg Phe Lys Ala His Lys Ser
545 550 555 560
Ile Ile Arg Ser Gln Glu Asn Ile Glu Tyr Asn Val Ala Phe Asp Lys
565 570 575
Ser Thr Asn Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe Ile Thr
580 585 590
Ile Ser Ser Arg Val Ala Thr Pro Lys Tyr Ser Tyr Lys Leu Asn Ile
595 600 605
Gly Asp Met Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro Cys Thr
610 615 620
Tyr Ser Ile Trp Arg Val Val Glu Lys Asp Thr Glu Gly Ser Phe Phe
625 630 635 640
His Asn Lys Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Ile Thr Ser
645 650 655
Ile Val Asp Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala Gly Ile
660 665 670
Glu Tyr Ser Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln Phe Leu
675 680 685
Arg Ser Ile Asn Glu Asp Tyr Val Ile Lys Ser Asp Asn Trp Arg Asn
690 695 700
Met Asn Leu Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu Leu Asp
705 710 715 720
Val Met Lys Val Asn Lys Asp Lys Asp Val Gln Asn Thr Phe Arg Ala
725 730 735
Glu Ile Glu Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu Gly Ser
740 745 750
Leu Phe His His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser Leu Ile
755 760 765
Ser Ser Tyr Phe Met Leu Asn Asn Lys Lys Glu Asp Tyr Asp Gln Glu
770 775 780
Leu Phe Asp Ser Asp Phe Phe Arg Leu Met Lys Ser Ile Gly Asp Lys
785 790 795 800
Arg Val Arg Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser Thr Val
805 810 815
Leu Gln Ile Ala Arg Glu Asn Asn Ile Lys Ser Leu Cys Val Glu Gly
820 825 830
Tyr Leu Pro Thr Ser Thr Lys Lys Thr Lys Pro Lys Gln Asn Gln Lys
835 840 845
Ser Ile Asp Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn Asp Gly
850 855 860
Cys Lys Val Leu Gly Ile Asn Leu Gln Ala Ile Asp Pro Arg Asp Thr
865 870 875 880
Ser His Leu Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr Lys Val
885 890 895
Gly Lys Glu Ala Arg Tyr Thr Ile Val Glu Pro Ser Asn Ile Lys Glu
900 905 910
Tyr Met Thr Asn Arg Phe Asp Asp Trp His Arg Gly Val Thr Lys Lys
915 920 925
Ser Lys Lys Gly Asp Val Gln Thr Ser Thr Thr Val Pro Leu Tyr Gln
930 935 940
Glu Ala Leu Arg Gln Phe Ala Ser His Tyr Glu Leu Asp Phe Asp Ser
945 950 955 960
Leu Pro Lys Met Lys Phe Tyr Asp Leu Ala Lys Arg Leu Gly Asp His
965 970 975
Glu Lys Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr Leu Ser Thr
980 985 990
Tyr Pro Val Thr Lys Asp Ser Ser Lys Ile Thr Phe Asn Gly Arg Glu
995 1000 1005
Arg Trp Tyr Asn Glu Ser Asp Val Val Ala Ala Val Asn Ile Val Leu
1010 1015 1020
Arg Gly Ile Lys Asp Glu Asp Glu Gln Pro Asp Asp Val Lys Lys Gln
1025 1030 1035 1040
Ala Leu Ala Arg Thr Lys
1045
<210> 11
<211> 1054
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 11
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Gly Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg
500 505 510
Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
515 520 525
Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg
530 535 540
Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys
545 550 555 560
Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile
565 570 575
Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly
580 585 590
Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr
595 600 605
Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly
610 615 620
Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu
625 630 635 640
Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr
645 650 655
Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu
660 665 670
Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu
675 680 685
Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr
690 695 700
Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly
705 710 715 720
Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe
725 730 735
Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser
740 745 750
Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe
755 760 765
Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp Glu Gln
770 775 780
Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys Leu Glu
785 790 795 800
Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile Ala Asn
805 810 815
Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile Arg Gly
820 825 830
Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys Ala Asn
835 840 845
Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys Ile Arg
850 855 860
Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly Ser Leu
865 870 875 880
Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp Lys Ala
885 890 895
Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly Asp Trp
900 905 910
Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile Gly Thr
915 920 925
Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His Tyr Glu
930 935 940
Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp Arg Lys
945 950 955 960
Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu Lys Leu
965 970 975
Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly Arg Ile
980 985 990
Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile Val Phe
995 1000 1005
Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala Ala Ala
1010 1015 1020
Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser Asp Glu
1025 1030 1035 1040
Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050
<210> 12
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 12
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Gly Gly Ala Pro Ser Thr Leu Glu Lys
225 230 235 240
Phe Ile Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln
245 250 255
Thr Asp Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp
260 265 270
Cys Cys Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln
275 280 285
Tyr Asp Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala
290 295 300
Leu Lys Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu
305 310 315 320
Glu Gln Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys
325 330 335
Lys Leu Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr
340 345 350
Tyr Thr Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu
355 360 365
Tyr Lys Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val
370 375 380
Val Leu Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg
385 390 395 400
Asn Ile Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln
405 410 415
Asp Ile Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys
420 425 430
Ser Gln Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp
435 440 445
Thr Asn Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro
450 455 460
Asn Ser Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro
465 470 475 480
Asp Gly Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe
485 490 495
Phe Gln Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln
500 505 510
Phe Arg Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln
515 520 525
Thr Ala Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu
530 535 540
Ala Arg Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn
545 550 555 560
Leu Lys Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val
565 570 575
Arg Ile Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln
580 585 590
Ile Gly Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His
595 600 605
Ala Tyr Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu
610 615 620
Leu Gly Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile
625 630 635 640
Thr Glu Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu
645 650 655
Ala Tyr Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val
660 665 670
Ser Leu Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr
675 680 685
Val Glu Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg
690 695 700
Leu Tyr Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val
705 710 715 720
Arg Gly Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe
725 730 735
Arg Phe Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser
740 745 750
Leu Ser Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser
755 760 765
Tyr Phe Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp
770 775 780
Glu Gln Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys
785 790 795 800
Leu Glu Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile
805 810 815
Ala Asn Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile
820 825 830
Arg Gly Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys
835 840 845
Ala Asn Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys
850 855 860
Ile Arg Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly
865 870 875 880
Ser Leu Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp
885 890 895
Lys Ala Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly
900 905 910
Asp Trp Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 13
<211> 1054
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 13
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Gly Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg
500 505 510
Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
515 520 525
Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg
530 535 540
Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys
545 550 555 560
Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile
565 570 575
Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly
580 585 590
Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr
595 600 605
Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly
610 615 620
Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu
625 630 635 640
Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr
645 650 655
Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu
660 665 670
Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu
675 680 685
Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr
690 695 700
Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly
705 710 715 720
Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe
725 730 735
Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser
740 745 750
Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe
755 760 765
Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp Glu Gln
770 775 780
Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys Leu Glu
785 790 795 800
Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile Ala Asn
805 810 815
Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile Arg Gly
820 825 830
Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys Ala Asn
835 840 845
Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys Ile Arg
850 855 860
Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly Ser Leu
865 870 875 880
Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp Lys Ala
885 890 895
Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly Asp Trp
900 905 910
Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile Gly Thr
915 920 925
Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His Tyr Glu
930 935 940
Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp Arg Lys
945 950 955 960
Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu Lys Leu
965 970 975
Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly Arg Ile
980 985 990
Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile Val Phe
995 1000 1005
Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala Ala Ala
1010 1015 1020
Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser Asp Glu
1025 1030 1035 1040
Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050
<210> 14
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 14
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Gly Gly Asn Gln Gly Phe Thr Trp
435 440 445
Thr Asn Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro
450 455 460
Asn Ser Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro
465 470 475 480
Asp Gly Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe
485 490 495
Phe Gln Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln
500 505 510
Phe Arg Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln
515 520 525
Thr Ala Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu
530 535 540
Ala Arg Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn
545 550 555 560
Leu Lys Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val
565 570 575
Arg Ile Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln
580 585 590
Ile Gly Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His
595 600 605
Ala Tyr Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu
610 615 620
Leu Gly Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile
625 630 635 640
Thr Glu Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu
645 650 655
Ala Tyr Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val
660 665 670
Ser Leu Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr
675 680 685
Val Glu Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg
690 695 700
Leu Tyr Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val
705 710 715 720
Arg Gly Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe
725 730 735
Arg Phe Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser
740 745 750
Leu Ser Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser
755 760 765
Tyr Phe Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp
770 775 780
Glu Gln Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys
785 790 795 800
Leu Glu Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile
805 810 815
Ala Asn Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile
820 825 830
Arg Gly Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys
835 840 845
Ala Asn Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys
850 855 860
Ile Arg Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly
865 870 875 880
Ser Leu Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp
885 890 895
Lys Ala Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly
900 905 910
Asp Trp Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 15
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 15
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Gly Gly Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe
485 490 495
Phe Gln Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln
500 505 510
Phe Arg Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln
515 520 525
Thr Ala Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu
530 535 540
Ala Arg Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn
545 550 555 560
Leu Lys Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val
565 570 575
Arg Ile Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln
580 585 590
Ile Gly Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His
595 600 605
Ala Tyr Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu
610 615 620
Leu Gly Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile
625 630 635 640
Thr Glu Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu
645 650 655
Ala Tyr Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val
660 665 670
Ser Leu Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr
675 680 685
Val Glu Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg
690 695 700
Leu Tyr Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val
705 710 715 720
Arg Gly Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe
725 730 735
Arg Phe Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser
740 745 750
Leu Ser Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser
755 760 765
Tyr Phe Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp
770 775 780
Glu Gln Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys
785 790 795 800
Leu Glu Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile
805 810 815
Ala Asn Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile
820 825 830
Arg Gly Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys
835 840 845
Ala Asn Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys
850 855 860
Ile Arg Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly
865 870 875 880
Ser Leu Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp
885 890 895
Lys Ala Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly
900 905 910
Asp Trp Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 16
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 16
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Gly Gly Asn Ser Pro Val Asp Thr Cys Gln
500 505 510
Phe Arg Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln
515 520 525
Thr Ala Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu
530 535 540
Ala Arg Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn
545 550 555 560
Leu Lys Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val
565 570 575
Arg Ile Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln
580 585 590
Ile Gly Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His
595 600 605
Ala Tyr Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu
610 615 620
Leu Gly Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile
625 630 635 640
Thr Glu Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu
645 650 655
Ala Tyr Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val
660 665 670
Ser Leu Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr
675 680 685
Val Glu Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg
690 695 700
Leu Tyr Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val
705 710 715 720
Arg Gly Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe
725 730 735
Arg Phe Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser
740 745 750
Leu Ser Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser
755 760 765
Tyr Phe Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp
770 775 780
Glu Gln Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys
785 790 795 800
Leu Glu Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile
805 810 815
Ala Asn Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile
820 825 830
Arg Gly Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys
835 840 845
Ala Asn Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys
850 855 860
Ile Arg Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly
865 870 875 880
Ser Leu Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp
885 890 895
Lys Ala Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly
900 905 910
Asp Trp Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 17
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 17
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg
500 505 510
Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
515 520 525
Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg
530 535 540
Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys
545 550 555 560
Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile
565 570 575
Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly
580 585 590
Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr
595 600 605
Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly
610 615 620
Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu
625 630 635 640
Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr
645 650 655
Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu
660 665 670
Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu
675 680 685
Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr
690 695 700
Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly
705 710 715 720
Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe
725 730 735
Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser
740 745 750
Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe
755 760 765
Ser Thr Ala Leu Asn Ala Gly Gly Ser Lys Asn Asn Pro Ile Ser Asp
770 775 780
Glu Gln Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys
785 790 795 800
Leu Glu Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile
805 810 815
Ala Asn Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile
820 825 830
Arg Gly Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys
835 840 845
Ala Asn Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys
850 855 860
Ile Arg Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly
865 870 875 880
Ser Leu Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp
885 890 895
Lys Ala Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly
900 905 910
Asp Trp Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 18
<211> 1054
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 18
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg
500 505 510
Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
515 520 525
Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg
530 535 540
Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys
545 550 555 560
Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile
565 570 575
Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly
580 585 590
Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr
595 600 605
Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly
610 615 620
Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu
625 630 635 640
Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr
645 650 655
Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu
660 665 670
Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu
675 680 685
Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr
690 695 700
Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly
705 710 715 720
Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe
725 730 735
Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser
740 745 750
Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe
755 760 765
Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp Glu Gln
770 775 780
Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys Leu Glu
785 790 795 800
Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile Ala Asn
805 810 815
Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile Arg Gly
820 825 830
Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys Ala Asn
835 840 845
Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys Ile Arg
850 855 860
Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly Ser Leu
865 870 875 880
Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp Lys Ala
885 890 895
Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly Asp Trp
900 905 910
Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Gly Gly Thr
915 920 925
Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His Tyr Glu
930 935 940
Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp Arg Lys
945 950 955 960
Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu Lys Leu
965 970 975
Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly Arg Ile
980 985 990
Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile Val Phe
995 1000 1005
Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala Ala Ala
1010 1015 1020
Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser Asp Glu
1025 1030 1035 1040
Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050
<210> 19
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 19
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg
500 505 510
Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
515 520 525
Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg
530 535 540
Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys
545 550 555 560
Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile
565 570 575
Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly
580 585 590
Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr
595 600 605
Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly
610 615 620
Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu
625 630 635 640
Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr
645 650 655
Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu
660 665 670
Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu
675 680 685
Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr
690 695 700
Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly
705 710 715 720
Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe
725 730 735
Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser
740 745 750
Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe
755 760 765
Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp Glu Gln
770 775 780
Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys Leu Glu
785 790 795 800
Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile Ala Asn
805 810 815
Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile Arg Gly
820 825 830
Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys Ala Asn
835 840 845
Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys Ile Arg
850 855 860
Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly Ser Leu
865 870 875 880
Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp Lys Ala
885 890 895
Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly Asp Trp
900 905 910
Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile Gly Gly
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 20
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 20
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Gly Gly Asn Gln Gly Phe Thr Trp
435 440 445
Thr Asn Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro
450 455 460
Asn Ser Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro
465 470 475 480
Asp Gly Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe
485 490 495
Phe Gln Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln
500 505 510
Phe Arg Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln
515 520 525
Thr Ala Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu
530 535 540
Ala Arg Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn
545 550 555 560
Leu Lys Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val
565 570 575
Arg Ile Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln
580 585 590
Ile Gly Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His
595 600 605
Ala Tyr Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu
610 615 620
Leu Gly Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile
625 630 635 640
Thr Glu Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu
645 650 655
Ala Tyr Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val
660 665 670
Ser Leu Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr
675 680 685
Val Glu Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg
690 695 700
Leu Tyr Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val
705 710 715 720
Arg Gly Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe
725 730 735
Arg Phe Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser
740 745 750
Leu Ser Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser
755 760 765
Tyr Phe Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp
770 775 780
Glu Gln Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys
785 790 795 800
Leu Glu Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile
805 810 815
Ala Asn Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile
820 825 830
Arg Gly Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys
835 840 845
Ala Asn Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys
850 855 860
Ile Arg Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly
865 870 875 880
Ser Leu Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp
885 890 895
Lys Ala Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly
900 905 910
Asp Trp Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Gly
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 21
<211> 3
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 21
Thr Thr Asn
1
<210> 22
<211> 3
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 22
Thr Thr His
1
<210> 23
<211> 3
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 23
Thr Thr Tyr
1
<210> 24
<211> 3
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 24
Thr Thr Cys
1
<210> 25
<211> 1087
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 25
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 26
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 26
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Gly Gly Glu Ser Leu
35 40 45
Ala Leu Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg
50 55 60
Arg Lys His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly
65 70 75 80
Ile Leu Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu
85 90 95
Ile Asp Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn
100 105 110
Asn Asp Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly
115 120 125
Phe Lys Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr
130 135 140
Met Leu Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg
145 150 155 160
Thr Val Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys
165 170 175
Arg Asn Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu
180 185 190
Glu Arg Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn
195 200 205
Met Ser Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala
210 215 220
Ser Gln Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly
225 230 235 240
Phe Cys Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr
245 250 255
Thr Phe Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu
260 265 270
Ile Ser Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu
275 280 285
Leu Tyr Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile
290 295 300
Arg Thr Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val
305 310 315 320
Tyr Asp Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu
325 330 335
Glu Leu Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr
340 345 350
Gly Lys Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe
355 360 365
Gly Tyr Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr
370 375 380
Leu Arg Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala
385 390 395 400
Asn Lys Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser
405 410 415
Phe Thr Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu
420 425 430
Pro Tyr Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala
435 440 445
Gly Tyr Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu
450 455 460
Pro Asn Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr
465 470 475 480
Gln Ala Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro
485 490 495
Val Ser Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp
500 505 510
Glu Arg Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 27
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 27
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Gly Gly Ser Asn Lys Glu Asn Ser Thr
130 135 140
Met Leu Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg
145 150 155 160
Thr Val Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys
165 170 175
Arg Asn Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu
180 185 190
Glu Arg Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn
195 200 205
Met Ser Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala
210 215 220
Ser Gln Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly
225 230 235 240
Phe Cys Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr
245 250 255
Thr Phe Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu
260 265 270
Ile Ser Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu
275 280 285
Leu Tyr Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile
290 295 300
Arg Thr Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val
305 310 315 320
Tyr Asp Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu
325 330 335
Glu Leu Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr
340 345 350
Gly Lys Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe
355 360 365
Gly Tyr Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr
370 375 380
Leu Arg Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala
385 390 395 400
Asn Lys Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser
405 410 415
Phe Thr Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu
420 425 430
Pro Tyr Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala
435 440 445
Gly Tyr Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu
450 455 460
Pro Asn Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr
465 470 475 480
Gln Ala Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro
485 490 495
Val Ser Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp
500 505 510
Glu Arg Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 28
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 28
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Gly Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu
180 185 190
Glu Arg Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn
195 200 205
Met Ser Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala
210 215 220
Ser Gln Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly
225 230 235 240
Phe Cys Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr
245 250 255
Thr Phe Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu
260 265 270
Ile Ser Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu
275 280 285
Leu Tyr Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile
290 295 300
Arg Thr Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val
305 310 315 320
Tyr Asp Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu
325 330 335
Glu Leu Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr
340 345 350
Gly Lys Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe
355 360 365
Gly Tyr Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr
370 375 380
Leu Arg Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala
385 390 395 400
Asn Lys Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser
405 410 415
Phe Thr Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu
420 425 430
Pro Tyr Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala
435 440 445
Gly Tyr Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu
450 455 460
Pro Asn Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr
465 470 475 480
Gln Ala Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro
485 490 495
Val Ser Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp
500 505 510
Glu Arg Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 29
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 29
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Gly Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu
275 280 285
Leu Tyr Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile
290 295 300
Arg Thr Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val
305 310 315 320
Tyr Asp Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu
325 330 335
Glu Leu Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr
340 345 350
Gly Lys Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe
355 360 365
Gly Tyr Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr
370 375 380
Leu Arg Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala
385 390 395 400
Asn Lys Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser
405 410 415
Phe Thr Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu
420 425 430
Pro Tyr Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala
435 440 445
Gly Tyr Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu
450 455 460
Pro Asn Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr
465 470 475 480
Gln Ala Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro
485 490 495
Val Ser Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp
500 505 510
Glu Arg Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 30
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 30
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Gly Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe
355 360 365
Gly Tyr Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr
370 375 380
Leu Arg Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala
385 390 395 400
Asn Lys Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser
405 410 415
Phe Thr Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu
420 425 430
Pro Tyr Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala
435 440 445
Gly Tyr Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu
450 455 460
Pro Asn Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr
465 470 475 480
Gln Ala Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro
485 490 495
Val Ser Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp
500 505 510
Glu Arg Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 31
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 31
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Gly Gly Lys Arg Met Pro Asn Leu Ala
385 390 395 400
Asn Lys Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser
405 410 415
Phe Thr Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu
420 425 430
Pro Tyr Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala
435 440 445
Gly Tyr Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu
450 455 460
Pro Asn Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr
465 470 475 480
Gln Ala Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro
485 490 495
Val Ser Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp
500 505 510
Glu Arg Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 32
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 32
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Gly Gly Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 33
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 33
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
Gly Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 34
<211> 1087
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 34
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Gly Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 35
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 35
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro Gly Gly His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 36
<211> 1087
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 36
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Gly Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 37
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 37
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Gly Gly Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 38
<211> 1087
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 38
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Gly Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 39
<211> 1087
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 39
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Gly Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 40
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 40
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Gly Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 41
<211> 1087
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 41
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Gly Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 42
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 42
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Gly Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 43
<211> 1087
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 43
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Gly Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 44
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 44
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly Gly Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 45
<211> 1087
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 45
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Gly Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 46
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 46
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Gly Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 47
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 47
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Gly Gly Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 48
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 48
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Gly Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 49
<211> 1087
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 49
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Gly Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 50
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 50
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 51
<211> 1087
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 51
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Gly Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 52
<211> 1089
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 52
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Gly Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 53
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 53
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys
740 745 750
Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys
755 760 765
Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu
770 775 780
Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr
785 790 795 800
Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys
805 810 815
Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His
820 825 830
His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr
835 840 845
Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn
850 855 860
Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys
865 870 875 880
Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp
885 890 895
Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro
900 905 910
Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr
915 920 925
Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn
930 935 940
Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln
945 950 955 960
Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn
965 970 975
Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg
980 985 990
Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn
995 1000 1005
Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys
1010 1015 1020
Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035 1040
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly Gly Ser
1045 1050 1055
<210> 54
<211> 1058
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 54
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Gly
35 40 45
Gly Arg Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg
50 55 60
His Arg Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu
65 70 75 80
Thr Asp His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val
85 90 95
Lys Gly Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu
100 105 110
Leu His Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu
115 120 125
Glu Asp Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn
130 135 140
Ser Lys Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg
145 150 155 160
Leu Lys Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr
165 170 175
Ser Asp Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala
180 185 190
Tyr His Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu
195 200 205
Glu Thr Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe
210 215 220
Gly Trp Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys
225 230 235 240
Thr Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala
245 250 255
Asp Leu Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg
260 265 270
Asp Glu Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu
275 280 285
Asn Val Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys
290 295 300
Glu Ile Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser
305 310 315 320
Thr Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys
325 330 335
Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp
340 345 350
Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln
355 360 365
Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu
370 375 380
Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 55
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 55
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Gly Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys
740 745 750
Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys
755 760 765
Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu
770 775 780
Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr
785 790 795 800
Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys
805 810 815
Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His
820 825 830
His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr
835 840 845
Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn
850 855 860
Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys
865 870 875 880
Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp
885 890 895
Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro
900 905 910
Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr
915 920 925
Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn
930 935 940
Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln
945 950 955 960
Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn
965 970 975
Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg
980 985 990
Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn
995 1000 1005
Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys
1010 1015 1020
Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035 1040
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly Gly Ser
1045 1050 1055
<210> 56
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 56
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Gly Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys
740 745 750
Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys
755 760 765
Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu
770 775 780
Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr
785 790 795 800
Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys
805 810 815
Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His
820 825 830
His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr
835 840 845
Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn
850 855 860
Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys
865 870 875 880
Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp
885 890 895
Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro
900 905 910
Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr
915 920 925
Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn
930 935 940
Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln
945 950 955 960
Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn
965 970 975
Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg
980 985 990
Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn
995 1000 1005
Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys
1010 1015 1020
Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035 1040
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly Gly Ser
1045 1050 1055
<210> 57
<211> 1058
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 57
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Gly Gly Ile Asn Pro Tyr Glu Ala Arg Val
85 90 95
Lys Gly Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu
100 105 110
Leu His Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu
115 120 125
Glu Asp Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn
130 135 140
Ser Lys Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg
145 150 155 160
Leu Lys Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr
165 170 175
Ser Asp Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala
180 185 190
Tyr His Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu
195 200 205
Glu Thr Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe
210 215 220
Gly Trp Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys
225 230 235 240
Thr Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala
245 250 255
Asp Leu Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg
260 265 270
Asp Glu Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu
275 280 285
Asn Val Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys
290 295 300
Glu Ile Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser
305 310 315 320
Thr Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys
325 330 335
Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp
340 345 350
Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln
355 360 365
Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu
370 375 380
Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 58
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 58
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Gly His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys
740 745 750
Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys
755 760 765
Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu
770 775 780
Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr
785 790 795 800
Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys
805 810 815
Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His
820 825 830
His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr
835 840 845
Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn
850 855 860
Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys
865 870 875 880
Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp
885 890 895
Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro
900 905 910
Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr
915 920 925
Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn
930 935 940
Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln
945 950 955 960
Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn
965 970 975
Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg
980 985 990
Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn
995 1000 1005
Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys
1010 1015 1020
Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035 1040
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly Gly Ser
1045 1050 1055
<210> 59
<211> 1058
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 59
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Gly Gly Val His Asn Val Asn Glu Val Glu
115 120 125
Glu Asp Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn
130 135 140
Ser Lys Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg
145 150 155 160
Leu Lys Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr
165 170 175
Ser Asp Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala
180 185 190
Tyr His Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu
195 200 205
Glu Thr Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe
210 215 220
Gly Trp Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys
225 230 235 240
Thr Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala
245 250 255
Asp Leu Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg
260 265 270
Asp Glu Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu
275 280 285
Asn Val Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys
290 295 300
Glu Ile Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser
305 310 315 320
Thr Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys
325 330 335
Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp
340 345 350
Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln
355 360 365
Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu
370 375 380
Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 60
<211> 1058
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 60
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Gly Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn
130 135 140
Ser Lys Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg
145 150 155 160
Leu Lys Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr
165 170 175
Ser Asp Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala
180 185 190
Tyr His Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu
195 200 205
Glu Thr Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe
210 215 220
Gly Trp Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys
225 230 235 240
Thr Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala
245 250 255
Asp Leu Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg
260 265 270
Asp Glu Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu
275 280 285
Asn Val Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys
290 295 300
Glu Ile Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser
305 310 315 320
Thr Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys
325 330 335
Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp
340 345 350
Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln
355 360 365
Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu
370 375 380
Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 61
<211> 1058
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 61
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Gly Gly Ser Pro Phe
210 215 220
Gly Trp Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys
225 230 235 240
Thr Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala
245 250 255
Asp Leu Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg
260 265 270
Asp Glu Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu
275 280 285
Asn Val Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys
290 295 300
Glu Ile Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser
305 310 315 320
Thr Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys
325 330 335
Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp
340 345 350
Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln
355 360 365
Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu
370 375 380
Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 62
<211> 1058
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 62
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Gly Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys
325 330 335
Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp
340 345 350
Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln
355 360 365
Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu
370 375 380
Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 63
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 63
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Gly Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys
740 745 750
Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys
755 760 765
Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu
770 775 780
Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr
785 790 795 800
Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys
805 810 815
Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His
820 825 830
His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr
835 840 845
Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn
850 855 860
Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys
865 870 875 880
Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp
885 890 895
Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro
900 905 910
Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr
915 920 925
Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn
930 935 940
Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln
945 950 955 960
Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn
965 970 975
Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg
980 985 990
Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn
995 1000 1005
Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys
1010 1015 1020
Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035 1040
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly Gly Ser
1045 1050 1055
<210> 64
<211> 1058
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 64
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Gly Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 65
<211> 1058
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 65
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Gly Gly Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 66
<211> 1058
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 66
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Gly Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 67
<211> 1058
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 67
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Gly Gly Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 68
<211> 1058
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 68
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Gly Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 69
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 69
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Gly Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys
740 745 750
Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys
755 760 765
Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu
770 775 780
Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr
785 790 795 800
Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys
805 810 815
Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His
820 825 830
His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr
835 840 845
Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn
850 855 860
Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys
865 870 875 880
Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp
885 890 895
Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro
900 905 910
Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr
915 920 925
Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn
930 935 940
Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln
945 950 955 960
Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn
965 970 975
Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg
980 985 990
Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn
995 1000 1005
Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys
1010 1015 1020
Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035 1040
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly Gly Ser
1045 1050 1055
<210> 70
<211> 1058
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 70
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Gly Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 71
<211> 1058
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 71
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Gly Gly Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 72
<211> 1369
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 72
Met Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser
1 5 10 15
Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys
20 25 30
Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu
35 40 45
Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg
50 55 60
Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile
65 70 75 80
Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp
85 90 95
Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys
100 105 110
Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala
115 120 125
Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val
130 135 140
Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala
145 150 155 160
His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn
165 170 175
Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr
180 185 190
Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp
195 200 205
Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu
210 215 220
Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly
225 230 235 240
Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn
245 250 255
Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr
260 265 270
Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala
275 280 285
Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser
290 295 300
Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala
305 310 315 320
Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu
325 330 335
Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe
340 345 350
Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala
355 360 365
Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met
370 375 380
Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu
385 390 395 400
Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His
405 410 415
Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro
420 425 430
Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg
435 440 445
Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala
450 455 460
Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu
465 470 475 480
Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met
485 490 495
Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His
500 505 510
Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val
515 520 525
Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu
530 535 540
Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val
545 550 555 560
Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe
565 570 575
Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu
580 585 590
Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu
595 600 605
Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu
610 615 620
Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr
625 630 635 640
Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg
645 650 655
Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg
660 665 670
Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
675 680 685
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr
690 695 700
Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser
705 710 715 720
Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys
725 730 735
Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met
740 745 750
Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn
755 760 765
Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg
770 775 780
Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His
785 790 795 800
Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr
805 810 815
Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn
820 825 830
Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu
835 840 845
Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn
850 855 860
Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met
865 870 875 880
Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg
885 890 895
Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu
900 905 910
Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
915 920 925
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr
930 935 940
Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys
945 950 955 960
Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val
965 970 975
Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
980 985 990
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu
995 1000 1005
Phe 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 Tyr
1025 1030 1035 1040
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly
1045 1050 1055
Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu
1060 1065 1070
Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu
1075 1080 1085
Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly
1090 1095 1100
Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu
1105 1110 1115 1120
Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp
1125 1130 1135
Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys
1140 1145 1150
Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1155 1160 1165
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu
1170 1175 1180
Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro
1185 1190 1195 1200
Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala
1205 1210 1215
Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1220 1225 1230
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly
1235 1240 1245
Ser 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 Arg
1265 1270 1275 1280
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn
1285 1290 1295
Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His
1300 1305 1310
Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe
1315 1320 1325
Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu
1330 1335 1340
Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg
1345 1350 1355 1360
Ile Asp Leu Ser Gln Leu Gly Gly Asp
1365
<210> 73
<211> 1082
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 73
Met Ala Ala Phe Lys Pro Asn Pro Ile Asn Tyr Ile Leu Gly Leu Asp
1 5 10 15
Ile Gly Ile Ala Ser Val Gly Trp Ala Met Val Glu Ile Asp Glu Glu
20 25 30
Glu Asn Pro Ile Arg Leu Ile Asp Leu Gly Val Arg Val Phe Glu Arg
35 40 45
Ala Glu Val Pro Lys Thr Gly Asp Ser Leu Ala Met Ala Arg Arg Leu
50 55 60
Ala Arg Ser Val Arg Arg Leu Thr Arg Arg Arg Ala His Arg Leu Leu
65 70 75 80
Arg Ala Arg Arg Leu Leu Lys Arg Glu Gly Val Leu Gln Ala Ala Asp
85 90 95
Phe Asp Glu Asn Gly Leu Ile Lys Ser Leu Pro Asn Thr Pro Trp Gln
100 105 110
Leu Arg Ala Ala Ala Leu Asp Arg Lys Leu Thr Pro Leu Glu Trp Ser
115 120 125
Ala Val Leu Leu His Leu Ile Lys His Arg Gly Tyr Leu Ser Gln Arg
130 135 140
Lys Asn Glu Gly Glu Thr Ala Asp Lys Glu Leu Gly Ala Leu Leu Lys
145 150 155 160
Gly Val Ala Asn Asn Ala His Ala Leu Gln Thr Gly Asp Phe Arg Thr
165 170 175
Pro Ala Glu Leu Ala Leu Asn Lys Phe Glu Lys Glu Ser Gly His Ile
180 185 190
Arg Asn Gln Arg Gly Asp Tyr Ser His Thr Phe Ser Arg Lys Asp Leu
195 200 205
Gln Ala Glu Leu Ile Leu Leu Phe Glu Lys Gln Lys Glu Phe Gly Asn
210 215 220
Pro His Val Ser Gly Gly Leu Lys Glu Gly Ile Glu Thr Leu Leu Met
225 230 235 240
Thr Gln Arg Pro Ala Leu Ser Gly Asp Ala Val Gln Lys Met Leu Gly
245 250 255
His Cys Thr Phe Glu Pro Ala Glu Pro Lys Ala Ala Lys Asn Thr Tyr
260 265 270
Thr Ala Glu Arg Phe Ile Trp Leu Thr Lys Leu Asn Asn Leu Arg Ile
275 280 285
Leu Glu Gln Gly Ser Glu Arg Pro Leu Thr Asp Thr Glu Arg Ala Thr
290 295 300
Leu Met Asp Glu Pro Tyr Arg Lys Ser Lys Leu Thr Tyr Ala Gln Ala
305 310 315 320
Arg Lys Leu Leu Gly Leu Glu Asp Thr Ala Phe Phe Lys Gly Leu Arg
325 330 335
Tyr Gly Lys Asp Asn Ala Glu Ala Ser Thr Leu Met Glu Met Lys Ala
340 345 350
Tyr His Ala Ile Ser Arg Ala Leu Glu Lys Glu Gly Leu Lys Asp Lys
355 360 365
Lys Ser Pro Leu Asn Leu Ser Ser Glu Leu Gln Asp Glu Ile Gly Thr
370 375 380
Ala Phe Ser Leu Phe Lys Thr Asp Glu Asp Ile Thr Gly Arg Leu Lys
385 390 395 400
Asp Arg Val Gln Pro Glu Ile Leu Glu Ala Leu Leu Lys His Ile Ser
405 410 415
Phe Asp Lys Phe Val Gln Ile Ser Leu Lys Ala Leu Arg Arg Ile Val
420 425 430
Pro Leu Met Glu Gln Gly Lys Arg Tyr Asp Glu Ala Cys Ala Glu Ile
435 440 445
Tyr Gly Asp His Tyr Gly Lys Lys Asn Thr Glu Glu Lys Ile Tyr Leu
450 455 460
Pro Pro Ile Pro Ala Asp Glu Ile Arg Asn Pro Val Val Leu Arg Ala
465 470 475 480
Leu Ser Gln Ala Arg Lys Val Ile Asn Gly Val Val Arg Arg Tyr Gly
485 490 495
Ser Pro Ala Arg Ile His Ile Glu Thr Ala Arg Glu Val Gly Lys Ser
500 505 510
Phe Lys Asp Arg Lys Glu Ile Glu Lys Arg Gln Glu Glu Asn Arg Lys
515 520 525
Asp Arg Glu Lys Ala Ala Ala Lys Phe Arg Glu Tyr Phe Pro Asn Phe
530 535 540
Val Gly Glu Pro Lys Ser Lys Asp Ile Leu Lys Leu Arg Leu Tyr Glu
545 550 555 560
Gln Gln His Gly Lys Cys Leu Tyr Ser Gly Lys Glu Ile Asn Leu Val
565 570 575
Arg Leu Asn Glu Lys Gly Tyr Val Glu Ile Asp His Ala Leu Pro Phe
580 585 590
Ser Arg Thr Trp Asp Asp Ser Phe Asn Asn Lys Val Leu Val Leu Gly
595 600 605
Ser Glu Asn Gln Asn Lys Gly Asn Gln Thr Pro Tyr Glu Tyr Phe Asn
610 615 620
Gly Lys Asp Asn Ser Arg Glu Trp Gln Glu Phe Lys Ala Arg Val Glu
625 630 635 640
Thr Ser Arg Phe Pro Arg Ser Lys Lys Gln Arg Ile Leu Leu Gln Lys
645 650 655
Phe Asp Glu Asp Gly Phe Lys Glu Cys Asn Leu Asn Asp Thr Arg Tyr
660 665 670
Val Asn Arg Phe Leu Cys Gln Phe Val Ala Asp His Ile Leu Leu Thr
675 680 685
Gly Lys Gly Lys Arg Arg Val Phe Ala Ser Asn Gly Gln Ile Thr Asn
690 695 700
Leu Leu Arg Gly Phe Trp Gly Leu Arg Lys Val Arg Ala Glu Asn Asp
705 710 715 720
Arg His His Ala Leu Asp Ala Val Val Val Ala Cys Ser Thr Val Ala
725 730 735
Met Gln Gln Lys Ile Thr Arg Phe Val Arg Tyr Lys Glu Met Asn Ala
740 745 750
Phe Asp Gly Lys Thr Ile Asp Lys Glu Thr Gly Lys Val Leu His Gln
755 760 765
Lys Thr His Phe Pro Gln Pro Trp Glu Phe Phe Ala Gln Glu Val Met
770 775 780
Ile Arg Val Phe Gly Lys Pro Asp Gly Lys Pro Glu Phe Glu Glu Ala
785 790 795 800
Asp Thr Pro Glu Lys Leu Arg Thr Leu Leu Ala Glu Lys Leu Ser Ser
805 810 815
Arg Pro Glu Ala Val His Glu Tyr Val Thr Pro Leu Phe Val Ser Arg
820 825 830
Ala Pro Asn Arg Lys Met Ser Gly Ala His Lys Asp Thr Leu Arg Ser
835 840 845
Ala Lys Arg Phe Val Lys His Asn Glu Lys Ile Ser Val Lys Arg Val
850 855 860
Trp Leu Thr Glu Ile Lys Leu Ala Asp Leu Glu Asn Met Val Asn Tyr
865 870 875 880
Lys Asn Gly Arg Glu Ile Glu Leu Tyr Glu Ala Leu Lys Ala Arg Leu
885 890 895
Glu Ala Tyr Gly Gly Asn Ala Lys Gln Ala Phe Asp Pro Lys Asp Asn
900 905 910
Pro Phe Tyr Lys Lys Gly Gly Gln Leu Val Lys Ala Val Arg Val Glu
915 920 925
Lys Thr Gln Glu Ser Gly Val Leu Leu Asn Lys Lys Asn Ala Tyr Thr
930 935 940
Ile Ala Asp Asn Gly Asp Met Val Arg Val Asp Val Phe Cys Lys Val
945 950 955 960
Asp Lys Lys Gly Lys Asn Gln Tyr Phe Ile Val Pro Ile Tyr Ala Trp
965 970 975
Gln Val Ala Glu Asn Ile Leu Pro Asp Ile Asp Cys Lys Gly Tyr Arg
980 985 990
Ile Asp Asp Ser Tyr Thr Phe Cys Phe Ser Leu His Lys Tyr Asp Leu
995 1000 1005
Ile Ala Phe Gln Lys Asp Glu Lys Ser Lys Val Glu Phe Ala Tyr Tyr
1010 1015 1020
Ile Asn Cys Asp Ser Ser Asn Gly Arg Phe Tyr Leu Ala Trp His Asp
1025 1030 1035 1040
Lys Gly Ser Lys Glu Gln Gln Phe Arg Ile Ser Thr Gln Asn Leu Val
1045 1050 1055
Leu Ile Gln Lys Tyr Gln Val Asn Glu Leu Gly Lys Glu Ile Arg Pro
1060 1065 1070
Cys Arg Leu Lys Lys Arg Pro Pro Val Arg
1075 1080
<210> 74
<211> 1307
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 74
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
<210> 75
<211> 1228
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 75
Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr
1 5 10 15
Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp
20 25 30
Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys
35 40 45
Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp
50 55 60
Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu
65 70 75 80
Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn
85 90 95
Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn
100 105 110
Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu
115 120 125
Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe
130 135 140
Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn
145 150 155 160
Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile
165 170 175
Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys
180 185 190
Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys
195 200 205
Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe
210 215 220
Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile
225 230 235 240
Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn
245 250 255
Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys
260 265 270
Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser
275 280 285
Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe
290 295 300
Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys
305 310 315 320
Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile
325 330 335
Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe
340 345 350
Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp
355 360 365
Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp
370 375 380
Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu
385 390 395 400
Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu
405 410 415
Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser
420 425 430
Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys
435 440 445
Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys
450 455 460
Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr
465 470 475 480
Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile
485 490 495
Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr
500 505 510
Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro
515 520 525
Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala
530 535 540
Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys
545 550 555 560
Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly
565 570 575
Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met
580 585 590
Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro
595 600 605
Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly
610 615 620
Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys
625 630 635 640
Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn
645 650 655
Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu
660 665 670
Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys
675 680 685
Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile
690 695 700
Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His
705 710 715 720
Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile
725 730 735
Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys
740 745 750
Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys
755 760 765
Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr
770 775 780
Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile
785 790 795 800
Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
805 810 815
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp
820 825 830
Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
835 840 845
Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
850 855 860
Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu
865 870 875 880
Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile
885 890 895
Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys
900 905 910
Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
915 920 925
Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
930 935 940
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys
945 950 955 960
Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
965 970 975
Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
980 985 990
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser
1010 1015 1020
Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu Glu
1025 1030 1035 1040
Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp
1045 1050 1055
Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg Ile
1060 1065 1070
Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe Asp Trp Glu Glu
1075 1080 1085
Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys Tyr Gly Ile
1090 1095 1100
Asn Tyr Gln Gln Gly Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser Asp
1105 1110 1115 1120
Lys Ala Phe Tyr Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu Gln
1125 1130 1135
Met Arg Asn Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu Ile Ser
1140 1145 1150
Pro Val Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu
1155 1160 1165
Ala Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala
1170 1175 1180
Tyr Asn Ile Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys
1185 1190 1195 1200
Ala Glu Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys
1205 1210 1215
Glu Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 76
<211> 986
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 76
Met Glu Lys Arg Ile Asn Lys Ile Arg Lys Lys Leu Ser Ala Asp Asn
1 5 10 15
Ala Thr Lys Pro Val Ser Arg Ser Gly Pro Met Lys Thr Leu Leu Val
20 25 30
Arg Val Met Thr Asp Asp Leu Lys Lys Arg Leu Glu Lys Arg Arg Lys
35 40 45
Lys Pro Glu Val Met Pro Gln Val Ile Ser Asn Asn Ala Ala Asn Asn
50 55 60
Leu Arg Met Leu Leu Asp Asp Tyr Thr Lys Met Lys Glu Ala Ile Leu
65 70 75 80
Gln Val Tyr Trp Gln Glu Phe Lys Asp Asp His Val Gly Leu Met Cys
85 90 95
Lys Phe Ala Gln Pro Ala Ser Lys Lys Ile Asp Gln Asn Lys Leu Lys
100 105 110
Pro Glu Met Asp Glu Lys Gly Asn Leu Thr Thr Ala Gly Phe Ala Cys
115 120 125
Ser Gln Cys Gly Gln Pro Leu Phe Val Tyr Lys Leu Glu Gln Val Ser
130 135 140
Glu Lys Gly Lys Ala Tyr Thr Asn Tyr Phe Gly Arg Cys Asn Val Ala
145 150 155 160
Glu His Glu Lys Leu Ile Leu Leu Ala Gln Leu Lys Pro Glu Lys Asp
165 170 175
Ser Asp Glu Ala Val Thr Tyr Ser Leu Gly Lys Phe Gly Gln Arg Ala
180 185 190
Leu Asp Phe Tyr Ser Ile His Val Thr Lys Glu Ser Thr His Pro Val
195 200 205
Lys Pro Leu Ala Gln Ile Ala Gly Asn Arg Tyr Ala Ser Gly Pro Val
210 215 220
Gly Lys Ala Leu Ser Asp Ala Cys Met Gly Thr Ile Ala Ser Phe Leu
225 230 235 240
Ser Lys Tyr Gln Asp Ile Ile Ile Glu His Gln Lys Val Val Lys Gly
245 250 255
Asn Gln Lys Arg Leu Glu Ser Leu Arg Glu Leu Ala Gly Lys Glu Asn
260 265 270
Leu Glu Tyr Pro Ser Val Thr Leu Pro Pro Gln Pro His Thr Lys Glu
275 280 285
Gly Val Asp Ala Tyr Asn Glu Val Ile Ala Arg Val Arg Met Trp Val
290 295 300
Asn Leu Asn Leu Trp Gln Lys Leu Lys Leu Ser Arg Asp Asp Ala Lys
305 310 315 320
Pro Leu Leu Arg Leu Lys Gly Phe Pro Ser Phe Pro Val Val Glu Arg
325 330 335
Arg Glu Asn Glu Val Asp Trp Trp Asn Thr Ile Asn Glu Val Lys Lys
340 345 350
Leu Ile Asp Ala Lys Arg Asp Met Gly Arg Val Phe Trp Ser Gly Val
355 360 365
Thr Ala Glu Lys Arg Asn Thr Ile Leu Glu Gly Tyr Asn Tyr Leu Pro
370 375 380
Asn Glu Asn Asp His Lys Lys Arg Glu Gly Ser Leu Glu Asn Pro Lys
385 390 395 400
Lys Pro Ala Lys Arg Gln Phe Gly Asp Leu Leu Leu Tyr Leu Glu Lys
405 410 415
Lys Tyr Ala Gly Asp Trp Gly Lys Val Phe Asp Glu Ala Trp Glu Arg
420 425 430
Ile Asp Lys Lys Ile Ala Gly Leu Thr Ser His Ile Glu Arg Glu Glu
435 440 445
Ala Arg Asn Ala Glu Asp Ala Gln Ser Lys Ala Val Leu Thr Asp Trp
450 455 460
Leu Arg Ala Lys Ala Ser Phe Val Leu Glu Arg Leu Lys Glu Met Asp
465 470 475 480
Glu Lys Glu Phe Tyr Ala Cys Glu Ile Gln Leu Gln Lys Trp Tyr Gly
485 490 495
Asp Leu Arg Gly Asn Pro Phe Ala Val Glu Ala Glu Asn Arg Val Val
500 505 510
Asp Ile Ser Gly Phe Ser Ile Gly Ser Asp Gly His Ser Ile Gln Tyr
515 520 525
Arg Asn Leu Leu Ala Trp Lys Tyr Leu Glu Asn Gly Lys Arg Glu Phe
530 535 540
Tyr Leu Leu Met Asn Tyr Gly Lys Lys Gly Arg Ile Arg Phe Thr Asp
545 550 555 560
Gly Thr Asp Ile Lys Lys Ser Gly Lys Trp Gln Gly Leu Leu Tyr Gly
565 570 575
Gly Gly Lys Ala Lys Val Ile Asp Leu Thr Phe Asp Pro Asp Asp Glu
580 585 590
Gln Leu Ile Ile Leu Pro Leu Ala Phe Gly Thr Arg Gln Gly Arg Glu
595 600 605
Phe Ile Trp Asn Asp Leu Leu Ser Leu Glu Thr Gly Leu Ile Lys Leu
610 615 620
Ala Asn Gly Arg Val Ile Glu Lys Thr Ile Tyr Asn Lys Lys Ile Gly
625 630 635 640
Arg Asp Glu Pro Ala Leu Phe Val Ala Leu Thr Phe Glu Arg Arg Glu
645 650 655
Val Val Asp Pro Ser Asn Ile Lys Pro Val Asn Leu Ile Gly Val Asp
660 665 670
Arg Gly Glu Asn Ile Pro Ala Val Ile Ala Leu Thr Asp Pro Glu Gly
675 680 685
Cys Pro Leu Pro Glu Phe Lys Asp Ser Ser Gly Gly Pro Thr Asp Ile
690 695 700
Leu Arg Ile Gly Glu Gly Tyr Lys Glu Lys Gln Arg Ala Ile Gln Ala
705 710 715 720
Ala Lys Glu Val Glu Gln Arg Arg Ala Gly Gly Tyr Ser Arg Lys Phe
725 730 735
Ala Ser Lys Ser Arg Asn Leu Ala Asp Asp Met Val Arg Asn Ser Ala
740 745 750
Arg Asp Leu Phe Tyr His Ala Val Thr His Asp Ala Val Leu Val Phe
755 760 765
Glu Asn Leu Ser Arg Gly Phe Gly Arg Gln Gly Lys Arg Thr Phe Met
770 775 780
Thr Glu Arg Gln Tyr Thr Lys Met Glu Asp Trp Leu Thr Ala Lys Leu
785 790 795 800
Ala Tyr Glu Gly Leu Thr Ser Lys Thr Tyr Leu Ser Lys Thr Leu Ala
805 810 815
Gln Tyr Thr Ser Lys Thr Cys Ser Asn Cys Gly Phe Thr Ile Thr Thr
820 825 830
Ala Asp Tyr Asp Gly Met Leu Val Arg Leu Lys Lys Thr Ser Asp Gly
835 840 845
Trp Ala Thr Thr Leu Asn Asn Lys Glu Leu Lys Ala Glu Gly Gln Ile
850 855 860
Thr Tyr Tyr Asn Arg Tyr Lys Arg Gln Thr Val Glu Lys Glu Leu Ser
865 870 875 880
Ala Glu Leu Asp Arg Leu Ser Glu Glu Ser Gly Asn Asn Asp Ile Ser
885 890 895
Lys Trp Thr Lys Gly Arg Arg Asp Glu Ala Leu Phe Leu Leu Lys Lys
900 905 910
Arg Phe Ser His Arg Pro Val Gln Glu Gln Phe Val Cys Leu Asp Cys
915 920 925
Gly His Glu Val His Ala Asp Glu Gln Ala Ala Leu Asn Ile Ala Arg
930 935 940
Ser Trp Leu Phe Leu Asn Ser Asn Ser Thr Glu Phe Lys Ser Tyr Lys
945 950 955 960
Ser Gly Lys Gln Pro Phe Val Gly Ala Trp Gln Ala Phe Tyr Lys Arg
965 970 975
Arg Leu Lys Glu Val Trp Lys Pro Asn Ala
980 985
<210> 77
<211> 978
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 77
Met Gln Glu Ile Lys Arg Ile Asn Lys Ile Arg Arg Arg Leu Val Lys
1 5 10 15
Asp Ser Asn Thr Lys Lys Ala Gly Lys Thr Gly Pro Met Lys Thr Leu
20 25 30
Leu Val Arg Val Met Thr Pro Asp Leu Arg Glu Arg Leu Glu Asn Leu
35 40 45
Arg Lys Lys Pro Glu Asn Ile Pro Gln Pro Ile Ser Asn Thr Ser Arg
50 55 60
Ala Asn Leu Asn Lys Leu Leu Thr Asp Tyr Thr Glu Met Lys Lys Ala
65 70 75 80
Ile Leu His Val Tyr Trp Glu Glu Phe Gln Lys Asp Pro Val Gly Leu
85 90 95
Met Ser Arg Val Ala Gln Pro Ala Pro Lys Asn Ile Asp Gln Arg Lys
100 105 110
Leu Ile Pro Val Lys Asp Gly Asn Glu Arg Leu Thr Ser Ser Gly Phe
115 120 125
Ala Cys Ser Gln Cys Cys Gln Pro Leu Tyr Val Tyr Lys Leu Glu Gln
130 135 140
Val Asn Asp Lys Gly Lys Pro His Thr Asn Tyr Phe Gly Arg Cys Asn
145 150 155 160
Val Ser Glu His Glu Arg Leu Ile Leu Leu Ser Pro His Lys Pro Glu
165 170 175
Ala Asn Asp Glu Leu Val Thr Tyr Ser Leu Gly Lys Phe Gly Gln Arg
180 185 190
Ala Leu Asp Phe Tyr Ser Ile His Val Thr Arg Glu Ser Asn His Pro
195 200 205
Val Lys Pro Leu Glu Gln Ile Gly Gly Asn Ser Cys Ala Ser Gly Pro
210 215 220
Val Gly Lys Ala Leu Ser Asp Ala Cys Met Gly Ala Val Ala Ser Phe
225 230 235 240
Leu Thr Lys Tyr Gln Asp Ile Ile Leu Glu His Gln Lys Val Ile Lys
245 250 255
Lys Asn Glu Lys Arg Leu Ala Asn Leu Lys Asp Ile Ala Ser Ala Asn
260 265 270
Gly Leu Ala Phe Pro Lys Ile Thr Leu Pro Pro Gln Pro His Thr Lys
275 280 285
Glu Gly Ile Glu Ala Tyr Asn Asn Val Val Ala Gln Ile Val Ile Trp
290 295 300
Val Asn Leu Asn Leu Trp Gln Lys Leu Lys Ile Gly Arg Asp Glu Ala
305 310 315 320
Lys Pro Leu Gln Arg Leu Lys Gly Phe Pro Ser Phe Pro Leu Val Glu
325 330 335
Arg Gln Ala Asn Glu Val Asp Trp Trp Asp Met Val Cys Asn Val Lys
340 345 350
Lys Leu Ile Asn Glu Lys Lys Glu Asp Gly Lys Val Phe Trp Gln Asn
355 360 365
Leu Ala Gly Tyr Lys Arg Gln Glu Ala Leu Leu Pro Tyr Leu Ser Ser
370 375 380
Glu Glu Asp Arg Lys Lys Gly Lys Lys Phe Ala Arg Tyr Gln Phe Gly
385 390 395 400
Asp Leu Leu Leu His Leu Glu Lys Lys His Gly Glu Asp Trp Gly Lys
405 410 415
Val Tyr Asp Glu Ala Trp Glu Arg Ile Asp Lys Lys Val Glu Gly Leu
420 425 430
Ser Lys His Ile Lys Leu Glu Glu Glu Arg Arg Ser Glu Asp Ala Gln
435 440 445
Ser Lys Ala Ala Leu Thr Asp Trp Leu Arg Ala Lys Ala Ser Phe Val
450 455 460
Ile Glu Gly Leu Lys Glu Ala Asp Lys Asp Glu Phe Cys Arg Cys Glu
465 470 475 480
Leu Lys Leu Gln Lys Trp Tyr Gly Asp Leu Arg Gly Lys Pro Phe Ala
485 490 495
Ile Glu Ala Glu Asn Ser Ile Leu Asp Ile Ser Gly Phe Ser Lys Gln
500 505 510
Tyr Asn Cys Ala Phe Ile Trp Gln Lys Asp Gly Val Lys Lys Leu Asn
515 520 525
Leu Tyr Leu Ile Ile Asn Tyr Phe Lys Gly Gly Lys Leu Arg Phe Lys
530 535 540
Lys Ile Lys Pro Glu Ala Phe Glu Ala Asn Arg Phe Tyr Thr Val Ile
545 550 555 560
Asn Lys Lys Ser Gly Glu Ile Val Pro Met Glu Val Asn Phe Asn Phe
565 570 575
Asp Asp Pro Asn Leu Ile Ile Leu Pro Leu Ala Phe Gly Lys Arg Gln
580 585 590
Gly Arg Glu Phe Ile Trp Asn Asp Leu Leu Ser Leu Glu Thr Gly Ser
595 600 605
Leu Lys Leu Ala Asn Gly Arg Val Ile Glu Lys Thr Leu Tyr Asn Arg
610 615 620
Arg Thr Arg Gln Asp Glu Pro Ala Leu Phe Val Ala Leu Thr Phe Glu
625 630 635 640
Arg Arg Glu Val Leu Asp Ser Ser Asn Ile Lys Pro Met Asn Leu Ile
645 650 655
Gly Ile Asp Arg Gly Glu Asn Ile Pro Ala Val Ile Ala Leu Thr Asp
660 665 670
Pro Glu Gly Cys Pro Leu Ser Arg Phe Lys Asp Ser Leu Gly Asn Pro
675 680 685
Thr His Ile Leu Arg Ile Gly Glu Ser Tyr Lys Glu Lys Gln Arg Thr
690 695 700
Ile Gln Ala Ala Lys Glu Val Glu Gln Arg Arg Ala Gly Gly Tyr Ser
705 710 715 720
Arg Lys Tyr Ala Ser Lys Ala Lys Asn Leu Ala Asp Asp Met Val Arg
725 730 735
Asn Thr Ala Arg Asp Leu Leu Tyr Tyr Ala Val Thr Gln Asp Ala Met
740 745 750
Leu Ile Phe Glu Asn Leu Ser Arg Gly Phe Gly Arg Gln Gly Lys Arg
755 760 765
Thr Phe Met Ala Glu Arg Gln Tyr Thr Arg Met Glu Asp Trp Leu Thr
770 775 780
Ala Lys Leu Ala Tyr Glu Gly Leu Pro Ser Lys Thr Tyr Leu Ser Lys
785 790 795 800
Thr Leu Ala Gln Tyr Thr Ser Lys Thr Cys Ser Asn Cys Gly Phe Thr
805 810 815
Ile Thr Ser Ala Asp Tyr Asp Arg Val Leu Glu Lys Leu Lys Lys Thr
820 825 830
Ala Thr Gly Trp Met Thr Thr Ile Asn Gly Lys Glu Leu Lys Val Glu
835 840 845
Gly Gln Ile Thr Tyr Tyr Asn Arg Tyr Lys Arg Gln Asn Val Val Lys
850 855 860
Asp Leu Ser Val Glu Leu Asp Arg Leu Ser Glu Glu Ser Val Asn Asn
865 870 875 880
Asp Ile Ser Ser Trp Thr Lys Gly Arg Ser Gly Glu Ala Leu Ser Leu
885 890 895
Leu Lys Lys Arg Phe Ser His Arg Pro Val Gln Glu Lys Phe Val Cys
900 905 910
Leu Asn Cys Gly Phe Glu Thr His Ala Asp Glu Gln Ala Ala Leu Asn
915 920 925
Ile Ala Arg Ser Trp Leu Phe Leu Arg Ser Gln Glu Tyr Lys Lys Tyr
930 935 940
Gln Thr Asn Lys Thr Thr Gly Asn Thr Asp Lys Arg Ala Phe Val Glu
945 950 955 960
Thr Trp Gln Ser Phe Tyr Arg Lys Lys Leu Lys Glu Val Trp Lys Pro
965 970 975
Ala Val
<210> 78
<211> 757
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 78
Met Pro Lys Pro Ala Val Glu Ser Glu Phe Ser Lys Val Leu Lys Lys
1 5 10 15
His Phe Pro Gly Glu Arg Phe Arg Ser Ser Tyr Met Lys Arg Gly Gly
20 25 30
Lys Ile Leu Ala Ala Gln Gly Glu Glu Ala Val Val Ala Tyr Leu Gln
35 40 45
Gly Lys Ser Glu Glu Glu Pro Pro Asn Phe Gln Pro Pro Ala Lys Cys
50 55 60
His Val Val Thr Lys Ser Arg Asp Phe Ala Glu Trp Pro Ile Met Lys
65 70 75 80
Ala Ser Glu Ala Ile Gln Arg Tyr Ile Tyr Ala Leu Ser Thr Thr Glu
85 90 95
Arg Ala Ala Cys Lys Pro Gly Lys Ser Ser Glu Ser His Ala Ala Trp
100 105 110
Phe Ala Ala Thr Gly Val Ser Asn His Gly Tyr Ser His Val Gln Gly
115 120 125
Leu Asn Leu Ile Phe Asp His Thr Leu Gly Arg Tyr Asp Gly Val Leu
130 135 140
Lys Lys Val Gln Leu Arg Asn Glu Lys Ala Arg Ala Arg Leu Glu Ser
145 150 155 160
Ile Asn Ala Ser Arg Ala Asp Glu Gly Leu Pro Glu Ile Lys Ala Glu
165 170 175
Glu Glu Glu Val Ala Thr Asn Glu Thr Gly His Leu Leu Gln Pro Pro
180 185 190
Gly Ile Asn Pro Ser Phe Tyr Val Tyr Gln Thr Ile Ser Pro Gln Ala
195 200 205
Tyr Arg Pro Arg Asp Glu Ile Val Leu Pro Pro Glu Tyr Ala Gly Tyr
210 215 220
Val Arg Asp Pro Asn Ala Pro Ile Pro Leu Gly Val Val Arg Asn Arg
225 230 235 240
Cys Asp Ile Gln Lys Gly Cys Pro Gly Tyr Ile Pro Glu Trp Gln Arg
245 250 255
Glu Ala Gly Thr Ala Ile Ser Pro Lys Thr Gly Lys Ala Val Thr Val
260 265 270
Pro Gly Leu Ser Pro Lys Lys Asn Lys Arg Met Arg Arg Tyr Trp Arg
275 280 285
Ser Glu Lys Glu Lys Ala Gln Asp Ala Leu Leu Val Thr Val Arg Ile
290 295 300
Gly Thr Asp Trp Val Val Ile Asp Val Arg Gly Leu Leu Arg Asn Ala
305 310 315 320
Arg Trp Arg Thr Ile Ala Pro Lys Asp Ile Ser Leu Asn Ala Leu Leu
325 330 335
Asp Leu Phe Thr Gly Asp Pro Val Ile Asp Val Arg Arg Asn Ile Val
340 345 350
Thr Phe Thr Tyr Thr Leu Asp Ala Cys Gly Thr Tyr Ala Arg Lys Trp
355 360 365
Thr Leu Lys Gly Lys Gln Thr Lys Ala Thr Leu Asp Lys Leu Thr Ala
370 375 380
Thr Gln Thr Val Ala Leu Val Ala Ile Asp Leu Gly Gln Thr Asn Pro
385 390 395 400
Ile Ser Ala Gly Ile Ser Arg Val Thr Gln Glu Asn Gly Ala Leu Gln
405 410 415
Cys Glu Pro Leu Asp Arg Phe Thr Leu Pro Asp Asp Leu Leu Lys Asp
420 425 430
Ile Ser Ala Tyr Arg Ile Ala Trp Asp Arg Asn Glu Glu Glu Leu Arg
435 440 445
Ala Arg Ser Val Glu Ala Leu Pro Glu Ala Gln Gln Ala Glu Val Arg
450 455 460
Ala Leu Asp Gly Val Ser Lys Glu Thr Ala Arg Thr Gln Leu Cys Ala
465 470 475 480
Asp Phe Gly Leu Asp Pro Lys Arg Leu Pro Trp Asp Lys Met Ser Ser
485 490 495
Asn Thr Thr Phe Ile Ser Glu Ala Leu Leu Ser Asn Ser Val Ser Arg
500 505 510
Asp Gln Val Phe Phe Thr Pro Ala Pro Lys Lys Gly Ala Lys Lys Lys
515 520 525
Ala Pro Val Glu Val Met Arg Lys Asp Arg Thr Trp Ala Arg Ala Tyr
530 535 540
Lys Pro Arg Leu Ser Val Glu Ala Gln Lys Leu Lys Asn Glu Ala Leu
545 550 555 560
Trp Ala Leu Lys Arg Thr Ser Pro Glu Tyr Leu Lys Leu Ser Arg Arg
565 570 575
Lys Glu Glu Leu Cys Arg Arg Ser Ile Asn Tyr Val Ile Glu Lys Thr
580 585 590
Arg Arg Arg Thr Gln Cys Gln Ile Val Ile Pro Val Ile Glu Asp Leu
595 600 605
Asn Val Arg Phe Phe His Gly Ser Gly Lys Arg Leu Pro Gly Trp Asp
610 615 620
Asn Phe Phe Thr Ala Lys Lys Glu Asn Arg Trp Phe Ile Gln Gly Leu
625 630 635 640
His Lys Ala Phe Ser Asp Leu Arg Thr His Arg Ser Phe Tyr Val Phe
645 650 655
Glu Val Arg Pro Glu Arg Thr Ser Ile Thr Cys Pro Lys Cys Gly His
660 665 670
Cys Glu Val Gly Asn Arg Asp Gly Glu Ala Phe Gln Cys Leu Ser Cys
675 680 685
Gly Lys Thr Cys Asn Ala Asp Leu Asp Val Ala Thr His Asn Leu Thr
690 695 700
Gln Val Ala Leu Thr Gly Lys Thr Met Pro Lys Arg Glu Glu Pro Arg
705 710 715 720
Asp Ala Gln Gly Thr Ala Pro Ala Arg Lys Thr Lys Lys Ala Ser Lys
725 730 735
Ser Lys Ala Pro Pro Ala Glu Arg Glu Asp Gln Thr Pro Ala Gln Glu
740 745 750
Pro Ser Gln Thr Ser
755
<210> 79
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 79
Pro Lys Lys Lys Arg Lys Val Pro Gly
1 5
<210> 80
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 80
Ala Ser Pro Lys Lys Lys Arg Lys Val
1 5
<210> 81
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 81
Pro Tyr Gly Glu Arg
1 5
<210> 82
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 82
Asp Leu Ser Asn Tyr Asn Pro Tyr Gly Glu Arg
1 5 10
<210> 83
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 83
Pro Gly Gly Glu Arg
1 5
<210> 84
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 84
Gly Asn Leu Gly Ala
1 5
<210> 85
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 85
Leu Gly Asn Gln Gly
1 5
<210> 86
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 86
Pro Asp Gly Arg Trp
1 5
<210> 87
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 87
Ala Ala Gly Asn Ser
1 5
<210> 88
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 88
Ser Lys Asn Asn Pro
1 5
<210> 89
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 89
Asn Ile Gly Thr Gly
1 5
<210> 90
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 90
Ala Gly Asn Leu Gly Ala Pro
1 5
<210> 91
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 91
Leu Gly Asn Gln Gly Phe Thr
1 5
<210> 92
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 92
His Pro Asp Gly Arg Trp
1 5
<210> 93
<211> 28
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 93
Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg Thr Pro Arg Phe
1 5 10 15
Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
20 25
<210> 94
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 94
Ser Lys Asn Asn Pro Ile Ser
1 5
<210> 95
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 95
Lys Asn Ile Gly Thr
1 5
<210> 96
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 96
Gly Asn Gly Gly Ala
1 5
<210> 97
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 97
Gly Asn Leu Gly Gly Gly Ala
1 5
<210> 98
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 98
Gly Gly Asn Gln Gly
1 5
<210> 99
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 99
Leu Gly Gly Gly Asn Gln Gly
1 5
<210> 100
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 100
Pro Asp Gly Arg Gly
1 5
<210> 101
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 101
Pro Asp Gly Gly Gly Arg Trp
1 5
<210> 102
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 102
Ala Ala Gly Gly Gly Asn Ser
1 5
<210> 103
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 103
Gly Gly Ser Lys Asn Asn Pro
1 5
<210> 104
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 104
Asn Gly Gly Thr Gly
1 5
<210> 105
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 105
Asn Ile Gly Gly Gly Thr Gly
1 5
<210> 106
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 106
Asn Pro Gln Thr Gly
1 5
<210> 107
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 107
Arg Ser Asn Lys Glu
1 5
<210> 108
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 108
Asn Lys Gly Glu Asn
1 5
<210> 109
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 109
Ser Gly Ala Arg Gly
1 5
<210> 110
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 110
Gly Lys Gly Lys Ser
1 5
<210> 111
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 111
Gln Asn Gly Lys Arg
1 5
<210> 112
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 112
Glu Asn Arg Lys Lys
1 5
<210> 113
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 113
Asn Pro Thr Gly His
1 5
<210> 114
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 114
Thr Glu Pro His Phe
1 5
<210> 115
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 115
Leu Ser Lys His Pro
1 5
<210> 116
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 116
Asn Leu Gly Asn Tyr
1 5
<210> 117
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 117
Val Thr Gly Ala Ala
1 5
<210> 118
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 118
Ala Ser Gly His Phe
1 5
<210> 119
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 119
Tyr Gly Lys Glu Ser
1 5
<210> 120
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 120
His Asn Asn Asp Pro
1 5
<210> 121
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 121
Lys Asn Gly Glu Pro
1 5
<210> 122
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 122
Ala Asn Gly Gly Leu
1 5
<210> 123
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 123
Gly Val Gly Ser Arg
1 5
<210> 124
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 124
Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu
1 5 10
<210> 125
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 125
Arg Gly Phe Lys Ser Asn Arg Lys Ser Glu Arg Ser Asn
1 5 10
<210> 126
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 126
Asp Pro Lys Phe Ala Leu His Lys Arg Asn Lys Gly Glu Asn Tyr Thr
1 5 10 15
Asn Thr Ile Ala
20
<210> 127
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 127
Ser Pro Ser Gly Ala Arg Gly Leu Thr
1 5
<210> 128
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 128
Gly Lys Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp
1 5 10
<210> 129
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 129
Glu Gln Asn Gly Lys Arg Met
1 5
<210> 130
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 130
Tyr Gly Leu Thr Leu Asn Pro Thr
1 5
<210> 131
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 131
Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His
1 5 10
<210> 132
<211> 3
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 132
Lys His Pro
1
<210> 133
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 133
Leu Asn Leu Gly Asn Tyr Asp
1 5
<210> 134
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 134
Met Pro Lys Arg Ser Val Thr Gly
1 5
<210> 135
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 135
Ile Lys Leu Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser
1 5 10 15
Asp
<210> 136
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 136
His Asn Asn Asp
1
<210> 137
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 137
Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly Pro Val
1 5 10 15
<210> 138
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 138
Asn Pro Gln Thr Gly Gly Gly
1 5
<210> 139
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 139
Arg Gly Gly Ser Asn Lys Glu
1 5
<210> 140
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 140
Asn Lys Gly Gly Gly Glu Asn
1 5
<210> 141
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 141
Ser Gly Gly Gly Ala Arg Gly
1 5
<210> 142
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 142
Gly Lys Gly Gly Gly Lys Ser
1 5
<210> 143
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 143
Gln Asn Gly Gly Gly Lys Arg
1 5
<210> 144
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 144
Glu Gly Gly Asn Arg Lys Lys
1 5
<210> 145
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 145
Asn Pro Thr Gly Gly Gly His
1 5
<210> 146
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 146
Thr Glu Pro His Gly
1 5
<210> 147
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 147
Thr Glu Pro Gly Gly His Phe
1 5
<210> 148
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 148
Gly Ser Lys His Pro
1 5
<210> 149
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 149
Leu Gly Gly Ser Lys His Pro
1 5
<210> 150
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 150
Asn Gly Gly Asn Tyr
1 5
<210> 151
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 151
Asn Leu Gly Asn Gly
1 5
<210> 152
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 152
Asn Leu Gly Gly Gly Asn Tyr
1 5
<210> 153
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 153
Gly Thr Gly Ala Ala
1 5
<210> 154
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 154
Val Thr Gly Gly Gly Ala Ala
1 5
<210> 155
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 155
Ala Ser Gly His Gly
1 5
<210> 156
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 156
Ala Ser Gly Gly Gly His Phe
1 5
<210> 157
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 157
Gly Gly Lys Glu Ser
1 5
<210> 158
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 158
Tyr Gly Gly Gly Lys Glu Ser
1 5
<210> 159
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 159
His Asn Gly Gly Asn Asp Pro
1 5
<210> 160
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 160
Lys Asn Gly Gly Gly Glu Pro
1 5
<210> 161
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 161
Ala Asn Gly Gly Gly
1 5
<210> 162
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 162
Ala Asn Gly Gly Gly Gly Leu
1 5
<210> 163
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 163
Gly Gly Gly Ser Arg
1 5
<210> 164
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 164
Gly Val Gly Gly Gly Ser Arg
1 5
<210> 165
<211> 23
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 165
atttttgtgc ccatcgttgg cac 23
<210> 166
<211> 23
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 166
agaaatccgt ctttcattga cgg 23
<210> 167
<211> 35
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 167
atttaccctg acgatagagt gcaacttcac aagct 35
<210> 168
<211> 35
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 168
agcttgtgaa gttgcactct atcgtcaggg taaat 35
<210> 169
<211> 35
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 169
ggttctgatt ggagttgtcc aggtttttgg aagct 35
<210> 170
<211> 35
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 170
agcttccaaa aacctggaca actccaatca gaacc 35
<210> 171
<211> 23
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 171
atttttgtgc ccatcgttgg cac 23
<210> 172
<211> 23
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 172
atctttgtgc ccatcgttgg cac 23
<210> 173
<211> 23
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 173
attcttgtgc ccatcgttgg cac 23
<210> 174
<211> 23
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 174
ataattgtgc ccatcgttgg cac 23
<210> 175
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 175
Asn Glu Gly Arg Arg
1 5
<210> 176
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 176
Leu Ser Gly Ile Asn
1 5
<210> 177
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 177
Arg Arg Gly Val His
1 5
<210> 178
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 178
Asp Thr Gly Asn Glu
1 5
<210> 179
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 179
Pro Gly Glu Gly Ser
1 5
<210> 180
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 180
Ser Thr Gly Lys Pro
1 5
<210> 181
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 181
Leu Lys Gly Tyr Thr
1 5
<210> 182
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 182
Lys Arg Asn Arg Gln
1 5
<210> 183
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 183
Lys Lys Gly Asn Arg
1 5
<210> 184
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 184
Ser Ser Ser Asp Ser
1 5
<210> 185
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 185
Lys Gly Lys Gly Arg
1 5
<210> 186
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 186
Asn Lys Gly Tyr Lys
1 5
<210> 187
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 187
Ala Glu Ser Met Pro
1 5
<210> 188
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 188
Asn Leu Leu Thr Asp His Ser Glu Leu Ser Gly Ile
1 5 10
<210> 189
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 189
Arg Gly Val His
1
<210> 190
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 190
Glu Glu Asp Thr Gly Asn Glu Leu
1 5
<210> 191
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 191
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
1 5 10 15
Lys
<210> 192
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 192
Gly Tyr Arg Val Thr Ser Thr Gly Lys Pro Glu Phe Thr Asn
1 5 10
<210> 193
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 193
Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu
1 5 10
<210> 194
<211> 2
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 194
Asn Arg
1
<210> 195
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 195
Ser Ser Asp Ser Lys Ile
1 5
<210> 196
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 196
Lys Gly Lys Gly Arg Ile
1 5
<210> 197
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 197
Lys Phe Lys Lys Glu Arg Asn Lys Gly
1 5
<210> 198
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 198
Asn Gln Met Phe Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu
1 5 10 15
<210> 199
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 199
Asn Glu Gly Gly Gly Arg Arg
1 5
<210> 200
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 200
Gly Ser Gly Ile Asn
1 5
<210> 201
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 201
Leu Ser Gly Gly Asn
1 5
<210> 202
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 202
Leu Ser Gly Gly Gly Ile Asn
1 5
<210> 203
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 203
Arg Arg Gly Gly His
1 5
<210> 204
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 204
Arg Arg Gly Gly Gly Val His
1 5
<210> 205
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 205
Asp Thr Gly Gly Gly Asn Glu
1 5
<210> 206
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 206
Pro Gly Glu Gly Gly Gly Ser
1 5
<210> 207
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 207
Ser Thr Gly Gly Gly Lys Pro
1 5
<210> 208
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 208
Gly Lys Gly Tyr Thr
1 5
<210> 209
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 209
Leu Lys Gly Gly Thr
1 5
<210> 210
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 210
Leu Lys Gly Gly Gly Tyr Thr
1 5
<210> 211
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 211
Lys Arg Gly Gly Asn Arg Gln
1 5
<210> 212
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 212
Lys Lys Gly Gly Gly Asn Arg
1 5
<210> 213
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 213
Ser Ser Gly Gly Ser Asp Ser
1 5
<210> 214
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 214
Lys Gly Lys Gly Gly Gly Arg
1 5
<210> 215
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 215
Asn Lys Gly Gly Lys
1 5
<210> 216
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 216
Asn Lys Gly Gly Gly Tyr Lys
1 5
<210> 217
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 217
Ala Glu Gly Gly Ser Met Pro
1 5
<210> 218
<211> 229
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 218
Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg
1 5 10 15
Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu
20 25 30
Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His
35 40 45
Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val
50 55 60
Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr
65 70 75 80
Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys
85 90 95
Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu
100 105 110
Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg
115 120 125
Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met
130 135 140
Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser
145 150 155 160
Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg
165 170 175
Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys
180 185 190
Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile
195 200 205
Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp
210 215 220
Ala Thr Gly Leu Lys
225
<210> 219
<211> 167
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 219
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 Arg Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala
35 40 45
Ile Gly Leu 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 Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr 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 Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp
165
<210> 220
<211> 677
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 220
Thr Leu Asn Ile Glu Asp Glu Tyr Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro
675
<210> 221
<211> 677
<212> PRT
<213> 人工序列
<220>
<223> 人工的
<400> 221
Thr Leu Asn Ile Glu Asp Glu Tyr Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asn Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Lys Ala Gly Phe Cys Arg Leu Phe Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Pro Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Trp Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro
675
<210> 222
<211> 25
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 222
tcagtctgaa gagcagagcc aggaa 25
<210> 223
<211> 25
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 223
ttatattgtt cctccgtgcg tcagt 25
<210> 224
<211> 25
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 224
cttgtcatgg tcatctgcta ctcgg 25
<210> 225
<211> 25
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 225
atatttcctg ctccccagtg gatcg 25
<210> 226
<211> 25
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 226
ttgaaatgag tttggtcagg gatgg 25
<210> 227
<211> 25
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 227
aactgtgtat ttccgtgctg attcc 25
<210> 228
<211> 25
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 228
ggagctgtag gcgattatag ttgaa 25
<210> 229
<211> 24
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 229
ttctcaaacc ctggaaagca cttt 24
<210> 230
<211> 100
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 230
ttaaagtagt tctttgatga atatgaatga gtagggaaag gattctttgt gatagtgata 60
cctctgtggt aagagaaggg tggtatgtga gttttagtct 100
<210> 231
<211> 20
<212> DNA/RNA
<213> 人工序列
<220>
<223> 人工的
<400> 231
gtgatagtga tacctctgtg 20

Claims (23)

1.工程化的Cas核酸酶，其在柔性区包含一个或多个突变，所述突变增加了参比Cas核酸酶的包含至少5个连续氨基酸残基的柔性区的柔性，其中所述工程化的Cas核酸酶与所述参比Cas核酸酶相比具有提高的活性，所述柔性区使用DynaMine程序来确定时，最高柔性的氨基酸残基的柔性得分S2 预测小于或等于0.8，其中，所述参比Cas核酸酶选自下组：Cas9、Cas12a、Cas12b、Cas12c、Cas12d、Cas12f、Cas12g、Cas12h、Cms1、Cas12i、Cas12j、Cas12k和CasX。
2.如权利要求1所述的工程化的Cas核酸酶，其中所述柔性区具有5个连续的氨基酸残基，其中第3个氨基酸残基具有最低的柔性得分，并且其中较高的柔性得分表示较低的构象柔性。
3.如权利要求1的工程化的Cas核酸酶，其为工程化的Cas12b核酸酶，其包含一个或多个突变，所述突变增加了对应于参比Cas12b核酸酶中氨基酸残基835至839的柔性区柔性，其中所述氨基酸残基编号基于SEQ ID NO: 1，其中所述工程化的Cas12b核酸酶与所述参比Cas12b核酸酶相比具有提高的活性。
4.如权利要求1的工程化的Cas核酸酶，其为工程化的Cas12i核酸酶，其包含一个或多个突变，所述突变增加了参比Cas12i核酸酶中的柔性区的柔性，所述柔性区选自对应于以下的区的组：氨基酸残基228-232、氨基酸残基439-443、氨基酸残基478-482、氨基酸残基500-504、氨基酸残基775-779和氨基酸残基925-929，其中所述氨基酸残基编号基于SEQ IDNO: 8，其中所述工程化的Cas12i核酸酶与所述参比Cas12i核酸酶相比具有增加的活性。
5.如权利要求1的工程化的Cas核酸酶，其为工程化的Cas9核酸酶，所述柔性区选自对应于以下的区的组：氨基酸残基39-43，氨基酸残基135-139，氨基酸残基176-180、氨基酸残基274-278、氨基酸残基351-355、氨基酸残基389-393、氨基酸残基521-525、氨基酸残基541-545、氨基酸残基755-759、氨基酸残基774-778、氨基酸残基786-790、氨基酸残基811-815、氨基酸残基848-852、氨基酸残基855-859、氨基酸残基874-878、氨基酸残基891-895、氨基酸残基1019-1023和氨基酸残基1036-1040，其中所述氨基酸残基编号基于SEQ ID NO:25，其中所述工程化的Cas9核酸酶与所述参比Cas9核酸酶相比具有增强的活性。
6.如权利要求1的工程化的Cas核酸酶，其为工程化的Cas9核酸酶，所述柔性区选自对应于以下的区的组：氨基酸残基45-49、氨基酸残基84-88、氨基酸残基116-120、氨基酸残基128-132、氨基酸残基216-220、氨基酸残基318-322、氨基酸残基387-391、氨基酸残基497-501、氨基酸残基583-587、氨基酸残基594-598、氨基酸残基614-618、氨基酸残基696-700和氨基酸残基739-743，其中所述氨基酸残基编号基于SEQ ID NO: 53，其中所述工程化的Cas9核酸酶与所述参比Cas9核酸酶相比具有增加的活性。
7.如权利要求1中所述的工程化的Cas核酸酶，其中所述一个或多个突变包括在所述柔性区中插入一个或多个G残基。
8.如权利要求1中所述的工程化的Cas核酸酶，其中所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：亮氨酸(L)、异亮氨酸(I)、缬氨酸(V)、半胱氨酸(C)、酪氨酸(Y)、苯丙氨酸(F)和色氨酸(W)。
9.如权利要求1所述的工程化的Cas核酸酶，其中所述活性是位点特异性核酸酶活性。
10.如权利要求9的工程化的Cas核酸酶，其中所述工程化的Cas核酸酶在真核细胞中一个或多个基因组位点的基因编辑效率比所述参比Cas核酸酶高至少约20%。
11.如权利要求10所述的工程化的Cas核酸酶，其中所述基因编辑效率是使用如下方法来测量的：T7核酸内切酶1 (T7E1)测定、靶DNA的测序、由分解追踪插入缺失(TIDE)测定或通过扩增子分析进行插入缺失检测(IDAA)测定。
12.如权利要求1所述的工程化的Cas核酸酶，其包含选自下组SEQ ID NOs: 2、14、18、20、27、28、33、34、41、54、58-59的氨基酸序列。
13.一种工程化的Cas效应蛋白，其包含权利要求1-12中任一项所述的工程化的Cas核酸酶。
14.如权利要求13所述的工程化的Cas效应蛋白，其中所述效应蛋白能够诱导DNA分子中的双链断裂或单链断裂。
15.如权利要求13所述的工程化的Cas效应蛋白，其还包含与所述工程化的Cas核酸酶融合的功能结构域。
16.如权利要求15所述的工程化的Cas效应蛋白，其中所述功能结构域选自下组：翻译起始结构域、转录阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域、逆转录酶结构域、报告分子结构域和核酸酶结构域。
17.如权利要求13所述的工程化的Cas效应蛋白，其包含：含有所述工程化的Cas核酸酶的N末端部分的第一多肽和含有所述工程化的Cas核酸酶或其功能衍生物的C末端部分的第二多肽，其中所述第一多肽和所述第二多肽能够在包含指导序列的指导RNA的存在下彼此缔合，以形成与靶核酸特异性结合的成簇规律间隔短回文重复序列(CRISPR)复合物，所述靶核酸包含与所述指导序列互补的靶序列。
18.一种工程化的CRISPR-Cas系统，包括：
(a)权利要求13-17中任一项所述的工程化的Cas效应蛋白；以及
(b)包含与靶序列互补的指导序列的指导RNA，或编码所述指导RNA的一种或多种核酸，
其中所述工程化的Cas效应蛋白和所述指导RNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合包含所述靶序列的靶核酸并诱导所述靶核酸的修饰。
19.检测样品中靶核酸的方法，包括：
(a)使样品与权利要求18中的工程化的CRISPR-Cas系统以及加标签的检测核酸接触，该检测核酸为单链且不与所述指导RNA的指导序列杂交；以及
(b)测量通过所述工程化的Cas效应蛋白切割所述加标签的检测核酸而产生的可检测信号，从而检测所述靶核酸。
20.修饰包含靶序列的靶核酸的方法，包括使所述靶核酸与权利要求18所述的工程化的CRISPR-Cas系统接触。
21.如权利要求20所述的方法，其中通过所述工程化的CRISPR-Cas系统切割所述靶核酸、改变所述靶核酸中的靶序列或改变所述靶核酸的表达。
22.一种工程化的细胞，包含使用如权利要求20或21中所述的方法修饰过的所述靶核酸。
23.如权利要求18所述的工程化的CRISPR-Cas系统在制备治疗与个体的细胞中靶核酸相关的疾病或病症的药物中的用途。
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