CN113337488B - 一种分离的Cas13蛋白 - Google Patents

一种分离的Cas13蛋白 Download PDF

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CN113337488B
CN113337488B CN202010136188.8A CN202010136188A CN113337488B CN 113337488 B CN113337488 B CN 113337488B CN 202010136188 A CN202010136188 A CN 202010136188A CN 113337488 B CN113337488 B CN 113337488B
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陈玲玲
杨力
杨良中
李斯琪
王滢
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Abstract

本发明涉及生物技术领域，特别是涉及一种分离的Cas13蛋白。本发明提供一种分离的Cas13蛋白，所述Cas13蛋白的氨基酸序列包括：如SEQ ID NO.1~3其中之一所示的氨基酸序列；或，与SEQ ID NO.1~3具有80%以上序列相似性的氨基酸序列。本发明所提供的新型Cas13蛋白，具有结合RNA的能力，且可以与gRNA相配的情况下具有RNA靶向切割活性，从而可以可用于RNA编辑、核酸检测等，基于新型Cas13蛋白的碱基编辑体系可以在37℃实现高灵敏、高精度的分子检测，具有很好的特异性和兼容性，检测灵敏度高，且检测成本低廉、操作方便、快捷、应用范围广，在核酸检测方面具有很好的应用前景。

Description

一种分离的Cas13蛋白
技术领域
本发明涉及生物技术领域，特别是涉及一种分离的Cas13蛋白。
背景技术
规律成簇间隔短回文重复系统(clustered regularly interspacedshortpalindromic repeat；CRISPR-associated，CRISPR-Cas)是古菌和细菌的抵抗外源核酸侵染的重要免疫防御系统，用来抵抗噬菌体病毒等的入侵。
CRISPR/Cas系统可以特异性识别并切割外源DNA或RNA，从而沉默外源基因的表达。目前已知的CRISPR-Cas系统具有非常多的类别，其中CRISPR-Cas9系统被广泛应用于基因敲除，敲入，碱基编辑等，克服了传统基因编辑技术步骤繁琐、耗时长、效率低等缺点，以其较少的成分、便捷的操作以及较高的效率满足了大多数领域的基因编辑需求，并有着潜在且巨大的临床应用价值。CRISPR/Cas13是靶向RNA的CRISPR系统，共有VI-A(Cas13a/C2c2)，VI-B(Cas13b)，VI-C(Cas13c)和VI-D(Cas13d)四种亚型，从15年开始被发现以来已经被应用于RNA敲低，RNA碱基编辑和RNA剪接调控等多方面。除了其RNA特异性切割活性之外，CRISPR/Cas13蛋白被激活后还能非特异性地切割其他RNA(附属切割)。这一新的特性，为基于CRISPR/Cas13系统的核酸检测平台提供了关键的理论支持。利用这种新的CRISPR技术：CRISPR-Cas13可以高灵敏度检测MERS等RNA病毒感染在内的疾病，并且不同Cas13蛋白连用可以实现多种核酸的同时检测。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种分离的Cas13蛋白，用于解决现有技术中的问题。
为实现上述目的及其他相关目的，本发明提供一种分离的Cas13蛋白，所述Cas13蛋白的氨基酸序列包括：
a)如SEQ ID NO.1～3其中之一所示的氨基酸序列；或，
b)与SEQ ID NO.1～3具有80％以上序列相似性的氨基酸序列、且具有a)所限定的氨基酸序列的功能。
在本发明一些实施方式中，所述分离的Cas13蛋白能够特异性地切割目的RNA。
在本发明一些实施方式中，所述目的RNA源自新型冠状病毒2019-nCoV。
在本发明一些实施方式中，所述分离的Cas13蛋白被激活后能够非特异性地切割其他RNA。
在本发明一些实施方式中，所述分离的Cas13蛋白来源于Bacteroidesgraminisolvens；
和/或，所述分离的Cas13蛋白来源于Prevotella intermedia；
和/或，所述分离的Cas13蛋白来源于Prevotellacae bacterium。
在本发明一些实施方式中，所述分离的Cas13蛋白的RNA酶活结构域HEPN motif突变后丧失切割RNA的活性、但所述分离的Cas13蛋白仍保留结合RNA的能力。
本发明另一方面提供一种分离的多核苷酸，编码所述的分离的Cas13蛋白。
本发明另一方面提供一种构建体，所述构建体含有、所述的分离的多核苷酸。
本发明另一方面提供一种表达系统，所述表达系统含有所述的构建体或基因组中整合有外源的所述的多核苷酸。
在本发明一些实施方式中，所述表达系统的宿主细胞选自真核细胞或原核细胞，优选选自人细胞。
本发明另一方面提供所述的分离的Cas13蛋白、所述的分离的多核苷酸、所述的构建体或所述的表达系统在RNA水平的基因编辑中的用途。
在本发明一些实施方式中，所述用途具体为在真核生物的RNA水平的基因编辑、或核酸检测中的用途。
本发明另一方面提供一种碱基编辑体系，包括所述的分离的Cas13蛋白，所述碱基编辑体系还包括gRNA。
本发明另一方面提供一种RNA水平的基因编辑方法，包括：通过所述的分离的Cas13蛋白、或所述的碱基编辑体系进行基因编辑。
本发明另一方面提供一种核酸检测方法，包括：通过所述的分离的Cas13蛋白、或所述的碱基编辑体系进行核酸检测。
在本发明一些实施方式中，所述核酸检测方法包括：
1)提供扩增的目的片段；
2)通过所述的分离的Cas13蛋白、或所述的碱基编辑体系对步骤1)所提供的扩增产物进行结合、切割；
和/或，所述核酸检测方法为小胖威利综合征、或新型冠状病毒2019-nCoV的检测方法。
在本发明一些实施方式中，所述核酸检测方法还包括：通过探针，判断检测结果。
附图说明
图1显示为本发明实施例1中基因组数据分析流程示意图。
图2显示为本发明实施例2中蛋白纯化结果示意图。
图3显示为本发明实施例2中northern blot鉴定结果示意图。
图4a显示为本发明实施例3中Pin4Cas13b的附属切割活性示意图。
图4b显示为本发明实施例3中BgrCas13a的附属切割活性示意图。
图5显示为本发明实施例4中敲低RNA活性鉴定结果示意图。
图6显示为本发明实施例5中标记RNA能力鉴定结果示意图。
图7a显示为本发明实施例6中Cas13蛋白用于检测sno-lncRNA1结果示意图。
图7b显示为本发明实施例6中Cas13蛋白用于检测sno-lncRNA2结果示意图。
图7c显示为本发明实施例6中Cas13蛋白用于检测sno-lncRNA3结果示意图。
图7d显示为本发明实施例6中Cas13蛋白用于检测sno-lncRNA4结果示意图。
图7e显示为本发明实施例6中Cas13蛋白用于检测sno-lncRNA5结果示意图。
图8显示为本发明实施例7中底物RNA荧光变化示意图。
图9显示为本发明实施例8中试纸检测结果示意图。
图10显示为本发明实施例8中RNA检测比较示意图。
图11显示为本发明实施例9中新型冠状病毒特异性靶标构建示意图。
图12显示为本发明实施例9中新型冠状病毒gRNA筛选结果示意图。
图13显示为本发明实施例9中试纸检测结果示意图。
具体实施方式
为了使本发明的发明目的、技术方案和有益技术效果更加清晰，以下结合实施例对本发明进行进一步详细说明，熟悉此技术的人士可由本说明书所揭露的内容容易地了解本申请发明的其他优点及功效。
本发明发明人经过大量研究，提供了一种新型的分离的Cas13蛋白，所述Cas13蛋白具有结合RNA的能力，且可以与gRNA相配的情况下具有RNA靶向切割活性，从而可用于RNA水平的基因编辑、核酸检测等，在此基础上完成了本发明。
本发明第一方面提供一种分离的Cas13蛋白，所述Cas13蛋白的氨基酸序列包括：
a)如SEQ ID NO.1～3其中之一所示的氨基酸序列；或，
b)与SEQ ID NO.1～3具有80％以上序列相似性的氨基酸序列、且具有a)所限定的氨基酸序列的功能。具体的，所述b)中的氨基酸序列具体指：如SEQ ID No.1～3其中之一所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID No.1～3其中之一所示的多肽片段的功能的多肽片段，例如，可以是具有能够与gRNA相配合，对RNA(特异或者非特异)进行切割的功能。所述b)中的氨基酸序列可与SEQ ID NO.1～3其中之一所示的氨基酸序列具有80％、85％、90％、93％、95％、97％、或99％以上的相似性。所述分离的Cas13蛋白源自Bacteroidesgraminisolvens、Prevotella intermedia、或Prevotellacae bacterium。
本发明还提供了具有上述氨基酸序列的分离的多肽作为Cas13蛋白的用途。本发明所提供的分离的Cas13蛋白可以与特定的gRNA相配合，从而可以使所述分离的Cas13蛋白能够特异性地切割目的RNA，同时所述分离的Cas13蛋白可以被激活并非特异切割其他RNA，例如，Cas13识别到特异的靶序列之后即被激活。在本发明一具体实施例中，所述目的RNA可以源自新型冠状病毒2019-nCoV。在本发明另一具体实施例中，所述目的RNA可以源自患有小胖威利综合征的个体。
本发明所提供的分离的Cas13蛋白可以具有结合RNA的能力，当所述分离的Cas13蛋白丧失切割RNA的活性、且所述分离的Cas13蛋白保留结合RNA的能力时，所述分离的Cas13蛋白可以用来进行RNA标记。在本发明另一具体实施例中，所述分离的Cas13蛋白的RNA酶活结构域HEPN motif具有突变。
本发明第二方面提供一种分离的多核苷酸，编码本发明第一方面所提供的分离的Cas13蛋白。
本发明第三方面提供一种构建体，所述构建体含有本发明第二方面所提供的分离的多核苷酸。所述构建体通常可以通过将所述分离的多核苷酸插入合适的表达载体中构建获得，本领域技术人员可选择合适的表达载体，例如，所述表达载体可以是包括但不限于质粒、病毒以及噬菌体等表达载体。在本发明一具体实施例中，所述表达载体可以是pCMV载体等。
本发明第四方面提供一种表达系统，所述表达系统含有本发明第三方面所提供的构建体或基因组中整合有外源的本发明第二方面所提供的分离的多核苷酸。所述表达系统可以是宿主细胞，所述宿主细胞可以表达如上所述的分离的Cas13蛋白，所述分离的Cas13蛋白可以与gRNA相配合，从而可以将所述分离的Cas13蛋白定位到目标区域，实现目标区域的结合，并可以进一步实现对核酸的目标区域的切割。所述宿主细胞可以是真核细胞和/或原核细胞等，例如，可以是人细胞等。在本发明另一具体实施例中，所述宿主细胞可以是HeLa、293FT等细胞系。
本发明第五方面提供本发明第一方面所提供的分离的Cas13蛋白、或本发明第二方面所提供的分离的多核苷酸、或本发明第三方面所提供的构建体、或本发明第四方面所提供的表达系统在RNA编辑、或核酸检测中的用途，优选为真核生物的RNA编辑、或核酸检测中的用途，所述真核生物具体可以是后生动物，具体可以是包括但不限于人、小鼠、线虫、涡虫等。所述核酸可以是DNA和/或RNA。所述用途具体可以是通过所述分离的Cas13蛋白与特定的gRNA相配合，从而可以使所述分离的Cas13蛋白能够特异性地切割目的RNA，从而实现靶标片段的RNA编辑(例如，RNA敲低、敲除等)，或者实现靶标片段的核酸的检测。所述用途具体还可以是通过丧失切割RNA的活性、且保留结合RNA的能力的分离的Cas13蛋白，从而实现分离的Cas13蛋白对靶标片段的RNA标记，从而实现靶标片段的核酸的检测。所述用途也可以是将本发明所提供的分离的Cas13蛋白、分离的多核苷酸、构建体、表达系统与其他技术相配合(例如，RPA技术等)，对目的DNA进行扩增，同时在上游引入T7启动子，这样可以通过T7 RNA聚合酶将其转录成RNA，从而可以使其被Cas13检测到，以实现DNA检测的检测。
本发明第六方面提供一种碱基编辑体系，包括本发明第一方面所提供的分离的Cas13蛋白，所述碱基编辑体系还包括gRNA。本领域技术人员可以根据基因的目标编辑区域，选择合适的靶向特异性位点的gRNA。例如，所述gRNA的序列通常可以与目标区域至少部分互补，从而可以与所述分离的Cas13蛋白相配合，将所述Cas13蛋白定位到目标区域，实现Cas13蛋白与目标区域的结合，并可以进一步实现对核酸的目标区域的切割。在本发明一具体实施例中，gRNA可以靶向新型冠状病毒2019-nCoV的核酸，所述gRNA的多核苷酸序列具体可以包括SEQ ID NO.53～62其中之一所示序列。所述碱基编辑体系还可以包括其他CRISPR-Cas13体系所需要的的各种试剂，例如，还可以包括RNA酶灭活剂、重组酶聚合酶扩增反应试剂、T7 RNA聚合酶、rNTP、Cas13蛋白反应缓冲液和yeast tRNA等。RNA酶灭活剂可以用于抑制样品中RNA酶活性，从而抑制其对RNA的降解，有效的保护待检测RNA。所述rNTP可以为核糖核苷三磷酸，在RNA合成中起原料作用。所述yeast tRNA可以为酵母tRNA，用于提高特异性。
本发明第七方面提供一种RNA编辑方法，包括：通过本发明第一方面所提供的分离的Cas13蛋白、或如本发明第六方面所提供的碱基编辑体系进行编辑。例如，所述RNA编辑方法可以包括：在适当条件下培养本发明第四方面所提供的表达系统，从而表达所述分离的Cas13蛋白，所述分离的Cas13蛋白可以在与其配合的靶向目标区域的gRNA存在的条件下，对靶标区域进行碱基编辑。提供所述gRNA存在的条件的方法对于本领域技术人员来说应该是已知的，例如，可以是在适当条件下培养能够表达所述gRNA的表达系统，所述表达系统可以是包括含有编码所述gRNA的多核苷酸的表达载体的宿主细胞、或染色体中整合有编码所述gRNA的多核苷酸的宿主细胞。在本发明一具体实施例中，所述gRNA与所述分离的Cas13蛋白可以在同一宿主细胞中表达，所述宿主细胞可以为靶细胞。在本发明另一具体实施例中，所述基因编辑为体外基因编辑。
本发明第八方面提供一种核酸检测方法，包括：通过本发明第一方面所提供的分离的Cas13蛋白、或如本发明第六方面所提供的碱基编辑体系进行核酸检测。例如，所述核酸检测方法可以包括：在适当条件下培养本发明第四方面所提供的表达系统，从而表达所述分离的Cas13蛋白，所述分离的Cas13蛋白可以在与其配合的靶向目标区域的gRNA存在的条件下，对靶标区域进行结合、或切割。在本发明一具体实施例中，所述gRNA与所述分离的Cas13蛋白可以在同一宿主细胞中表达，所述宿主细胞可以为靶细胞。在本发明另一具体实施例中，所述核酸检测为体外核酸检测。再例如，所述核酸检测方法可以包括：1)提供扩增的目的片段；2)通过本发明第一方面所提供的分离的Cas13蛋白、或本发明第六方面所提供的碱基编辑体系对步骤1)所提供的扩增产物进行结合、或切割。合适的提供扩增的目的片段的方法对于本领域技术人员来说应该是已知的，例如，可以通过多核苷酸如SEQ IDNO.51～SEQ ID NO.52所示的引物对靶标片段进行扩增，所述靶标片段可以是新型冠状病毒2019-nCoV的核酸序列。在本发明一具体实施例中，所述核酸检测方法为小胖威利综合征、或新型冠状病毒2019-nCoV的检测方法。在本发明另一具体实施例中，所述核酸检测为体外核酸检测。
本发明所提供的核酸检测方法中，还可以包括：通过探针，判断检测结果。合适的探针对于本领域技术人员来说应该是已知的，例如，所述探针可以是报告基因，所述报告基因可以与所述分离的Cas13蛋白结合，从而可以用于判断检测结果。再例如，所述探针可以是能够与活化的Cas13蛋白反应的核酸，且可以被标记有的报告系统，所述报告系统可以是荧光报告试剂、生物素等，活化的Cas13蛋白可以与探针反应，从而可以用于判断检测结果，在本发明一具体实施例中，所述探针可以是连接有生物素和荧光报告试剂的核酸序列，所述核酸序列通常包括碱基U。
本发明所提供的新型Cas13蛋白，具有结合RNA的能力，且可以与gRNA相配的情况下具有RNA靶向切割活性，从而可以可用于RNA编辑、核酸检测等，基于新型Cas13蛋白的碱基编辑体系可以在37℃实现高灵敏、高精度的分子检测，具有很好的特异性和兼容性，检测灵敏度高，且检测成本低廉、操作方便、快捷、应用范围广，在核酸检测方面具有很好的应用前景。
下面通过实施例对本申请的发明予以进一步说明，但并不因此而限制本申请的范围。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring HarborLaboratory Press，1989 and Third edition，2001；Ausubel等，CURRENT PROTOCOLS INMOLECULAR BIOLOGY，John Wiley&Sons，New York，1987 and periodic updates；theseries METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATINSTRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS INENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，AcademicPress，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，ChromatinProtocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
实施例1
Cas13蛋白的发现
按图1所示流程图对基因组数据进行了分析，通过已发表Cas13蛋白序列，两两比对找出保守特征(seed)，然后从NCBI所有原核基因组数据，提取具有潜在编码蛋白能力的所有开放阅读框，并筛选出含有seed的开放阅读框作为潜在的Cas13蛋白。进一步比对聚类分析发现了了一类新的Cas13蛋白(SEQ ID NO.1-3)(如表1所示)。
表1
实施例2
Cas13蛋白鉴定
选取表1中的三个Cas13蛋白，分别为BgrCas13a(来自于物种Bacteroidesgraminisolvens isolate UBA4I89)(SEQ ID NO.1)、PbaCas13b(来自于物种Prevotellaintermedia strain KCOM 2698)(SEQ ID NO.2)、Pin4Cas13b(来自于物种Prevotellacaebacterium UBA776)(SEQ ID NO.3)，并进行了纯化，纯化结果如图2所示。之后对这三个Cas13蛋白的RNA切割活性进行了鉴定。RNA体外切割反应体系如下：
表2
实验所用BgrCas13a的gRNA序列BgrCas13a-gssRNA1如SEQ ID NO.4所示，实验所用Pin4Cas13b的gRNA序列Pin4Cas13b-gssRNA1如SEQ ID NO.5所示，实验所用底物ssRNA1序列如SEQ ID NO.6所示。
对反应产物进行northern blot鉴定，结果可以看到BgrCas13a和Pin4Cas13b对目的RNA具有明显的切割活性，具体结果如图3所示。
实施例3
Cas13蛋白附属切割活性鉴定
进一步，在gRNA的“引导”下，Cas13与靶向RNA结合发挥其切割活性，“活化”的Cas13除了切割靶向RNA外，还会非特异切割其他RNA。实验鉴定了Cas13蛋白BgrCas13a(SEQID NO.1)、Pin4Cas13b(SEQ ID NO.3)的附属切割活性。RNA附属切割反应体系如下：
表3
37°反应，3h，用多功能微孔板检测仪(BioTek SynergyNEO)实时监测底物RNA荧光变化，具体结果如图4a和图4b所示，基于荧光报告系统的BgrCas13a和Pin4Cas13b都可以有效检测ssRNA1。
实施例4
Cas13蛋白哺乳动物细胞内敲低RNA活性鉴定
Cas13的RNA特异性切割应用到哺乳动物细胞中可以用于特定RNA的降解敲低，为基因功能性研究提供了简单方便的工具。为了验证新型Cas13蛋白对哺乳动物细胞RNA的敲低能力，实验构建了Cas13蛋白和针对ACTB的gRNA的293FT细胞表达体系，所述Cas13蛋白表达载体为pHAGE-IRES-puro载体，编码Cas13蛋白的序列插入到载体中，插入位点为NheI和XbaI，插入蛋白序列见SEQ ID NO.1～3，所述ACTB gRNA表达载体为pUC19在HindIII和KpnI位点插入U6启动子和gRNA序列，所述gRNA序列见(SEQ ID NO.7～12)。并转染进293FT细胞，48小时后收细胞抽提RNA，并进行RT-qPCR检验。与gNC(所述对照组gNC的序列如SEQ IDNO.13～15所示)的对照组相比，BgrCas13a和PbaCas13b对在gRNA2的介导下对ACTB有一定的敲低作用，而Pin4Cas13b的敲低效果不是很明显，具体结果如图5所示(其中，各组数据中，左侧柱形为对照组，其余两个为实验组，分别是两个gRNA)，由图5可见，只有第二条gRNA有明显作用。
实施例5
Cas13蛋白哺乳动物细胞内标记RNA能力鉴定
通过突变Cas13蛋白的RNA酶活结构域HEPN motif可以使Cas13蛋白丧失切割RNA的活性，但是仍保留其结合RNA的能力，形成的dCas13蛋白常用来进行RNA标记，RNA定点编辑，RNA剪接调控以及RNA纯化等。本实验为验证新型Cas13蛋白的RNA标记能力，分别对BgrCas13a、PbaCas13b、Pin4Cas13b进行了HEPN motif的突变，并融合上绿色荧光蛋白EGFP，形成的dBgrCas13a-EGFP如序列如SEQ ID NO.16所示，dPbaCas13b-EGFP如序列如SEQID NO.17所示，形成的dPin4Cas13b-EGFP如序列如SEQ ID NO.18所示，蛋白和gRNAs表达载体同实施例4，与针对具有高度重复序列的MUC4 RNA的gRNA共同瞬转入HeLa细胞，BgrCas13a-gMUC4序列如SEQ ID NO.19所示，PbaCas13b-gMUC4序列如SEQ ID NO.20所示，Pin4Cas13b-gMUC4序列如SEQ ID NO.21所示，24小时后，细胞传到35mm玻底皿中(Cellvis)，36-48小时后，换成无血清的活细胞培养基，在Delta-vision显微镜下60倍镜观察信号并以50％的曝光强度和0.2秒的曝光时间拍照，处理并分析结果。具体结果如图6所示。发现dBgrCas13a-EGFP和dPin4Cas13b-EGFP均没有形成明显的MUC4单分子信号，而dPbaCas13b-EGFP虽然有信号，更像蛋白的异常聚集，为了降低蛋白本身聚集产生的影响，实验在dPbaCas13b-EGFP后面继续融合一段可溶性标签GB1，重新转染成像，可以看到dPbaCas13b-EGFP-GB1形成的信号更加真实，且与单分子荧光原位杂交技术smFISH所得到的信号共定位，显示出其RNA标记的可行性。
实施例6
Cas13蛋白用于检测小胖威利综合征(PWS)病人分子标志物核酸sno-lncRNAs
小胖威利综合征sno-lncRNAs分子标志物组合包括5条长非编码RNA，分别为sno-lncRNA1(SEQ ID NO.22)，sno-lncRNA2(SEQ ID NO.23)，sno-lncRNA3(SEQ ID NO.24)，sno-lncRNA4(SEQ ID NO.25)，sno-lncRNA5(SEQ ID NO.26)。序列是在NCBI GeneExpression Omnibus数据库(https://www.ncbi.nlm.nih.gov/geo/；accession numberGSE38541)中获得的。为了实现微量检测，本发明利用重组酶聚合酶扩增sno-lncRNAs分子标志物。
gRNA的核苷酸序列如下：
表4
RNA荧光报告检测实验如同实施例2中RNA附属切割实验。结果如图7a-图7e所示，其中，图7a-图7e分别为Cas13蛋白用于检测sno-lncRNA1、sno-lncRNA2、sno-lncRNA3、sno-lncRNA4、sno-lncRNA5的结果示意图，由图7可知，对于所选gRNA，Pin4Cas13b能够有效地检测出sno-lncRNA3、sno-lncRNA4、sno-lncRNA5。
实施例7
基于Cas13蛋白的小胖威利综合征综合征的检测
1)全血样品采集及RNA抽提
全血样品采集自浙江大学医学院附属儿童医院，采集时间为2018年7月。小胖威利综合征患者全血样品4例，正常人全血样品4例，对应作为小胖威利综合征组与正常对照组。上述所有标本的取得均获得试验对象同意及均通过组织伦理委员会的批准。
取新鲜抗凝血液按1:3的比例加入血液RNA稳定剂(TIANGEN)(如取300μl健康人新鲜全血加入900μl血液RNA稳定剂)立即盖上管盖，上下颠倒混匀8-10次，室温放置2h。利用RNAprep Pure血液总RNA提取试剂盒(TIANGEN)抽提全血RNA(详细步骤及注意事项参考RNAprep Pure血液总RNA提取试剂盒说明书)。
2)RPA扩增样品RNA中的sno-lnRNA4
将RNA酶灭活后的样品上清加入一管式重组酶聚合酶扩增反应体系中，所述反应体系包含sno-lncRNA1的重组酶聚合酶扩增特异性引物sno-lncRNA4-RPA-F序列如SEQ IDNO.42所示和sno-lncRNA4-RPA-R序列如SEQ ID NO.43所示。37°反应30min，反应体系及条件如下：
表5
3)sno-lnRNA4 RPA产物的Cas13检测
将重组酶聚合酶扩增反应产物加入一管式小胖威利综合征检测反应体系中，
反应体系及条件如下：
表6
Fluorescence Component 50μl Reaction
2xRNA Cleavage Buffer 25μl
RNase inhibitor 1μl
Cas13a 1μl
RPA product 1μl
rNTP(25Mm ATP,CTP,GTP,UTP) 2μl
T7 Enzyme 1μl
yeast tRNA 1μl
SL4-gRNA4 1μl
RNAse Alert v2 5μl
DEPC-H20 12μl
用多功能微孔板检测仪(BioTek SynergyNEO)监测底物RNA荧光变化(如图8所示)。正常组能够检测到明显的荧光信号，而病人组信号在很低的水平，表明Pin4Cas13b附属荧光报告底物系统能够有效的检测临床样品(血液)sno-lncRNA4分子标志物的表达情况。
实施例8
利用基于Cas13的试纸检测方法检测正常人与小胖威利综合征患者
本实施例提供了小胖威利综合征基于新型Cas13蛋白的试纸检测方法，所述检测方法包括如下步骤：
1)样品RNA提取(见实施例7)
2)重组酶聚合酶扩增RNA
由于样品中RNA含量低，本检测方法利用了恒温扩增反应RPA对目的RNA进行了扩增，从而放大信号；
3)Cas13经目的RNA活化后切割报告复合物
当检测样品中存在sno-lncRNA4时，Pin4Cas13b识别sno-lncRNA4后会被活化，活化的Pin4Cas13b可以非特异性切割荧光标记物-RNA-生物素标记物复合体。
4)试纸检测
荧光标记物-RNA-生物素标记物复合体被切割后可以产生游离的荧光标记物，反应产物在试纸上层析时，完整的荧光标记物-RNA-生物素标记物复合体在试纸上会被限定在质控线C，使质控线出现条带，而切割后产生的游离的荧光素会继续层析到检测线T带从而被检测出来。
将重组酶聚合酶扩增反应产物加入一管式小胖威利综合征检测反应体系中，37°反应30min，反应体系如下：
表7
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其中荧光标记物-RNA-生物素标记物复合体由上海生工生物工程(上海)股份有限公司合成，其中的RNA序列信息如SEQ ID NO.44所示：
荧光标记物-RNA-生物素标记物复合体结构如下所示：
表10
将80μl HybriDetect Assay Buffer加入上述反应管中，混匀，加入HybriDetectDipsticks插入反应管，5min，结果及说明如图9所示。上述实验表明，本发明提供小胖威利综合征检测试剂盒用于检测小胖威利综合征具有以下特点：检测及操作性能优异，准确度和灵敏度高，特异性强。
5)新型Cas13蛋白与常用LwaCas13a蛋白RNA检测比较
为了比较新发现Cas13蛋白与以往常用Cas13蛋白LwaCas13a在PWS检测方面的灵敏度。实验比较了基于荧光报告系统Pin4Cas13b和LwaCas13a在检测sno-lncRNA4时的灵敏度。所用LwaCas13a的序列如SEQ ID NO.59所示，所用Lwa对应Lwa-SL4-gRNA如SEQ IDNO.60所示。荧光报告系统实验体系和步骤与实施例7相同，结果如图10所示，Pin4Cas13b在优化gRNA后检测sno-lncRNA4的灵敏度不低于LwaCas13a.
实施例9
利用基于Cas13系统检测新型冠状病毒2019-nCoV
1)新型冠状病毒标准品制备
对新型冠状病毒和SARS冠状病毒以及蝙蝠SARS样冠状病毒进行基因组比对，找出序列差异的地方作为新型冠状病毒特异性靶标如图11。所用新型冠状病毒(EPI_ISL_402119)，SARS冠状病毒(SARS-CoV NC_004718.3SARS coronavirus)，MERS冠状病毒(MERS-CoV NC_019843.3Middle East respiratory syndrome coronavirus)，蝙蝠样冠状病毒(bat-SL-CoVZC45 MG772933.1Bat SARS-like coronavirus isolate bat-SL-CoVZC45)。得到新型冠状病毒的特异性靶标序列nCoV-ssRNA1为SEQ ID NO.46所示。合成该靶标序列，并进行DNA扩增。扩增产物进行体外转录，体系为：
表11
37℃反应2h后10％Urea-PAGE凝胶电泳回收。
2)针对nCoV-ssRNA1分子靶标的重组酶聚合酶高效扩增
为了实现微量检测，本发明利用重组酶聚合酶扩增nCoV-ssRNA1分子靶标。引物由北京擎科新业生物技术有限公司合成。特异性重组酶聚合酶扩增引物的核苷酸序列如下：
表12
用RT RAA核酸扩增试剂(基础型)(杭州众测生物科技有限公司)对目的RNA进行重组酶聚合酶扩增引物。重组酶聚合酶扩增反应体系及过程如下：
表13
3)高效、特异靶向新型冠状病毒gRNA筛选
以重组酶聚合酶扩增产物作为底物进行RNA筛选，gRNA的核苷酸序列如下：
表14
在gRNA的“引导”下，Cas13a与靶向RNA结合发挥其切割活性，“活化”的Cas13a除了切割靶向RNA外，还会非特异切割其他RNA。因此通过Cas13附属切割荧光RNA底物的能力来筛选出能高效率的gRNA。荧光RNA底物RNAse Alert v2购买自公司(Integrated DNATechnologies)。Cas13切割RNA反应体系如下：
表15
37°反应，3h，用多功能微孔板检测仪(BioTek SynergyNEO)实时监测底物RNA荧光变化(如图13所示)。由图12可知，用nCoV-ssRNA1-gRNA10可以得到最好的效果。
4)利用基于Cas13的试纸检测方法检测2019-nCoV标准品
为了更方便利用新型Cas13蛋白对2019-nCoV进行检测，实验对2019-nCoV病毒标准品nCoV-ssRNA1进行了试纸检测，检测步骤与实施例8相同，实验所用gRNA为nCoV-ssRNA1-gRNA10，结果如图13所示。由图13可知，nCoV-ssRNA1-gRNA10可以准确地检出病毒标准品。
综上所述，本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。
序列表
<110> 中国科学院上海生命科学研究院
<120> 一种分离的Cas13蛋白
<160> 60
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1130
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Lys Val Ser Lys Val Lys Val Lys Val Gly Ala Gly Arg Ser Ser
1 5 10 15
Glu Arg Met Val Phe Met Arg Arg Thr Ser Lys Ile Gly Ser Leu Val
20 25 30
Tyr Glu Asp Glu Gln Arg Asn Gly Lys Pro Glu Thr Asp Asp Lys Thr
35 40 45
Thr Ser Ile Leu Pro Asp Lys Lys Arg Asp Ser Phe Ile Leu Ser Ile
50 55 60
Val Asn Lys Thr Ile Pro Lys Lys Glu Ile Val Lys Lys Asn Leu Gly
65 70 75 80
Lys Gly Phe Val Asn Glu Tyr Tyr Asn Ala Ile Ala Gly Ile Ile Asp
85 90 95
Ser Phe Leu Glu Lys Lys Ile Val Asp Arg Lys His Tyr Ile Ile Val
100 105 110
Asn Lys Leu Thr Glu Glu Glu Ile Lys Gln Tyr Leu Asn His Arg Phe
115 120 125
Gln Glu Ala Asn Tyr Lys Tyr Val Arg Asp Lys Glu Glu Val Asn Phe
130 135 140
Asn Leu Pro Lys Leu Leu Lys Glu Ser Ala Lys Ser Asn Ser Thr Ala
145 150 155 160
Pro Leu Gln Pro Tyr Lys Glu Trp Ala Glu Trp His Ile Glu Thr Lys
165 170 175
Ser Val Arg Leu Ile Arg Ser Ile Gln Asn Asn Arg Leu Val Ile Asp
180 185 190
Thr Gln Glu Glu Ala Glu Asn Met Ser Pro Arg Lys Arg Ala Leu Leu
195 200 205
Lys Trp Glu Asn Glu Phe Leu Leu Ser His Lys Leu Asp Leu Gln Asp
210 215 220
Val Glu Lys Thr Tyr Leu Ile Asp Asp Leu Ile His Ala Leu His Glu
225 230 235 240
Val Thr Tyr Thr Thr Asn Asp Lys Gly Phe Ile Asn Gly Asn Glu Tyr
245 250 255
His Arg Phe Leu Lys Lys Ala Leu Gln Ser His Gln Gln Asn Ile Phe
260 265 270
Gly Ser Arg Glu Thr Pro Asn Lys Val Asn Arg Glu Asn Ala Glu Leu
275 280 285
Tyr Ser Tyr Asn Met Glu Val Val Lys Tyr Leu Glu His Tyr Phe Pro
290 295 300
Ile Lys Lys Thr Asn Arg Arg Asn Thr Leu Asp Thr Lys Asp Tyr Tyr
305 310 315 320
Leu Asn Gly Ile Asn Ile Lys Asp Arg Val Arg Lys Gln Leu Glu Asn
325 330 335
Ala Val Arg Asn Asn Leu Val Arg Gln Gly Lys Tyr Thr Leu His Thr
340 345 350
Leu Thr Thr Asp Thr Ala Asn Ser Asp Asn Leu Ser Lys Ile Lys Ala
355 360 365
Asp Glu Gly Phe Ala Leu Thr Met Leu Asn Gln Cys Ala Phe Ala Ala
370 375 380
Asn Asn Val Arg Asn Ile Ile Asp Pro Thr Gln Val Glu Asp Ile Leu
385 390 395 400
Leu Asp Arg Pro Phe Asn Glu Ser Leu Glu Lys Phe Asn Ser Ala Gln
405 410 415
Met Leu His Leu Ser Ser Phe Phe Asp Val Lys Glu Phe Asn Glu Pro
420 425 430
Leu Arg Ala Ile Arg Asp Ala Val Ala Lys Ile Arg His Asn Ile Ile
435 440 445
His Tyr Lys Val Asn Ala Leu Asn Val Ile Phe Lys Ile Glu Thr Phe
450 455 460
Gly Ser Thr Glu Lys Gln Tyr Lys Asp Thr Ile Phe Gly Ser Leu Leu
465 470 475 480
Gln Ala Asp Met Met Asn Val Ser Glu Ser Leu Ala Lys Gln Leu Met
485 490 495
Thr Gly Asn Val Leu Glu Tyr Tyr Pro Met Leu Glu Leu Lys Ser Phe
500 505 510
Phe Ser Lys Asn Ser Ile Ser Leu Tyr Arg Ser Val Ile Pro Phe Ala
515 520 525
Pro Gly Phe Lys Arg Val Met Lys Lys Gly Glu Asn Tyr Gln Asn Ala
530 535 540
Asn Asn Lys Asp Asp Lys Ser Lys Tyr Tyr Asn Leu Lys Ile Glu Ser
545 550 555 560
Phe Leu Pro Gln Glu Ser Phe Thr Lys Glu Ala Tyr Asp Ala Arg Tyr
565 570 575
Phe Leu Leu Lys Leu Ile Tyr Asn Asn Ile Phe Leu Pro Lys Phe Thr
580 585 590
Glu Ser Thr Asp Trp Phe Lys Ser Thr Val Asn Gly Val Ile Ala Leu
595 600 605
Asn Arg Glu Glu Asn Val Arg Lys Gly Lys Lys His Lys Ile Ala Phe
610 615 620
Ala Glu Ile Arg Leu Met Asp Ser Arg Asp Thr Ile Gly Thr Tyr Ala
625 630 635 640
Ala Tyr Leu Gln Ser Cys Ala Ile Ile Glu Ala Asn Lys Lys Pro Asp
645 650 655
Ala Ala Lys Asp Glu Thr Val Leu Leu Asn Phe Glu Lys Leu Ile Leu
660 665 670
Gln Ile Phe Ile Lys Gly Phe Asp Asp Phe Leu Arg Arg Tyr Gly Met
675 680 685
Leu Lys Phe Val Gln Gln Pro Ser Leu Gln Phe Lys Ser Gly Ala Lys
690 695 700
Asp Glu Thr Lys Gln Arg Lys Asp Trp Ala Lys Glu Val Glu Gly Ser
705 710 715 720
Leu His Ala Pro Val Asn Asp Ile Asn Pro Thr Glu Ser Ser His Ile
725 730 735
Ser Phe Tyr Val Phe Cys Lys Leu Leu Asp Ala Pro His Leu Asn Asp
740 745 750
Leu Arg Asn Glu Leu Ile Lys Tyr Tyr Arg Ser Gln Ser His Arg Pro
755 760 765
Gln Lys Ile Lys Asn Ile Lys Tyr Leu Leu Ser Ile Ile Glu Leu Cys
770 775 780
Leu Leu Ser Val Asp Ala Ser Glu Leu Asn Lys Leu Phe Thr Asp Ala
785 790 795 800
Asp Asn Ser Ile Asn Asp Arg Leu Leu Ala Phe Leu Glu Lys Asn Lys
805 810 815
Thr Val Ala Asp Tyr Glu Asp Leu Tyr Thr Gln Ser Asp Asn Glu Thr
820 825 830
Met Val Leu His Arg Thr Ile Ala Lys Ala Tyr Lys Tyr Gly Thr Arg
835 840 845
Lys Val Leu Glu Gln Val Ile Asp Ser Asp Pro Ser Tyr Arg Val Thr
850 855 860
Val Ser Glu Tyr Glu Glu Trp Lys Asn Leu Lys Glu Asn Val Glu Glu
865 870 875 880
Trp Ile Ala Cys Arg Glu Lys Leu His Ala Lys Trp Val Lys Val Glu
885 890 895
Arg Val Pro Lys Met Lys Glu Gly Glu Met Lys Asp Asp Met Lys Glu
900 905 910
Tyr Val Glu Val Cys Ser Thr Ile Asp Arg Tyr Asn Trp Leu Asp Asn
915 920 925
Lys Val His Leu Glu His Ile Asn Lys Leu His Asn Leu Thr Ile Asp
930 935 940
Leu Leu Gly Arg Met Ala Gly Phe Asn Thr Leu Phe Glu Arg Asp Leu
945 950 955 960
Gln Tyr Tyr Cys Asn Cys Asn Glu Lys Val Arg Ser Leu Leu Lys Pro
965 970 975
Glu Ser Ser Glu Gly Phe Asp Phe Ser Lys Gly Leu Pro Lys Phe Thr
980 985 990
Glu Glu Gln Asn Ser Ile Leu Phe Gln Thr Phe Leu Val Glu Gly Tyr
995 1000 1005
Arg Asp Thr Arg Asn Phe Val Ala His Phe Asn Phe Leu Asn Ser Phe
1010 1015 1020
Glu Asp Ser Tyr Ser Lys Asn Arg Tyr Ser Leu Ile Asp Val Ile Asn
1025 1030 1035 1040
Arg Leu Arg Lys Gln Phe Lys Tyr Asp Arg Lys Leu Lys Asn Ala Val
1045 1050 1055
Ser Lys Ala Ile Ile Lys Leu Phe Glu Lys His Gly Met Val Leu Thr
1060 1065 1070
Phe Ser Phe Glu Gly Asp His Glu Leu Val Val Lys Asp Val Phe Pro
1075 1080 1085
Arg Lys Ile Ile His Phe Ser Lys Leu Lys Leu Asp Lys Lys Ile Glu
1090 1095 1100
Gln Lys Pro Lys Val Glu Thr Asp Met Val Pro Lys Glu Phe Cys Ala
1105 1110 1115 1120
Met Cys Lys Phe Leu Leu Glu Leu Lys Lys
1125 1130
<210> 2
<211> 1091
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Phe Val Met Lys Ile Pro Asp Leu Ile Asn Asn Tyr Lys Glu Gln
1 5 10 15
Phe Gly Thr Tyr Ala Val Met Ala Leu Met Asn Ala Gln Thr Val Leu
20 25 30
Asp His Ile Gln Lys Leu Ala Cys Ile Gly Asn Ser Glu Ser Tyr Lys
35 40 45
Glu Asp Leu Trp Lys His Pro Val Met Leu His Ile Lys Asn Ala Ala
50 55 60
Tyr Glu Thr Asp Asn Asn Pro Glu Thr Thr Leu Phe Val Met Glu Lys
65 70 75 80
Leu Gln Phe Tyr Phe Pro Phe Leu Lys Ile Met Ala Glu Arg Gln Arg
85 90 95
Glu Tyr Ser Asn Lys Lys Tyr Asn Glu Asn Arg Leu Ala Val Asn Ser
100 105 110
Arg Asp Leu Phe Phe Val Leu Asp Lys Val Phe Arg Val Leu Lys Asn
115 120 125
Tyr Arg Asn Thr Thr Ala His Phe Met Thr Lys Ser Leu Ser Glu Asn
130 135 140
Glu Glu Tyr Leu Phe Leu Lys Asn Glu Gln Pro Leu Ala Asn Met Val
145 150 155 160
Asn Glu Tyr Tyr Thr Val Ala Leu Arg Asp Met Lys Asp Arg Tyr Ser
165 170 175
Tyr Ser Thr Glu Asp Leu Ala Phe Ile Gln Asp Asn Arg Tyr Lys Gln
180 185 190
Thr Arg Gly Asp Asp Gly Lys Arg Lys Ala Thr Val Asn Leu Asp Phe
195 200 205
Phe Leu Ser Leu Gln Ser His Asn Gly Asp Pro Ala Lys Lys Leu His
210 215 220
Leu Ser Gly Val Gly Val Ala Leu Leu Ile Cys Leu Phe Leu Glu Lys
225 230 235 240
Gln Tyr Val Asn Leu Phe Ile Ser Lys Leu Ser Ile Phe Ser Thr Tyr
245 250 255
Lys Pro Asn Ser Glu Gln His Arg Ile Ile Cys Arg Ser Met Gly Ile
260 265 270
His Ser Ile Lys Leu Pro Lys Asp Arg Ile His Ser Leu Lys Asp Gly
275 280 285
Met Ser Val Ala Met Asp Met Leu Asn Glu Thr Lys Arg Cys Pro Lys
290 295 300
Glu Leu Phe Asp Thr Leu Ser Ala Asp Lys Gln Ala Arg Phe Arg Thr
305 310 315 320
Ile Ser Asp Asp His Asn Glu Val Leu Leu Met Arg Ser Ser Asp Arg
325 330 335
Phe Ala Gln Leu Leu Leu Gln Tyr Ile Asp Tyr Asn Lys Leu Phe Lys
340 345 350
Gly Ile Arg Phe His Val Asn Met Gly Lys Leu Arg Tyr Leu Phe Asn
355 360 365
Ala Ala Lys Asn Cys Ile Asp Gly Gln Thr Arg Val Arg Val Ile Glu
370 375 380
His Gln Leu Asn Gly Tyr Gly Arg Ile Asp Glu Ile Glu Ala Glu Arg
385 390 395 400
Lys Ser Glu Asp Gly Lys Phe Ala Asn Ser Gly Ile Ser Ile Arg Asp
405 410 415
Phe Glu Asn Val Arg Arg Asp Asp Gly Asn Pro Glu Asn Tyr Pro Tyr
420 425 430
Val Val Asp Thr Tyr Ser His Tyr Ile Leu Glu Asn Asn Lys Val Glu
435 440 445
Met Gln Phe Thr Asp Ser Cys Ile Leu Pro Phe Ile Glu Glu Tyr Gly
450 455 460
Gly Lys Trp Tyr Val Ala Lys Asp Val Pro Ala Cys Arg Ile Ser Thr
465 470 475 480
Leu Glu Leu Pro Ala Leu Ala Phe His Met Leu Leu Leu Gly Ser Lys
485 490 495
Lys Thr Glu Glu Arg Ile Lys Glu Val Tyr Gly Lys Tyr Met Lys Leu
500 505 510
Phe Glu Ala Met Ser Asn Asp Ala Val Thr Lys Glu Asn Ile Glu Ser
515 520 525
Phe Gly Ile Ala Leu Trp Asn Leu Pro Gln Lys Ala Ile Asp Ala Ile
530 535 540
Asn Gly Cys Ala His Ala Lys Asn Val Asn Ala Phe Ile Lys Asn Thr
545 550 555 560
Val Glu Glu Met Leu Ala Asp Thr Glu Lys Arg Leu Glu Arg Leu Lys
565 570 575
Glu Asp Arg Lys Gly Ile Ser Ser His Asp Asn Lys Met Gly Lys Pro
580 585 590
Asn Phe Arg Gln Ile Ser Thr Gly Arg Leu Ala Asp Phe Leu Ala Lys
595 600 605
Asp Ile Val Lys Leu Gln Pro Ala Thr Thr Glu Asp Gly Ser Asp Lys
610 615 620
Met Thr Gly Leu Asn Tyr Arg Val Met Gln Ala Ala Ile Ala Thr Tyr
625 630 635 640
Asn Ser Asn Gly Asn Pro Glu Ala Glu Gln Gln Phe Arg Gly Met Phe
645 650 655
Glu Lys Ala Arg Leu Ile Gly Lys Ser Pro Asn Ala His Pro Phe Leu
660 665 670
Tyr Lys Val Phe Ala Ser Ala Leu Pro Lys Asn Thr Ile Glu Phe Tyr
675 680 685
Asp Lys Tyr Leu Arg Glu Arg Lys Arg Tyr Leu Ser Asn Leu Thr Lys
690 695 700
Asp Ile Glu Ser Gly Lys Thr Ala Ser Val Pro Phe Val Asn Arg Asn
705 710 715 720
Gln Asn Lys Trp Lys Lys Pro Asn Gln Glu Tyr Leu Gly Gln Thr Tyr
725 730 735
Gly Asn Ser Gln Asn Leu Ala Ile Glu Leu Pro Arg Gln Met Phe Asp
740 745 750
Ala Asp Ile Lys Ala Phe Leu Lys Gly Leu Pro Glu Met Lys Asp Val
755 760 765
Asp Phe Asp Asn Ala Asn Val Thr Phe Leu Ile Gly Glu Tyr Met Lys
770 775 780
Arg Val Leu Ala Asp Asp Phe Gln Asn Phe Tyr Ala Trp Lys Arg Asn
785 790 795 800
Tyr Arg Tyr Met Asp Met Leu Met Gly Glu Thr Asp Ser Lys Asp Gly
805 810 815
Leu Arg His Met Phe Thr Ser Val Glu Glu Arg Glu Lys Leu Trp Arg
820 825 830
Glu Arg Glu Gly Arg Thr Ser Ser Tyr Arg Lys Trp Ala Ala Arg Lys
835 840 845
Asn Gln Gly Cys Gly Tyr Ser Lys Arg Leu Pro Asp Asn Val Leu Asp
850 855 860
Glu Ile Leu Ala Lys Arg Leu Ser Gly Ser Arg Asn Asp Tyr Gln Lys
865 870 875 880
Cys Glu Lys Ala Ile Arg Arg Gln Lys Val Gln Asp Ala Met Leu Phe
885 890 895
Met Leu Ala Lys Lys Ala Phe Thr Gln His Ala Asp Phe Glu Gly Lys
900 905 910
Asp Phe Lys Leu Lys Asn Ile Met Pro Asp Ala Asp Lys Gly Ile Leu
915 920 925
Ser Glu Thr Met Pro Met Thr Phe Thr Phe Glu Lys Gly Gly Lys Gln
930 935 940
Tyr Thr Ile Glu Ser Gly Gly Met Lys Leu Lys Asn Tyr Gly Asp Phe
945 950 955 960
His Ala Leu Ala Asn Asp Arg Arg Ile Gly Cys Leu Met Lys Leu Ile
965 970 975
Gly Thr Asn Thr Ala Gln Lys Glu Asp Leu Ile Ser Glu Phe Asn Lys
980 985 990
Tyr Asp Gln Cys Arg Pro Gln Val Val Lys Leu Val Leu Asp Leu Glu
995 1000 1005
Lys Trp Ala Phe Ala Lys Tyr Pro Glu Leu Gly Ala Ser Glu Glu Met
1010 1015 1020
Ser Tyr Val Ser Phe Asp Lys Ile Leu Asn Lys Leu Thr Gln His Arg
1025 1030 1035 1040
Asn Leu Gly Asn Arg Gln Ser Asn Ile Leu Arg Lys Ile Arg Asn Ala
1045 1050 1055
Phe Asp His Asn Ser Tyr Pro Glu Ser Gly Ile Val Glu Ile Thr Thr
1060 1065 1070
Leu Pro Glu Ile Ala Gln Asn Met Lys Asp Met Phe Gly Arg Tyr Ala
1075 1080 1085
Ile Val Lys
1090
<210> 3
<211> 1140
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ile Ile Lys Ser Tyr Ile Val Lys Met Glu Asp Asp Lys Lys Thr
1 5 10 15
Thr Glu Ser Thr Tyr Thr Leu Asp Asn Lys His Phe Gly Ala Ala Phe
20 25 30
Leu Asn Leu Ala Arg His Asn Val Tyr Ile Thr Val Asn His Ile Asn
35 40 45
Lys Ile Leu Glu Glu Gly Glu Ile Asn Arg Asp Gly Tyr Glu Thr Thr
50 55 60
Leu Lys Asn Ser Trp Asp Glu Ile Lys Asp Ile Asn Lys Lys Asp Arg
65 70 75 80
Leu Ser Lys Leu Ile Ile Lys His Phe Ser Phe Leu Glu Ala Ala Thr
85 90 95
Tyr Arg Gln Asn Pro Thr Asp Thr Thr Lys Gln Lys Glu Glu Lys Gln
100 105 110
Ala Glu Ala Gln Ser Leu Glu Ser Leu Lys Lys Ser Phe Phe Val Phe
115 120 125
Ile Tyr Lys Leu Arg Asp Leu Arg Asn His Tyr Ser His Tyr Lys His
130 135 140
Ser Lys Ser Leu Glu Arg Pro Lys Phe Glu Glu Asp Leu Leu Lys Lys
145 150 155 160
Met Tyr Asn Ile Phe Asn Ala Ser Ile Arg Leu Val Lys Glu Asp Tyr
165 170 175
Lys His Asn Lys Asn Ile Asn Pro Lys Lys Asp Phe Lys His Leu Asn
180 185 190
Arg Lys Arg Lys Gly Lys Phe Tyr Tyr Ser Phe Ala Asp Asn Glu Gly
195 200 205
Asn Ile Thr Glu Ser Gly Leu Leu Phe Phe Val Ser Leu Phe Leu Glu
210 215 220
Lys Lys Asp Ala Ile Trp Val Gln Lys Lys Leu Glu Gly Phe Lys Cys
225 230 235 240
Ser Asn Glu Ser Tyr Gln Lys Met Thr Asn Glu Val Phe Cys Arg Ser
245 250 255
Arg Met Leu Leu Pro Lys Leu Arg Leu Glu Ser Thr Gln Thr Gln Asp
260 265 270
Trp Ile Leu Leu Asp Met Leu Asn Glu Leu Ile Arg Cys Pro Lys Ser
275 280 285
Leu Tyr Glu Arg Leu Arg Gly Asp Asp Arg Glu Lys Phe Arg Val Pro
290 295 300
Leu Asp Pro Ile Asp Glu Asp Tyr Asp Ala Glu Gln Glu Pro Phe Lys
305 310 315 320
Asn Thr Leu Val Arg His Gln Asp Arg Phe Pro Tyr Phe Ala Leu Arg
325 330 335
Tyr Phe Asp Tyr Asn Glu Ile Phe Ser Asn Leu Arg Phe Gln Ile Asp
340 345 350
Leu Gly Thr Tyr His Phe Ser Ile Tyr Lys Lys Leu Ile Gly Gly Gln
355 360 365
Lys Glu Asp Arg His Leu Thr His Lys Leu Tyr Gly Phe Glu Arg Ile
370 375 380
Gln Glu Phe Asp Lys Gln Asn Arg Thr Asp Glu Trp Lys Ser Ile Val
385 390 395 400
Lys Asp Leu Asp Thr Tyr Glu Thr Ser Asn Glu Arg Tyr Ile Ser Glu
405 410 415
Thr Thr Pro His Tyr His Leu Glu Asn Gln Lys Ile Gly Ile Arg Phe
420 425 430
Arg Asn Gly Asn Asn Asp Ile Trp Pro Ser Leu Glu Thr Asn Gly Glu
435 440 445
Asn Asn Glu Lys Ser Lys Tyr Lys Leu Asp Lys Gln Tyr Gln Ala Glu
450 455 460
Ala Phe Leu Ser Val His Glu Leu Leu Pro Met Met Phe Tyr Tyr Leu
465 470 475 480
Leu Leu Lys Thr Glu Asn Thr Asp Asn Asp Asn Glu Ile Glu Thr Lys
485 490 495
Lys Lys Glu Asn Lys Asn Asp Lys Gln Glu Lys His Lys Ile Glu Glu
500 505 510
Ile Ile Glu Asn Lys Ile Lys Asp Ile Tyr Ala Leu Tyr Asp Ala Phe
515 520 525
Ala Asn Gly Glu Ile Asn Ser Lys Glu Glu Leu Lys Glu Tyr Leu Lys
530 535 540
Gly Lys Asp Ile Glu Ile Val His Leu Pro Lys Gln Met Ile Ala Ile
545 550 555 560
Leu Glu Ser Lys Pro Lys Asp Met Val Glu Glu Ala Glu Arg Lys Gln
565 570 575
Arg Lys Met Val Lys Asp Thr Lys Lys Leu Leu Ala Ala Leu Glu Lys
580 585 590
Gln Thr Gln Glu Glu Ile Glu Asp Gly Gly Arg Asn Ile Arg Leu Leu
595 600 605
Lys Ser Gly Glu Ile Ala Arg Trp Leu Val Asn Asp Met Met Arg Phe
610 615 620
Gln Pro Val Gln Lys Asp Asn Glu Gly Asn Pro Leu Asn Asn Ser Lys
625 630 635 640
Ala Asn Ser Thr Glu Tyr Gln Met Leu Gln Arg Ser Leu Ala Leu Tyr
645 650 655
Asn Lys Glu Glu Lys Pro Thr Arg Tyr Phe Arg Gln Val Asn Leu Ile
660 665 670
Asn Ser Ser Asn Pro His Pro Phe Leu Lys Trp Thr Lys Trp Glu Glu
675 680 685
Cys Asn Asn Ile Leu Ser Phe Tyr Arg Ser Tyr Leu Thr Lys Lys Ile
690 695 700
Glu Phe Leu Asn Lys Leu Lys Pro Glu Asp Trp Glu Lys Asn Gln Tyr
705 710 715 720
Phe Leu Lys Leu Lys Glu Pro Lys Thr Asn Arg Glu Thr Leu Val Gln
725 730 735
Gly Trp Lys Asn Gly Phe Asn Leu Pro Arg Gly Ile Phe Thr Glu Pro
740 745 750
Ile Arg Glu Trp Phe Lys Arg His Gln Asn Asn Ser Glu Glu Tyr Glu
755 760 765
Lys Val Glu Ala Leu Asp Arg Val Gly Leu Val Thr Lys Val Ile Pro
770 775 780
Leu Phe Phe Lys Glu Glu Tyr Phe Lys Glu Asp Ala Gln Lys Glu Ile
785 790 795 800
Asn Asn Cys Val Gln Pro Phe Tyr Ser Phe Pro Tyr Asn Val Gly Asn
805 810 815
Ile His Lys Pro Lys Glu Glu Asp Phe Leu His Arg Glu Glu Arg Ile
820 825 830
Glu Leu Trp Asp Lys Lys Lys Asp Lys Phe Lys Gly Tyr Lys Ala Lys
835 840 845
Lys Lys Phe Lys Glu Met Thr Asp Lys Glu Lys Glu Glu His Arg Ser
850 855 860
Tyr Leu Glu Phe Gln Ser Trp Asn Lys Phe Glu Arg Glu Leu Arg Leu
865 870 875 880
Val Arg Asn Gln Asp Ile Val Thr Trp Leu Leu Cys Thr Glu Leu Ile
885 890 895
Asp Lys Leu Lys Val Glu Gly Leu Asn Val Glu Glu Leu Lys Lys Leu
900 905 910
Arg Leu Lys Asp Ile Asp Thr Asp Thr Ala Lys Gln Glu Lys Asn Asn
915 920 925
Ile Leu Asn Arg Val Met Pro Met Gln Leu Pro Val Thr Val Tyr Glu
930 935 940
Ile Asp Asp Ser His Asn Ile Val Lys Asp Arg Pro Leu His Thr Val
945 950 955 960
Tyr Ile Glu Glu Thr Lys Thr Lys Leu Leu Lys Gln Gly Asn Phe Lys
965 970 975
Ala Leu Val Lys Asp Arg Arg Leu Asn Gly Leu Phe Ser Phe Val Lys
980 985 990
Thr Pro Ser Glu Ala Glu Ser Glu Ser Asn Pro Ile Ser Lys Ser Arg
995 1000 1005
Val Glu Ser Glu Leu Gly Lys Tyr Gln Asn Ala Arg Leu Asn Ile Ile
1010 1015 1020
Glu Asp Met Leu Ala Leu Glu Lys Thr Leu Ile Asp Asn Tyr Asn Ser
1025 1030 1035 1040
Leu Asp Thr Asp Asn Phe His Asn Met Leu Thr Gly Trp Leu Lys Leu
1045 1050 1055
Lys Gly Lys Ala Asn Lys Ala Arg Phe Gln Asn Glu Val Asp Leu Leu
1060 1065 1070
Val Ala Val Arg Asn Ala Phe Ser His Asn Gln Tyr Pro Met Tyr Asn
1075 1080 1085
Glu Asn Leu Phe Gly Asn Ile Glu Arg Phe Ser Leu Ser Ser Ser Asn
1090 1095 1100
Ile Ile Glu Ser Lys Gly Leu Asp Ile Ala Val Lys Leu Lys Glu Glu
1105 1110 1115 1120
Val Ser Lys Ala Val Glu Lys Ile Gln Asn Glu Glu Asp Asp Lys Lys
1125 1130 1135
Glu Lys Glu Thr
1140
<210> 4
<211> 64
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 4
gttgtagttc ccttcattaa tgggatcatt tgcatctaga ttgctgttct accaagtaat 60
ccat 64
<210> 5
<211> 67
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 5
gctaccaagt aatccatatt tctagaggat cgttgttttt accttgcaaa cagcaggcag 60
atgcaac 67
<210> 6
<211> 124
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggccagtgaa ttcgagctcg gtacccgggg atcctctaga aatatggatt acttggtaga 60
acagcaatct actcgacctg caggcatgca agcttggcgt aatcatggtc atagctgttt 120
cctg 124
<210> 7
<211> 64
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 7
gttgtagttc ccttcattaa tgggatcatt tgcatcctgg cggcgggtgt ggacgggcgg 60
cgga 64
<210> 8
<211> 64
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 8
gttgtagttc ccttcattaa tgggatcatt tgcatcgagc cacacgcagc tcattgtaga 60
aggt 64
<210> 9
<211> 66
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggcggcgggt gtggacgggc ggcggatcgg gttgtagaag ccctcatttt gagagggtat 60
tacaac 66
<210> 10
<211> 64
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 10
gagccacacg cagctcattg tagaaggtgt tgtagaagcc ctcattttga gagggtatta 60
caac 64
<210> 11
<211> 66
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 11
ggcggcgggt gtggacgggc ggcggatcgg gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 12
<211> 64
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 12
gagccacacg cagctcattg tagaaggtgt tgtttttacc ttgcaaacag caggcagatg 60
caac 64
<210> 13
<211> 64
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 13
gttgtagttc ccttcattaa tgggatcatt tgcatcaggg ttttcccagt cacgacgttg 60
taaa 64
<210> 14
<211> 67
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 14
gcagggtttt cccagtcacg acgttgtaaa agttgtagaa gccctcattt tgagagggta 60
ttacaac 67
<210> 15
<211> 67
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 15
gcagggtttt cccagtcacg acgttgtaaa agttgttttt accttgcaaa cagcaggcag 60
atgcaac 67
<210> 16
<211> 1409
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Met Pro Lys Lys Lys Arg Lys Val Lys Val Ser Lys Val Lys Val Lys
1 5 10 15
Val Gly Ala Gly Arg Ser Ser Glu Arg Met Val Phe Met Arg Arg Thr
20 25 30
Ser Lys Ile Gly Ser Leu Val Tyr Glu Asp Glu Gln Arg Asn Gly Lys
35 40 45
Pro Glu Thr Asp Asp Lys Thr Thr Ser Ile Leu Pro Asp Lys Lys Arg
50 55 60
Asp Ser Phe Ile Leu Ser Ile Val Asn Lys Thr Ile Pro Lys Lys Glu
65 70 75 80
Ile Val Lys Lys Asn Leu Gly Lys Gly Phe Val Asn Glu Tyr Tyr Asn
85 90 95
Ala Ile Ala Gly Ile Ile Asp Ser Phe Leu Glu Lys Lys Ile Val Asp
100 105 110
Arg Lys His Tyr Ile Ile Val Asn Lys Leu Thr Glu Glu Glu Ile Lys
115 120 125
Gln Tyr Leu Asn His Arg Phe Gln Glu Ala Asn Tyr Lys Tyr Val Arg
130 135 140
Asp Lys Glu Glu Val Asn Phe Asn Leu Pro Lys Leu Leu Lys Glu Ser
145 150 155 160
Ala Lys Ser Asn Ser Thr Ala Pro Leu Gln Pro Tyr Lys Glu Trp Ala
165 170 175
Glu Trp His Ile Glu Thr Lys Ser Val Arg Leu Ile Arg Ser Ile Gln
180 185 190
Asn Asn Arg Leu Val Ile Asp Thr Gln Glu Glu Ala Glu Asn Met Ser
195 200 205
Pro Arg Lys Arg Ala Leu Leu Lys Trp Glu Asn Glu Phe Leu Leu Ser
210 215 220
His Lys Leu Asp Leu Gln Asp Val Glu Lys Thr Tyr Leu Ile Asp Asp
225 230 235 240
Leu Ile His Ala Leu His Glu Val Thr Tyr Thr Thr Asn Asp Lys Gly
245 250 255
Phe Ile Asn Gly Asn Glu Tyr His Arg Phe Leu Lys Lys Ala Leu Gln
260 265 270
Ser His Gln Gln Asn Ile Phe Gly Ser Arg Glu Thr Pro Asn Lys Val
275 280 285
Asn Arg Glu Asn Ala Glu Leu Tyr Ser Tyr Asn Met Glu Val Val Lys
290 295 300
Tyr Leu Glu His Tyr Phe Pro Ile Lys Lys Thr Asn Arg Arg Asn Thr
305 310 315 320
Leu Asp Thr Lys Asp Tyr Tyr Leu Asn Gly Ile Asn Ile Lys Asp Arg
325 330 335
Val Arg Lys Gln Leu Glu Asn Ala Val Arg Asn Asn Leu Val Arg Gln
340 345 350
Gly Lys Tyr Thr Leu His Thr Leu Thr Thr Asp Thr Ala Asn Ser Asp
355 360 365
Asn Leu Ser Lys Ile Lys Ala Asp Glu Gly Phe Ala Leu Thr Met Leu
370 375 380
Asn Gln Cys Ala Phe Ala Ala Asn Asn Val Arg Asn Ile Ile Asp Pro
385 390 395 400
Thr Gln Val Glu Asp Ile Leu Leu Asp Arg Pro Phe Asn Glu Ser Leu
405 410 415
Glu Lys Phe Asn Ser Ala Gln Met Leu His Leu Ser Ser Phe Phe Asp
420 425 430
Val Lys Glu Phe Asn Glu Pro Leu Arg Ala Ile Arg Asp Ala Val Ala
435 440 445
Lys Ile Ala His Asn Ile Ile Ala Tyr Lys Val Asn Ala Leu Asn Val
450 455 460
Ile Phe Lys Ile Glu Thr Phe Gly Ser Thr Glu Lys Gln Tyr Lys Asp
465 470 475 480
Thr Ile Phe Gly Ser Leu Leu Gln Ala Asp Met Met Asn Val Ser Glu
485 490 495
Ser Leu Ala Lys Gln Leu Met Thr Gly Asn Val Leu Glu Tyr Tyr Pro
500 505 510
Met Leu Glu Leu Lys Ser Phe Phe Ser Lys Asn Ser Ile Ser Leu Tyr
515 520 525
Arg Ser Val Ile Pro Phe Ala Pro Gly Phe Lys Arg Val Met Lys Lys
530 535 540
Gly Glu Asn Tyr Gln Asn Ala Asn Asn Lys Asp Asp Lys Ser Lys Tyr
545 550 555 560
Tyr Asn Leu Lys Ile Glu Ser Phe Leu Pro Gln Glu Ser Phe Thr Lys
565 570 575
Glu Ala Tyr Asp Ala Arg Tyr Phe Leu Leu Lys Leu Ile Tyr Asn Asn
580 585 590
Ile Phe Leu Pro Lys Phe Thr Glu Ser Thr Asp Trp Phe Lys Ser Thr
595 600 605
Val Asn Gly Val Ile Ala Leu Asn Arg Glu Glu Asn Val Arg Lys Gly
610 615 620
Lys Lys His Lys Ile Ala Phe Ala Glu Ile Arg Leu Met Asp Ser Arg
625 630 635 640
Asp Thr Ile Gly Thr Tyr Ala Ala Tyr Leu Gln Ser Cys Ala Ile Ile
645 650 655
Glu Ala Asn Lys Lys Pro Asp Ala Ala Lys Asp Glu Thr Val Leu Leu
660 665 670
Asn Phe Glu Lys Leu Ile Leu Gln Ile Phe Ile Lys Gly Phe Asp Asp
675 680 685
Phe Leu Arg Arg Tyr Gly Met Leu Lys Phe Val Gln Gln Pro Ser Leu
690 695 700
Gln Phe Lys Ser Gly Ala Lys Asp Glu Thr Lys Gln Arg Lys Asp Trp
705 710 715 720
Ala Lys Glu Val Glu Gly Ser Leu His Ala Pro Val Asn Asp Ile Asn
725 730 735
Pro Thr Glu Ser Ser His Ile Ser Phe Tyr Val Phe Cys Lys Leu Leu
740 745 750
Asp Ala Pro His Leu Asn Asp Leu Arg Asn Glu Leu Ile Lys Tyr Tyr
755 760 765
Arg Ser Gln Ser His Arg Pro Gln Lys Ile Lys Asn Ile Lys Tyr Leu
770 775 780
Leu Ser Ile Ile Glu Leu Cys Leu Leu Ser Val Asp Ala Ser Glu Leu
785 790 795 800
Asn Lys Leu Phe Thr Asp Ala Asp Asn Ser Ile Asn Asp Arg Leu Leu
805 810 815
Ala Phe Leu Glu Lys Asn Lys Thr Val Ala Asp Tyr Glu Asp Leu Tyr
820 825 830
Thr Gln Ser Asp Asn Glu Thr Met Val Leu His Arg Thr Ile Ala Lys
835 840 845
Ala Tyr Lys Tyr Gly Thr Arg Lys Val Leu Glu Gln Val Ile Asp Ser
850 855 860
Asp Pro Ser Tyr Arg Val Thr Val Ser Glu Tyr Glu Glu Trp Lys Asn
865 870 875 880
Leu Lys Glu Asn Val Glu Glu Trp Ile Ala Cys Arg Glu Lys Leu His
885 890 895
Ala Lys Trp Val Lys Val Glu Arg Val Pro Lys Met Lys Glu Gly Glu
900 905 910
Met Lys Asp Asp Met Lys Glu Tyr Val Glu Val Cys Ser Thr Ile Asp
915 920 925
Arg Tyr Asn Trp Leu Asp Asn Lys Val His Leu Glu His Ile Asn Lys
930 935 940
Leu His Asn Leu Thr Ile Asp Leu Leu Gly Arg Met Ala Gly Phe Asn
945 950 955 960
Thr Leu Phe Glu Arg Asp Leu Gln Tyr Tyr Cys Asn Cys Asn Glu Lys
965 970 975
Val Arg Ser Leu Leu Lys Pro Glu Ser Ser Glu Gly Phe Asp Phe Ser
980 985 990
Lys Gly Leu Pro Lys Phe Thr Glu Glu Gln Asn Ser Ile Leu Phe Gln
995 1000 1005
Thr Phe Leu Val Glu Gly Tyr Arg Asp Thr Ala Asn Phe Val Ala Ala
1010 1015 1020
Phe Asn Phe Leu Asn Ser Phe Glu Asp Ser Tyr Ser Lys Asn Arg Tyr
1025 1030 1035 1040
Ser Leu Ile Asp Val Ile Asn Arg Leu Arg Lys Gln Phe Lys Tyr Asp
1045 1050 1055
Arg Lys Leu Lys Asn Ala Val Ser Lys Ala Ile Ile Lys Leu Phe Glu
1060 1065 1070
Lys His Gly Met Val Leu Thr Phe Ser Phe Glu Gly Asp His Glu Leu
1075 1080 1085
Val Val Lys Asp Val Phe Pro Arg Lys Ile Ile His Phe Ser Lys Leu
1090 1095 1100
Lys Leu Asp Lys Lys Ile Glu Gln Lys Pro Lys Val Glu Thr Asp Met
1105 1110 1115 1120
Val Pro Lys Glu Phe Cys Ala Met Cys Lys Phe Leu Leu Glu Leu Lys
1125 1130 1135
Lys Leu Glu Ala Leu Pro Val Ala Thr Met Val Ser Lys Gly Glu Glu
1140 1145 1150
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
1155 1160 1165
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
1170 1175 1180
Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro
1185 1190 1195 1200
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys
1205 1210 1215
Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser
1220 1225 1230
Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
1235 1240 1245
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
1250 1255 1260
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly
1265 1270 1275 1280
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val
1285 1290 1295
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys
1300 1305 1310
Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr
1315 1320 1325
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn
1330 1335 1340
His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys
1345 1350 1355 1360
Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr
1365 1370 1375
Leu Gly Met Asp Glu Leu Tyr Lys Ser Asn Ser Lys Pro Gly Phe His
1380 1385 1390
Met Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys
1395 1400 1405
Lys
<210> 17
<211> 1371
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Met Pro Lys Lys Lys Arg Lys Val Met Phe Val Met Lys Ile Pro Asp
1 5 10 15
Leu Ile Asn Asn Tyr Lys Glu Gln Phe Gly Thr Tyr Ala Val Met Ala
20 25 30
Leu Met Asn Ala Gln Thr Val Leu Asp His Ile Gln Lys Leu Ala Cys
35 40 45
Ile Gly Asn Ser Glu Ser Tyr Lys Glu Asp Leu Trp Lys His Pro Val
50 55 60
Met Leu His Ile Lys Asn Ala Ala Tyr Glu Thr Asp Asn Asn Pro Glu
65 70 75 80
Thr Thr Leu Phe Val Met Glu Lys Leu Gln Phe Tyr Phe Pro Phe Leu
85 90 95
Lys Ile Met Ala Glu Arg Gln Arg Glu Tyr Ser Asn Lys Lys Tyr Asn
100 105 110
Glu Asn Arg Leu Ala Val Asn Ser Arg Asp Leu Phe Phe Val Leu Asp
115 120 125
Lys Val Phe Arg Val Leu Lys Asn Tyr Ala Asn Thr Thr Ala Ala Phe
130 135 140
Met Thr Lys Ser Leu Ser Glu Asn Glu Glu Tyr Leu Phe Leu Lys Asn
145 150 155 160
Glu Gln Pro Leu Ala Asn Met Val Asn Glu Tyr Tyr Thr Val Ala Leu
165 170 175
Arg Asp Met Lys Asp Arg Tyr Ser Tyr Ser Thr Glu Asp Leu Ala Phe
180 185 190
Ile Gln Asp Asn Arg Tyr Lys Gln Thr Arg Gly Asp Asp Gly Lys Arg
195 200 205
Lys Ala Thr Val Asn Leu Asp Phe Phe Leu Ser Leu Gln Ser His Asn
210 215 220
Gly Asp Pro Ala Lys Lys Leu His Leu Ser Gly Val Gly Val Ala Leu
225 230 235 240
Leu Ile Cys Leu Phe Leu Glu Lys Gln Tyr Val Asn Leu Phe Ile Ser
245 250 255
Lys Leu Ser Ile Phe Ser Thr Tyr Lys Pro Asn Ser Glu Gln His Arg
260 265 270
Ile Ile Cys Arg Ser Met Gly Ile His Ser Ile Lys Leu Pro Lys Asp
275 280 285
Arg Ile His Ser Leu Lys Asp Gly Met Ser Val Ala Met Asp Met Leu
290 295 300
Asn Glu Thr Lys Arg Cys Pro Lys Glu Leu Phe Asp Thr Leu Ser Ala
305 310 315 320
Asp Lys Gln Ala Arg Phe Arg Thr Ile Ser Asp Asp His Asn Glu Val
325 330 335
Leu Leu Met Arg Ser Ser Asp Arg Phe Ala Gln Leu Leu Leu Gln Tyr
340 345 350
Ile Asp Tyr Asn Lys Leu Phe Lys Gly Ile Arg Phe His Val Asn Met
355 360 365
Gly Lys Leu Arg Tyr Leu Phe Asn Ala Ala Lys Asn Cys Ile Asp Gly
370 375 380
Gln Thr Arg Val Arg Val Ile Glu His Gln Leu Asn Gly Tyr Gly Arg
385 390 395 400
Ile Asp Glu Ile Glu Ala Glu Arg Lys Ser Glu Asp Gly Lys Phe Ala
405 410 415
Asn Ser Gly Ile Ser Ile Arg Asp Phe Glu Asn Val Arg Arg Asp Asp
420 425 430
Gly Asn Pro Glu Asn Tyr Pro Tyr Val Val Asp Thr Tyr Ser His Tyr
435 440 445
Ile Leu Glu Asn Asn Lys Val Glu Met Gln Phe Thr Asp Ser Cys Ile
450 455 460
Leu Pro Phe Ile Glu Glu Tyr Gly Gly Lys Trp Tyr Val Ala Lys Asp
465 470 475 480
Val Pro Ala Cys Arg Ile Ser Thr Leu Glu Leu Pro Ala Leu Ala Phe
485 490 495
His Met Leu Leu Leu Gly Ser Lys Lys Thr Glu Glu Arg Ile Lys Glu
500 505 510
Val Tyr Gly Lys Tyr Met Lys Leu Phe Glu Ala Met Ser Asn Asp Ala
515 520 525
Val Thr Lys Glu Asn Ile Glu Ser Phe Gly Ile Ala Leu Trp Asn Leu
530 535 540
Pro Gln Lys Ala Ile Asp Ala Ile Asn Gly Cys Ala His Ala Lys Asn
545 550 555 560
Val Asn Ala Phe Ile Lys Asn Thr Val Glu Glu Met Leu Ala Asp Thr
565 570 575
Glu Lys Arg Leu Glu Arg Leu Lys Glu Asp Arg Lys Gly Ile Ser Ser
580 585 590
His Asp Asn Lys Met Gly Lys Pro Asn Phe Arg Gln Ile Ser Thr Gly
595 600 605
Arg Leu Ala Asp Phe Leu Ala Lys Asp Ile Val Lys Leu Gln Pro Ala
610 615 620
Thr Thr Glu Asp Gly Ser Asp Lys Met Thr Gly Leu Asn Tyr Arg Val
625 630 635 640
Met Gln Ala Ala Ile Ala Thr Tyr Asn Ser Asn Gly Asn Pro Glu Ala
645 650 655
Glu Gln Gln Phe Arg Gly Met Phe Glu Lys Ala Arg Leu Ile Gly Lys
660 665 670
Ser Pro Asn Ala His Pro Phe Leu Tyr Lys Val Phe Ala Ser Ala Leu
675 680 685
Pro Lys Asn Thr Ile Glu Phe Tyr Asp Lys Tyr Leu Arg Glu Arg Lys
690 695 700
Arg Tyr Leu Ser Asn Leu Thr Lys Asp Ile Glu Ser Gly Lys Thr Ala
705 710 715 720
Ser Val Pro Phe Val Asn Arg Asn Gln Asn Lys Trp Lys Lys Pro Asn
725 730 735
Gln Glu Tyr Leu Gly Gln Thr Tyr Gly Asn Ser Gln Asn Leu Ala Ile
740 745 750
Glu Leu Pro Arg Gln Met Phe Asp Ala Asp Ile Lys Ala Phe Leu Lys
755 760 765
Gly Leu Pro Glu Met Lys Asp Val Asp Phe Asp Asn Ala Asn Val Thr
770 775 780
Phe Leu Ile Gly Glu Tyr Met Lys Arg Val Leu Ala Asp Asp Phe Gln
785 790 795 800
Asn Phe Tyr Ala Trp Lys Arg Asn Tyr Arg Tyr Met Asp Met Leu Met
805 810 815
Gly Glu Thr Asp Ser Lys Asp Gly Leu Arg His Met Phe Thr Ser Val
820 825 830
Glu Glu Arg Glu Lys Leu Trp Arg Glu Arg Glu Gly Arg Thr Ser Ser
835 840 845
Tyr Arg Lys Trp Ala Ala Arg Lys Asn Gln Gly Cys Gly Tyr Ser Lys
850 855 860
Arg Leu Pro Asp Asn Val Leu Asp Glu Ile Leu Ala Lys Arg Leu Ser
865 870 875 880
Gly Ser Arg Asn Asp Tyr Gln Lys Cys Glu Lys Ala Ile Arg Arg Gln
885 890 895
Lys Val Gln Asp Ala Met Leu Phe Met Leu Ala Lys Lys Ala Phe Thr
900 905 910
Gln His Ala Asp Phe Glu Gly Lys Asp Phe Lys Leu Lys Asn Ile Met
915 920 925
Pro Asp Ala Asp Lys Gly Ile Leu Ser Glu Thr Met Pro Met Thr Phe
930 935 940
Thr Phe Glu Lys Gly Gly Lys Gln Tyr Thr Ile Glu Ser Gly Gly Met
945 950 955 960
Lys Leu Lys Asn Tyr Gly Asp Phe His Ala Leu Ala Asn Asp Arg Arg
965 970 975
Ile Gly Cys Leu Met Lys Leu Ile Gly Thr Asn Thr Ala Gln Lys Glu
980 985 990
Asp Leu Ile Ser Glu Phe Asn Lys Tyr Asp Gln Cys Arg Pro Gln Val
995 1000 1005
Val Lys Leu Val Leu Asp Leu Glu Lys Trp Ala Phe Ala Lys Tyr Pro
1010 1015 1020
Glu Leu Gly Ala Ser Glu Glu Met Ser Tyr Val Ser Phe Asp Lys Ile
1025 1030 1035 1040
Leu Asn Lys Leu Thr Gln His Arg Asn Leu Gly Asn Arg Gln Ser Asn
1045 1050 1055
Ile Leu Arg Lys Ile Ala Asn Ala Phe Asp Ala Asn Ser Tyr Pro Glu
1060 1065 1070
Ser Gly Ile Val Glu Ile Thr Thr Leu Pro Glu Ile Ala Gln Asn Met
1075 1080 1085
Lys Asp Met Phe Gly Arg Tyr Ala Ile Val Lys Leu Glu Ala Leu Pro
1090 1095 1100
Val Ala Thr Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val
1105 1110 1115 1120
Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser
1125 1130 1135
Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu
1140 1145 1150
Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu
1155 1160 1165
Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp
1170 1175 1180
His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr
1185 1190 1195 1200
Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr
1205 1210 1215
Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu
1220 1225 1230
Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys
1235 1240 1245
Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys
1250 1255 1260
Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu
1265 1270 1275 1280
Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
1285 1290 1295
Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln
1300 1305 1310
Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
1315 1320 1325
Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu
1330 1335 1340
Tyr Lys Ser Asn Ser Lys Pro Gly Phe His Met Lys Arg Pro Ala Ala
1345 1350 1355 1360
Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys
1365 1370
<210> 18
<211> 1420
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Met Pro Lys Lys Lys Arg Lys Val Met Ile Ile Lys Ser Tyr Ile Val
1 5 10 15
Lys Met Glu Asp Asp Lys Lys Thr Thr Glu Ser Thr Tyr Thr Leu Asp
20 25 30
Asn Lys His Phe Gly Ala Ala Phe Leu Asn Leu Ala Arg His Asn Val
35 40 45
Tyr Ile Thr Val Asn His Ile Asn Lys Ile Leu Glu Glu Gly Glu Ile
50 55 60
Asn Arg Asp Gly Tyr Glu Thr Thr Leu Lys Asn Ser Trp Asp Glu Ile
65 70 75 80
Lys Asp Ile Asn Lys Lys Asp Arg Leu Ser Lys Leu Ile Ile Lys His
85 90 95
Phe Ser Phe Leu Glu Ala Ala Thr Tyr Arg Gln Asn Pro Thr Asp Thr
100 105 110
Thr Lys Gln Lys Glu Glu Lys Gln Ala Glu Ala Gln Ser Leu Glu Ser
115 120 125
Leu Lys Lys Ser Phe Phe Val Phe Ile Tyr Lys Leu Arg Asp Leu Ala
130 135 140
Asn His Tyr Ser Ala Tyr Lys His Ser Lys Ser Leu Glu Arg Pro Lys
145 150 155 160
Phe Glu Glu Asp Leu Leu Lys Lys Met Tyr Asn Ile Phe Asn Ala Ser
165 170 175
Ile Arg Leu Val Lys Glu Asp Tyr Lys His Asn Lys Asn Ile Asn Pro
180 185 190
Lys Lys Asp Phe Lys His Leu Asn Arg Lys Arg Lys Gly Lys Phe Tyr
195 200 205
Tyr Ser Phe Ala Asp Asn Glu Gly Asn Ile Thr Glu Ser Gly Leu Leu
210 215 220
Phe Phe Val Ser Leu Phe Leu Glu Lys Lys Asp Ala Ile Trp Val Gln
225 230 235 240
Lys Lys Leu Glu Gly Phe Lys Cys Ser Asn Glu Ser Tyr Gln Lys Met
245 250 255
Thr Asn Glu Val Phe Cys Arg Ser Arg Met Leu Leu Pro Lys Leu Arg
260 265 270
Leu Glu Ser Thr Gln Thr Gln Asp Trp Ile Leu Leu Asp Met Leu Asn
275 280 285
Glu Leu Ile Arg Cys Pro Lys Ser Leu Tyr Glu Arg Leu Arg Gly Asp
290 295 300
Asp Arg Glu Lys Phe Arg Val Pro Leu Asp Pro Ile Asp Glu Asp Tyr
305 310 315 320
Asp Ala Glu Gln Glu Pro Phe Lys Asn Thr Leu Val Arg His Gln Asp
325 330 335
Arg Phe Pro Tyr Phe Ala Leu Arg Tyr Phe Asp Tyr Asn Glu Ile Phe
340 345 350
Ser Asn Leu Arg Phe Gln Ile Asp Leu Gly Thr Tyr His Phe Ser Ile
355 360 365
Tyr Lys Lys Leu Ile Gly Gly Gln Lys Glu Asp Arg His Leu Thr His
370 375 380
Lys Leu Tyr Gly Phe Glu Arg Ile Gln Glu Phe Asp Lys Gln Asn Arg
385 390 395 400
Thr Asp Glu Trp Lys Ser Ile Val Lys Asp Leu Asp Thr Tyr Glu Thr
405 410 415
Ser Asn Glu Arg Tyr Ile Ser Glu Thr Thr Pro His Tyr His Leu Glu
420 425 430
Asn Gln Lys Ile Gly Ile Arg Phe Arg Asn Gly Asn Asn Asp Ile Trp
435 440 445
Pro Ser Leu Glu Thr Asn Gly Glu Asn Asn Glu Lys Ser Lys Tyr Lys
450 455 460
Leu Asp Lys Gln Tyr Gln Ala Glu Ala Phe Leu Ser Val His Glu Leu
465 470 475 480
Leu Pro Met Met Phe Tyr Tyr Leu Leu Leu Lys Thr Glu Asn Thr Asp
485 490 495
Asn Asp Asn Glu Ile Glu Thr Lys Lys Lys Glu Asn Lys Asn Asp Lys
500 505 510
Gln Glu Lys His Lys Ile Glu Glu Ile Ile Glu Asn Lys Ile Lys Asp
515 520 525
Ile Tyr Ala Leu Tyr Asp Ala Phe Ala Asn Gly Glu Ile Asn Ser Lys
530 535 540
Glu Glu Leu Lys Glu Tyr Leu Lys Gly Lys Asp Ile Glu Ile Val His
545 550 555 560
Leu Pro Lys Gln Met Ile Ala Ile Leu Glu Ser Lys Pro Lys Asp Met
565 570 575
Val Glu Glu Ala Glu Arg Lys Gln Arg Lys Met Val Lys Asp Thr Lys
580 585 590
Lys Leu Leu Ala Ala Leu Glu Lys Gln Thr Gln Glu Glu Ile Glu Asp
595 600 605
Gly Gly Arg Asn Ile Arg Leu Leu Lys Ser Gly Glu Ile Ala Arg Trp
610 615 620
Leu Val Asn Asp Met Met Arg Phe Gln Pro Val Gln Lys Asp Asn Glu
625 630 635 640
Gly Asn Pro Leu Asn Asn Ser Lys Ala Asn Ser Thr Glu Tyr Gln Met
645 650 655
Leu Gln Arg Ser Leu Ala Leu Tyr Asn Lys Glu Glu Lys Pro Thr Arg
660 665 670
Tyr Phe Arg Gln Val Asn Leu Ile Asn Ser Ser Asn Pro His Pro Phe
675 680 685
Leu Lys Trp Thr Lys Trp Glu Glu Cys Asn Asn Ile Leu Ser Phe Tyr
690 695 700
Arg Ser Tyr Leu Thr Lys Lys Ile Glu Phe Leu Asn Lys Leu Lys Pro
705 710 715 720
Glu Asp Trp Glu Lys Asn Gln Tyr Phe Leu Lys Leu Lys Glu Pro Lys
725 730 735
Thr Asn Arg Glu Thr Leu Val Gln Gly Trp Lys Asn Gly Phe Asn Leu
740 745 750
Pro Arg Gly Ile Phe Thr Glu Pro Ile Arg Glu Trp Phe Lys Arg His
755 760 765
Gln Asn Asn Ser Glu Glu Tyr Glu Lys Val Glu Ala Leu Asp Arg Val
770 775 780
Gly Leu Val Thr Lys Val Ile Pro Leu Phe Phe Lys Glu Glu Tyr Phe
785 790 795 800
Lys Glu Asp Ala Gln Lys Glu Ile Asn Asn Cys Val Gln Pro Phe Tyr
805 810 815
Ser Phe Pro Tyr Asn Val Gly Asn Ile His Lys Pro Lys Glu Glu Asp
820 825 830
Phe Leu His Arg Glu Glu Arg Ile Glu Leu Trp Asp Lys Lys Lys Asp
835 840 845
Lys Phe Lys Gly Tyr Lys Ala Lys Lys Lys Phe Lys Glu Met Thr Asp
850 855 860
Lys Glu Lys Glu Glu His Arg Ser Tyr Leu Glu Phe Gln Ser Trp Asn
865 870 875 880
Lys Phe Glu Arg Glu Leu Arg Leu Val Arg Asn Gln Asp Ile Val Thr
885 890 895
Trp Leu Leu Cys Thr Glu Leu Ile Asp Lys Leu Lys Val Glu Gly Leu
900 905 910
Asn Val Glu Glu Leu Lys Lys Leu Arg Leu Lys Asp Ile Asp Thr Asp
915 920 925
Thr Ala Lys Gln Glu Lys Asn Asn Ile Leu Asn Arg Val Met Pro Met
930 935 940
Gln Leu Pro Val Thr Val Tyr Glu Ile Asp Asp Ser His Asn Ile Val
945 950 955 960
Lys Asp Arg Pro Leu His Thr Val Tyr Ile Glu Glu Thr Lys Thr Lys
965 970 975
Leu Leu Lys Gln Gly Asn Phe Lys Ala Leu Val Lys Asp Arg Arg Leu
980 985 990
Asn Gly Leu Phe Ser Phe Val Lys Thr Pro Ser Glu Ala Glu Ser Glu
995 1000 1005
Ser Asn Pro Ile Ser Lys Ser Arg Val Glu Ser Glu Leu Gly Lys Tyr
1010 1015 1020
Gln Asn Ala Arg Leu Asn Ile Ile Glu Asp Met Leu Ala Leu Glu Lys
1025 1030 1035 1040
Thr Leu Ile Asp Asn Tyr Asn Ser Leu Asp Thr Asp Asn Phe His Asn
1045 1050 1055
Met Leu Thr Gly Trp Leu Lys Leu Lys Gly Lys Ala Asn Lys Ala Arg
1060 1065 1070
Phe Gln Asn Glu Val Asp Leu Leu Val Ala Val Ala Asn Ala Phe Ser
1075 1080 1085
Ala Asn Gln Tyr Pro Met Tyr Asn Glu Asn Leu Phe Gly Asn Ile Glu
1090 1095 1100
Arg Phe Ser Leu Ser Ser Ser Asn Ile Ile Glu Ser Lys Gly Leu Asp
1105 1110 1115 1120
Ile Ala Val Lys Leu Lys Glu Glu Val Ser Lys Ala Val Glu Lys Ile
1125 1130 1135
Gln Asn Glu Glu Asp Asp Lys Lys Glu Lys Glu Thr Leu Glu Ala Leu
1140 1145 1150
Pro Val Ala Thr Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val
1155 1160 1165
Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe
1170 1175 1180
Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr
1185 1190 1195 1200
Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr
1205 1210 1215
Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro
1220 1225 1230
Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly
1235 1240 1245
Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys
1250 1255 1260
Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile
1265 1270 1275 1280
Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His
1285 1290 1295
Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp
1300 1305 1310
Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile
1315 1320 1325
Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro
1330 1335 1340
Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr
1345 1350 1355 1360
Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val
1365 1370 1375
Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu
1380 1385 1390
Leu Tyr Lys Ser Asn Ser Lys Pro Gly Phe His Met Lys Arg Pro Ala
1395 1400 1405
Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys
1410 1415 1420
<210> 19
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gttgtagttc ccttcattaa tgggatcatt tgcatcgtga cctgtggatg ctgagg 56
<210> 20
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gtgacctgtg gatgctgagg gttgtagaag ccctcatttt gagagggtat tacaac 56
<210> 21
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gtgacctgtg gatgctgagg gttgttttta ccttgcaaac agcaggcaga tgcaac 56
<210> 22
<211> 2905
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tggatcgatg atgagtcctc caaaaaaaac attccttgga aaagctgaac aaaatgagtg 60
aaaactcata ccgtcattct catcggaact gaggtccagc acattgcttc ctctggggcc 120
tttactagtg agggacaact tccactagtc cctataggtt ctaacaccca tggcgttcac 180
aggtgaaggt ctcctgtcat attacaaaca ttccccccac tcccctccca aaaaaaggaa 240
catttcctct gtgtcaggta cacaggtcag agagtcatag ggtgactggc tgctctgtga 300
tggcatagtg gccgtggaag tggatggaaa tgtcgcaggc ttaggggagt gtggggctgg 360
cctcggcttg gtgagctccc aggaaggggg acctggaaat cgctgcgggc atccttgcat 420
agaggaccag gtacgtgttc cagttctgct ttcaaggaga gcatgaggcc caggcctcag 480
acagtccttg gtcttgatct ttggttagct gaggacaggg gagtgccaag ggtgataggg 540
gagagcgcac agcaatggat gttgtggcag acagctaaag cctttctgtc cctcctttca 600
cggtcagtcc cagggggccc ccagcacaga cttttttcac ctctcgagct gtgagattcc 660
tgtggtgttg tggccaagcc tgggcaggaa ggtaccccag agaaaaggtt gtagagggct 720
cacgtggaca tgcatgccat cagtggtctt gggctgtcat ttggtggtga tgggctttcc 780
agggatttct ctggtggatg acaggttggc cattgggggt cttcaagggc aggtcctagg 840
gagcatgagg gtcgggctga tgcctgtggt cctgggtcac tccattcttc ctggcagagt 900
agtagaagtg tccatcttcc tggaaatggt ctcttggccc gccattgcca ggcggcccca 960
cctgctgcag ctcgtgtgag caaatactgc tctggatccc atttcccgct ttgaaagcag 1020
cttccatccc aaaggcgtcc agagggaatg tgccctggcc ctggccctgg cctgggactc 1080
tccgatgggg tccagtcaca cttggcgtat tcatggaggt ccttgtgcat tccaggcaac 1140
acatgtgcaa taccaggggc actgccgtgg agagtctctt gatttgggat gcctcctcgc 1200
ctttgtgtgt tggcgatgtt ccagtattgg attctgctac atcctcaggg caatgaagcg 1260
tttcagctca cttagccggt ttgacatcat agtgcagcca ggacagccgt gccacattgg 1320
catggcacat agttgactta ggacagtcgg aaagggttgg tggttctatg tctcgcagct 1380
cacttcatag ctgtttgcat gtaaacctgg tgatggtgtt gagatggtgt tgacttgctg 1440
acgttggggt ggttggtagt gcagttaagt tggctgcatg atgggatgtt gcctcagatg 1500
tcggtgcagc gaagccctgt aggtgtgtcc aatgcaagga aaatgaaagg tggtgtgtcc 1560
tgtccacctt gtctttaggg gttcaggcag gaatgtcctg ttggaaaact agccattccc 1620
tttgtgtgtc ctgaaggcat ggtgcgggca tgtgaactga ttggaggaag gaagtgtgtg 1680
tcttgaggtc ctttggctgg gttcagaaga ctgtctctgg ccttgatgcc gtgtgccgtg 1740
tttaagacca acgtggtggc cattgcaaag gtgccttcca gtgcaagttt tggttggttt 1800
ggtgtcaggt gtgccttacc tgggagagcg gcgagagaca gtgtctggtc acgacattat 1860
atgccccgta aggaggactg tgtgcttgca ccgccagggg tgctggctgt tagtcgaggg 1920
acatcaggcc aatgtctgtc tgacccctag gtcaaaattc gcatcctact gattgaaggc 1980
cttgggtctc gagccttttg gttgcaggtg tggggatggt ttccaggtct ggaggctgcc 2040
aactatgaca acccgttgtg ctgttgggaa acatctgtgt gggggagaca catacacata 2100
gacacactca ctctgacata cacacctaca catgcacagg cgtgcacagc cacaggcaga 2160
tagatgcagc aatgggtgtg tgctattgtt gtggtcacat gtgcttccta ggtgtccaca 2220
gagtgacatg ggtcaggtat attcatgaag atggaggtct gtcatggagc ccaaggagaa 2280
gccggtgtgc aagtttccct gtgggaaaga atgggattga agtggtgacc agtggacttg 2340
ggcccgtttc tcaagcatgc atttgatttc tttacacttc tcagcagcag ttgtcatgag 2400
aggtctgccg cttgctggca aggaagatgg tcaattcttc tgaccaacag aggctgcctt 2460
gagtcctgat ttggtgtggc ctggcatcta tggtgcaccc aatgctggtg agtggctcag 2520
ggtggtaagc attcctctgc cctgtgtgca gaggtgtctt ttgaattttg agacctgcaa 2580
agagctgttt ctcagcaggc cactaatggc atggcgagtt ccactcccag agctgattgt 2640
ctgtgttcat tgctcagtgg tgtggctctg ggttcccccc aggatgctga aggtgtttaa 2700
ggtcatcagc tctcccactg cccagcctgt ggtgtcaaat gttctgcctc ttcgaacgtg 2760
cttggatcga tgatgagtcc cccataaaaa cattccttgg aaaagctgaa caaaatgagt 2820
gagaactcat accgtcgttc tcatcagaac tgaggtccag cacgttgcct cctctgggga 2880
ctgtaggagc gagggacaac ttcca 2905
<210> 23
<211> 1191
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tggaccaatg atgacttcca tacatgcatt ccttggaaag ctgaacaaaa tgagtgggaa 60
ctctgtacta tcatcttagt tgaactgagg tccaccgggg gctatagagg gacaactgtc 120
attttttttg atgcccgttg catgaagcat tcttccaggc ttaggaaggg attccgtttg 180
ggtgaaggag agtcacaggg gagttgaggc tatgtgcaca ggacttctgt ctacaaggct 240
ggggtagcac acatgcagtc cgtgggacta ggtgaatgtg caggttgctc agggagagtt 300
aacctgtagc ctgcctctgg gtggccaaga ttgtccttga ctcctggctc tgggttccca 360
caacccctgg aggaagacaa gtaattgtgc ttgggaagag agaacaaggc catacttttg 420
cctttctttt tttcattctt agtttgagaa cagtggagtt ccaatcacct ggcatagctt 480
tcatgttgtg ttccatgcat tgtgagtaca tgcatatggc aggaacacac aggttcccac 540
tgcaagctga gcctgatagg caaggagtct ctagcttaac cccagcccag tgtacgtcaa 600
tggcttcctc attaaaattg catggccctg tagcttttgt gagtgacatt ataggtatcg 660
atgtaagggt gttgagaggg atgctcaatg tcagtgctct ttagtgtgct gatgcactac 720
tacaagggca cctgagccct tgtccatgca tttctgtgtc caaaatcagg taaagccaga 780
gttgggtatc aaccagatac actgcagggg atcagaggac atggcctttg tgtactgcac 840
atctgtcaga ttttctcaag cacacccgtg tttcctcttc acctcccacg tggaagtgat 900
gaattggcca ggaccatgtt ggtgagggac aatcatcatc tcatgttaac ctcagagaag 960
ccaggaagct cctggacaca catggtgtag gcttccttgg aggctgttgg atctctcctg 1020
aatgtaagca attccttccc agtgcaccct gattttcctc atttgcagaa acaaagattg 1080
tgtgtggatc gatgatgact tccatatata cattccttgg aaagctgaac aaaatgagtg 1140
aaaactctat accgtcattc tcgtcgaact gaggtccagc acattactcc a 1191
<210> 24
<211> 1249
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
tggatcgatg atgacttcct tatatacatt ccttggaaag ctgaacaaaa tgagtgaaaa 60
ctctataccg tcatcctcgt cgaactgagg tccagcacat tgctcttaca ggggctagag 120
agagagggac aaatttcatt tgatgatgcc cattgcacca aggggttctg tccaggctta 180
ggatggggtc tcgtttgggc aaaggagaat ggcaggggag tggaggctat gtacacagga 240
gattccttgt ttgaaggact ctatttgtga ggccagggta ccacacatgc tgtccgcagg 300
agtaggtgaa tgtgcagttg cccaggaaga gttagcctgt agcctgcctc tgcatggcag 360
tttgtccttg ggtcctggct ctggatttcc atgttccttg gaggaggata ggtgattttg 420
cttgagaaga cagcacagta ccatactttt gttgtttttc tgctcatttc atcgtccatt 480
gaggacaatg aagttgtggt cagcaggcat agctttcagg ccagcgtgcc catgttgtgt 540
cccatgcatt gtgagcacat gcatgtggca tgaacacata ggctgccact ccaagctgag 600
tctgataggc aatgagactc tggcttatcc tgatcccggt gtagatcaaa gtcttcccag 660
taggattgca tggccccgag gctattgtga gctgcattgc aggtgtggaa gcaagggtgt 720
tgagagggat gctcaacatt agtgctcttt agcgagatga tgcactataa gggcaccctg 780
aacccagacg tgcatcccta tgtacgtgca tttctgtgtc cataaatagt tgaagccaga 840
cagccagatt ccagatgtat cgcagggggc tggatgacat ggcccttgtc acctgtgtac 900
ctgtctgcct ttctgaagca cgcttgtgtt tcctctacac ctcccaggta gcattggcat 960
ggaaggcagg cccatgttgg tgagggacaa ttgttatctt gtgtgagccg cagggatacc 1020
aggaaacccc tggacacaaa tggcaaaggc ttctttggaa gttgttggat cccttctgca 1080
tgtaagcagt tctttcccag agcgctctga ttttcctcat ttgcagggac aaacactgtg 1140
cgtggatcga tgatgacttc catatataca ttccttggaa agctgaacaa aatgagtgaa 1200
aactctatac cgtcatcctc gtcgaactga ggtccagcac attactcca 1249
<210> 25
<211> 1249
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tggatcgatg atgacttcca tatatacatt ccttggaaag ctgaacaaaa tgagtgaaaa 60
ctctatactg tcatcctcgt cgaactgagg tccagcacat tactccaaca ggggctagac 120
agagaaggcc aacatccgtt tgttgacatg ggttatatca aggcgtctgt tcaggcttag 180
aatgtggtct cttatgggtg atgggggtca caggagagtg gtggctccca tgtataggaa 240
atttcttgtt tgaaggactg tcagtgaggg tgggtaacac atgcattgtc tgcaggacta 300
ggtgaatgtc catgtggcct agcaagagtt agctggtagc ccgcctctgg ttgccaattt 360
gttcttgagt ccttgttctg ggttctcagg tcccacggag gaaaacagat ctgtgtggtt 420
gagaggtggg tacaaggccg catctttgtc atttgttggc taactttgtc cttggttgag 480
gacattagag ttttggtcac caggcatagc ctatgtgcct ttgtgcccgt gttgtatccc 540
acgtgttttg aggacatgta ttttgcacgt aaaggtgagc tcctgctcca agctggttct 600
gataccaaag gagtccctgg cttatcctaa actcatggta ggttaaagcc ttcctcctta 660
ggggttcagg gccgcaaggc ttttgtgagt ggcattgcag gcgttgaagc agtgatgttg 720
agagggatgg tcaatgtcag tgctctttag caggatggtg tactgcaggg gcccccagcc 780
ccgagacgag catccctgca tccatgcatt tctgcctcca tgaacagggg aggccagaga 840
caggcagata gtagataaat tgcaggggac tggatgacat ggccctcgtg acctgtgcac 900
ctgtctgtct ttctgaagca cgcctgtgtt aactctgcac ctcccaggta gcactggcat 960
ggagggcagg cacatgttgg tgagggacaa ttgttacctt gtgtgagctg cggagatacc 1020
aggaagcccc tggacacaaa tggcaaaggc tccttcggaa gttgttggat cccttctgaa 1080
tgtaagcact tctttcccag agcactctga gtttcctcat ttgcagggac aaatactgtg 1140
cgtggatcga tgatgacttc cacatataca ttccttggaa agctgaacaa aatgagtgaa 1200
aactctatac cgtcatcctc gtcgaactga ggtccagcac attactcca 1249
<210> 26
<211> 2187
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
tggatcgatg atgactataa aaaaaatgga tctcatcgga atctgaacaa aatgagtgac 60
caaatcattt ctgtgccact tctgtgagct gaggtccagc actgggacca tgccaggcaa 120
acatgaggag ggagtttaca gggactaggg tattggaaaa ctaacctacc catatatatt 180
agagcaaatg aattgttctc agttctgtaa atatccagct gtggtttgac atagcaactc 240
tctttgtatt tgcttggcag catttataga aacagctggt agaccctggg gaaccagggc 300
atagtgagat tcatgggacg ttatttcacc tactcttgag ccattcacac atttgagctc 360
gcagaattat ctggtcaatg agcagtctct tgagggtaat gagatggtca acatccaaat 420
ccaaagatac taatgagatt ttactttttt ttttgagatg gagtctcatt ctgttgccca 480
ggctggagtg cagtggcata atctcggctc actgcaacct ccacctcctg ggttcaagag 540
attctcccgc cccagcatcc tgagtagctg ggattacagg cgcccgccac cacccctggc 600
taatttttgt gtttgtagta gagatggagt ttcaccatat tggccaagct ggtctcgaac 660
tcccaacctc aggtgatcca gccactgcag cctctcaaag tgttgggatt acaggcatga 720
gccgccgtgc ccggccttac tttttatttt caattaagtg aaacaattac atggaaattg 780
aaaaaaaaaa aaggtactct aagtagacta ctctagtgta ttttaatttt cttgccaatt 840
cagccactag gattgtgtca cttgtttttt tgattttagt ttttcgtatc aaaaagcaaa 900
ccactgttta tatttgagtg ctggtttcaa ctgtatgctg cctcaaataa acttagatta 960
tattacaaat tcttttttac atagatttca ttggctatat tcatatatcc tcaaaggttt 1020
ccattgtgtt tgaagaggta tcctaattgt aataaatgct tttgattggg gatgccaggg 1080
aagctttgag actaatgtct cagatttttt ttaaaaaagt ttatattcta tagtgatcaa 1140
agatgtaaga ataatttttc ttgtcattca ttggatagtc cagtgagtgt ctactgtgaa 1200
ttcttatatt cacttgtaac taaacattcc atttggcaaa tttcgtttca taacttccct 1260
aatttcctct acactttgta gattgggtga gatacaattc catggaattt taccactgaa 1320
ccagtggagg gctaattttg tcatttgtac cagttaacta acttgggagg aaacagaaca 1380
acacactcct gaggtagtat ccaattttta gtaaatttat ttttattatt tttcaaactg 1440
tcagtgattt ggtttggtga acaataggtt aatacagaca atgttaggaa ttattaatct 1500
gatgcgctat ctgaagcatc agtttccttt atttttaaga tttttttctt ctctgtagtt 1560
tggataatat ttaaaacatt tccaagagtt ataaattagt atattttcta tgctgattaa 1620
taatgaaacc ttttgtctta tacagatttt taaaaaatgg acgtgtgcgt gcatgtgtat 1680
atatataaat atatatatat ataaaatctc ttctcacaag atgatgcaga ctaaaactgc 1740
attttacatt ttatatggct aagtttattg tttccttgtg ttgtaatatg atggaaagta 1800
atgggaataa ttctgcttcc tgtccttagg gaccagtagg aattttccca tgataagcat 1860
tttgtcctta aaatactgac atcctgttaa gagatcatga cactccaatg accatgagac 1920
cctggctatg ctagtcagcc agggggctga ccattgacag tgatgctggt ctccatgatt 1980
aaatagtgtg ggtcagcatg tcccagggtg acagaaagct gtgtgatcag tgtctgtcac 2040
atatctcaga ctcagggttc ctttttaaaa tgtgcctgga tcgatgatga cttaaagatt 2100
tatctaattt aaatctgaac aaaatgagtg accaaaacac ttctgtacca cttctgtgag 2160
ctgaggtcca gcacagagac cactcca 2187
<210> 27
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gtgacctgtg gatgctgagg gttgttttta ccttgcaaac agcaggcaga tgcaac 56
<210> 28
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
gccatcacag agcagccagt caccctatga gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 29
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
gccatcacag agcagccagt caccctatga gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 30
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
gggagctcac caagccgagg ccagccccac gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 31
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
ggtgcccttg tagtagtgca tcagcacact gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 32
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
gcacactaaa gagcactgac attgagcatc gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 33
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gcctggacag aaccccttgg tgcaatgggc gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 34
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
gagaccccat cctaagcctg gacagaaccc gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 35
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
gtgaccccca tcacccataa gagaccacat gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 36
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
gaacagacgc cttgatataa cccatgtcaa gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 37
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
gcctgaacag acgccttgat ataacccatg gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 38
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
gagaccacat tctaagcctg aacagacgcc gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 39
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
ggtccctaag gacaggaagc agaattattc gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 40
<211> 67
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
aggacaaaat gcttatcatg ggaaaattcc tgttgttttt accttgcaaa cagcaggcag 60
atgcaac 67
<210> 41
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
ggcaagaaaa ttaaaataca ctagagtagt gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 42
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
gaaattaata cgactcacta tagggacatc cgtttgttga catgggttat atcaag 56
<210> 43
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
cttcaaacaa gaaatttcct atacatggga gc 32
<210> 44
<211> 13
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 44
augucuuaug ucu 13
<210> 45
<211> 13
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 45
augucuuaug ucu 13
<210> 46
<211> 159
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 46
agaagattgg ttagatgatg atagtcaaca aactgttggt caacaagacg gcagtgagga 60
caatcagaca actactattc aaacaattgt tgaggttcaa cctcaattag agatggaact 120
tacaccagtt gttcagacta ttgaagtgaa tagttttag 159
<210> 47
<211> 57
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 47
gaaattaata cgactcacta tagggagaag attggttaga tgatgatagt caacaaa 57
<210> 48
<211> 32
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 48
ctaaaactat tcacttcaat agtctgaaca ac 32
<210> 49
<211> 66
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 49
gccgtcttgt tgaccaacag tttgttgact gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 50
<211> 66
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 50
gtcctcactg ccgtcttgtt gaccaacagt gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 51
<211> 66
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 51
gattgtcctc actgccgtct tgttgaccaa gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 52
<211> 66
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 52
gtagttgtct gattgtcctc actgccgtct gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 53
<211> 66
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 53
gaatagtagt tgtctgattg tcctcactgc gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 54
<211> 66
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 54
gtttgaatag tagttgtctg attgtcctca gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 55
<211> 66
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 55
gaggttgaac ctcaacaatt gtttgaatag gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 56
<211> 66
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 56
gttccatctc taattgaggt tgaacctcaa gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 57
<211> 66
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 57
ggtgtaagtt ccatctctaa ttgaggttga gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 58
<211> 66
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 58
gtctgaacaa ctggtgtaag ttccatctct gttgttttta ccttgcaaac agcaggcaga 60
tgcaac 66
<210> 59
<211> 1152
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 59
Met Lys Val Thr Lys Val Asp Gly Ile Ser His Lys Lys Tyr Ile Glu
1 5 10 15
Glu Gly Lys Leu Val Lys Ser Thr Ser Glu Glu Asn Arg Thr Ser Glu
20 25 30
Arg Leu Ser Glu Leu Leu Ser Ile Arg Leu Asp Ile Tyr Ile Lys Asn
35 40 45
Pro Asp Asn Ala Ser Glu Glu Glu Asn Arg Ile Arg Arg Glu Asn Leu
50 55 60
Lys Lys Phe Phe Ser Asn Lys Val Leu His Leu Lys Asp Ser Val Leu
65 70 75 80
Tyr Leu Lys Asn Arg Lys Glu Lys Asn Ala Val Gln Asp Lys Asn Tyr
85 90 95
Ser Glu Glu Asp Ile Ser Glu Tyr Asp Leu Lys Asn Lys Asn Ser Phe
100 105 110
Ser Val Leu Lys Lys Ile Leu Leu Asn Glu Asp Val Asn Ser Glu Glu
115 120 125
Leu Glu Ile Phe Arg Lys Asp Val Glu Ala Lys Leu Asn Lys Ile Asn
130 135 140
Ser Leu Lys Tyr Ser Phe Glu Glu Asn Lys Ala Asn Tyr Gln Lys Ile
145 150 155 160
Asn Glu Asn Asn Val Glu Lys Val Gly Gly Lys Ser Lys Arg Asn Ile
165 170 175
Ile Tyr Asp Tyr Tyr Arg Glu Ser Ala Lys Arg Asn Asp Tyr Ile Asn
180 185 190
Asn Val Gln Glu Ala Phe Asp Lys Leu Tyr Lys Lys Glu Asp Ile Glu
195 200 205
Lys Leu Phe Phe Leu Ile Glu Asn Ser Lys Lys His Glu Lys Tyr Lys
210 215 220
Ile Arg Glu Tyr Tyr His Lys Ile Ile Gly Arg Lys Asn Asp Lys Glu
225 230 235 240
Asn Phe Ala Lys Ile Ile Tyr Glu Glu Ile Gln Asn Val Asn Asn Ile
245 250 255
Lys Glu Leu Ile Glu Lys Ile Pro Asp Met Ser Glu Leu Lys Lys Ser
260 265 270
Gln Val Phe Tyr Lys Tyr Tyr Leu Asp Lys Glu Glu Leu Asn Asp Lys
275 280 285
Asn Ile Lys Tyr Ala Phe Cys His Phe Val Glu Ile Glu Met Ser Gln
290 295 300
Leu Leu Lys Asn Tyr Val Tyr Lys Arg Leu Ser Asn Ile Ser Asn Asp
305 310 315 320
Lys Ile Lys Arg Ile Phe Glu Tyr Gln Asn Leu Lys Lys Leu Ile Glu
325 330 335
Asn Lys Leu Leu Asn Lys Leu Asp Thr Tyr Val Arg Asn Cys Gly Lys
340 345 350
Tyr Asn Tyr Tyr Leu Gln Val Gly Glu Ile Ala Thr Ser Asp Phe Ile
355 360 365
Ala Arg Asn Arg Gln Asn Glu Ala Phe Leu Arg Asn Ile Ile Gly Val
370 375 380
Ser Ser Val Ala Tyr Phe Ser Leu Arg Asn Ile Leu Glu Thr Glu Asn
385 390 395 400
Glu Asn Gly Ile Thr Gly Arg Met Arg Gly Lys Thr Val Lys Asn Asn
405 410 415
Lys Gly Glu Glu Lys Tyr Val Ser Gly Glu Val Asp Lys Ile Tyr Asn
420 425 430
Glu Asn Lys Gln Asn Glu Val Lys Glu Asn Leu Lys Met Phe Tyr Ser
435 440 445
Tyr Asp Phe Asn Met Asp Asn Lys Asn Glu Ile Glu Asp Phe Phe Ala
450 455 460
Asn Ile Asp Glu Ala Ile Ser Ser Ile Arg His Gly Ile Val His Phe
465 470 475 480
Asn Leu Glu Leu Glu Gly Lys Asp Ile Phe Ala Phe Lys Asn Ile Ala
485 490 495
Pro Ser Glu Ile Ser Lys Lys Met Phe Gln Asn Glu Ile Asn Glu Lys
500 505 510
Lys Leu Lys Leu Lys Ile Phe Lys Gln Leu Asn Ser Ala Asn Val Phe
515 520 525
Asn Tyr Tyr Glu Lys Asp Val Ile Ile Lys Tyr Leu Lys Asn Thr Lys
530 535 540
Phe Asn Phe Val Asn Lys Asn Ile Pro Phe Val Pro Ser Phe Thr Lys
545 550 555 560
Leu Tyr Asn Lys Ile Glu Asp Leu Arg Asn Thr Leu Lys Phe Phe Trp
565 570 575
Ser Val Pro Lys Asp Lys Glu Glu Lys Asp Ala Gln Ile Tyr Leu Leu
580 585 590
Lys Asn Ile Tyr Tyr Gly Glu Phe Leu Asn Lys Phe Val Lys Asn Ser
595 600 605
Lys Val Phe Phe Lys Ile Thr Asn Glu Val Ile Lys Ile Asn Lys Gln
610 615 620
Arg Asn Gln Lys Thr Gly His Tyr Lys Tyr Gln Lys Phe Glu Asn Ile
625 630 635 640
Glu Lys Thr Val Pro Val Glu Tyr Leu Ala Ile Ile Gln Ser Arg Glu
645 650 655
Met Ile Asn Asn Gln Asp Lys Glu Glu Lys Asn Thr Tyr Ile Asp Phe
660 665 670
Ile Gln Gln Ile Phe Leu Lys Gly Phe Ile Asp Tyr Leu Asn Lys Asn
675 680 685
Asn Leu Lys Tyr Ile Glu Ser Asn Asn Asn Asn Asp Asn Asn Asp Ile
690 695 700
Phe Ser Lys Ile Lys Ile Lys Lys Asp Asn Lys Glu Lys Tyr Asp Lys
705 710 715 720
Ile Leu Lys Asn Tyr Glu Lys His Asn Arg Asn Lys Glu Ile Pro His
725 730 735
Glu Ile Asn Glu Phe Val Arg Glu Ile Lys Leu Gly Lys Ile Leu Lys
740 745 750
Tyr Thr Glu Asn Leu Asn Met Phe Tyr Leu Ile Leu Lys Leu Leu Asn
755 760 765
His Lys Glu Leu Thr Asn Leu Lys Gly Ser Leu Glu Lys Tyr Gln Ser
770 775 780
Ala Asn Lys Glu Glu Thr Phe Ser Asp Glu Leu Glu Leu Ile Asn Leu
785 790 795 800
Leu Asn Leu Asp Asn Asn Arg Val Thr Glu Asp Phe Glu Leu Glu Ala
805 810 815
Asn Glu Ile Gly Lys Phe Leu Asp Phe Asn Glu Asn Lys Ile Lys Asp
820 825 830
Arg Lys Glu Leu Lys Lys Phe Asp Thr Asn Lys Ile Tyr Phe Asp Gly
835 840 845
Glu Asn Ile Ile Lys His Arg Ala Phe Tyr Asn Ile Lys Lys Tyr Gly
850 855 860
Met Leu Asn Leu Leu Glu Lys Ile Ala Asp Lys Ala Lys Tyr Lys Ile
865 870 875 880
Ser Leu Lys Glu Leu Lys Glu Tyr Ser Asn Lys Lys Asn Glu Ile Glu
885 890 895
Lys Asn Tyr Thr Met Gln Gln Asn Leu His Arg Lys Tyr Ala Arg Pro
900 905 910
Lys Lys Asp Glu Lys Phe Asn Asp Glu Asp Tyr Lys Glu Tyr Glu Lys
915 920 925
Ala Ile Gly Asn Ile Gln Lys Tyr Thr His Leu Lys Asn Lys Val Glu
930 935 940
Phe Asn Glu Leu Asn Leu Leu Gln Gly Leu Leu Leu Lys Ile Leu His
945 950 955 960
Arg Leu Val Gly Tyr Thr Ser Ile Trp Glu Arg Asp Leu Arg Phe Arg
965 970 975
Leu Lys Gly Glu Phe Pro Glu Asn His Tyr Ile Glu Glu Ile Phe Asn
980 985 990
Phe Asp Asn Ser Lys Asn Val Lys Tyr Lys Ser Gly Gln Ile Val Glu
995 1000 1005
Lys Tyr Ile Asn Phe Tyr Lys Glu Leu Tyr Lys Asp Asn Val Glu Lys
1010 1015 1020
Arg Ser Ile Tyr Ser Asp Lys Lys Val Lys Lys Leu Lys Gln Glu Lys
1025 1030 1035 1040
Lys Asp Leu Tyr Ile Arg Asn Tyr Ile Ala His Phe Asn Tyr Ile Pro
1045 1050 1055
His Ala Glu Ile Ser Leu Leu Glu Val Leu Glu Asn Leu Arg Lys Leu
1060 1065 1070
Leu Ser Tyr Asp Arg Lys Leu Lys Asn Ala Ile Met Lys Ser Ile Val
1075 1080 1085
Asp Ile Leu Lys Glu Tyr Gly Phe Val Ala Thr Phe Lys Ile Gly Ala
1090 1095 1100
Asp Lys Lys Ile Glu Ile Gln Thr Leu Glu Ser Glu Lys Ile Val His
1105 1110 1115 1120
Leu Lys Asn Leu Lys Lys Lys Lys Leu Met Thr Asp Arg Asn Ser Glu
1125 1130 1135
Glu Leu Cys Glu Leu Val Lys Val Met Phe Glu Tyr Lys Ala Leu Glu
1140 1145 1150
<210> 60
<211> 68
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 60
ggggatttag actaccccaa aaacgaaggg gactaaaact cctactggtc cctaaggaca 60
ggaagcag 68

Claims (18)

1.一种分离的Cas13蛋白，所述Cas13蛋白的氨基酸序列为如SEQ ID NO.1～3其中之一所示的氨基酸序列。
2.如权利要求1所述的分离的Cas13蛋白，其特征在于，所述分离的Cas13蛋白能够特异性地切割目的RNA。
3.如权利要求2所述的分离的Cas13蛋白，其特征在于，所述目的RNA源自新型冠状病毒2019-nCoV。
4.如权利要求1所述的分离的Cas13蛋白，其特征在于，所述分离的Cas13蛋白被激活后能够非特异性地切割其他RNA。
5.如权利要求1所述的分离的Cas13蛋白，其特征在于，所述分离的Cas13蛋白来源于Bacteroides graminisolvens isolate UBA4I89；
和/或，所述分离的Cas13蛋白来源于Prevotella intermedia strain KCOM 2698；
和/或，所述分离的Cas13蛋白来源于Prevotellacae bacterium UBA776。
6.如权利要求1所述的分离的Cas13蛋白，其特征在于，所述分离的Cas13蛋白的RNA酶活结构域HEPN motif突变后丧失切割RNA的活性、但所述分离的Cas13蛋白仍保留结合RNA的能力。
7.一种分离的多核苷酸，编码如权利要求1～6任一权利要求所述的分离的Cas13蛋白。
8.一种构建体，所述构建体含有如权利要求7所述的分离的多核苷酸。
9.一种表达系统，所述表达系统含有如权利要求8所述的构建体或基因组中整合有外源的如权利要求7所述的多核苷酸。
10.如权利要求9所述的表达系统，其特征在于，所述表达系统的宿主细胞选自真核细胞或原核细胞。
11.如权利要求10所述的表达系统，其特征在于，所述表达系统的宿主细胞选自人细胞。
12.如权利要求1～6任一权利要求所述的分离的Cas13蛋白、如权利要求7所述的分离的多核苷酸、如权利要求8所述的构建体或如权利要求9～11任一权利要求所述的表达系统在制备RNA水平的基因编辑的系统中的用途。
13.如权利要求12所述的用途，其特征在于，所述用途为在制备真核生物的RNA水平的基因编辑、或核酸检测的系统中的用途。
14.一种碱基编辑体系，包括如权利要求1～6任一权利要求所述的分离的Cas13蛋白，所述碱基编辑体系还包括gRNA；
其中，所述的分离的Cas13蛋白为BgrCas13a，BgrCas13a的氨基酸序列如SEQ ID NO.1所示，所述gRNA的多核苷酸序列如SEQ ID NO.4所示；或所述的分离的Cas13蛋白为Pin4Cas13b，Pin4Cas13b的氨基酸序列如SEQ ID NO.3所示，
所述gRNA的多核苷酸序列如SEQ ID NO.5所示，或如SEQ ID NO.32-37任一所示，或如SEQ ID NO.39-41任一所示，或如SEQ ID NO.58所示。
15.一种非疾病诊断和治疗目的的RNA水平的基因编辑方法，包括：通过如权利要求1～6任一权利要求所述的分离的Cas13蛋白、或如权利要求14所述的碱基编辑体系进行基因编辑。
16.一种核酸检测试剂盒，包括：权利要求1～6任一权利要求所述的分离的Cas13蛋白、或权利要求14所述的碱基编辑体系。
17.如权利要求16所述的核酸检测试剂盒，其特征在于，所述核酸检测试剂盒的检测方法包括：
1)提供扩增的目的片段；
2)通过如权利要求1～6任一权利要求所述的分离的Cas13蛋白、或如权利要求14所述的碱基编辑体系对步骤1)所提供的扩增产物进行结合、切割；
和/或，所述核酸检测试剂盒的检测方法为小胖威利综合征、或新型冠状病毒2019-nCoV的检测方法。
18.如权利要求17所述的核酸检测试剂盒，其特征在于，所述核酸检测试剂盒的检测方法还包括：通过探针，判断检测结果。
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