CN116917335A - 用于阻遏α-突触核蛋白(SYNUCLEIN)表达的新颖锌指蛋白转录因子 - Google Patents

用于阻遏α-突触核蛋白(SYNUCLEIN)表达的新颖锌指蛋白转录因子 Download PDF

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CN116917335A
CN116917335A CN202180079361.8A CN202180079361A CN116917335A CN 116917335 A CN116917335 A CN 116917335A CN 202180079361 A CN202180079361 A CN 202180079361A CN 116917335 A CN116917335 A CN 116917335A
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A·哈塔米
B·蔡特勒
L·张
D·E·帕雄
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Abstract

本公开提供抑制神经系统中的α‑突触核蛋白表达的锌指融合蛋白，及使用该蛋白质治疗帕金森氏症(Parkinson’s disease)、路易体痴呆(Lewy body dementia)、多系统萎缩、阿尔茨海默氏症(Alzheimer’s disease)及其他神经变性疾病的方法。

Description

用于阻遏α-突触核蛋白(SYNUCLEIN)表达的新颖锌指蛋白转 录因子
对相关申请的交叉引用
本申请要求2020年10月2日提交的美国临时申请号63/087,164的优先权，其公开内容以全文引用之方式并入本文中。
序列表
本临时申请含有序列表，该序列表已以ASCII格式以电子方式提交且以全文引用的方式并入本文中。将2021年9月30日创建的序列表的电子副本命名为025297_WO035_SL.txt，且大小为163,291字节。
发明背景
帕金森氏症(PD)为特征在于运动缺陷之神经变性病症。大致50％ PD患者最终产生痴呆。PD为仅次于阿尔茨海默氏症的第二大常见神经变性疾病。在美国，有大约一百万名PD患者且每年有50,000至60,000例新病例。散发性PD之典型发病年龄为60至70岁。自初始诊断至死于PD并发症通常具有15至20年之时间间隔。
PD患者显示一系列运动症状，诸如动作迟缓，僵硬，驼背姿势，面部表情蒙面，躯干前倾，手臂摆动减少，肘、腕、髋部及膝部屈曲，姿势不稳，静止时四肢震颤，以及拖着脚步，步履蹒跚。患者通常亦具有非运动症状，包括嗅觉丧失、睡眠障碍、肠道蠕动减少、神经痛及痴呆。参见例如Jeanjean and Aubert,Lancet(2011)378(9805):1773-4；Kalia and Lang,Lancet(2015)386(9996):896-912。
PD患者脑部之特征在于在称为黑质之区域中丧失产生多巴胺(多巴胺能)之神经元。PD患者脑部之特征亦在于路易体及路易神经突之存在，路易体为在神经元内部形成之蛋白质聚集体或凝集块，路易神经突为含有类似于路易体之蛋白质聚集体的神经突(神经元之过程)。路易体首先于1912年由Friedrich Lewy在PD患者脑部中发现，且稍后发现含有聚集及不可溶形式之α-突触核蛋白之原纤维(Goedert and Spillantini,MolPsychiatry.(1998)3(6):462-5；Spillantini等人,Neurosci Lett.(1998)251(3):205-8；Spillantini等人,Nature(1997)388(6645):839-40)。在1997年在PD家族中鉴定出α-突触核蛋白基因(SNCA)之突变(参见例如Polymeropoulos等人,Science(1997)276(5321):2045-7)。随后，SNCA基因之复制及三倍化复制以及α-突触核蛋白中之额外点突变展示与PD之遗传或家族性形式相关。此外，控制α-突触核蛋白表达水平之基因组之部分的变化已展示与大型无偏倚人群研究(全基因组关联研究，GWAS)中PD之风险增加相关。
α-突触核蛋白是与神经元突触前末端之囊泡释放有关的膜结合蛋白质。其亦可在DNA修复中起一定作用。成熟α-突触核蛋白为中央核心区(残基61-95)含有疏水性氨基酸之小14kD蛋白质，称为NAC(阿尔茨海默氏症淀粉样蛋白之非A-β组分)区。NAC引起蛋白质聚集。错误折叠α-突触核蛋白多肽聚集成寡聚物及基原纤维，其随后聚集在一起形成路易体中发现之大型不溶性聚集体。累积证据表明α-突触核蛋白错误折叠及聚集在PD中所发生之细胞损伤中起主要作用，且最终导致黑质中之神经元死亡。此外，已显示α-突触核蛋白之较小聚集体自细胞移动至细胞且在整个脑部中扩散，类似于朊病毒疾病中所见。抑制α-突触核蛋白聚集可减少对神经元之损伤且减缓或甚至停止PD之进展。
减少α-突触核蛋白水平之当前方法包括使用反义寡核苷酸(ASO)，其在RNA水平靶向α-突触核蛋白；及单克隆抗体(mAb)，其靶向细胞外部之α-突触核蛋白之特定3D形状或构象。然而，仍迫切需要藉由靶向α-突触核蛋白治疗PD之临床上有效的方法。
发明内容
本公开提供靶向人SNCA基因中或附近之位点的锌指蛋白(ZFP)域。本公开的ZFP域可与转录因子融合以在DNA水平特异性抑制人SNCA基因之表达。此等融合蛋白含有(i)特异性结合至SNCA基因中之靶区域的ZFP域及(ii)减少基因转录之转录阻遏物域。
在一个方面中，本公开提供一种融合蛋白，其包含锌指蛋白(ZFP)域及转录阻遏物域，其中ZFP域结合至人α-突触核蛋白基因(SNCA基因)的靶区域。在一些实施例中，靶区域(亦即靶位点)在SNCA基因中之转录起始位点(TSS，例如TSS 1、2a或2b)之约1kb内。在其他实施例中，靶区域位于SNCA基因之TSS 2a上游约500bp内、TSS 2b下游约500bp内，和/或TSS1上游或下游约500bp内，如图2B和/或4中所示。靶区域之非限制性实例展示于表1中。
在一些实施例中，融合蛋白包含一个或多个(例如两个、三个、四个、五个或六个)锌指。其可以优选地在没有可检测之脱靶结合或活性或在最小可检测之脱靶结合或活性(例如，与并非SNCA基因之基因结合)下将SNCA基因之表达阻遏至少约40％、75％、90％、95％或99％。锌指域之非限制性实例展示于表1中。在一些实施例中，融合蛋白包含表1中所示之一个或多个识别螺旋序列。在其他实施例中，融合蛋白包含来自表格之单行的一些或所有识别螺旋序列，具有或不具有指定主链突变。在某些实施例中，融合蛋白包含表2中所示之氨基酸序列。
在一些实施例中，融合蛋白之转录阻遏物域来自KOX1蛋白之KRAB域。锌指域可经由肽接头连接至转录阻遏物域。在另一方面中，本公开提供一种核酸构建体，其包含本公开融合蛋白之编码序列，其中编码序列可操作地连接至转录调控元件，诸如在脑细胞中为组成性活性或诱导性的哺乳动物启动子，且其中该启动子任选地为人突触蛋白I启动子。本公开亦提供一种包含该核酸构建体之宿主细胞。该宿主细胞为诸如脑细胞或多能干细胞之人细胞，其中该干细胞任选地为胚胎干细胞或诱导性多能干细胞(iPSC)。
在另一方面中，本公开提供一种抑制人脑细胞中α-突触核蛋白的表达的方法，其包括将本公开融合蛋白引入细胞中(例如，经由引入核酸构建体或重组病毒，诸如AAV(例如，AAV2、AAV6、AAV9或其杂合体)，由此抑制细胞中α-突触核蛋白的表达。脑细胞可为神经元、胶质细胞、室管膜细胞或神经上皮细胞。细胞可在罹患帕金森氏症、路易体痴呆、阿尔茨海默氏症、多系统萎缩或另一突触核蛋白病或处于罹患该等疾病之风险下的患者脑部中。
本公开亦提供一种治疗患者之突触核蛋白病(例如减缓突触核蛋白病进展)的方法，其包括向该患者施用编码本公开之融合蛋白的重组AAV。在一些实施例中，该AAV经由静脉内、鞘内、脑室内、大池内、纹状体内或黑质内注射或注射至任何脑部区域引入该患者。患者可患有帕金森氏症、路易体痴呆、阿尔茨海默氏症或多系统萎缩。
本公开亦提供用于以上方法中的融合蛋白及本融合蛋白在制造用于以上方法中的药物中的用途。
本公开之其他特征、目标及优势在以详细描述中显而易见。然而，应理解，详细描述虽然指示本公开之实施例及方面，但仍仅藉助于说明(而非限制)给出。对于本领域技术人员而言，在本公开之范畴内的各种改变及修饰将自详细描述变得显而易见。
附图说明
图1说明藉由工程化6-指锌指蛋白转录因子(ZFP-TF)特异性靶向SNCA基因，其识别基因中之18个碱基对。ZFP-TF与基因之结合导致SNCA转录减少，其又导致SNCA mRNA及α-突触核蛋白蛋白质水平减少。该图揭示例示性工程化ZFP(SBS-82110)之基因组靶序列(结合序列；SEQ ID NO:226)。
图2A是说明人SNCA基因之基因组结构的图式。该基因具有7个外显子(2个非蛋白质编码及5个蛋白质编码)，其中各转录物含有5个内含子。存在三个转录起始位点(TSS)，命名为TSS 1、TSS 2a及TSS 2b，其分别以非编码外显子1、2a及2b开始产生转录物。所有转录物中之第一蛋白质编码外显子为外显子3。
图2B是说明人SNCA基因之上游基因组区域的图式。SNCA mRNA序列展示为红色条。基因下面的簇中之小三角形描绘本文例示之416种代表性ZFP-TF所靶向之SNCA基因中之区域。该图亦展示ZFP-TF中之每一者对减少用ZFP-TF mRNA转染之后24小时收集之SK-N-MC人神经母细胞瘤细胞中之人SNCA mRNA表达之影响。信使RNA水平藉由RT-qPCR测量。归一化SNCA表达水平由梯度条“SNCA mRNA”指示。最深(红)色表示100％减少。最浅色(白色)表示0％减少。RT-qPCR数据相对于两个管家基因(ATP5B及EIF4A2)之mRNA含量之平均值归一化。指向右之三角形指示ZFP-TF结合基因之有义链。指向左之三角形指示ZFN-TF结合基因之反义链。
图3A-3E为展示自如本文中所描述之416种ZFP-TF库之筛选结果的图。在SK-N-MC人神经上皮细胞系中进行筛选。各图中之y轴是α-突触核蛋白mRNA表达，其相对于两个管家基因(EIF4A2及ATP5B)之几何平均值归一化且在用编码不同ZFP-TF之RNA转染之后24小时进行评估。RNA剂量自左至右增加(3、10、30、100、300及1,000ng)。长条表示四个技术复制品之平均值且误差条表示标准偏差。图下方之编号为ZFP-TF之内部参考编号。
图4是说明人SNCA基因之上游基因组区域的图式。SNCA mRNA序列展示为红色条。基因下面的簇中之小三角形描绘具有1、2或3个磷酸酯接触突变之27种代表性ZFP-TF所靶向之SNCA基因中之区域。该图亦展示ZFP-TF中之每一者对减少用ZFP-TF mRNA转染之后24小时收集之SK-N-MC人神经母细胞瘤细胞中之人SNCA mRNA表达之影响。信使RNA水平藉由RT-qPCR测量。归一化SNCA表达水平由梯度条“SNCA mRNA”指示。最深(红)色表示100％减少。最浅色(白色)表示0％减少。RT-qPCR数据相对于两个管家基因(ATP5B及EIF4A2)之mRNA含量之平均值归一化。指向右之三角形指示ZFP-TF结合基因之有义链。指向左之三角形指示ZFN-TF结合基因之反义链。
图5展示自具有1、2或3个磷酸酯接触突变之27种代表性ZFP-TF库之说明性筛选结果。在SK-N-MC人神经上皮细胞系中进行筛选。各图中之y轴是α-突触核蛋白mRNA表达，其相对于两个管家基因(EIF4A2及ATP5B)之几何平均值归一化且在用编码不同ZFP-TF之RNA转染之后24小时进行评估。RNA剂量自左至右增加(3、10、30、100、300及1,000ng)。长条表示四个技术复制品之平均值且误差条表示标准偏差。图下方之编号为ZFP-TF之内部参考编号。
图6A及6B为展示40种α-突触核蛋白ZFP-TF在SK-N-MC人神经母细胞瘤细胞及人iPSC衍生之神经元中显示一系列α-突触核蛋白阻遏活性之图组。表1及表2中所述之二十种说明性ZFP-TF展示于A图中。在图3A-3E中表征但未描述于表1及2中之说明性ZFP-TF展示于B图中。y轴为α-突触核蛋白mRNA表达，其相对于两个管家基因(ATP5B及EIF4A2)之几何平均值归一化且在用编码不同ZFP-TF之RNA转染SK-N-MC细胞之后24小时或在用编码不同ZFP-TF之AAV6转导iPSC衍生之神经元之后28天进行评估。所用RNA或AAV6之量以x轴指示，其中RNA(3、10、30、100、300及1,000ng)或AAV6(1E3、3E3、1E4、3E4、1E5及3E5)剂量自左至右增加。蓝条及橙条表示四个技术复制品之平均值且误差条表示标准偏差。放大版之滴定尺度示出于图的底部。
图7A-7D为描绘40种α-突触核蛋白ZFP-TF在小鼠原代神经元(A及B)及人iPSC衍生之神经元(C及D)中之脱靶活性的火山图组。为容易参考起见，来自绘示ZFP-TF在人iPSC衍生之神经元中之阻遏活性的图6A-6B之条形图紧邻相应ZFP-TF之火山图展示。火山图概述微阵列数据，其展示在转导之后7天小鼠原代神经元(A及B)或转导之后19天人iPSC衍生神经元(C及D)之转录物组的变化。黄色圆圈指示人α-突触核蛋白。绿色圆圈(各火山图之右侧)表示显著上调>2倍之基因。此等基因之数量以绿色文字指示。红色圆圈表示显著下调>2倍之基因。此等基因之数量以红色文字指示。
图8A及8B为展示以下之图组：雌性PAC突触核蛋白小鼠之不同脑部区域中之(A)ZFP-TF及α-突触核蛋白mRNA表达水平或(B)胶质原纤维酸性蛋白(GFAP)、离子化钙结合衔接分子1(IBA1)及NeuN之mRNA表达水平(Kuo等人，Hum Mol Genet.(2010)19(9):1633-50)。对动物纹状体中之两个部位双侧施用编码指定测试物或媒剂之AAV9。所有基因表达数据均相对于3个管家基因(ATP5B、EIF4A2及GAPDH)之几何平均值归一化。y轴为指定基因之mRNA表达量且x轴为脑部区域。红色方块指示施用ZFP-TF 82195之n＝3动物之平均表达数据，且绿色三角形指示施用ZFP-TF 82264之n＝3动物之平均表达数据。在图A中，α-突触核蛋白mRNA表达相对于3个媒剂处理之动物之平均值归一化，且ZFP-TF表达以对数尺度提供为每ng输入RNA之拷贝。在图B中，所有表达数据均相对于3个媒剂处理之动物之平均值归一化。藉由单向方差分析(ANOVA)随后Dunnett多重比较检验来进行统计分析。*p<0.05，**p<0.01，***p<0.001，***p<0.0001。
发明详述
本发明提供靶向人SNCA基因中或附近之位点(即，序列)的ZFP域。如本文所描述之ZFP域可连接或融合至另一功能性分子或域。本发明之ZFP域可与转录因子融合以阻遏人SNCA基因转录为RNA。融合蛋白称作锌指蛋白转录因子(ZFP-TF)。此等ZFP-TF含有特异性结合至SNCA基因中或附近之靶区域的锌指蛋白(ZFP)域及减少基因转录之转录阻遏物域。预期藉由将ZFP-TF引入患者脑部中来降低神经元中α-突触核蛋白之水平来抑制(例如减少或停止)α-突触核蛋白组装成寡聚体(较小可溶性聚集物)或原纤维(较大不溶性聚集物)形式。在α-突触核蛋白聚集减少之情况下，脑细胞将有能力用其细胞质量控制机制及时移除错误折叠及有毒形式之α-突触核蛋白。因此，将减少或阻止α-突触核蛋白之聚集及细胞间繁殖。
我们的α-突触核蛋白抑制之ZFP-TF方法比藉由其他人测试之当前方法具有若干优点。ZFP-TF可能需要仅一次施用(藉由向患者引入ZFP-TF表达构建体)，而ASO需要重复给药。另外，ZFP-TF方法仅需要接合各细胞之基因组中之SNCA基因之两个等位基因。相比之下，ASO需要接合各细胞中之SNCA mRNA之多个拷贝。
我们的ZFP-TF方法优于抗体方法，因为抗体仅可结合α-突触核蛋白形状或构象之子集。此可能不足以产生强大的治疗效果。相比之下，ZFP-TF在DNA水平下阻遏α-突触核蛋白表达且导致降低所有形式α-突触核蛋白之水平。因此，不同于抗体，ZFP-TF对毒性物质之形式不可知。另外，认为抗体主要作用于细胞上或细胞外部之α-突触核蛋白，而ZFP-TF可直接减少细胞内部α-突触核蛋白且间接降低细胞外α-突触核蛋白水平。因此，预期ZFP-TF方法更有效，因为α-突触核蛋白主要为细胞内蛋白质。此外，抗体需要重复施用，而ZFP-TF仅需要单次递送其表达构建体。
I.ZFP域之靶标
本融合蛋白之ZFP域特异性结合至人SNCA基因中或附近之靶区域。图1说明ZFP域与靶SNCA基因序列之结合。该图中之ZFP域具有六个锌指；然而，如下文进一步描述，亦可使用具有更少或更多锌指之ZFP域。
人SNCA基因跨越约117kb且已定位至chr4:89,724,099-89,838,315(GRCh38/hg38)。其核苷酸序列可以GenBank登录号NC_000004版本000004.12获得。基因具有7个外显子(2个非蛋白质编码及5个蛋白质编码)，其中各转录物具有5个内含子(图2A)。基因存在三个转录起始位点(TSS)：一个在外显子1之起点，且两个在外显子2中，引起外显子2a及2b之表达。第一蛋白质编码外显子为外显子3。亦参见Touchman等人,Genome Res.(2001)11:78-86。人α-突触核蛋白之同功型1(全长)下文展示：
同功型2-4与同功型1的不同之处在于缺失氨基酸残基103-130。同功型2-5与同功型1的不同之处在于缺失氨基酸残基41-54。α-突触核蛋白之基因分析已指向基因拷贝扩增(参见例如Brueggemann等人，Neurology(2008)71:1294；Troiano等人，Neurology(2008)71:1295；Uchiyama等人，Neurology(2008)71:1289-90)及某些点突变作为诸如PD及路易体痴呆之突触核蛋白病的潜在原因。举例而言，已在一些PD患者中鉴定出以下α-突触核蛋白点突变：A30P(Kruger等人，Nature Genet.(1998)18:106-8)；E46K(Zarranz等人，AnnNeurol.(2004)55:164-73；Choi等人，FEBS Lett.(2004)576:363-8)；H50Q(Khalaf等人，JBiol Chem.(2014)289:21856-76)；G51D(Lesage等人，Ann Neurol.(2013)73:459-71)；及A53T(Polymeropoulos等人，Science(1997)276:2045-7)。
ZFP-TF之DNA结合ZFP域将融合蛋白引导至SNCA基因之靶区域且将融合蛋白之转录阻遏物域带到靶区域。阻遏物域接着藉由RNA聚合酶阻遏SNCA基因转录。ZFP-TF之靶区域可为SNCA基因中或附近允许阻遏基因表达的任何适合位点。举例而言，靶区域包括SNCATSS或SNCA转录调控元件(例如，启动子、增强子、RNA聚合酶暂停位点及其类似物)或与SNCATSS或SNCA转录调控元件相邻(在其下游或上游)。
如上文所描述，人SNCA基因具有三个转录起始位点(TSS)。他们自5’至3’为TSS 1、TSS 2a及TSS 2b，其定位于外显子1(TSS 1)、外显子2a及外显子2b(TSS 2a及2b)之5’端处(图2B)。三个TSS处之转录产生具有不同长度之RNA同工型。然而，因为α-突触核蛋白之翻译开始于外显子3处，所以由不同TSS产生之RNA导致形成相同蛋白质。在一些实施例中，本ZFP-TF之基因组靶区域跨越TSS 1(例如碱基对529-1529)或TSS 2a及2b(例如碱基对1613-2949)或在其附近。在某些实施例中，靶区域在TSS 1之上游或下游约500bp内，和/或在TSS2a之上游或下游约500bp内，和/或在TSS 2b之上游或下游约500bp内。
在一些实施例中，基因组靶区域长度为至少8bp。举例而言，靶区域长度可为8bp至40bp，诸如长度为12、15、18、21、24、27、30、33或36bp。靶向序列可在基因之有义链或基因之反义链上。为了确保靶向精确性且减少ZFP-TF之脱靶结合或活性，所选SNCA靶区域之序列与其他基因中之序列优选具有小于75％同源性(例如小于70％、小于65％、小于60％或小于50％)。在某些实施例中，本发明ZFP-TF之靶区域的长度为15-18bp且驻存于TSS 1、2a或2b之500bp内。靶区域之实例展示于图2B及表1中。
在一些实施例中，本发明之工程化ZFP结合至如表1之单行中所示之靶位点(亦即结合序列)，优选没有或几乎没有可检测之脱靶结合或活性。
用于进一步评估靶区段之其他准则包括：ZFP结合至此类区段或相关区段之先前可用性；设计新颖ZFP以结合给定靶区段之容易性；及脱靶结合风险。
II.ZFP域
“锌指蛋白”或“ZFP”是指具有藉由锌稳定化之DNA结合域的蛋白质。ZFP以序列特异性方式结合于DNA。ZFP之个别DNA结合单元称为锌“指”。各锌指含有通常由七个氨基酸残基构成且决定DNA结合特异性之DNA结合“识别螺旋”。ZFP域具有至少一个指，各指结合两个至四个DNA碱基对，通常结合三个或四个DNA碱基对。各锌指通常包含大致30个氨基酸及螯合物锌。工程化ZFP与天然存在之ZFP相比可具有新颖结合特异性。工程化方法包括但不限于合理设计及各种类型之选择。合理设计包括例如使用包含三联体(或四联体)核苷酸序列及个别锌指氨基酸序列之数据库，其中各三联体或四联体核苷酸序列与结合特定三联体或四联体序列之锌指之一个或多个氨基酸序列相关联。参见例如详细描述于以下专利中之ZFP设计方法：美国专利5,789,538；5,925,523；6,007,988；6,013,453；6,140,081；6,200,759；6,453,242；6,534,261；6,979,539；及8,586,526；及国际专利公开WO 95/19431；WO96/06166；WO 98/53057；WO 98/53058；WO 98/53059；WO 98/53060；WO 98/54311；WO 00/27878；WO 01/60970；WO 01/88197；WO 02/016536；WO 02/099084；及WO 03/016496。如本文所描述之ZFP域可连接或融合至另一分子，例如蛋白质。此类ZFP-融合物可包含能够进行基因活化(例如活化域)、基因阻遏(例如阻遏域)、配位体结合(例如配位体结合域)、高产量筛选(例如配位体结合域)、局部超突变(例如活化诱导之胞苷脱氨酶域)、染色质修饰(例如组蛋白脱乙酰基酶域)、重组(例如重组酶域)、靶向整合(例如整合酶域)、DNA修饰(例如DNA甲基转移酶域)、碱基编辑(例如碱基编辑器域)或靶向DNA裂解(例如核酸酶域)之域。工程化ZFP域之实例展示于表1中。
本发明之工程化ZFP融合蛋白之ZFP域可包括至少一个(例如一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个、十二个、十三个或更多)锌指。具有一个锌指之ZFP域通常识别包括3或4个核苷酸之靶位点。具有两个锌指之ZFP域通常识别包括6或8个核苷酸之靶位点。具有三个锌指之ZFP域通常识别包括9或12个核苷酸之靶位点。具有四个锌指之ZFP域通常识别包括12至15个核苷酸之靶位点。具有五个锌指之ZFP域通常识别包括15至18个核苷酸之靶位点。具有六个锌指之ZFP域可以识别包括18至21个核苷酸之靶位点。
在一些实施例中，本发明之工程化ZFP包含表1中所示之DNA结合之识别螺旋序列。举例而言，工程化ZFP可包含如表1中所示之F1、F2、F3、F4、F5或F6之序列。
在一些实施例中，本发明之工程化ZFP包含表1之单行中所示之两个相邻DNA结合之识别螺旋序列。举例而言，工程化ZFP可包含如表1之单行中所示之F1-F2、F2-F3、F3-F4、F4-F5或F5-F6之序列。
在一些实施例中，本发明之工程化ZFP包含表1之单行中所示之DNA结合之识别螺旋序列。举例而言，工程化ZFP可包含如表1之单行中所示之F1、F2、F3、F4、F5及F6(例如F1-F6)之序列。
ZFP域之靶标特异性可藉由对如例如描述于美国专利公开2018/0087072中之ZFP主链序列进行突变来提高。突变包括对ZFP主链中之残基作出之彼等突变，该等残基可与DNA主链上之磷酸酯非特异性相互作用但不涉及核苷酸靶标特异性。在一些实施例中，此等突变包含使阳离子氨基酸残基突变为中性或阴离子氨基酸残基。在一些实施例中，此等突变包含使极性氨基酸残基突变为中性或非极性氨基酸残基。在其他实施例中，突变相对于DNA结合螺旋在位置(-5)、(-9)和/或(-14)进行。在一些实施例中，锌指可包含位置(-5)、(-9)和/或(-14)处之一个或多个突变。在其他实施例中，多指ZFP域中之一个或多个锌指可包含位置(-5)、(-9)和/或(-14)处之突变。在一些实施例中，位置(-5)、(-9)和/或(-14)处之氨基酸(例如，精氨酸(R)或赖氨酸(K))突变成丙氨酸(A)、亮氨酸(L)、丝氨酸(S)、天冬氨酸(N)、谷氨酸(E)、酪氨酸(Y)和/或谷氨酰胺(Q)。具有1、2或3个主链突变之工程化ZFP之实例显示于图4及5及表1及2中。表1中之符号“^”指示指定识别螺旋中之第1个氨基酸上游之第4位置处之精氨酸(R)残基变为谷氨酰胺(Q)。在各识别螺旋序列中，七个DNA结合氨基酸之位置编号为-1、+1、+2、+3、+4、+5及+6。因此，R取代为Q之位置编号为(-5)。
在一些实施例中，本发明之工程化ZFP包含如表1中所示之DNA结合之识别螺旋序列及相关主链突变。在一些实施例中，本发明之工程化ZFP包含如表1之单行中所示之DNA结合之识别螺旋序列及相关主链突变。
在一些实施例中，本文所述之工程化ZFP包含表2之单行中所示之序列之识别螺旋及主链部分。在一些实施例中，本文所述之工程化ZFP包含表2之单行中所示之序列之识别螺旋及主链部分，因为该序列将在翻译后修饰之后出现。举例而言，翻译后修饰可以自如表2中所示之序列移除起始甲硫氨酸残基。
在一些实施例中，本发明ZFP-TF包含一个或多个锌指域。该等域可经由可延伸柔性接头连接在一起，使得例如一个域包含一个或多个(例如4、5或6个)锌指且另一个域包含额外的一个或多个(例如4、5或6个)锌指。在一些实施例中，接头为标准指间接头，使得指阵列包含一个包含8、9、10、11或12或更多个指之DNA结合域。在其他实施例中，接头为非典型接头，诸如柔性接头。举例而言，两个ZFP域可以以配置(自N端至C端)ZFP-ZFP-TF、TF-ZFP-ZFP、ZFP-TF-ZFP或ZFP-TF-ZFP-TF(两种ZFP-TF融合蛋白经由接头融合在一起)连接至转录阻遏物TF。
在一些实施例中，ZFP-TF为“双手的”，亦即，它们含有两个藉由插入氨基酸隔开之锌指簇(两个ZFP域)，使得两个ZFP域结合至两个不连续的靶位点。双手型锌指结合蛋白之实例为SIP1，其中四个锌指之簇位于蛋白质之氨基端，且三指之簇位于羧基端。(参见Remacle等人，EMBO J.(1999)18(18):5073-84)。此等蛋白质中之各锌指簇能够结合至独特靶序列且两个靶序列之间的间距可包含多个核苷酸。
III.锌指蛋白质转录因子
本文所描述之ZFP域可与转录因子融合。在一些实施例中，本发明融合蛋白含有DNA结合锌指蛋白(ZFP)域及转录因子域(亦即ZFP-TF)。在一些实施例中，转录因子可为转录阻遏物域，其中ZFP及阻遏物域可藉由直接肽基联接或肽接头，或藉由二聚化(例如经由亮氨酸拉链、STAT蛋白N端域或FK506结合蛋白)彼此相关联。如本文所用，“融合蛋白”是指具有共价连接域之多肽以及经由非共价键彼此相关联的多肽之复合物。转录阻遏物域可在任何适合位置与ZFP域相关联，包括ZFP域之C端或N端。
在一些实施例中，本ZFP-TF以小于约25nM之KD结合至其靶标且将人SNCA基因之转录阻遏20％或更多(例如，30％、40％、50％、60％、70％、80％、90％或95％或更多)。在一些实施例中，本ZFP-TF中的两种或更多种在患者中同时使用，其中ZFP-TF结合至SNCA基因中之不同靶标区域，以便实现SNCA表达之最佳阻遏。
A.转录阻遏物域
本发明ZFP-TF包含如本文所述之工程化ZFP域及减弱SNCA基因之转录活性之一个或多个转录阻遏物域。一个或多个工程化ZFP域及一个或多个转录阻遏物域可藉由柔性接头连接。转录阻遏物域之非限制性实例为KOX1、KAP-1、MAD、FKHR、EGR-1、ERD、SID、TGF-β-诱导早期基因(TIEG)、v-ERB-A、MBD2、MBD3、TRa、组蛋白甲基转移酶、组蛋白脱乙酰基酶(HDAC)、核激素受体(例如雌激素受体或甲状腺激素受体)、DNMT家族成员(例如DNMT1、DNMT3A、DNMT3B)、Rb及MeCP2。参见例如Bird等人(1999)Cell 99:451-454；Tyler等人(1999)Cell 99:443-446；Knoepfler等人(1999)Cell99:447-450；及Robertson等人(2000)Nature Genet.25:338-342。其他例示性阻遏域包括但不限于ROM2及AtHD2A。参见例如Chem等人(1996)Plant Cell 8:305-321；及Wu等人(2000)Plant J.22:19-27。
在一些实施例中，转录阻遏物域包含来自人锌指蛋白10/KOX1(ZNF10/KOX1)之Kruppel相关盒(KRAB)域的序列(例如GenBank号NM_015394.4)。例示性KRAB域序列为：
亦可使用此KRAB序列之变体，只要其具有相同或类似的转录阻遏物功能即可。
在一些实施例中，本文所描述之工程化ZFP-TF结合至如表1之单行中所示之靶位点，优选没有或几乎没有可检测之脱靶结合或活性。可例如藉由测量脱靶基因处之ZFP-TF之活性测定脱靶结合。在一些实施例中，本文所述之工程化ZFP-TF包含表1中所示之DNA结合之识别螺旋序列。在一些实施例中，本文所述之工程化ZFP-TF包含表1之单行中所示之两个相邻DNA结合之识别螺旋序列。在一些实施例中，本文所述之工程化ZFP-TF包含表1之单行中所示之DNA结合之识别螺旋序列。在一些实施例中，本文所述之工程化ZFP-TF包含表2之单行中所示之序列之识别螺旋及主链部分。在一些实施例中，本文所述之工程化ZFP-TF包含如表2之单行中所示之氨基酸序列。在一些实施例中，本文所述之工程化ZFP-TF包含表2之单行中所示之序列之识别螺旋及主链部分，因为该序列将在翻译后修饰之后出现。在一些实施例中，本文所述之工程化ZFP-TF包含表2之单行中所示之氨基酸序列，因为该序列将在翻译后修饰之后出现。举例而言，翻译后修饰可以自如表2中所示之序列移除起始甲硫氨酸残基。
B.肽接头
本发明ZFP-TF之ZFP域及转录阻遏物域和/或ZFP域内之锌指可经由肽接头，例如具有约5至200个氨基酸(例如5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个氨基酸)之不可裂解肽接头连接。优选接头通常是以重组融合蛋白形式合成之柔性氨基酸子序列。参见例如以上描述；及美国专利6,479,626；6,903,185；7,153,949；8,772,453；及9,163,245；及WO 2011/139349。本文所述之蛋白质可包括适合接头之任何组合。接头之非限制性实例为DGGGS(SEQ ID NO:2)、TGEKP(SEQ ID NO:3)、LRQKDGERP(SEQ ID NO:4)、GGRR(SEQ ID NO:5)、GGRRGGGS(SEQ ID NO:6)、LRQRDGERP(SEQ ID NO:7)、LRQKDGGGSERP(SEQID NO:8)、LRQKD(G3S)2ERP(SEQ ID NO:9)及TGSQKP(SEQ ID NO:10)。
在一些实施例中，TGEKPFA(SEQ ID NO:15)和/或TGSQKPFQ(SEQ ID NO:16)连接ZFP域内之锌指，和/或LRQKDAARGSGG(SEQ ID NO:17)或LRGSGG(SEQ ID NO:18)将ZFP域连接至转录阻遏物域。
在一些实施例中，肽接头之长度为三至20个氨基酸残基且富含G和/或S。此类接头之非限制性实例为G4S型接头(SEQ ID NO:11)，亦即含有一个或多个(例如2、3或4个)GGGGS(SEQ ID NO:11)基序，或基序之变化的接头(诸如相比基序具有一个、两个或三个氨基酸插入、缺失及取代的接头)。
IV.ZFP-TF之表达
本发明之ZFP-TF可经由编码其之核酸分子引入患者。核酸分子可为RNA或cDNA分子。核酸分子可经由注射包含脂质:核酸复合物(例如脂质体)之组合物而引入患者脑部中。或者，ZFP-TF可经由包含编码ZFP-TF之序列之核酸表达载体引入患者。表达载体可包括表达控制序列，诸如启动子、增强子、转录信号序列及转录终止序列，其允许在神经系统之细胞中表达ZFP-TF之编码序列。在一些实施例中，表达载体保持以稳定游离基因组形式存在于细胞中。在其他实施例中，表达载体整合至细胞之基因组中。
在一些实施例中，用于引导脑部中ZFP-TF表达之载体上之启动子为组成性活性启动子或可诱导的启动子。适合启动子包括(但不限于)逆转录病毒RSV LTR启动子(任选地具有RSV增强子)、CMV启动子(任选地具有CMV增强子)、CMV即刻早期启动子、SV40启动子、二氢叶酸还原酶(DHFR)启动子、β-肌动蛋白启动子、磷酸甘油酸激酶(PGK)启动子、EFlα启动子、MoMLV LTR启动子、CK6启动子、甲状腺素运载蛋白启动子(TTR)、TK启动子、四环素反应启动子(TRE)、HBV启动子、hAAT启动子、嵌合肝特异性启动子(LSP)、E2F启动子、端粒酶(hTERT)启动子、CMV增强子/鸡β-肌动蛋白/家兔β-球蛋白启动子(CAG启动子；Niwa等人，Gene(1991)108(2):193-9)及RU-486反应性启动子。亦可使用诸如突触蛋白I启动子、MeCP2启动子、CAMKII启动子、PrP启动子、GFAP启动子，或表达限于神经元和/或胶质细胞之工程化或天然启动子的脑细胞特异性启动子。
可采用将核苷酸序列引入细胞中的任何方法，包括但不限于：电穿孔、磷酸钙沉淀、显微注射、阳离子或阴离子脂质体，及与核定位信号组合之脂质体、天然存在之脂质体(例如胞外体)或病毒转导。
关于表达载体之体内递送，可使用病毒转导。本领域中已知之多种病毒载体可由本领域技术人员调适用于本发明，例如牛痘载体、腺病毒载体、慢病毒载体、痘病毒载体、腺相关病毒(AAV)载体、逆转录病毒载体及杂合病毒载体。在一些实施例中，本文所用之病毒载体为重组AAV(rAAV)载体。AAV载体尤其适用于CNS基因递送，因为其感染分裂与非分裂细胞，以用于长期表达之稳定游离型结构形式存在，且具有极低免疫原性(Hadaczek等人，MolTher.(2010)18:1458-61；Zaiss,等人，Gene Ther.(2008)15:808-16)。可使用任何适合的AAV血清型。举例而言，AAV可为AAV1、AAV2、AAV3、AAV3B、AAV4、AAV5、AAV6、AAV7、AAV8、AAV8.2、AAV9或AAVrh10，或假型，诸如AAV2/8、AAV2/5、AAV2/6或AAV2/9，或为本文中列举之AAV血清型中之一者之变体或衍生物的血清型(亦即，衍生自多种血清型之AAV；例如rAAV在其基因组中包含AAV2反向末端重复(ITR)及AAV8、5、6或9蛋白壳)。在一些实施例中，表达载体为AAV病毒载体且藉由重组AAV病毒粒子引入靶标人细胞中，该重组AAV病毒粒子之基因组包含包括在两端具有AAV反向末端重复(ITR)序列以允许在诸如昆虫细胞/杆状病毒生产系统或哺乳动物细胞生产系统之生产系统中产生AAV病毒粒子的构建体。AAV可经工程化使得其蛋白壳蛋白在人或非人灵长类动物中具有减少之免疫原性或增强之转导能力。在一些实施例中，使用AAV9。可使用本领域中已知之方法产生本文所述之病毒载体。可采用任何适合之容许细胞或包装细胞来产生病毒粒子。举例而言，哺乳动物或昆虫细胞可用作包装细胞系。
V.医药应用
本发明ZFP-TF可用于治疗需要下调α-突触核蛋白表达之患者。患者罹患以下神经变性疾病或处于罹患以下神经变性疾病之风险下：诸如帕金森氏症、路易体痴呆、阿尔茨海默氏症、多系统萎缩及任何其他突触核蛋白病。处于风险下之患者包括有基因倾向之彼等患者、已罹患重复脑损伤(诸如震荡)之彼等患者及已暴露于环境神经毒素之彼等患者。本发明提供一种治疗受试者(诸如有需要之人患者)之神经疾病(例如神经变性疾病)的方法，包含向受试者之神经系统引入治疗有效量(例如，允许充分阻遏SNCA表达之量)之ZFP-TF(例如，表达ZFP-TF之rAAV载体)。术语“治疗”涵盖缓解症状、预防症状发作、减缓疾病进展、改善生活质量及增加存活期。
本发明提供一种药物组合物，其包含病毒载体，诸如重组AAV(rAAV)，其重组基因组包含ZFP-TF之表达盒。药物组合物可进一步包含药学上可接受之载剂，诸如水、盐水(例如，磷酸盐缓冲盐水)、右旋糖、甘油、蔗糖、乳糖、明胶、聚葡萄糖、白蛋白或果胶。另外，组合物可含有辅助物质，诸如润湿剂或乳化剂、pH缓冲剂、稳定剂或增强药物组合物之有效性的其他试剂。药物组合物可含有递送媒剂，诸如脂质体、纳米胶囊、微粒、微球体、脂质粒子及囊泡。
本发明之治疗剂靶向之细胞为脑部中之细胞，包括但不限于神经元细胞(例如运动神经元、感觉神经元、多巴胺能神经元、胆碱能神经元、谷氨酸能神经元、GABA能神经元或血清素能神经元)；胶质细胞(例如少突胶质细胞、星形胶质细胞、周细胞、施万细胞或小胶质细胞)；室管膜细胞；或神经上皮细胞。由治疗剂靶向之脑部区域可为在突触核蛋白病中最显著地受到影响之彼等区域，诸如纹状体、尾核、壳核、黑质、中脑、嗅球、小脑、蓝斑核、脑桥、髓质、脑干、苍白球、海马区及大脑皮质或其他脑部区域。此等区域可经由纹状体内注射、黑质内注射、大脑内注射、大池内(ICM)注射或更一般经由脑实质内注射、脑室内(ICV)注射、鞘内注射或静脉内注射直接到达。其他施用途径包括(但不限于)室内、鼻内或眼内施用。在一些实施例中，在例如经由鞘内和/或脑室内注射或大池内注射直接施用至脑脊髓液(CSF)中之后，病毒载体在整个CNS组织中传播。在其他实施例中，在静脉内施用后，病毒载体跨越血脑屏障且实现在整个受试者之CNS组织中之广泛分布。在其他实施例中，病毒载体经由脑实质内注射直接递送至靶标区域。在一些情况下，病毒载体在脑实质内递送之后可逆行或顺行转运至其他脑部区域。在一些方面中，病毒载体具有独特的CNS组织靶向能力(例如CNS组织向性)，其以高效率达成稳定及无毒性的基因转移。
举例而言，药物组合物可经由例如室内施用至患者前脑之脑室区域，诸如右侧脑室、左侧脑室、第三脑室或第四脑室而提供至患者。药物组合物可经由大脑内施用提供至患者，例如将组合物注射至脑部之纹状体、尾核、壳核、黑质、中脑、嗅球、大脑、小脑、蓝斑核、脑桥、髓质、脑干、苍白球、海马体、大脑皮质、颅内腔、脑膜、硬膜、蛛网膜或软膜中或附近。在一些情况下，大脑内施用可包括将药剂施用至脑部周围之蛛网膜下腔之脑脊髓液(CSF)中。
在一些情况下，大脑内施用涉及使用立体定位程序注射。立体定位程序在本领域中是众所周知的且通常涉及使用计算机及3维扫描装置，该等计算机及3维扫描装置一起用于引导注射至特定大脑内区域，例如脑室区域。亦可使用显微注射泵(例如来自WorldPrecision Instruments)。在一些情况下，使用显微注射泵来递送包含病毒载体之组合物。在一些情况下，组合物之输注速率在0.1μl/min至100μl/min范围内。如本领域技术人员所了解，输注速率将视各种因素而定，包括例如受试者物种、受试者年龄、受试者体重/体型、AAV血清型、所需剂量及所靶向之大脑内区域。因此，本领域技术人员可认为其他输注速率在某些情形下是适当的。
可例如藉由静脉内施用将rAAV递送至受试者。在某些情况下，可能需要将rAAV局部递送至脑部组织、脊髓、脑脊髓液(CSF)、神经元细胞、胶质细胞、脑膜、星形胶质细胞、少突胶质细胞、小胶质细胞、间质间隙及其类似物。在一些情况下，重组AAV可藉由注射至脑室区域以及纹状体、尾核、壳核、黑质、中脑、嗅球、小脑、蓝斑核、脑桥、髓质、脑干、苍白球、海马体、大脑皮质或其他脑部区域中而直接递送至CNS。AAV可使用本领域中已知之神经外科技术，诸如藉由立体定位注射以针、导管或相关装置来递送(参见例如Stein等人，J Vir.(1999)73:3424-9；Davidson等人，PNAS.(2000)97:3428-32；Davidson等人，Nat Genet.(1993)3:219-223；及Alisky and Davidson,Hum.Gene Ther.(2000)11:2315-29)。
除非本文另外定义，否则结合本发明使用之科学与技术术语将具有本领域普通技术人员通常理解之含义。尽管可使用类似于或等效于本文中所描述之彼等的方法及材料来实践或测试本发明，但下文描述例示性方法及材料。在有矛盾的情况下，将以本发明(包括定义)为准。一般而言，与本文所述之神经学、医学、药物及医药化学以及细胞生物学结合使用之命名法及其技术为本领域中熟知且常用者。酵素反应及纯化技术根据制造商之说明书如本领域中通常所实现或如本文所描述来进行。此外，除非上下文另外需要，否则单数术语应包括复数术语且复数术语应包括单数术语。在本说明书及实施例通篇中，词语“具有(have)”及“包含(comprise)”或诸如“具有(has/having)”、“包含(comprises/comprising)”之变化形式应理解为暗示包括陈述的整数或整数群，但不排除任何其他整数或整数群。本文所提及之所有公开及其他参考文献均以全文引用之方式并入本文中。尽管本文中引用多个文献，但此引用不构成此等文献中之任一者形成本领域中公知常识之部分的许可。如本文所使用，术语“大致”或“约”当应用于所关注之一个或多个值时是指类似于所陈述参考值的值。在某些实施例中，除非另外说明或另外自上下文显而易见，否则该术语是指在陈述参考值之任一方向(大于或小于)上在10％、9％、8％、7％、6％、5％、4％、3％、2％、1％或小于1％内的值范围。
为了能更好地理解本发明，阐述以下实例。此等实例仅为达成说明之目的且不应解释为以任何方式限制本发明之范畴。
实例
实例1：ZFP-TF之筛选
为了鉴定阻遏α-突触核蛋白表达之ZFP-TF，我们设计且筛选416种ZFP-TF之库，该等ZFP-TF经预测在人SNCA基因的跨越TSS 1上游500bp至TSS 1下游500bp或TSS 2a上游500bp至TSS 2b下游500bp之区域中结合15或18bp序列(图2B)。ZFN-TF之靶区域由图2B中之箭头表示，其中箭头之方向指示ZFP-TF结合之DNA链(5’至3’)。图4展示的具有1、2或3个磷酸酯接触变化且靶向SNCA基因之TSS 2a及2b之ZFP-TF之资料与图2B中所显示之彼等数据类似。在此研究中，使用KRAB域序列(SEQ ID NO:12)作为转录阻遏物且该序列融合至ZFP域之C端。47种代表性ZFP-TF之序列展示于下表2中。使用PCR(正向引物GCAGAGCTCTCTGGCTAACTAGAG(SEQ ID NO:13)；反向引物T(180)CTGGCAACTAGAAGGCACAG(SEQID NO:14)由pVAX-ZFP或pVAX-GFP质粒产生用于体外转录之模板。信使RNA根据制造商说明书使用mMESSAGE mMACHINE T7 ULTRA转录试剂盒(Thermo Fisher Scientific)合来成且使用RNeasy96管柱(Qiagen)纯化。接着将编码各ZFP-TF之RNA以6个剂量稀释度等分至96孔板中。
根据众所周知的方法在HEK293细胞中产生携有ZFP-TF编码序列之重组rAAV载体。在细胞经编码AAV辅助基因及rAAV基因组之质粒转染三天之后，收集细胞。接着藉由三轮冷冻/融化使细胞裂解且藉由离心移除细胞碎片。使用聚乙二醇使rAAV病毒粒子沉淀。再悬浮之后，藉由在氯化铯梯度上超速离心隔夜来纯化病毒粒子。病毒粒子藉由透析来调配，接着过滤灭菌。将AAV等分且储存于-80℃下直至使用。在解冻之后，AAV不再冷冻。
在SK-N-MC人神经上皮细胞系中进行筛选。SK-N-MC细胞以高含量表达人α-突触核蛋白且因此适于测试减少α-突触核蛋白表达之ZFP-TF。SK-N-MC细胞在组织培养瓶中培养直至汇合。细胞以150,000个细胞/孔铺板于96孔板上且再悬浮于SF溶液中。细胞随后与ZFP-TF RNA(6个剂量：3、10、30、100、300及1000ng)混合且转移至穿梭板孔。使用 装置(Lonza；程序CM-137)转染细胞。向板之各孔中添加伊格尔氏(Eagle’s)MEM细胞培养基。将细胞转移至96孔组织培养板中且在37℃下温育24小时。
亦在人iPSC衍生GABA能神经元(Cellular Dynamics International)中测试ZFP-TF。以40,000个细胞/孔之密度将细胞铺板于聚-L-鸟氨酸及层粘连蛋白包被之96孔板上，随后根据制造商说明书进行维护。铺板后48小时，用表达所需ZFP-TF之AAV6以6种不同MOI(1E3、3E3、1E4、3E4、1E5及3E5)转染细胞。经转导细胞维护长达32天(每3-5天更换50-75％培养基)。在AAV转染之后28-30天收集细胞。
将收集之细胞溶解且遵循制造商说明书使用C2CT试剂盒进行逆转录。TaqMan定量聚合酶链反应(qPCR)用于测量SNCA之表达水平。SNCA表达水平相对于管家基因EIF4A2、ATP5B及GAPDH之表达量的几何平均值归一化。使用模拟转染及用已知不靶向SNCA之ZFP-TF转染作为阴性对照。
用许多测试之ZFP-TF证实对α-突触核蛋白之剂量依赖性阻遏。达成之最大阻遏超过99％，但我们亦鉴定出阻遏α-突触核蛋白之程度较低(例如，在最高剂量下约90％、约75％或约40％)的ZFP-TF。图3A-E及5展示筛选数据。
实例2：α-突触核蛋白ZFP-TF之脱靶活性
为评估α-突触核蛋白ZFP-TF对整体基因表达之脱靶影响，我们对自人iPSC衍生神经元及原代小鼠皮质神经元分离之总RNA进行微阵列实验，该等神经元经编码代表性α-突触核蛋白ZFP-TF之AAV处理。
如实例1中所述处理人iPSC衍生神经元。对于微阵列分析，以260,000个细胞/孔之密度将细胞铺板于聚-L-鸟氨酸及层粘连蛋白包被之24孔板上，在铺板之后48小时以1E5VG/细胞进行转染，且在病毒转染之后19天收集。自收集之细胞分离之RNA用于微阵列分析。
原代小鼠皮质神经元购自Gibco。细胞以200,000个细胞/孔铺板于聚-D-赖氨酸包被之24孔板上且根据制造商规格使用含有GlutaMAXTM I补充剂、B27补充剂及青霉素/链霉素之Gibco神经基质培养基来维护。铺板后四十八小时(在DIV2下)，细胞以3E3 VG/细胞之MOI感染AAV6且7天后收集(在DIV9下；每3-4天进行50％培养基交换)。此随后为RNA分离及微阵列分析。
根据制造商说明书使用GeneTitanTM平台(Clariom S试剂盒)进行脱靶分析。使用TAC软件分析检定结果。分析中选出调控>2倍且FDR校正p值≤0.05之差异调控基因。使用已知具有最少脱靶之ZFP-TF及模拟转染作为阴性对照。
图7A-D展示人iPSC衍生神经元及原代小鼠皮质神经元中40种代表性α-突触核蛋白ZFP-TF之微阵列结果。存在一系列脱靶活性，其中一些ZFP-TF显示极低至不可检测之脱靶活性。
实例3：α-突触核蛋白mRNA表达之体内阻遏
将表达两种代表性ZFP-TF(82195及82264)之AAV9构建体用于证实人SNCA在PAC突触核蛋白小鼠模型中之体内阻遏，该等代表性ZFP-TF在人与小鼠神经元中具有最小至不可检测脱靶活性且在人iPSC衍生神经元中具有不同的最大阻遏活性(82195，约95％；82264，约80％)(Kuo等人，Hum Mol Genet.(2010)19(9):1633-50)。此小鼠模型在小鼠无α-突触核蛋白背景上表达完整人SNCA序列以及其上游调控序列。表达各ZFP-TF及媒剂之AAV9载体以0.5μL/min之速率双侧施用至8周龄雌性PAC突触核蛋白小鼠(n＝3只/组)中纹状体两个部位(2个部位/半球体：前侧纹状体5μL及后侧纹状体4μl，总共9μL/半球体及18μL/动物)。在输注之后，针留在原位5分钟以允许测试物扩散。接着，在1至2分钟内缓慢收回针。注射之立体定位坐标如下(喙侧纹状体-AP：+1.4mm，ML：+/-1.7mm，DV：-3.0mm；尾侧纹状体：AP：+0.2mm，ML：+/-2.3mm，DV：-2.7mm)。3周之后使小鼠安乐死且收集其脑部用于分子分析。在安乐死时，动物用0.9％盐水经贲门灌注，且移除脑部且切成对半。将左半球体进一步切成12个不同区域(嗅球；喙侧、内侧及尾侧皮质；喙侧、内侧及尾侧纹状体；海马体；丘脑；腹侧中脑；髓质；及小脑)。将切割之组织置放于RNALater中以保持RNA完整性。在24h之后，移除RNAlater且将组织在液氮中快速冷冻且保持在干冰上直至在-80℃下储存。
在冰上将脑部组织转移至含有0.6mL TRI试剂(Thermo Fisher)及两个3.2mm钢珠(BioSpec Products)之1.5mL埃彭道夫管(Eppendorf tube)。在4℃下使用QiagenTissueLyser使用以下参数溶解样品：5个循环，90s持续时间及25.1频率。短暂旋转之后，在室温下将70μL 1-溴-3-氯丙烷添加至各样品中。使样品涡旋10秒，在4℃下以12,000×g离心10分钟，且将来自各样品之120μL水相转移至96孔板之孔中。
将60微升异丙醇及12μL MagMax磁性珠粒(Thermo Scientific)添加至含有组织裂解物之水相的各孔中。遵循制造商之方案使用Kingfisher96机器人(ThermoScientific)及MagMax试剂盒(Thermo Fisher)自组织裂解物分离核糖核酸(RNA)。使用磁性台架自磁性珠粒分离100微升洗脱RNA。使用Nanodrop8000仪器(Thermo Scientific)评估RNA产量及质量。
利用高容量cDNA逆转录试剂盒(Applied Biosystems)制备互补脱氧核糖核酸(cDNA)，预设采用10μL RNA及10μL RT主混合物(10倍RT缓冲液,10倍随机引物，25倍dNTP混合物,Multiscribe酶及无RNA酶水)。必要时，调节RNA及RT主混合物体积以确保输入在100至1,000ng范围内。在C1000Touch Biorad热循环仪上使用以下程序进行逆转录：25℃保持10分钟，37℃保持120分钟，85℃保持5分钟，且保持在4℃下。
使用Biorad CFX384热循环仪使cDNA经历RT-qPCR。cDNA在无核酸酶水中稀释10倍，且将4μL经稀释cDNA添加至各10μL PCR反应中。各样品在技术上一式四份地测定。将2倍Fast Multiplex PCR(Qiagen)主混合物用于三重测定，且SsoAdvanced Universal ProbesSupermix(Biorad)用于其他测定。
使用以下循环条件：Qiagen Fast Multiplex主混合物→95℃持续5分钟，95℃持续45秒，60℃持续45秒，读板，40次循环；Biorad SsoAdvanced主混合物→95℃持续90秒，95℃持续12秒，60℃持续40秒，读取板，42次循环。
在RNA分离步骤之前对比之后掺入GFP RNA用于评估RNA回收％。来自研究中之动物之4个样品5倍稀释系列之合并RNA用作标准曲线。样品数据相对于三个管家基因ATP5B、EIF4A2及GAPDH之几何平均值归一化。
来自实验之α-突触核蛋白、ZFP-TF、GFAP、IBA1及NeuN mRNA表达数据展示于图8A及8B中。数据展示在具有显著ZFP-TF表达之脑部区域中存在α-突触核蛋白之阻遏。此外，GFAP、IBA1及NeuN表达数据表明ZFP-TF是良好耐受的，因为其施用不导致神经炎性标志物之表达升高或神经元标志物NeuN之表达减少。
序列表
下表1列出47种本发明之例示性工程化ZFP。对于各ZFP，ZFP域内各锌指之基因组靶序列(结合序列)及DNA结合之识别螺旋序列(亦即F1-F6)以单行显示。下表中之“^”指示指定螺旋中之第1个氨基酸上游之第4位置处之精氨酸(R)残基变为谷氨酰胺(Q)。各序列之SEQ ID NO在序列下方之括号中展示。在第2行中之核苷酸序列中，ZFP接触之核苷酸以大写字母展示。
表1例示性ZFP
下表2列出47种本发明之例示性ZFP-TF之完整氨基酸序列，其中DNA结合之识别螺旋序列呈粗体字，模块内及模块间接头加下划线。R(-5)Q主链突变由粗体字及下划线指示。ZFP与KRAB域之间的接头加双下划线。
表2例示性ZFP-TF序列
序列表
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<400> 14
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 180
ctggcaacta gaaggcacag 200
<210> 15
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 15
Thr Gly Glu Lys Pro Phe Ala
1 5
<210> 16
<211> 8
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 16
Thr Gly Ser Gln Lys Pro Phe Gln
1 5
<210> 17
<211> 12
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 17
Leu Arg Gln Lys Asp Ala Ala Arg Gly Ser Gly Gly
1 5 10
<210> 18
<211> 6
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 18
Leu Arg Gly Ser Gly Gly
1 5
<210> 19
<211> 21
<212> DNA
<213> 智人
<400> 19
atccgagata gggacgagga g 21
<210> 20
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 20
Gln Ser Gly His Leu Ala Arg
1 5
<210> 21
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 21
Gln Arg Lys Asp Leu Thr Gln
1 5
<210> 22
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 22
Arg Ser Asp His Leu Ser Arg
1 5
<210> 23
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 23
Thr Arg Asp Gly Leu Arg Gln
1 5
<210> 24
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 24
Arg Ser Ala Asn Leu Ala Arg
1 5
<210> 25
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 25
Asp Ser Ser Asp Arg Lys Lys
1 5
<210> 26
<211> 20
<212> DNA
<213> 智人
<400> 26
ccgagatagg gacgaggagc 20
<210> 27
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 27
Arg Ser Asp Asn Leu Ser Thr
1 5
<210> 28
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 28
Arg Ser Asp Asn Leu Thr Arg
1 5
<210> 29
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 29
Asp Arg Ser Asp Leu Ser Arg
1 5
<210> 30
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 30
Arg Arg Glu His Leu Arg Ala
1 5
<210> 31
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 31
Thr Ser Gly Asn Leu Thr Arg
1 5
<210> 32
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 32
Gln Arg Ser His Leu Ser Asp
1 5
<210> 33
<211> 21
<212> DNA
<213> 智人
<400> 33
cagggaaagc agcgagcgcc g 21
<210> 34
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 34
Glu Arg Gly Thr Leu Ala Arg
1 5
<210> 35
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 35
Met Ala Cys Cys Arg Tyr Ala
1 5
<210> 36
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 36
Arg Ser Ala Asp Leu Thr Arg
1 5
<210> 37
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 37
Gln Ser Gly Asp Leu Thr Arg
1 5
<210> 38
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 38
Gln Ser Gly Asn Leu Ala Arg
1 5
<210> 39
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 39
Arg Gln Glu His Arg Val Ala
1 5
<210> 40
<211> 20
<212> DNA
<213> 智人
<400> 40
gaaagcagcg agcgccggga 20
<210> 41
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 41
Arg Ser Ala His Leu Ser Arg
1 5
<210> 42
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 42
Met Ser Cys Cys Arg Ser Ala
1 5
<210> 43
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 43
Arg Ser Ala Asp Leu Ser Arg
1 5
<210> 44
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 44
Gln Arg Ser Asn Leu Asn Ile
1 5
<210> 45
<211> 20
<212> DNA
<213> 智人
<400> 45
ggacggcgac gaccagaagg 20
<210> 46
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 46
Arg Ser Asp Asn Leu Ser Val
1 5
<210> 47
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 47
Ala Ser Trp Thr Leu Thr Gln
1 5
<210> 48
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 48
Asp Arg Ser Asn Leu Thr Arg
1 5
<210> 49
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 49
Asp Arg Ser His Leu Thr Arg
1 5
<210> 50
<211> 20
<212> DNA
<213> 智人
<400> 50
cgcgacgcgg aagtgaggtg 20
<210> 51
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 51
Leu Arg His His Leu Thr Arg
1 5
<210> 52
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 52
Val Ala Glu Tyr Arg Tyr Lys
1 5
<210> 53
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 53
Arg His Ser His Leu Thr Ser
1 5
<210> 54
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 54
Arg Ser Asp Thr Leu Ser Ala
1 5
<210> 55
<211> 20
<212> DNA
<213> 智人
<400> 55
cgctcacgag ggtggaaagg 20
<210> 56
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 56
Gln Ser Gly His Leu Ser Arg
1 5
<210> 57
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 57
Asp Ala His His Leu His Arg
1 5
<210> 58
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 58
Arg Ser Ala Asn Leu Thr Arg
1 5
<210> 59
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 59
Asp Arg Ser Asn Arg Thr Thr
1 5
<210> 60
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 60
His Arg Ser Ser Leu Arg Arg
1 5
<210> 61
<211> 20
<212> DNA
<213> 智人
<400> 61
attcgacgac aggttagcgg 20
<210> 62
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 62
Arg Ser Asp Asp Leu Thr Arg
1 5
<210> 63
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 63
Gln Lys Phe Pro Arg Asp Ser
1 5
<210> 64
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 64
Arg Ser Asp His Leu Thr Gln
1 5
<210> 65
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 65
Asp Ser Ala Asn Leu Ser Arg
1 5
<210> 66
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 66
Asp Arg Ser Asn Leu Ser Arg
1 5
<210> 67
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 67
Asp Arg Thr Ser Leu Lys Trp
1 5
<210> 68
<211> 21
<212> DNA
<213> 智人
<400> 68
ggccgctgtg agccggcgac g 21
<210> 69
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 69
His Leu Cys Cys Leu Gly Arg
1 5
<210> 70
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 70
Arg Ser Cys Cys Leu His Leu
1 5
<210> 71
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 71
Arg Asn Ala Ser Arg Thr Arg
1 5
<210> 72
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 72
Gln Ser Ser Asp Leu Ser Arg
1 5
<210> 73
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 73
His Arg Lys Ser Leu Ser Arg
1 5
<210> 74
<211> 21
<212> DNA
<213> 智人
<400> 74
aggcggcacc cgggggggac t 21
<210> 75
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 75
Asp Arg Ser His Leu Ala Arg
1 5
<210> 76
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 76
Arg Ser Asp His Leu Ser Glu
1 5
<210> 77
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 77
Thr Ser Ser Asp Arg Thr Lys
1 5
<210> 78
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 78
Asp Arg Ser His Leu Ser Arg
1 5
<210> 79
<211> 21
<212> DNA
<213> 智人
<400> 79
gagcgcacag gaagggcgga g 21
<210> 80
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 80
Ala Lys Trp Asn Leu Asp Ala
1 5
<210> 81
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 81
Thr Gln Gly Tyr Leu Arg Lys
1 5
<210> 82
<211> 21
<212> DNA
<213> 智人
<400> 82
ggcggggagg agtcggagtt g 21
<210> 83
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 83
Thr Ser Gly Ser Leu Ser Arg
1 5
<210> 84
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 84
Gln Ala Gly His Leu Ala Lys
1 5
<210> 85
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 85
Asp Arg Ser Ala Arg Thr Arg
1 5
<210> 86
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 86
Gln Ser Ser Asp Leu Arg Arg
1 5
<210> 87
<211> 21
<212> DNA
<213> 智人
<400> 87
ggtcgtgggc accgggaggg g 21
<210> 88
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 88
Gln His Ala His Arg Asn Thr
1 5
<210> 89
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 89
Arg Pro Asp Thr Leu Arg Asp
1 5
<210> 90
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 90
Gln Asn Ala Thr Arg Thr Lys
1 5
<210> 91
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 91
Arg Ser Asp Ala Leu Ser Arg
1 5
<210> 92
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 92
Asp Arg Ser Ala Leu Ala Arg
1 5
<210> 93
<211> 21
<212> DNA
<213> 智人
<400> 93
ggctgcttct ccgggatccg c 21
<210> 94
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 94
Asn Asn Arg Asp Leu Ile Asn
1 5
<210> 95
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 95
Thr Ser Ser Asn Leu Ser Arg
1 5
<210> 96
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 96
Arg Ser Asp Thr Leu Ser Glu
1 5
<210> 97
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 97
Asn Asn Arg Asp Arg Thr Lys
1 5
<210> 98
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 98
His Arg Ser Thr Arg Asn Arg
1 5
<210> 99
<211> 21
<212> DNA
<213> 智人
<400> 99
ggaggacggc gacgaccaga a 21
<210> 100
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 100
Gln Asn Ala His Arg Lys Thr
1 5
<210> 101
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 101
Asp Asn Ser Thr Arg Ile Lys
1 5
<210> 102
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 102
Thr Ser Ala Asn Leu Ser Arg
1 5
<210> 103
<211> 20
<212> DNA
<213> 智人
<400> 103
agagggggcg agcgaccgag 20
<210> 104
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 104
Gln Arg Asn His Arg Thr Thr
1 5
<210> 105
<211> 21
<212> DNA
<213> 智人
<400> 105
cgtcgcggcg ctcggtcgct c 21
<210> 106
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 106
Asp Arg Ser Tyr Arg Asn Thr
1 5
<210> 107
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 107
Arg Arg Ser Asp Leu Lys Arg
1 5
<210> 108
<211> 21
<212> DNA
<213> 智人
<400> 108
ctgcgctgca gcccgcacgc a 21
<210> 109
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 109
Asp Ser Ser Thr Arg Lys Thr
1 5
<210> 110
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 110
Gln Ser Ala Thr Arg Thr Lys
1 5
<210> 111
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 111
Ala Ala Ser Asn Arg Ser Lys
1 5
<210> 112
<211> 21
<212> DNA
<213> 智人
<400> 112
cgtcgaatgg ccactcccag t 21
<210> 113
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 113
Arg Ser Asp Asn Leu Ser Gln
1 5
<210> 114
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 114
Ala Ser Asn Asp Arg Lys Lys
1 5
<210> 115
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 115
Tyr Asp Tyr Gly Arg Tyr Thr
1 5
<210> 116
<211> 21
<212> DNA
<213> 智人
<400> 116
gtacctgtgg atctaaacgg g 21
<210> 117
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 117
Gln Ser Asn His Arg Lys Thr
1 5
<210> 118
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 118
Val Tyr Glu Gly Leu Lys Lys
1 5
<210> 119
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 119
Arg Ser Asp Val Leu Ser Glu
1 5
<210> 120
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 120
Asp Arg Ser Asn Arg Ile Lys
1 5
<210> 121
<211> 21
<212> DNA
<213> 智人
<400> 121
aaagtggggg cgcttttgcg c 21
<210> 122
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 122
Arg Arg Ala Asp Leu Ser Arg
1 5
<210> 123
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 123
Tyr His Trp Tyr Leu Lys Lys
1 5
<210> 124
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 124
Arg Ser Asp His Leu Ser Gln
1 5
<210> 125
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 125
Thr Ser Ala Asn Arg Thr Thr
1 5
<210> 126
<211> 20
<212> DNA
<213> 智人
<400> 126
gcaaggcggg aaagtggggg 20
<210> 127
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 127
Arg Ser Asp His Leu Ser Thr
1 5
<210> 128
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 128
Arg Ser Asp Ala Leu Ala Arg
1 5
<210> 129
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 129
Gln Ser Ala Asn Arg Thr Thr
1 5
<210> 130
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 130
Gln Ser Gly Asn Leu His Val
1 5
<210> 131
<211> 21
<212> DNA
<213> 智人
<400> 131
gcgcaaggcg ggaaagtggg g 21
<210> 132
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 132
Ala Lys His His Arg Lys Thr
1 5
<210> 133
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 133
Arg Ser Asp Asn Leu Ser Ala
1 5
<210> 134
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 134
Gln Asn Arg Asp Arg Lys Asn
1 5
<210> 135
<211> 20
<212> DNA
<213> 智人
<400> 135
cgcgcaaggc gggaaagtgg 20
<210> 136
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 136
Arg Ser Asp Ser Leu Ser Val
1 5
<210> 137
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 137
Gln Asn Ala Asn Arg Lys Thr
1 5
<210> 138
<211> 20
<212> DNA
<213> 智人
<400> 138
ggagcacgct gcagggaaag 20
<210> 139
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 139
Asp Gln Ser Asn Leu Arg Ala
1 5
<210> 140
<211> 21
<212> DNA
<213> 智人
<400> 140
agcacgctgc agggaaagca g 21
<210> 141
<211> 20
<212> DNA
<213> 智人
<400> 141
tgcagggaaa gcagcgagcg 20
<210> 142
<211> 20
<212> DNA
<213> 智人
<400> 142
gtgggggcgg agagccgagg 20
<210> 143
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 143
Arg Ser Asp Asn Leu Ala Arg
1 5
<210> 144
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 144
Asp Arg Ser Val Leu His Arg
1 5
<210> 145
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 145
Gln Ser Ala His Arg Lys Asn
1 5
<210> 146
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 146
Arg Asn Asp His Arg Thr Thr
1 5
<210> 147
<211> 21
<212> DNA
<213> 智人
<400> 147
gagagggggc gagcgaccga g 21
<210> 148
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 148
Gln Gly Ala His Leu Gly Ala
1 5
<210> 149
<211> 21
<212> DNA
<213> 智人
<400> 149
gcagcgcaga ccccggcccg g 21
<210> 150
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 150
Arg Ser Asp Ala Leu Ser Val
1 5
<210> 151
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 151
Asp Ser Ser His Arg Thr Arg
1 5
<210> 152
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 152
Asp Asn Arg Asp Leu Ser Thr
1 5
<210> 153
<211> 21
<212> DNA
<213> 智人
<400> 153
tctcggaggg gccgggccgg g 21
<210> 154
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 154
Asp Asn Ser His Arg Thr Arg
1 5
<210> 155
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 155
Arg Ser Thr His Leu Val Arg
1 5
<210> 156
<211> 20
<212> DNA
<213> 智人
<400> 156
ggacgctctc ggaggggccg 20
<210> 157
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 157
Gln Glu Ser Asp Leu Val Arg
1 5
<210> 158
<211> 20
<212> DNA
<213> 智人
<400> 158
aaaaggagcg cacaggaagg 20
<210> 159
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 159
Arg Arg Ala His Leu Asn Gln
1 5
<210> 160
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 160
Gln Arg Val Ala Leu Gln Ala
1 5
<210> 161
<211> 21
<212> DNA
<213> 智人
<400> 161
ggaaaaggag cgcacaggaa g 21
<210> 162
<211> 21
<212> DNA
<213> 智人
<400> 162
tcaggtaagt acctgtggat c 21
<210> 163
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 163
Arg Ser Glu Leu Leu Gln Ser
1 5
<210> 164
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 164
Ala Arg Ser Thr Arg Ile Thr
1 5
<210> 165
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 165
Gln Ser Gly Ser Leu Thr Arg
1 5
<210> 166
<211> 20
<212> DNA
<213> 智人
<400> 166
tctgggaggt gagtacttgt 20
<210> 167
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 167
Arg Pro Tyr Thr Leu Arg Leu
1 5
<210> 168
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 168
Asp Asn Ser Asn Arg Ile Lys
1 5
<210> 169
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 169
Thr Ser Gly His Leu Ser Arg
1 5
<210> 170
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 170
Arg Lys Trp Thr Leu Gln Gly
1 5
<210> 171
<211> 20
<212> DNA
<213> 智人
<400> 171
ccaaagggac aagtactcac 20
<210> 172
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 172
Gln Ser Ala Asp Arg Thr Lys
1 5
<210> 173
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 173
Asp Ser Ala Asn Arg Ile Lys
1 5
<210> 174
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 174
Gln Asn Ala Asn Arg Ile Thr
1 5
<210> 175
<211> 21
<212> DNA
<213> 智人
<400> 175
cccaaaggga caagtactca c 21
<210> 176
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 176
Arg Ser Ala Asn Leu Ser Val
1 5
<210> 177
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 177
Asp Arg Ala Asn Leu Ser Arg
1 5
<210> 178
<211> 7
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成肽"
<400> 178
Gln Lys Ala Thr Arg Ile Thr
1 5
<210> 179
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 179
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser
20 25 30
Gly His Leu Ala Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Arg Lys Asp Leu Thr
50 55 60
Gln His Thr Lys Ile His Thr His Pro Arg Ala Pro Ile Pro Lys Pro
65 70 75 80
Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp His Leu
85 90 95
Ser Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp
100 105 110
Ile Cys Gly Arg Lys Phe Ala Thr Arg Asp Gly Leu Arg Gln His Thr
115 120 125
Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Arg Ser Ala Asn Leu Ala Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Asp Ser Ser Asp Arg Lys Lys His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 180
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 180
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser
20 25 30
Asp Asn Leu Ser Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Asp Asn Leu Thr
50 55 60
Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Asp Arg Ser Asp Leu Ser Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Arg Arg Glu His Leu Arg Ala His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Thr Ser Gly Asn Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Arg Ser His
165 170 175
Leu Ser Asp His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 181
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 181
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Glu Arg
20 25 30
Gly Thr Leu Ala Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Met Ala Cys Cys Arg Tyr
50 55 60
Ala His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Arg Ser Ala Asp Leu Thr Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Gln Ser Gly Asp Leu Thr Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Gln Ser Gly Asn Leu Ala Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Arg Gln Glu His Arg Val Ala His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 182
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 182
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser
20 25 30
Ala His Leu Ser Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Arg Ser Asp Leu Ser
50 55 60
Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Met Ser Cys Cys Arg Ser Ala His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Arg Ser Ala Asp Leu Ser Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Gln Ser Gly Asp Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Arg Ser Asn
165 170 175
Leu Asn Ile His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 183
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 183
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser
20 25 30
Asp Asn Leu Ser Val His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Ala Ser Trp Thr Leu Thr
50 55 60
Gln His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Asp Arg Ser Asp Leu Ser Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Asp Arg Ser Asn Leu Thr Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Asp Arg Ser His Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Arg Ser Asn
165 170 175
Leu Thr Arg His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 184
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 184
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Leu Arg
20 25 30
His His Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Val Ala Glu Tyr Arg Tyr
50 55 60
Lys His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Arg Ser Asp Asn Leu Ser Val His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Arg His Ser His Leu Thr Ser His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Arg Ser Asp Thr Leu Ser Ala His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Ala Asp
165 170 175
Leu Ser Arg His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 185
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 185
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser
20 25 30
Asp Asn Leu Ser Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Ser Gly His Leu Ser
50 55 60
Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Asp Ala His His Leu His Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Arg Ser Ala Asn Leu Thr Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Asp Arg Ser Asn Arg Thr Thr His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala His Arg Ser Ser
165 170 175
Leu Arg Arg His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 186
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 186
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser
20 25 30
Asp Asp Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Lys Phe Pro Arg Asp
50 55 60
Ser His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Arg Ser Asp His Leu Thr Gln His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Asp Ser Ala Asn Leu Ser Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Asp Arg Ser Asn Leu Ser Arg His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Arg Thr Ser
165 170 175
Leu Lys Trp His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 187
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 187
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Asp Arg
20 25 30
Ser Asn Leu Ser Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala His Leu Cys Cys Leu Gly
50 55 60
Arg His Thr Lys Ile His Thr His Pro Arg Ala Pro Ile Pro Lys Pro
65 70 75 80
Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Cys Cys Leu
85 90 95
His Leu His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp
100 105 110
Ile Cys Gly Arg Lys Phe Ala Arg Asn Ala Ser Arg Thr Arg His Thr
115 120 125
Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Gln Ser Ser Asp Leu Ser Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
His Arg Lys Ser Leu Ser Arg His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 188
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 188
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Asp Arg
20 25 30
Ser Asn Leu Ser Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Arg Ser His Leu Ala
50 55 60
Arg His Thr Lys Ile His Thr His Pro Arg Ala Pro Ile Pro Lys Pro
65 70 75 80
Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp His Leu
85 90 95
Ser Glu His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp
100 105 110
Ile Cys Gly Arg Lys Phe Ala Thr Ser Ser Asp Arg Thr Lys His Thr
115 120 125
Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Asp Arg Ser His Leu Ser Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Asp Arg Ser His Leu Ala Arg His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 189
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 189
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Asp Arg
20 25 30
Ser His Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Arg Ser His Leu Ala
50 55 60
Arg His Thr Lys Ile His Thr His Pro Arg Ala Pro Ile Pro Lys Pro
65 70 75 80
Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser Gly Asn Leu
85 90 95
Ala Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp
100 105 110
Ile Cys Gly Arg Lys Phe Ala Ala Lys Trp Asn Leu Asp Ala His Thr
115 120 125
Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Gln Ser Gly Asp Leu Thr Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Thr Gln Gly Tyr Leu Arg Lys His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 190
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 190
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Thr Ser
20 25 30
Gly Ser Leu Ser Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Ala Gly His Leu Ala
50 55 60
Lys His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Asp Arg Ser Ala Arg Thr Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Gln Ser Gly His Leu Ser Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Arg Ser Ala His Leu Ser Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Gln Ser Ser Asp Leu Arg Arg His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 191
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 191
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser
20 25 30
Asp His Leu Ser Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln His Ala His Arg Asn
50 55 60
Thr His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Arg Pro Asp Thr Leu Arg Asp His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Gln Asn Ala Thr Arg Thr Lys His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Arg Ser Asp Ala Leu Ser Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Asp Arg Ser Ala Leu Ala Arg His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 192
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 192
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Asn Asn
20 25 30
Arg Asp Leu Ile Asn His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Thr Ser Ser Asn Leu Ser
50 55 60
Arg His Thr Lys Ile His Thr His Pro Arg Ala Pro Ile Pro Lys Pro
65 70 75 80
Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp Thr Leu
85 90 95
Ser Glu His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp
100 105 110
Ile Cys Gly Arg Lys Phe Ala Asn Asn Arg Asp Arg Thr Lys His Thr
115 120 125
Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Gln Ser Ser Asp Leu Ser Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
His Arg Ser Thr Arg Asn Arg His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 193
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 193
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Asn
20 25 30
Ala His Arg Lys Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Asn Ser Thr Arg Ile
50 55 60
Lys His Thr Lys Ile His Thr His Pro Arg Ala Pro Ile Pro Lys Pro
65 70 75 80
Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Asp Arg Ser Asn Leu
85 90 95
Ser Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp
100 105 110
Ile Cys Gly Arg Lys Phe Ala His Leu Cys Cys Leu Gly Arg His Thr
115 120 125
Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Asp Arg Ser Asn Leu Ser Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Thr Ser Ala Asn Leu Ser Arg His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 194
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 194
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Arg
20 25 30
Asn His Arg Thr Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Arg Ser Asn Leu Thr
50 55 60
Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Met Ser Cys Cys Arg Ser Ala His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Arg Ser Ala Asp Leu Ser Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Arg Ser Ala His Leu Ser Arg His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Thr Ser Ala Asn
165 170 175
Leu Ser Arg His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 195
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 195
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser
20 25 30
Ser Asp Leu Ser Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Arg Ser Ala Leu Ala
50 55 60
Arg His Thr Lys Ile His Thr His Pro Arg Ala Pro Ile Pro Lys Pro
65 70 75 80
Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Asp Arg Ser Tyr Arg
85 90 95
Asn Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp
100 105 110
Ile Cys Gly Arg Lys Phe Ala Arg Arg Ser Asp Leu Lys Arg His Thr
115 120 125
Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Arg Ser Ala Asp Leu Thr Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Asp Arg Ser Ala Leu Ala Arg His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 196
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 196
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Asp Ser
20 25 30
Ser Thr Arg Lys Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Ser Ala Thr Arg Thr
50 55 60
Lys His Thr Lys Ile His Thr His Pro Arg Ala Pro Ile Pro Lys Pro
65 70 75 80
Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Glu Arg Gly Thr Leu
85 90 95
Ala Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp
100 105 110
Ile Cys Gly Arg Lys Phe Ala Gln Ser Gly Asp Leu Thr Arg His Thr
115 120 125
Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Gln Ser Ser Asp Leu Ser Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Ala Ala Ser Asn Arg Ser Lys His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 197
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 197
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser
20 25 30
Asp Asn Leu Ser Gln His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Ala Ser Asn Asp Arg Lys
50 55 60
Lys His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Tyr Asp Tyr Gly Arg Tyr Thr His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Asp Arg Ser His Leu Ala Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Gln Ser Gly Asn Leu Ala Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Asp Arg Ser Ala Leu Ala Arg His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 198
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 198
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser
20 25 30
Asp His Leu Ser Glu His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Ser Asn His Arg Lys
50 55 60
Thr His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Val Tyr Glu Gly Leu Lys Lys His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Gln Ser Gly His Leu Ser Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Arg Ser Asp Val Leu Ser Glu His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Asp Arg Ser Asn Arg Ile Lys His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 199
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 199
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Arg Arg
20 25 30
Ala Asp Leu Ser Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Tyr His Trp Tyr Leu Lys
50 55 60
Lys His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Gln Ser Ser Asp Leu Ser Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Asp Arg Ser His Leu Ala Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Gln Asn Phe Ser Arg Ser Asp His Leu Ser Gln His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Thr Ser Ala Asn Arg Thr Thr His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 200
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 200
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Arg Ser
20 25 30
Asp His Leu Ser Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Asp Ala Leu Ala
50 55 60
Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Gln Ser Ala Asn Arg Thr Thr His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Arg Ser Ala His Leu Ser Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Asp Arg Ser His Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Ser Gly Asn
165 170 175
Leu His Val His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 201
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 201
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Arg Ser
20 25 30
Asp His Leu Ser Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Asp Ala Leu Ala
50 55 60
Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Gln Ser Ala Asn Arg Thr Thr His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Arg Ser Ala His Leu Ser Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser
130 135 140
Asp Arg Ser His Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Ser Gly Asn
165 170 175
Leu His Val His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 202
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 202
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Arg Ser
20 25 30
Asp His Leu Ser Glu His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Ala Lys His His Arg Lys
50 55 60
Thr His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Gln Ser Gly Asn Leu Ala Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Arg Gln Glu His Arg Val Ala His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Arg Ser Asp Asn Leu Ser Ala His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Gln Asn Arg Asp Arg Lys Asn His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 203
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 203
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Arg Ser
20 25 30
Asp Ser Leu Ser Val His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Asn Ala Asn Arg Lys
50 55 60
Thr His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Arg Ser Ala His Leu Ser Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Asp Arg Ser His Leu Ala Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Gln Ser Ala Asn Arg Thr Thr His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Arg Ser Asp
165 170 175
Leu Lys Arg His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 204
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 204
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Arg Ser
20 25 30
Asp Ser Leu Ser Val His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Asn Ala Asn Arg Lys
50 55 60
Thr His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Arg Ser Ala His Leu Ser Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Asp Arg Ser His Leu Ala Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Gln Ser Ala Asn Arg Thr Thr His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Arg Ser Asp
165 170 175
Leu Lys Arg His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 205
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 205
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Gln Ser
20 25 30
Ala Asn Arg Thr Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Ala His Leu Ser
50 55 60
Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Gln Ser Gly Asp Leu Thr Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Gln Ser Ser Asp Leu Arg Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Asp Gln Ser Asn Leu Arg Ala His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Ala Asn
165 170 175
Leu Thr Arg His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 206
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 206
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Gln Ser
20 25 30
Ala Asn Arg Thr Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Ala His Leu Ser
50 55 60
Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Gln Ser Gly Asp Leu Thr Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Gln Ser Ser Asp Leu Arg Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser
130 135 140
Asp Gln Ser Asn Leu Arg Ala His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Ala Asn
165 170 175
Leu Thr Arg His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 207
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 207
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Gln Ser
20 25 30
Gly Asp Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Arg Ser Asn Leu Asn
50 55 60
Ile His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Arg Ser Ala His Leu Ser Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Gln Ser Gly Asp Leu Thr Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Asp Ser Ser Thr Arg Lys Thr His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Gln Ser Ala Thr Arg Thr Lys His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 208
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 208
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Gln Ser
20 25 30
Gly Asp Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Arg Ser Asn Leu Asn
50 55 60
Ile His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Arg Ser Ala His Leu Ser Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Gln Ser Gly Asp Leu Thr Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Asp Ser Ser Thr Arg Lys Thr His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Gln Ser Ala Thr Arg Thr Lys His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 209
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 209
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Met Ser
20 25 30
Cys Cys Arg Ser Ala His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Ala Asp Leu Ser
50 55 60
Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Gln Ser Gly Asp Leu Thr Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Gln Arg Ser Asn Leu Asn Ile His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser
130 135 140
Arg Ser Ala His Leu Ser Arg His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Ser Gly Asp
165 170 175
Leu Thr Arg His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 210
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 210
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Arg Ser
20 25 30
Asp Asn Leu Ala Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Arg Ser Val Leu His
50 55 60
Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Gln Ser Ala His Arg Lys Asn His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Arg His Ser His Leu Thr Ser His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Arg Ser Asp His Leu Ser Glu His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Asn Asp His
165 170 175
Arg Thr Thr His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 211
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 211
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Gln Arg
20 25 30
Asn His Arg Thr Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Arg Ser Asn Leu Thr
50 55 60
Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Met Ser Cys Cys Arg Ser Ala His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Arg Ser Ala Asp Leu Ser Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Arg Ser Asp His Leu Ser Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Gln Gly Ala His Leu Gly Ala His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 212
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 212
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Arg Ser
20 25 30
Asp Ala Leu Ser Val His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Ser Ser His Arg Thr
50 55 60
Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Asp Asn Arg Asp Leu Ser Thr His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Asp Arg Ser Asn Leu Thr Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Gln Ser Gly Asp Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Thr Gln Gly Tyr
165 170 175
Leu Arg Lys His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 213
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 213
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Arg Ser
20 25 30
Asp His Leu Ser Gln His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Asn Ser His Arg Thr
50 55 60
Arg His Thr Lys Ile His Thr His Pro Arg Ala Pro Ile Pro Lys Pro
65 70 75 80
Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Asp Arg Ser Asp Leu
85 90 95
Ser Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp
100 105 110
Ile Cys Gly Arg Lys Phe Ala Arg Ser Thr His Leu Val Arg His Thr
115 120 125
Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Asp Arg Ser His Leu Thr Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Asp Arg Ser Tyr Arg Asn Thr His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 214
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 214
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Asp Arg
20 25 30
Ser Asp Leu Ser Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Thr His Leu Val
50 55 60
Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Asp Arg Ser His Leu Thr Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Asp Arg Ser Tyr Arg Asn Thr His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Gln Glu Ser Asp Leu Val Arg His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Arg Ser Asn
165 170 175
Leu Thr Arg His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 215
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 215
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Arg Ser
20 25 30
Asp Asn Leu Ser Val His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Arg Ala His Leu Asn
50 55 60
Gln His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Asp Gln Ser Asn Leu Arg Ala His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Arg Arg Ser Asp Leu Lys Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Gln Ser Gly His Leu Ala Arg His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Arg Val Ala
165 170 175
Leu Gln Ala His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 216
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 216
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Gln Ser
20 25 30
Gly Asn Leu Ala Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Ala Lys Trp Asn Leu Asp
50 55 60
Ala His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Gln Ser Gly Asp Leu Thr Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Thr Gln Gly Tyr Leu Arg Lys His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Arg Ser Asp Asn Leu Ser Val His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Gln Ser Gly Asp Leu Thr Arg His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 217
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 217
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Thr Ser
20 25 30
Gly Asn Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Glu Leu Leu Gln
50 55 60
Ser His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Ala Arg Ser Thr Arg Ile Thr His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Gln Ser Gly Ser Leu Thr Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Gln Ser Gly Ser Leu Thr Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Ala Lys Trp Asn Leu Asp Ala His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 218
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 218
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Thr Ser
20 25 30
Gly Asn Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Glu Leu Leu Gln
50 55 60
Ser His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Ala Arg Ser Thr Arg Ile Thr His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Gln Ser Gly Ser Leu Thr Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Gln Ser Gly Ser Leu Thr Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Ala Lys Trp Asn Leu Asp Ala His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 219
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 219
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Thr Ser
20 25 30
Gly Asn Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Glu Leu Leu Gln
50 55 60
Ser His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Ala Arg Ser Thr Arg Ile Thr His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Gln Ser Gly Ser Leu Thr Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Gln Asn Phe Ser Gln Ser Gly Ser Leu Thr Arg His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Ala Lys Trp Asn Leu Asp Ala His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 220
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 220
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Arg Pro
20 25 30
Tyr Thr Leu Arg Leu His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Asn Ser Asn Arg Ile
50 55 60
Lys His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Arg Ser Ala Asn Leu Ala Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Thr Ser Gly His Leu Ser Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser
130 135 140
Gln Ser Gly His Leu Ala Arg His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Lys Trp Thr
165 170 175
Leu Gln Gly His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 221
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 221
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Arg Pro
20 25 30
Tyr Thr Leu Arg Leu His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Asn Ser Asn Arg Ile
50 55 60
Lys His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Arg Ser Ala Asn Leu Ala Arg His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Thr Ser Gly His Leu Ser Arg His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser
130 135 140
Gln Ser Gly His Leu Ala Arg His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Lys Trp Thr
165 170 175
Leu Gln Gly His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 222
<211> 294
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 222
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Gln Ser
20 25 30
Ala Asp Arg Thr Lys His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Asn Ser Asn Arg Ile
50 55 60
Lys His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Arg Ser Asp Asn Leu Ser Thr His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Asp Ser Ala Asn Arg Ile Lys His Thr Lys Ile His Thr
115 120 125
Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser
130 135 140
Arg Ser Asp His Leu Ser Glu His Ile Arg Thr His Thr Gly Glu Lys
145 150 155 160
Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Asn Ala Asn
165 170 175
Arg Ile Thr His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg
180 185 190
Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu
195 200 205
Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys
210 215 220
Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
225 230 235 240
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp
245 250 255
Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg
260 265 270
Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile
275 280 285
Lys Ser Ser Val Arg Ser
290
<210> 223
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 223
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Gln Ser
20 25 30
Ala Asp Arg Thr Lys His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Asn Ser Asn Arg Ile
50 55 60
Lys His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Arg Asn Phe Ser Arg Ser Ala Asn Leu Ser Val His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Asp Arg Ala Asn Leu Ser Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Arg Ser Asp Asn Leu Ser Val His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Gln Lys Ala Thr Arg Ile Thr His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 224
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 224
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Gln Ser
20 25 30
Ala Asp Arg Thr Lys His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Asn Ser Asn Arg Ile
50 55 60
Lys His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Arg Ser Ala Asn Leu Ser Val His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Asp Arg Ala Asn Leu Ser Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Arg Asn Phe Ser Arg Ser Asp Asn Leu Ser Val His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Gln Lys Ala Thr Arg Ile Thr His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 225
<211> 298
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /注释="人工序列的描述：合成多肽"
<400> 225
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Val Pro Ala Ala Met Ala
1 5 10 15
Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Gln Asn Phe Ser Gln Ser
20 25 30
Ala Asp Arg Thr Lys His Ile Arg Thr His Thr Gly Glu Lys Pro Phe
35 40 45
Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Asn Ser Asn Arg Ile
50 55 60
Lys His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg
65 70 75 80
Ile Cys Met Gln Asn Phe Ser Arg Ser Ala Asn Leu Ser Val His Ile
85 90 95
Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg
100 105 110
Lys Phe Ala Asp Arg Ala Asn Leu Ser Arg His Thr Lys Ile His Thr
115 120 125
His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln Cys Arg Ile Cys Met
130 135 140
Gln Asn Phe Ser Arg Ser Asp Asn Leu Ser Val His Ile Arg Thr His
145 150 155 160
Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala
165 170 175
Gln Lys Ala Thr Arg Ile Thr His Thr Lys Ile His Leu Arg Gln Lys
180 185 190
Asp Ala Ala Arg Gly Ser Gly Gly Asp Ala Lys Ser Leu Thr Ala Trp
195 200 205
Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg
210 215 220
Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn
225 230 235 240
Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu
245 250 255
Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp
260 265 270
Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr
275 280 285
Ala Phe Glu Ile Lys Ser Ser Val Arg Ser
290 295
<210> 226
<211> 18
<212> DNA
<213> 智人
<400> 226
aaagcagcga gcgccggg 18

Claims (27)

1.融合蛋白，其包含锌指蛋白(ZFP)域及转录阻遏物域，其中所述ZFP域结合至人α-突触核蛋白基因(SNCA基因)的靶区域。
2.如权利要求1的融合蛋白，其中所述靶区域在所述SNCA基因中的转录起始位点(TSS)的1kb内。
3.如权利要求2的融合蛋白，其中所述TSS为TSS1、2a或2b。
4.如权利要求3的融合蛋白，其中所述靶区域在所述SNCA基因的TSS 1上游500bp内、TSS 1下游500bp内、TSS 2a上游500bp内和/或TSS 2b下游500bp内。
5.如前述权利要求中任一项的融合蛋白，其中所述ZFP域包含六个锌指且所述融合蛋白任选地在没有可检测的脱靶结合或活性或在最小的可检测的脱靶结合或活性下将所述SNCA基因的表达阻遏至少约40％、75％、90％、95％或99％。
6.如前述权利要求中任一项的融合蛋白，其中所述转录阻遏物域包含来自人KOX1的KRAB域氨基酸序列。
7.如前述权利要求中任一项的融合蛋白，其中所述ZFP域经由肽接头连接至所述转录阻遏物。
8.如前述权利要求中任一项的融合蛋白，其中所述ZFP域包含表1中所示的DNA结合的识别螺旋序列。
9.如前述权利要求中任一项的融合蛋白，其中所述ZFP域包含如表1的单行中所示的DNA结合的识别螺旋序列。
10.核酸构建体，其包含如权利要求1至9中任一项的融合蛋白的编码序列，其中所述编码序列可操作地连接至转录调控元件。
11.如权利要求10的核酸构建体，其中所述转录调控元件是在脑细胞中为组成性活性或诱导性的哺乳动物启动子，且其中所述启动子任选地为人突触蛋白I启动子。
12.重组病毒，其包含如权利要求10或11的核酸构建体。
13.如权利要求12的重组病毒，其中所述重组病毒为腺相关病毒载体、腺病毒载体或慢病毒载体。
14.药物组合物，其包含如权利要求10或11的核酸构建体或如权利要求12或13的重组病毒及药学上可接受的载剂。
15.宿主细胞，其包含如权利要求10或11的核酸构建体或如权利要求12或13的重组病毒。
16.如权利要求15的宿主细胞，其中所述宿主细胞为人细胞。
17.如权利要求15的宿主细胞，其中所述宿主细胞为脑细胞或多能干细胞，其中所述干细胞任选地为胚胎干细胞或诱导性多能干细胞(iPSC)。
18.抑制人脑细胞中α-突触核蛋白的表达的方法，其包括任选地经由引入如权利要求10或11的核酸构建体或如权利要求12或13的重组病毒而将如权利要求1至9中任一项的融合蛋白引入所述细胞中，由此抑制所述细胞中所述α-突触核蛋白的表达。
19.如权利要求18的方法，其中所述人脑细胞为神经元、胶质细胞、室管膜细胞或神经上皮细胞。
20.如权利要求18或19的方法，其中所述细胞在罹患帕金森氏症(Parkinson’sdisease)、路易体痴呆(Lewy body dementia)、阿尔茨海默氏症(Alzheimer’s disease)、多系统萎缩或另一种突触核蛋白病或处于罹患所述疾病的风险下的患者的脑部中。
21.如权利要求18至20中任一项的方法，其包括将表达所述融合蛋白的重组病毒引入所述细胞中。
22.如权利要求21的方法，其中所述重组病毒为腺相关病毒(AAV)，任选地为血清型9的腺相关病毒。
23.治疗患者的突触核蛋白病的方法，其包括向所述患者施用编码如权利要求1至9中任一项的融合蛋白的重组AAV。
24.如权利要求23的方法，其中经由静脉内、鞘内、脑室内、大池内、纹状体内或黑质内注射或注射至任何脑部区域中来将所述AAV引入所述患者。
25.如权利要求23或24的方法，其中所述突触核蛋白病是帕金森氏症、路易体痴呆、阿尔茨海默氏症或多系统萎缩。
26.如权利要求1至9中任一项的融合蛋白、如权利要求10或11的核酸构建体、如权利要求12或13的重组病毒、如权利要求14的药物组合物，其用于如权利要求18至25中任一项的方法中。
27.如权利要求1至9中任一项的融合蛋白、如权利要求10或11的核酸构建体、如权利要求12或13的重组病毒在制造如权利要求18至25中任一项的方法中的药物中的用途。
CN202180079361.8A 2020-10-02 2021-10-01 用于阻遏α-突触核蛋白(SYNUCLEIN)表达的新颖锌指蛋白转录因子 Pending CN116917335A (zh)

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