CN113336854B - 一种精氨酸荧光探针及其制备方法和应用 - Google Patents
一种精氨酸荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种精氨酸荧光探针，包括对精氨酸响应的多肽B和对精氨酸进行表现的荧光蛋白A；所述的荧光蛋白A插入到多肽B中，将B分为B1和B2两个上下结构部分，形成B1‑A‑B2式的探针结构；同样在不同位置的截短和定点突变的优化突变体，所述多肽B和精氨酸特异性结合导致荧光蛋白A荧光信号改变；所述多肽B为精氨酸结合蛋白及其突变体。本发明提供的精氨酸荧光探针蛋白分子量相对较小且易于表达，荧光动态变化大，特异性好，并且能够通过基因操作在细胞的不同亚细胞器内表达，可在细胞内外高通量、定量检测精氨酸。
Description
技术领域
本发明涉及光学探针技术领域，尤其涉及一种精氨酸光学探针及其制备方法和应用。
背景技术
精氨酸作为20种天然氨基酸之一，最初是由Schlus于1886年从植物羽扇豆苗中分离提取，其分子结构于20世纪初已经清楚并能进行人工合成。精氨酸在生物体内以具有生理活性的L-型精氨酸形式发挥生物学功能。精氨酸不仅是机体蛋白质的组成成分，而且是多种生物活性物质的合成前体，如多胺和NO等，通过刺激部分激素分泌，参与内分泌调节和机体特异性免疫调节等生物学过程；此外还作为尿素循环的中间体，通过尿素循环解除氨中毒，避免由于氨过量造成的代谢紊乱；在机体的匀质代谢方面起着重要作用，可用于多种代谢途径，包括精氨酸酶、一氧化氮合酶、精氨酸/甘氨酸胍基转移酶、精氨酰-tRNA合成酶等。因此，精氨酸的多种生物学功能引起了科研工作者的广泛重视，成为目前氨基酸研究的热点之一。
哺乳动物中精氨酸的代谢主要有两个途径，一是在精氨酸酶作用下，精氨酸分解为鸟氨酸和尿素，鸟氨酸是合成多胺类物质的前体，多胺对于调节细胞生长和发育具有重要意义；二是在一氧化氮合酶作用下，精氨酸分解为等分子的瓜氨酸和NO，NO是细胞内、细胞间及神经递质作用的信使分子，广泛参与细胞间和细胞内的信号传递。另外，作为脒基转移反应的脒基供体，精氨酸、甘氨酸和甲硫氨酸共同合成胍基乙酸和磷酸肌酸。在神经组织，精氨酸也合成γ-胍基丁酸(Delforge J等,Eur J Biochem.1975,57(1):231-239；Fernandez ML等,J.Bacteriol.2004,186(18):6142-6149；Fernandez ML等,J.Bacteriol.2008,190(18):3018-3025)。
精氨酸是形成肌酸的三种底物之一，肌酸是一种重要的营养素(缺乏会导致智力迟钝)，也被用来形成腹水，也是一种体内的信号分子。精氨酸是尿素(含L-鸟氨酸、L-瓜氨酸和精氨琥珀酸)和一氧化氮循环(含鸟氨酸和精氨琥珀酸)中的一种中间产物，并通过鸟氨酸产生可调节细胞功能的多胺结构。精氨酸的缺乏(可通过增加精氨酸酶的活性来诱导，这种酶可将精氨酸转化为鸟氨酸)会损害B细胞功能(免疫性)并损害毛发和肌肉生长也可能损害神经肌肉功能。已知精氨酸酶在II型糖尿病患者中过度表达，是该人群队列中心血管疾病发展的危险因素，肾衰竭患者可能会降低精氨酸酶。
正是由于精氨酸具有上述重要的作用，因此精氨酸含量的检测也尤为重要。精氨酸的常用检测方法是毛细管电泳法(Li X-t等，Chem Res Chin Univ 2013,29(3):434-438；Meng J等，The Analyst 2010,135(7):1592-1599)、高效液相色谱法(Tateda N等，Analytical sciences:the international journal of the Japan Society forAnalytical Chemistry 2001,17(6):775-778；Wadud S等，Journal of chromatographyB,Analytical technologies in the biomedical and life sciences 2002,767(2):369-374)、酶联免疫吸附试验以及紫外可见光分光光度法(Hortpro MA等，J Am Chem So2003,125(1):20-21；Pu F等,Anal Chem 2010,82(19):8211-8216；Du J等，Chemicalcommunications(Cambridge,England)2013,49(47):5399-5401；Engeser M等，ChemicalCommunications 1999,(13):1191-1192)和荧光光谱测定法(Engeser M等，ChemicalCommunications 1999,(13):1191-1192)。
但是在活细胞研究中这些检测方法存在很大的缺陷，需要经过耗时的样品处理过程：细胞破碎、分离提取纯化等，不能在活细胞和亚细胞器中进行原位、实时、动态、高通量和高时空分辨率的检测。本领域仍需要能在细胞内、外实时原位、定量、高通量检测精氨酸的方法。
发明内容
有鉴于此，本发明的目的在于提供一种在细胞内外实时定位、高通量、定量检测精氨酸的精氨酸荧光探针。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供一种精氨酸光学探针，包含精氨酸敏感多肽或其功能变体和光学活性多肽或其功能变体，其中光学活性多肽或其功能变体位于精氨酸敏感多肽或其功能变体的序列内。精氨酸敏感多肽或其功能变体被光学活性多肽或其功能变体分为第一部分和第二部分。
本发明提供了一种精氨酸光学探针，包括精氨酸敏感多肽B和光学活性多肽A，其中光学活性多肽A位于精氨酸敏感多肽B的序列内，将精氨酸敏感多肽B分为第一部分B1和第二部分B2，形成B1-A-B2式的探针结构。
在一个实施方案中，精氨酸敏感多肽包括精氨酸结合蛋白的精氨酸结合域及其突变体。在一个实施方案中，精氨酸敏感多肽是精氨酸结合蛋白或其功能片段。在一个或多个实施方案中，所述精氨酸结合蛋白是STM4351蛋白。在一个实施方案中，精氨酸敏感多肽具有SEQ ID NO:1所示的序列，或与其有至少35％、至少40％、至少50％、至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少97％、至少99％序列相同性并保留对精氨酸敏感性的序列。
在一个实施方案中，光学活性多肽是荧光蛋白或其功能片段或变体。在一个实施方案中，荧光蛋白选自黄色荧光蛋白(如SEQ ID NO:2所示的cpYFP)、橘黄色荧光蛋白、红色荧光蛋白、绿色荧光蛋白(如SEQ ID NO:3所示的cpGFP)、蓝色荧光蛋白(如SEQ ID NO:4所示的cpBFP)、苹果红荧光蛋白(如SEQ ID NO:5所示的cpmApple)。优选地，光学活性多肽是cpYFP。在一个实施方案中，荧光蛋白具有SEQ ID NO:2-5中任一所示的序列。
在一个实施方案中，光学探针还包含侧接所述光学活性多肽的一个或多个接头。在一个实施方案中，光学活性多肽侧翼包含不超过5个氨基酸的接头，例如0、1、2、3、4个氨基酸的接头。在一个实施方案中，光学活性多肽侧翼的接头包含氨基酸Y。在一个实施方案中，接头Y位于光学活性多肽的N端和/或C端。在一个实施方案中，光学探针如下所示：精氨酸敏感多肽的第一部分B1-Y-光学活性多肽A-精氨酸敏感多肽的第二部分B2。在一个实施方案中，本发明光学探针不包含接头。
在一个实施方案中，光学活性多肽位于精氨酸敏感多肽的残基103-111和/或197-209之间，编号对应于精氨酸结合蛋白的全长。在一个实施方案中，光学活性多肽置换精氨酸敏感多肽的残基102-112和/或196-210之间的一个或多个氨基酸，编号对应于精氨酸结合蛋白的全长。
在一个实施方案中，光学活性多肽位于选自以下的精氨酸敏感多肽的一个或多个位点：103/104，103/105，103/106，103/107，103/108，103/109，103/110，103/111，104/105，104/106，104/107，104/108，104/109，104/110，104/111，105/106，105/107，105/108，105/109，105/110，105/111，106/107，106/108，106/109，106/110，106/111，107/108，107/109，107/110，107/111，108/109，108/110，108/111，109/110，109/111，110/111，197/198，197/199，197/200，197/201，197/202，197/203，197/204，197/205，197/206，197/207，197/208，197/209，198/199，198/200，198/201，198/202，198/203，198/204，198/205，198/206，198/207，198/208，198/209，199/200，199/201，199/202，199/203，199/204，199/205，199/206，199/207，199/208，199/209，200/201，200/202，200/203，200/204，200/205，200/206，200/207，200/208，200/209，201/202，201/203，201/204，201/205，201/206，201/207，201/208，201/209，202/203，202/204，202/205，202/206，202/207，202/208，202/209，203/204，203/205，203/206，203/207，203/208，203/209，204/205，204/206，204/207，204/208，204/209，205/206，205/207，205/208，205/209，206/207，206/208，206/209，207/208，207/209或208/209。
优选地，光学活性多肽位于选自以下的精氨酸敏感多肽的一个或多个位点：103/104，103/106，103/107，103/108，103/109，103/110，104/105，104/106，104/108，104/109，104/110，104/111，105/106，105/107，105/108，105/109，105/110，105/111，106/107，106/108，106/109，107/108，107/109，107/110，107/111，108/109，108/111，109/110，109/111，110/111，197/198，197/199，197/200，197/201，197/202，197/203，197/204，197/205，197/206，197/208，198/201，198/202，198/203，198/204，198/205，198/206，198/208，198/209，199/200，199/201，199/202，199/203，199/204，199/205，199/206，199/208，200/203，200/204，200/205，200/206，200/207，200/208，201/202，201/203，201/204，201/205，201/206，201/208，201/209，202/203，202/205，202/206，203/204，203/206，204/205，204/206，204/208，205/206，205/207，205/208或206/207。这些探针对精氨酸的响应超过对照。
优选地，光学活性多肽位于选自以下的精氨酸敏感多肽的一个或多个位点：104/109，104/110，104/111，105/106，105/107，105/110，106/108，106/109，107/109，107/111，110/111，197/199，197/203，197/204，198/202，198/203，198/204，199/200，199/201，199/202，199/204，199/205，200/203，200/204，200/205，200/206，201/202，201/203，201/204，201/205，201/206，202/205，203/206，204/205或204/206。这些探针对精氨酸的响应超过对照的1.5倍。
优选地，光学活性多肽位于选自以下的精氨酸敏感多肽的一个或多个位点：104/110，104/111，105/106，105/110，106/109，107/109，107/111，197/203，197/204，198/202，199/200，199/202，200/203，200/205，201/202，201/205，202/205，203/206，204/205或204/206。这些探针对精氨酸的响应超过对照的2倍。
在示例性实施方案中，本发明光学探针可为当cpYFP位于精氨酸结合蛋白的104/110，104/111，105/106，105/110，106/109，107/109，107/111，197/203，197/204，198/202，199/200，199/202，200/203，200/205，201/202，201/205，202/205，203/206，204/205或204/206处的探针。在一个实施方案中，本发明光学探针具有SEQ ID NO:6-39所示序列或由其组成。
本发明还提供具有一个或多个突变的精氨酸敏感多肽的突变体。所述氨基酸突变包括氨基酸的修饰、取代、缺失或序列的截短。在一个实施方案中，所述突变位于精氨酸结合蛋白的第30(S)，96(R)和第177(D)位中的1个、2个或3个。优选地，所述突变选自S30N，D177N，R96M，R96K中的1个、2个或3个。
本发明还提供包含具有一个或多个突变的精氨酸敏感多肽的光学探针。在一个或多个实施方案中，光学探针是如上所述的插入有光学活性多肽的任意光学探针，并且所述光学探针中的精氨酸敏感多肽在选自S30，R96和D177中的1个、2个或3个位点具有突变。在一个或多个实施方案中，包含突变的精氨酸敏感多肽的光学探针对精氨酸的响应不低于未突变的对应物。优选地，所述突变选自S30N，D177N，R96M，R96K中的1个、2个或3个。
在示例性实施方案中，本发明光学探针可为精氨酸结合蛋白的107/111位点插入有cpYFP并且具有选自S30N，D177N，R96M和R96K的一个或多个突变的探针。优选地，本发明光学探针为精氨酸结合蛋白的107/111位点插入有cpYFP并且具有选自S30N或D177N的探针。在一个实施方案中，本发明光学探针具有SEQ ID NO:40-41所示序列或由其组成。
本发明提供的光学探针包含氨基酸序列SEQ ID NO:6-41中任一或其变体。在一个实施方案中，本发明提供的光学探针包含与氨基酸序列SEQ ID NO:6-41中任一有至少35％、至少40％、至少50％、至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少99％序列相同性的序列。
本发明还提供融合多肽，包含本文所述光学探针和其它多肽。在一些实施方案中，其他多肽位于所述光学探针的N端和/或C端。在一些实施方案中，其他多肽包括将光学探针定位到不同细胞器或亚细胞器的多肽、用于纯化的标签或者用于免疫印迹的标签。
本发明还提供核酸序列，其包含本文所述多肽、探针或蛋白的编码序列或其互补序列或片段。在一个实施方案中，本发明的核酸序列选自(1)SEQ ID NO:6-41中任一所示氨基酸序列的编码序列或其互补序列，(2)与(1)具有至少99％、95％、90％、80％、70％或50％相同性的序列，(3)(1)或(2)的片段。在一个实施方案中，本发明核酸序列包含SEQ ID NO:42或其变体或片段。在一个或多个实施方案中，所述片段是引物。
本发明还涉及上述核酸序列的互补序列或其变体，包括编码本发明光学探针或融合蛋白的片段、类似物、衍生物、可溶性片段和变体的核酸序列或其互补序列。
本发明还提供包含本文所述核酸序列或其互补序列的核酸构建物，该核酸序列编码本发明所述光学探针或融合多肽。在一个或多个实施方案中，所述核酸构建物是克隆载体、表达载体或重组载体。在一个或多个实施方案中，所述核酸序列与表达控制序列操作性连接。在一些实施方案中，表达载体选自原核表达载体、真核表达载体和病毒载体。
本发明还提供包含本发明所述核酸序列或表达载体的细胞。在一个或多个实施方案中，所述细胞表达本文所述光学探针或融合多肽。
本发明还提供包括本文所述光学探针或融合多肽或多核苷酸或如本文所述方法制备的光学探针或融合多肽的检测试剂盒。
本发明提供制备本文所述光学探针的方法，包括：提供表达本文所述光学探针或融合多肽的细胞，在所述细胞表达的条件下培养所述细胞，和分离光学探针或融合多肽。
本发明还提供检测样品中精氨酸的方法，包括：使本文所述光学探针或融合多肽或如本文所述方法制备的光学探针或融合多肽与样品接触，和检测光学活性多肽的变化。所述检测可以在体内、体外、亚细胞或原位进行。所述样品例如血液。
本文还提供定量样品中精氨酸的方法，包括：使本文所述光学探针或融合多肽或如本文所述方法制备的光学探针或融合多肽与样品接触，检测光学活性多肽的变化，和根据光学活性多肽的变化定量样品中的精氨酸。
本发明还提供筛选化合物(例如药物)的方法，包括：使本文所述光学探针或融合多肽或如本文所述方法制备的光学探针或融合多肽与候选化合物接触，检测光学活性多肽的变化，和根据光学活性多肽的变化筛选化合物。所述方法可以高通量地筛选化合物。
本发明还提供本文所述精氨酸光学探针或融合多肽或如本文所述方法制备的精氨酸光学探针或融合多肽在精氨酸细胞内/外定位中的应用。在一个或多个实施方案中，所述定位是实时定位。
本发明的有益效果：本发明提供的精氨酸荧光探针，包括对精氨酸敏感的多肽B和荧光蛋白A；所述的荧光蛋白A插入到多肽B中，将B分为多肽B1和多肽B2两个部分，形成B1-A-B2式的探针结构；本发明提供的B1-A-B2式精氨酸荧光探针，易于成熟，荧光动态变化大，特异性好，并且能够通过基因操作的方法在细胞中表达，可在细胞内外实时定位、高通量、定量检测精氨酸。省去了耗时的处理样品步骤。实验效果表明本申请所提供的的精氨酸荧光探针对精氨酸的最高响应达到11倍以上，可以在细胞浆、线粒体、细胞核、高尔基体、过氧化物酶体和溶酶体等亚细胞结构中对细胞进行定位检测，并且可以进行高通量的化合物筛选与血液中精氨酸定量检测。
本发明还提供上述精氨酸光学探针的制备方法，包括以下步骤：1)将编码本文所述精氨酸光学探针的核酸序列纳入表达载体；2)将表达载体转移到宿主细胞中；2)在适合所述表达载体表达的条件下培养所述宿主细胞，3)分离精氨酸光学探针。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1为实施例2中精氨酸荧光探针的SDS-PAGE分析图。
图2为黄色荧光蛋白cpYFP在精氨酸结合蛋白不同插入位点形成的精氨酸荧光探针对精氨酸响应变化图。
图3为绿色荧光蛋白cpGFP在精氨酸结合蛋白不同插入位点形成的精氨酸荧光探针对精氨酸响应变化图。
图4为蓝色荧光蛋白cpBFP在精氨酸结合蛋白不同插入位点形成的精氨酸荧光探针对精氨酸响应变化图。
图5为苹果红荧光蛋白cpmApple在精氨酸结合蛋白不同插入位点形成的精氨酸荧光探针对精氨酸响应变化图。
图6为黄色荧光蛋白cpYFP在精氨酸结合蛋白107/111插入位点形成的精氨酸荧光探针基础上进行S30，R96，D177位点突变对精氨酸响应变化图。
图7A-B为不同插入位点104/110，105/106，105/110，106/108，106/109，107/109，197/199，197/203，197/204，198/201，198/202，198/203，199/200，199/201，199/202，199/205，200/202，200/203，200/204，200/205，200/206，201/202，201/203，201/205，202/205，203/206，204/205，204/206的精氨酸荧光探针对不同浓度精氨酸的滴定曲线。
图8为不同插入位点的104/110，107/109，107/111，197/203，199/200，199/201，199/202，200/202，200/203，200/204，200/205，201/202精氨酸荧光探针对20种氨基酸的特异性检测。
图9A-B为示例性精氨酸荧光探针的荧光光谱性质图。
图10为示例性精氨酸荧光探针在哺乳动物细胞中亚细胞器定位分析图。
图11为精氨酸荧光探针在哺乳动物细胞中对精氨酸跨膜运输进行动态监测。
图12为实施例11所述的在活细胞水平基于精氨酸荧光探针-进行高通量化合物筛选分析图。
图13为实施例12所述的精氨酸荧光探针对小鼠和人血液中的精氨酸定量分析图。
具体实施方案
在给出数值或范围时，本文所用术语“约”指该数值或范围在给定数值或范围的20％以内、10％以内和5％以内。
本文所用术语“包含”、“包括”和其等同形式包括“含有”以及“由……组成”的含义，例如“包含”X的组合物可仅由X组成或可含有其它物质，例如X+Y。
本文所用术语“精氨酸敏感多肽”或“精氨酸响应多肽”指对精氨酸产生响应的多肽，所述响应包括与敏感多肽的相互作用相关的多肽的化学，生物学，电学或生理学参数的任何响应。响应包括小的变化，例如，多肽的氨基酸或肽片段的方向的变化以及例如多肽的一级，二级或三级结构的变化，包括例如质子化，电化学势和/或构象的变化。“构象”是分子中包含侧基的分子的一级，二级和三级结构的三维排列；当分子的三维结构发生变化时，构象发生变化。构象变化的实例包括从α-螺旋转变为β-折叠或从β-折叠转变为α-螺旋。可以理解的是，只要荧光蛋白部分的荧光被改变，可检测到的改变不需要是构象改变。本文所述精氨酸敏感多肽还可包括其功能变体。精氨酸敏感多肽的功能变体包括但不限于可以与精氨酸相互作用从而发生与亲本精氨酸敏感多肽相同或相似变化的变体。
本发明所述精氨酸敏感多肽包括但不限于来源于鼠伤寒沙门氏菌(SalmonellaTyphimurium)的精氨酸结合蛋白STM4351或来源于大肠杆菌(Escherichia coli)的精氨酸周质结合蛋白或与其有90％以上同源性的变体。这些结合蛋白由两个结构域组成，两个结构域之间由两个柔性氨基酸肽链进行连接。精氨酸结合蛋白可以感应精氨酸浓度的变化，在精氨酸浓度动态变化的过程中精氨酸结合蛋白的空间构象也会发生很大改变。STM4351由精氨酸结合/调节域和DNA结合域组成。示例性STM4351蛋白如SEQ ID NO:1所示。在一个或多个实施方案中，精氨酸敏感多肽包含STM4351蛋白的精氨酸结合域，而不包括DNA结合域。
本文所用术语“光学探针”是指与光学活性多肽融合的精氨酸敏感多肽。发明人发现，精氨酸敏感多肽例如精氨酸结合蛋白专一性地对生理浓度的精氨酸结合后所产生的构象变化会引起光学活性多肽(例如荧光蛋白)的构象变化，进而导致光学活性多肽的光学性质发生改变。借助不同精氨酸浓度下测定的荧光蛋白的荧光绘制标准曲线，可以检测并分析精氨酸的存在和/或水平。当描述本发明光学探针时(例如描述插入位点或突变位点时)，提及氨基酸残基编号均参考SEQ ID NO:1。
在本发明的光学探针中，光学活性多肽(例如荧光蛋白)可操作地插入精氨酸敏感多肽中。基于蛋白质的“光学活性多肽”是具有发射荧光能力的多肽。荧光是光学活性多肽的一种光学性质，其可用作检测本发明的光学探针的响应性的手段。优选地，选择蛋白质底物以具有在未激活和活化的构象状态下容易区分的荧光特性。本文所述光学活性多肽还可为其功能变体。光学活性多肽的功能变体包括但不限于可以发生与亲本光学活性多肽相同或相似荧光性质变化的变体。
本文所用术语“荧光蛋白”指在激发光照射下发出荧光的蛋白质。荧光蛋白作为生物科学领域的基础检测手段，例如生物技术领域常用的绿色荧光蛋白GFP及由该蛋白突变衍生出的环状重排的蓝色荧光蛋白(cpBFP)、环状重排的绿色荧光蛋白(cpGFP)、环状重排的黄色荧光蛋白(cpYFP)等；还有本技术领域常用的红色荧光蛋白RFP，及由该蛋白衍生出来的环状重排的蛋白，如cpmApple，cpmOrange，cpmKate等。本领域知晓可用于本发明的荧光蛋白及其序列。示例性地，cpYFP如SEQ ID NO:2所示；cpGFP如SEQ ID NO:3所示；cpBFP如SEQ ID NO:4所示；cpmApple如SEQ ID NO:5所示。
“接头”或“连接区”指在本发明多肽、蛋白质或核酸中连接两个部分的氨基酸或核苷酸序列。示例性地，本发明中精氨酸敏感多肽与光学活性多肽的连接区氨基端的氨基酸数目选择的是0-3个，羧基端的氨基酸数目选择的是0-2个；当重组光学探针作为基本单元与功能蛋白连接时，可以融合在重组光学探针的氨基酸或羧基端。接头序列可为一个或多个柔性氨基酸组成的短肽链，如Y。
本发明所述的精氨酸光学探针包括精氨酸敏感多肽B，例如精氨酸结合蛋白或其变体，和光学活性多肽A，例如荧光蛋白。光学活性多肽A插入到精氨酸敏感多肽B中，将B分为B1和B2两个部分，形成B1-A-B2式的探针结构；精氨酸敏感多肽B和精氨酸相互作用导致光学活性多肽A的光学信号变强。
在本发明的光学探针中，光学活性多肽可以位于精氨酸敏感多肽的任何位置。在一个实施方案中，光学活性多肽以N-C方向位于N-C方向的精氨酸敏感多肽的任何位置。具体地，光学活性多肽位于精氨酸敏感多肽的柔性区域，所述的柔性区域是指蛋白质高级结构中存在的一些特定的如环状结构域等结构，这些结构域相比于蛋白质的其他高级结构具有更高的移动性和柔性，并且该区域可以在该蛋白质和配体结合后，空间结构构象发生动态变化。本发明中所述的柔性区域主要指精氨酸结合蛋白中的插入位点所在区域，如氨基酸残基102-112和196-210区域。在一个实施方案中，光学活性多肽位于精氨酸敏感多肽的选自以下的一个或多个位点：103/104，103/105，103/106，103/107，103/108，103/109，103/110，103/111，104/105，104/106，104/107，104/108，104/109，104/110，104/111，105/106，105/107，105/108，105/109，105/110，105/111，106/107，106/108，106/109，106/110，106/111，107/108，107/109，107/110，107/111，108/109，108/110，108/111，109/110，109/111，110/111，197/198，197/199，197/200，197/201，197/202，197/203，197/204，197/205，197/206，197/207，197/208，197/209，198/199，198/200，198/201，198/202，198/203，198/204，198/205，198/206，198/207，198/208，198/209，199/200，199/201，199/202，199/203，199/204，199/205，199/206，199/207，199/208，199/209，200/201，200/202，200/203，200/204，200/205，200/206，200/207，200/208，200/209，201/202，201/203，201/204，201/205，201/206，201/207，201/208，201/209，202/203，202/204，202/205，202/206，202/207，202/208，202/209，203/204，203/205，203/206，203/207，203/208，203/209，204/205，204/206，204/207，204/208，204/209，205/206，205/207，205/208，205/209，206/207，206/208，206/209，207/208，207/209或208/209。本文中，如果以“X/Y”形式表示的位点中的两个数字是连续的整数，则表示光学活性多肽位于该数字所述的氨基酸之间。例如插入位点93/94表示光学活性多肽位于精氨酸敏感多肽的氨基酸93与94之间。如果以“X/Y”形式表示的位点中的两个数字不是连续的整数，则表示光学活性多肽置换该数字所示氨基酸之间的氨基酸。例如插入位点93/97表示光学活性多肽置换精氨酸敏感多肽的氨基酸94-96。优选地，光学活性多肽位于选自以下的精氨酸敏感多肽的一个或多个位点：104/110，104/111，105/106，105/110，106/109，107/109，107/111，197/203，197/204，198/202，199/200，199/202，200/203，200/205，201/202，201/205，202/205，203/206，204/205或204/206。
提到某多肽或蛋白时，本发明所用术语“变体”或“突变体”包括具有所述多肽或蛋白相同功能、但序列不同的变体。多肽或蛋白的变体可包括：同源序列、保守性变体、等位变体、天然突变体、诱导突变体。这些变体包括但并不限于：在所述多肽或蛋白的序列中缺失、插入和/或取代一个或多个(通常为1-30个，较佳地1-20个，更佳地1-10个，最佳地1-5个)氨基酸，以及在其羧基末端和/或氨基末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸获得的序列。这些变体还可包含与所述多肽或蛋白的序列相同性为至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约98％、至少约99％或100％的多肽或蛋白。不希望受理论限制，氨基酸残基发生改变而不改变多肽或蛋白质的总体构型和功能，即功能保守突变。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变多肽或蛋白的功能。在本领域中，性能相似的氨基酸往往指具有相似侧链的氨基酸家族，在本领域已有明确定义。这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、精氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、具有β-分支侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)和具有芳香侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。又比如，在氨基末端和/或羧基末端添加一个或数个氨基酸通常也不会改变多肽或蛋白的功能。对于许多常见已知非遗传性编码氨基酸的保守氨基酸取代本领域已知。其他非编码氨基酸的保守取代可基于其物理性质与遗传上编码的氨基酸的性质的比较来确定。
在两种或多种多肽或核酸分子序列中，术语“相同性”或“相同性百分数”指在比较窗口或指定区域上，采用本领域已知方法如序列比较算法，通过手工比对和目测检查来比较和比对最大对应性时，两个或多个序列或子序列相同或其中在指定区域有一定百分数的氨基酸残基或核苷酸相同(例如，60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％相同)。例如，适合测定序列相同性百分数和序列相似性百分数的优选算法是BLAST和BLAST 2.0算法，分别可参见Altschul等(1977)Nucleic Acids Res.25:3389和Altschul等(1990)J.Mol.Biol.215:403。
本领域技术人员公知，在基因克隆操作中，常常需要设计合适的酶切位点，这势必在所表达的多肽或蛋白末端引入了一个或多个不相干的残基，而这并不影响目的多肽或蛋白的活性。又如为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化，常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内，例如，包括但不限于，适合的接头肽、信号肽、前导肽、末端延伸、谷胱甘肽S-转移酶(GST)、麦芽糖E结合蛋白、蛋白A、如6His或Flag的标签，或Xa因子或凝血酶或肠激酶的蛋白水解酶位点。
本发明光学探针可包含具有突变的精氨酸敏感多肽。在一个实施方案中，所述突变位于精氨酸结合蛋白的第30(S)，96(R)和第177(D)位。示例性地，所述突变为S30N，D177N，R96M，R96K。本发明光学探针中的精氨酸敏感多肽可为所述突变体。在示例性实施方案中，本发明光学探针可为精氨酸结合蛋白的107/111位点插入有cpYFP并且具有选自S30N，D177N，R96M和R96K的一个或多个突变的探针。
本文所用术语“功能片段”、“衍生物”和“类似物”是指基本上保持与原始多肽或蛋白(例如精氨酸结合蛋白或荧光蛋白)相同的生物学功能或活性的蛋白。本发明的多肽或蛋白(例如精氨酸结合蛋白或荧光蛋白)的功能变体、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的蛋白，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的蛋白，或(iii)成熟蛋白与另一个化合物(比如延长蛋白半衰期的化合物，例如聚乙二醇)融合所形成的蛋白，或(iv)附加的氨基酸序列融合到此蛋白序列而形成的蛋白(如分泌序列或用来纯化此蛋白的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些功能变体、衍生物和类似物属于本领域熟练技术人员公知的范围。
所述类似物与原始多肽或蛋白的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些蛋白包括天然或诱导的遗传变体。诱导变体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术得到。
所述类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的精氨酸敏感多肽并不限于上述列举的代表性蛋白、变体、衍生物和类似物。修饰(通常不改变一级结构)形式包括：体内或体外的蛋白的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在蛋白的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的蛋白。这种修饰可以通过将蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的蛋白。
本发明融合多肽包含本文所述光学探针和其它多肽。在一些实施方案中，本文所述光学探针还包含与之融合的其它多肽。本文所述其他多肽不影响光学探针的性质。其他多肽可位于所述光学探针的N端和/或C端。在一些实施方案中，其他多肽包括将光学探针定位到不同细胞器或亚细胞器的多肽、用于纯化的标签或者用于免疫印迹的标签。本文所述融合多肽中的光学探针和其它多肽之间可具有接头。
本文所述亚细胞器包括细胞浆、线粒体、细胞核、内质网、细胞膜、高尔基体、溶酶体和过氧化物酶体等。在一些实施方案中，用于纯化的标签或者用于免疫印迹的标签包括6组氨酸(6*His)、谷胱甘肽硫转移酶(GST)、Flag。
本发明表达载体包含与表达控制序列操作性连接的本发明所述的核酸序列或其互补序列，该核酸序列编码本发明所述光学探针或融合多肽。本发明所用术语“核酸”或“核苷酸”或“多核苷酸”或“核酸序列”可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。提到核酸时，本文所用术语“变体”可以是天然发生的等位变体或非天然发生的变体。这些核苷酸变体包括简并变体、取代变体、缺失变体和插入变体。如本领域所知的，等位变体是一个核酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的蛋白的功能。本发明核酸可包含与所述核酸序列的序列相同性为至少约50％、至少约60％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约98％、至少约99％或100％的核苷酸序列。本发明还涉及与上述的序列杂交的核酸片段。如本文所用，“核酸片段”的长度至少含15个核苷酸，较好是至少30个核苷酸，更好是至少50个核苷酸，最好是至少100个核苷酸以上。核酸片段可用于核酸的扩增技术(如PCR)。
本发明光学探针或融合蛋白的全长序列或其片段通常可以用PCR扩增法、人工合成法或重组法获得。对于PCR扩增法，可根据本发明所公开的核苷酸序列设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当核苷酸序列大于2500bp时，优选的进行2～6次PCR扩增，然后将各次扩增的片段按正确次序拼接在一起。本发明对所述的PCR扩增的程序和体系没有特殊限定，采用本领域常规的PCR扩增程序和体系即可。还可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离和纯化得到有关多肽或蛋白。此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。在本发明中，在光学探针的核苷酸序列小于2500bp时，可采用人工合成方法来合成。所述人工合成方法为本领域常规的DNA的人工合成方法，无其他特殊要求。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其功能变体、衍生物或类似物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(如载体)和细胞中。可通过突变PCR或化学合成等方法将突变引入本发明蛋白序列中。
本发明也涉及核酸构建物，该核酸构建物含有本文所述的多核苷酸，以及与这些序列操作性连接的一个或多个调控序列。本发明所述的多核苷酸可以多种方式被操作以保证所述多肽或蛋白的表达。在将核酸构建物插入载体之前可根据表达载体的不同或要求而对核酸构建物进行操作。利用重组DNA方法来改变多核苷酸序列的技术是本领域已知的。
在某些实施方案中，所述核酸构建物是载体。载体可以是克隆载体，也可以是表达载体，或者是同源重组载体。本发明的多核苷酸可被克隆入许多类型的载体，例如，质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。克隆载体可用于提供本发明蛋白或多肽的编码序列。表达载体可以以细菌载体或病毒载体形式提供给细胞。通常通过可操作地连接本发明的多核苷酸至启动子，并将构建体并入表达载体，实现本发明多核苷酸的表达。该载体对于复制和整合真核细胞可为合适的。典型的表达载体包含可用于调节期望核酸序列表达的表达控制序列。在一个或多个实施方案中，克隆载体和表达载体是一种载体，即克隆表达载体。同源重组载体用于将本文所述的表达框整合到宿主基因组中。
本文所用术语“表达控制序列”指调控目的基因的转录、翻译和表达的可以与目的基因操作性连接的元件，可以是复制起点、启动子、标记基因或翻译控制元件，包括增强子、操纵子、终止子、核糖体结合位点等，表达控制序列的选择取决于所用的宿主细胞。在重组表达载体中，“操作性连接”是指目的的核苷酸序列与调节序列以允许核苷酸序列表达的方式连接。本领域的技术人员熟知能用于构建含本发明融合蛋白编码序列和合适的转录/翻译控制信号的表达载体的方法。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTR和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。在一个实施方案中，表达载体可采用市售的pET28a载体，无其他特殊要求。示例性地，采用BamHI和EcoRI分别对编码所述光学探针的核苷酸序列和表达载体进行双酶切，然后将二者的酶切产物连接得到重组表达载体。本发明对酶切和连接的具体步骤和参数没有特殊限定，采用本领域常规的步骤和参数即可。
在获得重组表达载体后，将该载体转化到宿主细胞中，以产生包括融合蛋白的蛋白或肽。此种转移过程可用转化或转染等本领域技术人员熟知的常规技术进行。本发明所述的宿主细胞是指能够接收和容纳重组DNA分子的细胞，是重组基因扩增的场所，理想的受体细胞应该满足易于获取和增殖两个条件。本发明的“宿主细胞”可包括原核细胞和真核细胞，具体包括细菌细胞、酵母细胞、昆虫细胞和哺乳动物细胞。具体的可为大肠杆菌，链霉菌属，鼠伤寒沙门氏菌的细菌细胞，真菌细胞如酵母，植物细胞，果蝇S2或Sf9的昆虫细胞，CHO、COS、HEK293、HeLa细胞、或Bowes黑素瘤细胞的动物细胞等，其中包括但不限于上述的那些宿主细胞。所述宿主细胞优选各种利于基因产物表达或发酵生产的细胞，此类细胞已为本领域熟知并常用。在本发明实施例中所用的示例性宿主细胞为大肠杆菌JM109-DE3菌株。本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
本发明所述的转移到宿主细胞的方法为本领域常规的方法，包括磷酸钙或氯化钙共沉淀、DEAE-甘露聚糖-介导的转染、脂转染、天然感受态、化学介导的转移或电穿孔。当宿主为原核生物如大肠杆菌时，所述方法优选的为CaCl2法或MgCl2法处理，所用的步骤为本领域公知。当宿主细胞是真核细胞时，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
本发明在将表达载体转入宿主细胞后，对转入表达载体的宿主细胞进行扩增表达培养，分离得到精氨酸光学探针。所述宿主细胞扩增表达培养采用常规的方法即可。根据所用的宿主细胞种类，培养中所用的培养基可以是各种常规培养基。在适于宿主细胞生长的条件下进行培养。
在本发明中，光学探针在细胞内、细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离或纯化重组的蛋白。本发明对分离所述精氨酸荧光蛋白的方法没有特殊限定，采用本领域常规的融合蛋白的分离方法即可。这些方法是本领域技术人员所熟知的，包括但并不限于：常规的复性处理、盐析方法、离心、渗透破菌、超声处理、超离心、分子筛层析、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。在一个实施方案中，利用His标签的亲和层析法进行光学探针的分离。
本发明还提供了所述精氨酸光学探针在精氨酸实时定位、定量检测以及高通量化合物筛选中的应用。在一个方面，所述的精氨酸光学探针优选与细胞不同部位的信号肽连接，转入到细胞中，通过检测细胞中荧光信号的强弱，进行精氨酸的实时定位；通过精氨酸标准滴加曲线进行相应精氨酸的定量检测。本发明所述的精氨酸标准滴加曲线是根据精氨酸光学探针在不同浓度精氨酸的情况下的荧光信号绘制而成。本发明所述精氨酸光学探针直接转入细胞中，在精氨酸实时定位和定量检测过程中，不需要耗时的样品处理过程，更加准确。本发明精氨酸光学探针在进行高通量化合物筛选时，将不同的化合物添加到细胞培养液中，测定精氨酸含量的变化，从而筛选出对精氨酸含量变化有影响的化合物。在本发明中所述的精氨酸光学探针在精氨酸实时定位、定量检测以及高通量化合物筛选中的应用，均是非诊断和治疗目的，不涉及疾病的诊断和治疗。
在本文中，浓度、含量、百分数和其它数值均可用范围的形式表示。也应理解，使用这种范围形式只是为了方便和简洁，应该被弹性地解读为包括范围上下限所明确提及的数值，还应包括该范围内包括的所有单个数值或子范围。
实施例
下面结合实施例对本发明提供的精氨酸荧光探针进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
I.实验材料和试剂
实施例中主要采用常规的基因工程分子生物学克隆方法和细胞培养以及成像方法等，这些方法是本领域普通技术人员所熟知的，例如：简·罗斯凯姆斯等的《分子生物学实验参考手册》，J.萨姆布鲁克，D.W.拉塞尔著,黄培堂等译：《分子克隆实验指南》(第三版，2002年8月，科学出版社出版，北京)；费雷谢尼等的《动物细胞培养:基本技术指南》(第五版)，章静波，徐存拴等译；J.S.博尼费斯农，M.达索等的《精编细胞生物学实验指南》，章静波等译。
实施例中所用的基于pET28a-cpYFP，pET28a-精氨酸结合蛋白质粒由华东理工大学蛋白质实验室构建，pET28a质粒载体购自Invitrogen公司。所有用于PCR的引物均由上海捷瑞生物工程技术有限公司合成、纯化和经质谱法鉴定正确。实施例中构建的表达质粒都经过序列测定，序列测定由华大基因公司和杰李测序公司完成。各实施例所用的Taq DNA聚合酶购自东盛生物，pfu DNA聚合酶购自天根生化科技(北京)有限公司，primeSTAR DNA聚合酶购自TaKaRa公司，三种聚合酶购买时都附带赠送对应聚合酶缓冲液和dNTP。BamHI、BglII、HindIII、NdeI、XhoI、EcoRI、SpeI等限制性内切酶、T4连接酶、T4磷酸化酶(T4 PNK)购自Fermentas公司，购买时附带有相对应的缓冲液等。转染试剂Lip2000 Kit购于Invitrogen公司。精氨酸等均购自Sigma公司。除非特别声明，无机盐类等化学试剂均购自sigma-aldrich公司。HEPES盐，氨苄青霉素(Amp)和嘌呤霉素购自Ameresco公司；96孔检测黑板、384孔荧光检测黑板购自Grenier公司。
实施例中所用的DNA纯化试剂盒购自BBI公司(加拿大)，普通质粒小抽试剂盒购自天根生化科技(北京)有限公司。克隆菌株Mach1购自Invitrogen公司。镍柱亲和层析柱和脱盐柱填料均来自GE healthcare公司。
实施例中用到的主要仪器：Biotek Synergy 2多功能酶标仪(美国Bio-Tek公司)，X-15R高速冷冻离心机(美国Beckman公司)，Microfuge22R台式高速冷冻离心机(美国Beckman公司)，PCR扩增仪(德国Biometra公司)，超声破碎仪(宁波新芝公司)，核酸电泳仪(申能博彩公司)，荧光分光光度计(美国Varian公司)，CO2恒温细胞培养箱(SANYO)，倒置荧光显微镜(日本尼康公司)。
II.分子生物学方法和细胞实验方法
II.1聚合酶链式反应(PCR)：
1.目的片段扩增PCR：
该方法主要用于基因片段扩增和菌落PCR鉴定阳性克隆。所述PCR扩增的反应体系如下：模板序列0.5-1μL，正向引物(25μM)0.5μL，反向引物(25μM)0.5μL，10×pfu缓冲液5μL，pfu DNA聚合酶0.5μL，dNTP(10mM)1μL，灭菌超纯水(ddH2O)41.5-42μL，总体积50μL。PCR扩增程序如下：95℃变性2-10分钟，30轮循环(94-96℃持续30-45秒，50-65℃持续30-45秒，72℃持续一定时间(600bp/min))，72℃延伸10分钟。
2.长片段(>2500bp)扩增PCR：
本发明中使用的长片段扩增，主要是反向PCR扩增载体，在下述实施例中用于获得定点突变的一种技术。在变异部位设计反向PCR引物，其中一条引物的5’端包含变异的核苷酸序列。扩增后的产物就含有相应的突变位点。长片段扩增PCR反应体系如下：模板序列(10pg-1ng)1μL，正向引物(25μM)0.5μL，反向引物(25μM)0.5μL，5×PrimerSTAR缓冲液10μL，PrimerSTAR DNA聚合酶0.5μL，dNTP(2.5mM)4μL，灭菌超纯水(ddH2O)33.5μL，总体积50μL。PCR扩增程序如下：95℃变性5分钟，30轮循环(98℃持续10秒，50-68℃持续5-15秒，72℃持续一定时间(1000bp/min))，72℃延伸10分钟；或者95℃变性5分钟，30轮循环(98℃持续10秒，68℃持续一定时间(1000bp/min))，72℃延伸10分钟。
II.2核酸内切酶酶切反应：
对质粒载体进行双酶切的体系如下：质粒载体20μL(约1.5μg)，10×缓冲液5μL，限制性内切酶11-2μL，限制性内切酶21-2μL，用灭菌超纯水补至总体积50μL。反应条件37℃，1-7小时。
II.3 DNA片段5’端磷酸化反应
从微生物中抽提出的质粒或者基因组末端都含有磷酸基团，而PCR产物没有，故需对PCR产物的5’端碱基进行磷酸基团加成反应，只有末端含有磷酸基团DNA分子才能发生连接反应。磷酸化反应体系如下：PCR产物片段DNA序列5-8μL，10×T4连接酶缓冲液1μL，T4多聚核苷酸激酶(T4 PNK)1μL，灭菌超纯水0-3μL，总体积10μL。反应条件37℃，30分钟-2小时后72℃灭活20分钟。
II.4目的片段和载体的连接反应
不同的片段和载体之间的连接方法有所差异，本发明中使用了三种连接方法
1.平末端短片段和线性化载体的平末端连接
该方法的原理是PCR获得的平末端产物在T4 PNK作用下对DNA片段的5’末端进行磷酸化反应后，与线性化的载体在PEG4000和T4 DNA连接酶的作用下连接获得重组质粒。同源重组连接体系如下：T4 PNK处理的DNA片段4μL，线性化载体片段4μL，PEG4000 1μL，10×T4连接酶缓冲液1μL，T4 DNA连接酶1μL，总计10μL。反应条件22℃，30分钟。
2.含有粘性末端的DNA片段和含有粘性末端载体片段的连接
通过限制性内切酶切割的DNA片段通常会产生突出的粘性末端，因此可以和含有序列互补的粘性末端载体片段连接，形成重组质粒。连接反应体系如下：酶切后的PCR产物片段DNA 1-7μL，酶切后的质粒0.5-7μL，10×T4连接酶缓冲液1μL，T4 DNA连接酶1μL，灭菌超纯水补至总体积10μL。反应条件16℃，4-8小时。
3.反向PCR引入定点突变后5’端磷酸化的DNA片段产物自身环化的连接反应
将5’端磷酸化的DNA片段通过自身环化连接反应将线性化载体的3’端和5’端连接反应得到重组质粒。自身环化连接反应体系如下：磷酸化反应体系10μL，T4连接酶(5U/μL)0.5μL，总体积10.5μL。反应条件16℃，4-16小时。
II.5感受态细胞的制备与转化
感受态细胞的制备：
1.挑取单菌落(如Mach1)接种于5mL LB培养基中，37℃摇床过夜。
2.取0.5-1mL过夜培养的菌液转种到50mL LB培养基中，37℃，220rpm培养3至5小时，直到OD600达到0.5。
3.冰浴预冷细胞2小时。
4. 4℃，4000rpm离心10分钟。
5.弃上清，用5mL预冷的缓冲液重悬细胞，待均匀后再加入重悬缓冲液至终体积为50mL。
6.冰浴45分钟。
7. 4℃ 4000rpm离心10分钟，用5mL冰预冷的储存缓冲液重悬细菌。
8.每个EP管中放100μL菌液，-80℃或液氮冻存。
重悬缓冲液:CaCl2(100mM)、MgCl2(70mM)、NaAc(40mM)
储存缓冲液:0.5mL DMSO、1.9mL 80％甘油、1mL 10×CaCl2(1M)、1mL 10×MgCl2(700mM)、1mL 10×NaAc(400mM)、4.6mL ddH2O
感受态细胞的转化：
1.取100μL感受态细胞于冰浴上融化。
2.加入适当体积的连接产物，轻轻吹打混匀，冰浴30分钟。通常加入的连接产物的体积少于感受态细胞体积的1/10。
3.将菌液放入42℃水浴中热激90秒，迅速转移至冰浴中放置5分钟。
4.加入500μL LB，于37℃恒温摇床上200rpm培养1小时。
5.将菌液4000rpm离心3分钟，留200μL上清将菌体吹匀，均匀涂布于含适当抗生素的琼脂平板表面，平板于37℃恒温培养箱内倒置过夜。
II.6蛋白质的表达，纯化和荧光检测
1.将pET28a为基础的精氨酸探针质粒转化到JM109(DE3)中，倒置培养过夜，从平板上挑取克隆到250ml锥形瓶中，置于37℃摇床，220rpm培养至OD＝0.4-0.8，加入1/1000(v/v)的IPTG(1M)，18℃诱导表达24-36h。
2.诱导表达完成后，4000rpm，30分钟离心收菌，加入50mM的磷酸盐缓冲液重悬菌体沉淀，超声破碎至菌体澄清。9600rpm，4℃离心20分钟。
3.离心上清通过自装的镍柱亲和层析柱纯化获得蛋白，镍柱亲和层析后的蛋白再通过自装的脱盐柱获得溶解在20mM MOPS缓冲液(pH 7.4)或者磷酸盐缓冲液PBS中的蛋白。
4.纯化的精氨酸结合蛋白突变蛋白经过SDS-PAGE鉴定后，使用测定缓冲液(100mMHEPES，100mM NaCl，pH 7.3)或者磷酸盐缓冲液PBS稀释探针成终浓度为5～10μM的蛋白溶液。用测定缓冲液(20mM MOPS，pH 7.4)或者磷酸盐缓冲液PBS将精氨酸配制成终浓度为1M的储液。
5.取100μl 5μM的蛋白溶液，37℃温育5min，分别加入精氨酸混匀后至终浓度为100mM，利用多功能荧光酶标仪测定蛋白在340nm下的光吸收。
6.取100μl 1μM的荧光探针溶液，37℃温育5min，加入精氨酸滴定，测定蛋白的485nm荧光激发后528nm发射的荧光强度。对样品的荧光激发、发射测定利用多功能荧光酶标仪完成。
7.取100μl 1μM的荧光探针溶液，37℃温育5min，加入精氨酸，测定探针蛋白的吸收光谱和荧光光谱。对样品的吸收光谱和荧光光谱的测定是通过分光光度计和荧光分光光度计完成。
II.7哺乳动物细胞的转染和荧光检测
1.将PAAV为基础的精氨酸探针质粒通过转染试剂Lipofectamine2000(Invitrogen)转染到HeLa中，置于37℃，5％CO2的细胞培养箱中培养。待外源基因充分表达24～36h后进行荧光检测。
2.诱导表达完成后，将贴壁的HeLa细胞，用PBS冲洗三次，置于HBSS溶液中分别进行荧光显微镜和酶标仪检测。
实施例1：精氨酸结合蛋白质粒
通过PCR扩增土壤农杆菌基因中的STM4351基因，PCR产物凝胶电泳后回收后用BamHI和EcoRI酶切，同时对pET28a载体进行相同的双酶切。用T4 DNA ligase连接后，连接产物转化Trans5a，转化的Trans5a涂布于LB平板(卡那霉素100ug/mL)，置于37℃培养过夜。将生长Trans5a转化子进行质粒抽提后，进行PCR鉴定。阳性质粒经过测序正确后进行后续的质粒构建。
实施例2：不同插入位点的cpYFP光学探针的表达和检测
本实施例中，以pET28a-STM4351为基础根据精氨酸结合蛋白晶体结构选择了下述位点插入cpYFP，得到相应质粒：103/104，103/105，103/106，103/107，103/108，103/109，103/110，103/111，104/105，104/106，104/107，104/108，104/109，104/110，104/111，105/106，105/107，105/108，105/109，105/110，105/111，106/107，106/108，106/109，106/110，106/111，107/108，107/109，107/110，107/111，108/109，108/110，108/111，109/110，109/111，110/111，197/198，197/199，197/200，197/201，197/202，197/203，197/204，197/205，197/206，197/207，197/208，197/209，198/199，198/200，198/201，198/202，198/203，198/204，198/205，198/206，198/207，198/208，198/209，199/200，199/201，199/202，199/203，199/204，199/205，199/206，199/207，199/208，199/209，200/201，200/202，200/203，200/204，200/205，200/206，200/207，200/208，200/209，201/202，201/203，201/204，201/205，201/206，201/207，201/208，201/209，202/203，202/204，202/205，202/206，202/207，202/208，202/209，203/204，203/205，203/206，203/207，203/208，203/209，204/205，204/206，204/207，204/208，204/209，205/206，205/207，205/208，205/209，206/207，206/208，206/209，207/208，207/209或208/209。示例性的光学探针的序列如表1所示。
表1，光学探针的序列
| 序列 | 插入位点 |
| SEQ ID NO:6 | 104/109 |
| SEQ ID NO:7 | 104/110 |
| SEQ ID NO:8 | 104/111 |
| SEQ ID NO:9 | 105/106 |
| SEQ ID NO:10 | 105/107 |
| SEQ ID NO:11 | 105/110 |
| SEQ ID NO:12 | 106/108 |
| SEQ ID NO:13 | 106/109 |
| SEQ ID NO:14 | 107/109 |
| SEQ ID NO:15 | 107/111 |
| SEQ ID NO:16 | 110/111 |
| SEQ ID NO:17 | 197/199 |
| SEQ ID NO:18 | 197/203 |
| SEQ ID NO:19 | 197/204 |
| SEQ ID NO:20 | 198/202 |
| SEQ ID NO:21 | 198/203 |
| SEQ ID NO:22 | 199/200 |
| SEQ ID NO:23 | 199/201 |
| SEQ ID NO:24 | 199/202 |
| SEQ ID NO:25 | 199/204 |
| SEQ ID NO:26 | 199/205 |
| SEQ ID NO:27 | 200/203 |
| SEQ ID NO:28 | 200/204 |
| SEQ ID NO:29 | 200/205 |
| SEQ ID NO:30 | 200/206 |
| SEQ ID NO:31 | 201/202 |
| SEQ ID NO:32 | 201/203 |
| SEQ ID NO:33 | 201/204 |
| SEQ ID NO:34 | 201/205 |
| SEQ ID NO:35 | 201/206 |
| SEQ ID NO:36 | 202/205 |
| SEQ ID NO:37 | 203/206 |
| SEQ ID NO:38 | 204/205 |
| SEQ ID NO:39 | 204/206 |
利用PCR产生cpYFP的DNA片段，对该DNA片段使用5’末端的加磷操作后灭活，同时通过反向PCR扩增产生含有不同断裂位点的pET28a-精氨酸结合蛋白线性化载体，将线性化的pET28a-STM4351和5’末端磷酸化的cpYFP片段在PEG4000和T4 DNA ligase的作用下连接产生重组质粒，将这些平板在Kodak多功能活体成像系统，挑取在FITC通道激发下有黄色荧光的克隆，由北京六合华大基因科技股份有限公司上海分公司完成测序。
经过测序正确后，将重组质粒转化到JM109(DE3)中诱导表达，并纯化蛋白质，通过SDS-PAGE电泳大小在55Kda附近。该大小符合pET28a-STM4351-cpYFP表达出的含His-tag纯化标签的STM4351-cpYFP融合蛋白质的大小。结果如图1所示。
将纯化的STM4351-cpYFP融合蛋白质进行精氨酸响应筛选，将含有100mM精氨酸的融合荧光蛋白质的检测信号除以无精氨酸的融合荧光蛋白质的检测信号。结果如图2所示，检测结果显示对精氨酸响应超过2倍的有104/110，105/106，105/109，105/110，105/111，106/108，106/109，107/109，107/111，197/203，198/201，199/200，199/202，199/203，199/204，200/202，200/203，200/204，200/205，200/206，201/202，201/205，204/205位。
实施例3，不同插入位点的cpGFP光学探针的表达和检测
按照实施例1中的方法将cpYFP替换为绿色荧光蛋白cpGFP，融合到精氨酸结合蛋白中构建精氨酸绿色荧光蛋白荧光探针，按照实施例2中的方法表达并检测。结果如图3所示，荧光检测结果显示对精氨酸响应超过2倍的有104/110，105/106，105/109，105/110，105/111，106/108，106/109，107/109，107/111，197/203，198/201，199/200，199/202，199/203，199/204，200/202，200/203，200/204，200/205，200/206，201/202，201/205，204/205位点。
实施例4，不同插入位点的cpBFP光学探针的表达和检测
按照实施例1中的方法将cpYFP替换为蓝色荧光蛋白cpBFP，融合到精氨酸结合蛋白中构建精氨酸蓝色荧光蛋白荧光探针，按照实施例2中的方法表达并检测。结果如图4所示，荧光检测结果显示对精氨酸响应超过2倍的有104/110，105/106，105/109，105/110，105/111，106/108，106/109，107/109，107/111，197/203，198/201，199/200，199/202，199/203，199/204，200/202，200/203，200/204，200/205，200/206，201/202，201/205，204/205位点。
实施例5，不同插入位点的cpmApple光学探针的表达和检测
按照实施例1的方法将cpYFP替换为苹果红荧光蛋白cpmApple，融合到精氨酸结合蛋白中构建精氨酸红色荧光蛋白荧光探针，按照实施例2中的方法表达并检测。结果如图5所示，荧光检测结果显示对精氨酸响应超过2倍的有104/110，105/106，105/109，105/110，105/111，106/108，106/109，107/109，107/111，197/203，198/201，199/200，199/202，199/203，199/204，200/202，200/203，200/204，200/205，200/206，201/202，201/205，204/205位点。
实施例6，突变的cpYFP光学探针的表达和检测
通过反向PCR线性化质粒pET28a-STM4351-107/111-cpYFP，引物中含有所要突变位点的碱基序列，对得到的PCR产物在PNK、T4 DNA ligase和PEG4000的作用下加磷连接，得到S30，R96，D177，这3个位点的定点突变质粒，并由北京六合华大基因科技股份有限公司上海分公司完成测序。按照实施例2中的方法表达并检测。部分突变的光学探针的氨基酸序列如SEQ ID NO:40(107/111-S30N)和SEQ ID NO:41(107/111-D177N)所示；核酸序列如SEQID NO:42(S30N-107/111)所示。
结果如图6所示，荧光检测结果显示对精氨酸响应超过3倍的有S30N-107/111和D177N-107/111突变体。
实施例7，探针的滴定曲线和特异性
选择28个STM4351-cpYFP融合蛋白质104/110，105/106，105/110，106/108，106/109，107/109，197/199，197/203，197/204，198/201，198/202，198/203，199/200，199/201，199/202，199/205，200/202，200/203，200/204，200/205，200/206，201/202，201/203，201/205，202/205，203/206，204/205，204/206进行浓度梯度的精氨酸检测，检测420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值的变化，不同插入位点105/110，199/200，200/203和200/205精氨酸探针的Kd(结合常数)分别为5.4μM、11.7μM、58.5μM和15μM，变化幅度分别为4.3倍、11.0倍、8.6倍和3.0倍，结果如图7A-B所示。对响应倍数较大的插入位点104/110，107/109，107/111，197/203，199/200，199/201，199/202，200/202，200/203，200/204，200/205，201/202进行特异性检测，结果表明探针对精氨酸具有很好的专一性，如图8所示。
实施例8，探针的光谱性能
将响应倍数较高且特异性好的突变体STM4351-199/200-cpYFP进行纯化，将纯化的精氨酸荧光探针分别进行0mM和100mM精氨酸处理10min后，使用荧光分光光度计进行荧光谱的检测。对发射光谱的测定：固定激发波长分别为420nm和485nm，记录360-540nm的发射光谱，每5nm读取一次。精氨酸荧光探针STM4351-104/110-cpYFP的光谱曲线如图9A的第一图所示，探针在添加500mM精氨酸后，在420nm激发下荧光强度降低为添加0mM精氨酸的1.4倍；在485nm激发下荧光强度升高为添加0mM精氨酸的2.0倍。在相同条件下105/106，105/110，107/111，197/199，197/203，197/204，198/202，198/203，199/200，199/202，199/205，200/203，200/204，200/205，201/202，201/204，201/205，201/206，202/205和204/205探针做相同处理，其光谱曲线如图9A-B所示。
实施例9，探针的亚细胞器定位和在亚细胞器内的性能
本实施例中，我们使用不同的定位信号肽与精氨酸荧光探针STM4351-199/200-cpYFP的C端或N端进行融合，并且将精氨酸荧光探针STM4351-199/200-cpYFP定位到不同的细胞器中。
将融合不同定位信号肽的精氨酸荧光探针STM4351-199/200-cpYFP基因的质粒转染HeLa细胞36小时后，使用PBS冲洗之后，置于HBSS溶液中使用倒置荧光显微镜进行FITC通道下进行荧光检测。我们发现精氨酸荧光探针FLIPpro通过与不同的特异定位信号肽融合能够定位到包括细胞浆、线粒体、细胞核、高尔基体、内质网和细胞膜等亚细胞器中。结果如图10所示，不同的亚细胞结构中都显示有荧光，并且荧光的分布和强度各不相同。
实施例10：精氨酸跨膜运输的动态监测
将胞浆表达的STM4351-199/200-cpYFP基因的质粒转染HeLa细胞细胞36小时后，使用PBS冲洗之后，置于HBSS溶液中检测40min时间段内420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值的变化。结果如图11所示，在饥饿2个小时之后，添加10mM精氨酸后检测20min，ratio 485/420逐渐增加，最高可以达到2.2倍。同STM4351-105/110-cpYFP探针和STM4351-200/203-cpYFP探针。
实施例11，活细胞水平基于探进行高通量化合物筛选
本实施例中，我们使用精氨酸探针STM4351-199/200-cpYFP胞浆表达的HeLa细胞进行了高通量化合物筛选。
将经过转染STM4351-199/200-cpYFP基因的HeLa细胞，使用PBS冲洗之后，置于HBSS溶液中(无精氨酸)处理1hours后，使用10μM的化合物进行处理1hours。分别滴加精氨酸。使用酶标仪记录420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值变化。以未用任何化合物处理的样品为标准。结果如图12所示，我们发现使用2000种化合物处理的细胞中，绝大部分的化合物对精氨酸进入细胞影响极小。有16种化合物能够提高细胞对精氨酸的摄取能力，另外有11种化合物能够明显降低细胞对精氨酸的摄取。
实施例12，探针对血液中精氨酸进行定量检测
在本实施中，使用纯化的精氨酸荧光探针STM4351-199/200-cpYFP蛋白质对小鼠和人的血液上清中精氨酸进行了分析。
将精氨酸荧光探针STM4351-199/200-cpYFP荧光蛋白质与稀释处理的血液上清进行混合处理10min后，使用酶标仪检测420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值。结果如图13所示，发现小鼠血液中的精氨酸含量在56μM左右，人血液中的精氨酸含量在68μM左右。
由以上实施例可知，本发明提供的精氨酸荧光探针，蛋白分子量相对较小且易于成熟，荧光动态变化大，特异性好，并且能够通过基因操作的方法在细胞中表达，可在细胞内外实时定位、定量检测精氨酸；并且能够进行高通量的化合物筛选。
以上所述仅是本发明的优选实施方案，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 华东理工大学
<120> 一种精氨酸荧光探针及其制备方法和应用
<130> 197276
<160> 42
<170> SIPOSequenceListing 1.0
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<212> PRT
<213> Salmonella typhimurium
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Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
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Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
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Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr Gly Lys Gly Leu Gly
180 185 190
Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu Ile Asn Ala
195 200 205
Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln Met Gln Glu
210 215 220
Lys Trp Phe Thr Gln
225
<210> 2
<211> 246
<212> PRT
<213> Artificial Sequence
<400> 2
Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly
1 5 10 15
Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val
20 25 30
Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
35 40 45
Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser
50 55 60
Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val
65 70 75 80
Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp
85 90 95
Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly
100 105 110
Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys
115 120 125
Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu
130 135 140
Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
145 150 155 160
Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr
165 170 175
Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu
180 185 190
Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr
195 200 205
Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg
210 215 220
Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly
225 230 235 240
His Lys Leu Glu Tyr Asn
245
<210> 3
<211> 241
<212> PRT
<213> Artificial Sequence
<400> 3
Asn Val Tyr Ile Lys Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn
1 5 10 15
Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Tyr
20 25 30
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
35 40 45
Asp Asn His Tyr Leu Ser Val Gln Ser Ile Leu Ser Lys Asp Pro Asn
50 55 60
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
65 70 75 80
Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser
85 90 95
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Gln
100 105 110
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
115 120 125
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
130 135 140
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
145 150 155 160
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
165 170 175
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu
180 185 190
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
195 200 205
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
210 215 220
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
225 230 235 240
Asn
<210> 4
<211> 243
<212> PRT
<213> Artificial Sequence
<400> 4
Asn Val Tyr Ile Lys Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn
1 5 10 15
Phe Lys Ile Arg His Asn Ile Glu Gly Gly Gly Val Gln Leu Ala Tyr
20 25 30
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
35 40 45
Asp Asn His Tyr Leu Ser Val Gln Ser Ile Leu Ser Lys Asp Pro Asn
50 55 60
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
65 70 75 80
Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser
85 90 95
Glu Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro
100 105 110
Ile Gln Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val
115 120 125
Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys
130 135 140
Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val
145 150 155 160
Thr Thr Leu Ser His Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His
165 170 175
Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Gly Gly Tyr Ile
180 185 190
Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg
195 200 205
Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu
210 215 220
Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu
225 230 235 240
Glu Tyr Asn
<210> 5
<211> 242
<212> PRT
<213> Artificial Sequence
<400> 5
Val Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Ser Glu Ile
1 5 10 15
Lys Lys Gly Leu Arg Leu Lys Asp Gly Gly His Tyr Ala Ala Glu Val
20 25 30
Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr
35 40 45
Ile Val Asp Ile Lys Leu Asp Ile Val Ser His Asn Glu Asp Tyr Thr
50 55 60
Ile Val Glu Gln Cys Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly
65 70 75 80
Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser Leu Val Ser Lys
85 90 95
Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg Phe Lys
100 105 110
Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly
115 120 125
Glu Gly Glu Gly Arg Pro Tyr Glu Ala Phe Gln Thr Ala Lys Leu Lys
130 135 140
Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro
145 150 155 160
Gln Phe Met Tyr Gly Ser Lys Ala Tyr Ile Lys His Pro Ala Asp Ile
165 170 175
Pro Asp Tyr Phe Lys Leu Ser Phe Pro Glu Gly Phe Arg Trp Glu Arg
180 185 190
Val Met Asn Phe Glu Asp Gly Gly Ile Ile His Val Asn Gln Asp Ser
195 200 205
Ser Leu Gln Asp Gly Val Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr
210 215 220
Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp
225 230 235 240
Glu Ala
<210> 6
<211> 471
<212> PRT
<213> Artificial Sequence
<400> 6
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Tyr Asn Ser Asp Asn Val Tyr Ile Met
85 90 95
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
100 105 110
Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
115 120 125
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
130 135 140
Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
145 150 155 160
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
165 170 175
Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys
180 185 190
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp
195 200 205
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly
210 215 220
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly
225 230 235 240
Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly
245 250 255
Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe
260 265 270
Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe
275 280 285
Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu
290 295 300
Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys
305 310 315 320
Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Leu Ser
325 330 335
Ala Val Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu
340 345 350
Lys Gly Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr
355 360 365
Leu Gln Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr
370 375 380
Leu Asn Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe
385 390 395 400
Gly Asp Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr
405 410 415
Ala Ile Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr Gly Lys Gly
420 425 430
Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu Ile
435 440 445
Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln Met
450 455 460
Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 7
<211> 470
<212> PRT
<213> Artificial Sequence
<400> 7
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Tyr Asn Ser Asp Asn Val Tyr Ile Met
85 90 95
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
100 105 110
Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
115 120 125
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
130 135 140
Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
145 150 155 160
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
165 170 175
Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys
180 185 190
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp
195 200 205
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly
210 215 220
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly
225 230 235 240
Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly
245 250 255
Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe
260 265 270
Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe
275 280 285
Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu
290 295 300
Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys
305 310 315 320
Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Leu Ser Ala
325 330 335
Val Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys
340 345 350
Gly Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu
355 360 365
Gln Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu
370 375 380
Asn Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly
385 390 395 400
Asp Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala
405 410 415
Ile Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr Gly Lys Gly Leu
420 425 430
Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu Ile Asn
435 440 445
Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln Met Gln
450 455 460
Glu Lys Trp Phe Thr Gln
465 470
<210> 8
<211> 469
<212> PRT
<213> Artificial Sequence
<400> 8
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Tyr Asn Ser Asp Asn Val Tyr Ile Met
85 90 95
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
100 105 110
Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
115 120 125
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
130 135 140
Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
145 150 155 160
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
165 170 175
Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys
180 185 190
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp
195 200 205
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly
210 215 220
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly
225 230 235 240
Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly
245 250 255
Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe
260 265 270
Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe
275 280 285
Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu
290 295 300
Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys
305 310 315 320
Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Ser Ala Val
325 330 335
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
340 345 350
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
355 360 365
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
370 375 380
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
385 390 395 400
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
405 410 415
Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr Gly Lys Gly Leu Gly
420 425 430
Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu Ile Asn Ala
435 440 445
Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln Met Gln Glu
450 455 460
Lys Trp Phe Thr Gln
465
<210> 9
<211> 475
<212> PRT
<213> Artificial Sequence
<400> 9
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Asn Ser Asp Asn Val Tyr Ile
85 90 95
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg
100 105 110
His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln
115 120 125
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
130 135 140
Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
145 150 155 160
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
165 170 175
Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser
180 185 190
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
195 200 205
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
210 215 220
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr
225 230 235 240
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr
245 250 255
Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
260 265 270
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile
275 280 285
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
290 295 300
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe
305 310 315 320
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Tyr
325 330 335
Glu Gly Leu Ser Ala Val Val Val Thr Arg Lys Gly Ala Tyr His Thr
340 345 350
Phe Ala Asp Leu Lys Gly Lys Lys Val Gly Leu Glu Asn Gly Thr Thr
355 360 365
His Gln Arg Tyr Leu Gln Asp Lys Gln Gln Ala Ile Thr Pro Val Ala
370 375 380
Tyr Asp Ser Tyr Leu Asn Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu
385 390 395 400
Glu Gly Val Phe Gly Asp Val Ala Ala Ile Gly Lys Trp Leu Lys Asn
405 410 415
Asn Pro Asp Tyr Ala Ile Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr
420 425 430
Tyr Gly Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu
435 440 445
Leu Gln Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu
450 455 460
Tyr Ala Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470 475
<210> 10
<211> 474
<212> PRT
<213> Artificial Sequence
<400> 10
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Asn Ser Asp Asn Val Tyr Ile
85 90 95
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg
100 105 110
His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln
115 120 125
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
130 135 140
Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
145 150 155 160
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
165 170 175
Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser
180 185 190
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
195 200 205
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
210 215 220
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr
225 230 235 240
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr
245 250 255
Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
260 265 270
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile
275 280 285
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
290 295 300
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe
305 310 315 320
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Glu
325 330 335
Gly Leu Ser Ala Val Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe
340 345 350
Ala Asp Leu Lys Gly Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His
355 360 365
Gln Arg Tyr Leu Gln Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr
370 375 380
Asp Ser Tyr Leu Asn Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu
385 390 395 400
Gly Val Phe Gly Asp Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn
405 410 415
Pro Asp Tyr Ala Ile Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr
420 425 430
Gly Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu
435 440 445
Gln Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr
450 455 460
Ala Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 11
<211> 471
<212> PRT
<213> Artificial Sequence
<400> 11
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Asn Ser Asp Asn Val Tyr Ile
85 90 95
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg
100 105 110
His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln
115 120 125
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
130 135 140
Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
145 150 155 160
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
165 170 175
Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser
180 185 190
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
195 200 205
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
210 215 220
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr
225 230 235 240
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr
245 250 255
Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
260 265 270
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile
275 280 285
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
290 295 300
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe
305 310 315 320
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Leu Ser
325 330 335
Ala Val Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu
340 345 350
Lys Gly Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr
355 360 365
Leu Gln Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr
370 375 380
Leu Asn Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe
385 390 395 400
Gly Asp Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr
405 410 415
Ala Ile Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr Gly Lys Gly
420 425 430
Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu Ile
435 440 445
Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln Met
450 455 460
Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 12
<211> 474
<212> PRT
<213> Artificial Sequence
<400> 12
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Asn Ser Asp Asn Val Tyr
85 90 95
Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile
100 105 110
Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln
115 120 125
Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His
130 135 140
Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg
145 150 155 160
Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu
165 170 175
Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly
180 185 190
Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu
195 200 205
Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly
210 215 220
Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr
225 230 235 240
Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly
245 250 255
Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His
260 265 270
Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr
275 280 285
Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys
290 295 300
Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly
305 310 315 320
Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Glu
325 330 335
Gly Leu Ser Ala Val Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe
340 345 350
Ala Asp Leu Lys Gly Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His
355 360 365
Gln Arg Tyr Leu Gln Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr
370 375 380
Asp Ser Tyr Leu Asn Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu
385 390 395 400
Gly Val Phe Gly Asp Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn
405 410 415
Pro Asp Tyr Ala Ile Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr
420 425 430
Gly Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu
435 440 445
Gln Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr
450 455 460
Ala Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 13
<211> 473
<212> PRT
<213> Artificial Sequence
<400> 13
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Asn Ser Asp Asn Val Tyr
85 90 95
Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile
100 105 110
Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln
115 120 125
Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His
130 135 140
Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg
145 150 155 160
Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu
165 170 175
Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly
180 185 190
Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu
195 200 205
Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly
210 215 220
Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr
225 230 235 240
Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly
245 250 255
Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His
260 265 270
Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr
275 280 285
Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys
290 295 300
Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly
305 310 315 320
Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly
325 330 335
Leu Ser Ala Val Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala
340 345 350
Asp Leu Lys Gly Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln
355 360 365
Arg Tyr Leu Gln Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp
370 375 380
Ser Tyr Leu Asn Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly
385 390 395 400
Val Phe Gly Asp Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro
405 410 415
Asp Tyr Ala Ile Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr Gly
420 425 430
Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln
435 440 445
Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala
450 455 460
Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 14
<211> 474
<212> PRT
<213> Artificial Sequence
<400> 14
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Tyr Asn Ser Asp Asn Val
85 90 95
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys
100 105 110
Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr
115 120 125
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn
130 135 140
His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys
145 150 155 160
Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr
165 170 175
Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr
180 185 190
Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val
195 200 205
Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu
210 215 220
Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys
225 230 235 240
Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu
245 250 255
Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln
260 265 270
His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg
275 280 285
Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
290 295 300
Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
305 310 315 320
Gly Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn
325 330 335
Gly Leu Ser Ala Val Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe
340 345 350
Ala Asp Leu Lys Gly Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His
355 360 365
Gln Arg Tyr Leu Gln Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr
370 375 380
Asp Ser Tyr Leu Asn Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu
385 390 395 400
Gly Val Phe Gly Asp Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn
405 410 415
Pro Asp Tyr Ala Ile Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr
420 425 430
Gly Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu
435 440 445
Gln Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr
450 455 460
Ala Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 15
<211> 472
<212> PRT
<213> Artificial Sequence
<400> 15
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Tyr Asn Ser Asp Asn Val
85 90 95
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys
100 105 110
Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr
115 120 125
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn
130 135 140
His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys
145 150 155 160
Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr
165 170 175
Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr
180 185 190
Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val
195 200 205
Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu
210 215 220
Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys
225 230 235 240
Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu
245 250 255
Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln
260 265 270
His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg
275 280 285
Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
290 295 300
Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
305 310 315 320
Gly Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn
325 330 335
Ser Ala Val Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp
340 345 350
Leu Lys Gly Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg
355 360 365
Tyr Leu Gln Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser
370 375 380
Tyr Leu Asn Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val
385 390 395 400
Phe Gly Asp Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp
405 410 415
Tyr Ala Ile Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr Gly Lys
420 425 430
Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu
435 440 445
Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln
450 455 460
Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 16
<211> 475
<212> PRT
<213> Artificial Sequence
<400> 16
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Tyr Asn Ser
85 90 95
Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala
100 105 110
Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala
115 120 125
Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu
130 135 140
Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro
145 150 155 160
Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala
165 170 175
Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser
180 185 190
Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro
195 200 205
Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val
210 215 220
Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys
225 230 235 240
Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val
245 250 255
Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His
260 265 270
Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val
275 280 285
Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg
290 295 300
Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu
305 310 315 320
Lys Gly Ile Gly Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu
325 330 335
Glu Tyr Asn Ser Ala Val Val Val Thr Arg Lys Gly Ala Tyr His Thr
340 345 350
Phe Ala Asp Leu Lys Gly Lys Lys Val Gly Leu Glu Asn Gly Thr Thr
355 360 365
His Gln Arg Tyr Leu Gln Asp Lys Gln Gln Ala Ile Thr Pro Val Ala
370 375 380
Tyr Asp Ser Tyr Leu Asn Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu
385 390 395 400
Glu Gly Val Phe Gly Asp Val Ala Ala Ile Gly Lys Trp Leu Lys Asn
405 410 415
Asn Pro Asp Tyr Ala Ile Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr
420 425 430
Tyr Gly Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu
435 440 445
Leu Gln Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu
450 455 460
Tyr Ala Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470 475
<210> 17
<211> 474
<212> PRT
<213> Artificial Sequence
<400> 17
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys
180 185 190
Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu
195 200 205
Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
210 215 220
Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln
225 230 235 240
Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
245 250 255
Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu
260 265 270
Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu
275 280 285
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
290 295 300
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
305 310 315 320
Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro
325 330 335
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys
340 345 350
Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser
355 360 365
Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
370 375 380
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
385 390 395 400
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly
405 410 415
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Ser Asp Pro Asp Tyr Tyr
420 425 430
Gly Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu
435 440 445
Gln Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr
450 455 460
Ala Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 18
<211> 470
<212> PRT
<213> Artificial Sequence
<400> 18
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys
180 185 190
Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu
195 200 205
Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
210 215 220
Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln
225 230 235 240
Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
245 250 255
Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu
260 265 270
Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu
275 280 285
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
290 295 300
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
305 310 315 320
Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro
325 330 335
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys
340 345 350
Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser
355 360 365
Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
370 375 380
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
385 390 395 400
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly
405 410 415
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Tyr Gly Lys Gly Leu
420 425 430
Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu Ile Asn
435 440 445
Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln Met Gln
450 455 460
Glu Lys Trp Phe Thr Gln
465 470
<210> 19
<211> 469
<212> PRT
<213> Artificial Sequence
<400> 19
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys
180 185 190
Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu
195 200 205
Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
210 215 220
Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln
225 230 235 240
Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
245 250 255
Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu
260 265 270
Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu
275 280 285
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
290 295 300
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
305 310 315 320
Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro
325 330 335
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys
340 345 350
Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser
355 360 365
Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
370 375 380
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
385 390 395 400
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly
405 410 415
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Gly Lys Gly Leu Gly
420 425 430
Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu Ile Asn Ala
435 440 445
Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln Met Gln Glu
450 455 460
Lys Trp Phe Thr Gln
465
<210> 20
<211> 472
<212> PRT
<213> Artificial Sequence
<400> 20
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp
180 185 190
Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val
195 200 205
Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro
210 215 220
Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe
225 230 235 240
Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val
245 250 255
Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu
260 265 270
Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu
275 280 285
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp
290 295 300
Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala
305 310 315 320
Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu
325 330 335
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys
340 345 350
Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys
355 360 365
Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys
370 375 380
Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp
385 390 395 400
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp
405 410 415
Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp Tyr Tyr Gly Lys
420 425 430
Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu
435 440 445
Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln
450 455 460
Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 21
<211> 471
<212> PRT
<213> Artificial Sequence
<400> 21
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp
180 185 190
Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val
195 200 205
Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro
210 215 220
Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe
225 230 235 240
Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val
245 250 255
Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu
260 265 270
Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu
275 280 285
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp
290 295 300
Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala
305 310 315 320
Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu
325 330 335
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys
340 345 350
Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys
355 360 365
Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys
370 375 380
Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp
385 390 395 400
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp
405 410 415
Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Tyr Gly Lys Gly
420 425 430
Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu Ile
435 440 445
Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln Met
450 455 460
Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 22
<211> 475
<212> PRT
<213> Artificial Sequence
<400> 22
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala
180 185 190
Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn
195 200 205
Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr
210 215 220
Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
225 230 235 240
Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met
245 250 255
Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp
260 265 270
Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly
275 280 285
Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly
290 295 300
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp
305 310 315 320
Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys
325 330 335
Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu
340 345 350
Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe
355 360 365
Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe
370 375 380
Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly
385 390 395 400
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu
405 410 415
Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp Pro Asp Tyr
420 425 430
Tyr Gly Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu
435 440 445
Leu Gln Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu
450 455 460
Tyr Ala Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470 475
<210> 23
<211> 474
<212> PRT
<213> Artificial Sequence
<400> 23
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala
180 185 190
Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn
195 200 205
Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr
210 215 220
Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
225 230 235 240
Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met
245 250 255
Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp
260 265 270
Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly
275 280 285
Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly
290 295 300
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp
305 310 315 320
Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys
325 330 335
Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu
340 345 350
Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe
355 360 365
Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe
370 375 380
Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly
385 390 395 400
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu
405 410 415
Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Pro Asp Tyr Tyr
420 425 430
Gly Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu
435 440 445
Gln Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr
450 455 460
Ala Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 24
<211> 473
<212> PRT
<213> Artificial Sequence
<400> 24
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala
180 185 190
Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn
195 200 205
Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr
210 215 220
Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
225 230 235 240
Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met
245 250 255
Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp
260 265 270
Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly
275 280 285
Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly
290 295 300
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp
305 310 315 320
Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys
325 330 335
Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu
340 345 350
Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe
355 360 365
Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe
370 375 380
Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly
385 390 395 400
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu
405 410 415
Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp Tyr Tyr Gly
420 425 430
Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln
435 440 445
Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala
450 455 460
Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 25
<211> 471
<212> PRT
<213> Artificial Sequence
<400> 25
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala
180 185 190
Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn
195 200 205
Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr
210 215 220
Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
225 230 235 240
Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met
245 250 255
Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp
260 265 270
Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly
275 280 285
Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly
290 295 300
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp
305 310 315 320
Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys
325 330 335
Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu
340 345 350
Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe
355 360 365
Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe
370 375 380
Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly
385 390 395 400
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu
405 410 415
Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Gly Lys Gly
420 425 430
Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu Ile
435 440 445
Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln Met
450 455 460
Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 26
<211> 470
<212> PRT
<213> Artificial Sequence
<400> 26
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala
180 185 190
Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn
195 200 205
Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr
210 215 220
Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
225 230 235 240
Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met
245 250 255
Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp
260 265 270
Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly
275 280 285
Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly
290 295 300
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp
305 310 315 320
Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys
325 330 335
Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu
340 345 350
Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe
355 360 365
Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe
370 375 380
Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly
385 390 395 400
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu
405 410 415
Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Lys Gly Leu
420 425 430
Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu Ile Asn
435 440 445
Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln Met Gln
450 455 460
Glu Lys Trp Phe Thr Gln
465 470
<210> 27
<211> 473
<212> PRT
<213> Artificial Sequence
<400> 27
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Tyr Asn Ser Asp Asn Val Tyr Ile Met
180 185 190
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
195 200 205
Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
210 215 220
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
225 230 235 240
Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
245 250 255
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
260 265 270
Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys
275 280 285
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp
290 295 300
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly
305 310 315 320
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly
325 330 335
Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly
340 345 350
Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe
355 360 365
Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe
370 375 380
Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu
385 390 395 400
Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys
405 410 415
Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Tyr Gly
420 425 430
Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln
435 440 445
Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala
450 455 460
Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 28
<211> 472
<212> PRT
<213> Artificial Sequence
<400> 28
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Tyr Asn Ser Asp Asn Val Tyr Ile Met
180 185 190
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
195 200 205
Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
210 215 220
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
225 230 235 240
Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
245 250 255
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
260 265 270
Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys
275 280 285
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp
290 295 300
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly
305 310 315 320
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly
325 330 335
Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly
340 345 350
Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe
355 360 365
Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe
370 375 380
Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu
385 390 395 400
Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys
405 410 415
Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Gly Lys
420 425 430
Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu
435 440 445
Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln
450 455 460
Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 29
<211> 471
<212> PRT
<213> Artificial Sequence
<400> 29
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Tyr Asn Ser Asp Asn Val Tyr Ile Met
180 185 190
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
195 200 205
Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
210 215 220
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
225 230 235 240
Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
245 250 255
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
260 265 270
Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys
275 280 285
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp
290 295 300
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly
305 310 315 320
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly
325 330 335
Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly
340 345 350
Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe
355 360 365
Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe
370 375 380
Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu
385 390 395 400
Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys
405 410 415
Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Lys Gly
420 425 430
Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu Ile
435 440 445
Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln Met
450 455 460
Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 30
<211> 470
<212> PRT
<213> Artificial Sequence
<400> 30
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Tyr Asn Ser Asp Asn Val Tyr Ile Met
180 185 190
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
195 200 205
Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
210 215 220
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
225 230 235 240
Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
245 250 255
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
260 265 270
Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys
275 280 285
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp
290 295 300
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly
305 310 315 320
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly
325 330 335
Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly
340 345 350
Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe
355 360 365
Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe
370 375 380
Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu
385 390 395 400
Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys
405 410 415
Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Lys Gly Leu
420 425 430
Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu Ile Asn
435 440 445
Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln Met Gln
450 455 460
Glu Lys Trp Phe Thr Gln
465 470
<210> 31
<211> 475
<212> PRT
<213> Artificial Sequence
<400> 31
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Pro Tyr Asn Ser Asp Asn Val Tyr Ile
180 185 190
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg
195 200 205
His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln
210 215 220
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
225 230 235 240
Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
245 250 255
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
260 265 270
Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser
275 280 285
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
290 295 300
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
305 310 315 320
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr
325 330 335
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr
340 345 350
Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
355 360 365
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile
370 375 380
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
385 390 395 400
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe
405 410 415
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp Tyr
420 425 430
Tyr Gly Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu
435 440 445
Leu Gln Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu
450 455 460
Tyr Ala Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470 475
<210> 32
<211> 474
<212> PRT
<213> Artificial Sequence
<400> 32
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Pro Tyr Asn Ser Asp Asn Val Tyr Ile
180 185 190
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg
195 200 205
His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln
210 215 220
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
225 230 235 240
Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
245 250 255
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
260 265 270
Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser
275 280 285
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
290 295 300
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
305 310 315 320
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr
325 330 335
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr
340 345 350
Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
355 360 365
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile
370 375 380
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
385 390 395 400
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe
405 410 415
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Tyr
420 425 430
Gly Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu
435 440 445
Gln Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr
450 455 460
Ala Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 33
<211> 473
<212> PRT
<213> Artificial Sequence
<400> 33
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Pro Tyr Asn Ser Asp Asn Val Tyr Ile
180 185 190
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg
195 200 205
His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln
210 215 220
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
225 230 235 240
Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
245 250 255
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
260 265 270
Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser
275 280 285
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
290 295 300
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
305 310 315 320
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr
325 330 335
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr
340 345 350
Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
355 360 365
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile
370 375 380
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
385 390 395 400
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe
405 410 415
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Gly
420 425 430
Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln
435 440 445
Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala
450 455 460
Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 34
<211> 472
<212> PRT
<213> Artificial Sequence
<400> 34
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Pro Tyr Asn Ser Asp Asn Val Tyr Ile
180 185 190
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg
195 200 205
His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln
210 215 220
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
225 230 235 240
Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
245 250 255
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
260 265 270
Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser
275 280 285
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
290 295 300
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
305 310 315 320
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr
325 330 335
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr
340 345 350
Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
355 360 365
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile
370 375 380
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
385 390 395 400
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe
405 410 415
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Lys
420 425 430
Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu
435 440 445
Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln
450 455 460
Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 35
<211> 471
<212> PRT
<213> Artificial Sequence
<400> 35
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Pro Tyr Asn Ser Asp Asn Val Tyr Ile
180 185 190
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg
195 200 205
His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln
210 215 220
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
225 230 235 240
Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
245 250 255
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
260 265 270
Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser
275 280 285
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
290 295 300
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
305 310 315 320
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr
325 330 335
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr
340 345 350
Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
355 360 365
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile
370 375 380
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
385 390 395 400
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe
405 410 415
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Lys Gly
420 425 430
Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu Ile
435 440 445
Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln Met
450 455 460
Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 36
<211> 473
<212> PRT
<213> Artificial Sequence
<400> 36
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Asn Ser Asp Asn Val Tyr
180 185 190
Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile
195 200 205
Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln
210 215 220
Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His
225 230 235 240
Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg
245 250 255
Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu
260 265 270
Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly
275 280 285
Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu
290 295 300
Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly
305 310 315 320
Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr
325 330 335
Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly
340 345 350
Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His
355 360 365
Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr
370 375 380
Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys
385 390 395 400
Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly
405 410 415
Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly
420 425 430
Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln
435 440 445
Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala
450 455 460
Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 37
<211> 473
<212> PRT
<213> Artificial Sequence
<400> 37
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr Asn Ser Asp Asn Val
180 185 190
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys
195 200 205
Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr
210 215 220
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn
225 230 235 240
His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys
245 250 255
Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr
260 265 270
Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr
275 280 285
Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val
290 295 300
Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu
305 310 315 320
Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys
325 330 335
Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu
340 345 350
Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln
355 360 365
His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg
370 375 380
Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
385 390 395 400
Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
405 410 415
Gly Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn
420 425 430
Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln
435 440 445
Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala
450 455 460
Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 38
<211> 475
<212> PRT
<213> Artificial Sequence
<400> 38
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr Tyr Asn Ser Asp Asn
180 185 190
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
195 200 205
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
210 215 220
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
225 230 235 240
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
245 250 255
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
260 265 270
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
275 280 285
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
290 295 300
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
305 310 315 320
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
325 330 335
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
340 345 350
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
355 360 365
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
370 375 380
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
385 390 395 400
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
405 410 415
Ile Gly Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
420 425 430
Asn Gly Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu
435 440 445
Leu Gln Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu
450 455 460
Tyr Ala Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470 475
<210> 39
<211> 474
<212> PRT
<213> Artificial Sequence
<400> 39
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Glu Gly Leu Ser Ala Val
85 90 95
Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp Leu Lys Gly
100 105 110
Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg Tyr Leu Gln
115 120 125
Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser Tyr Leu Asn
130 135 140
Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val Phe Gly Asp
145 150 155 160
Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp Tyr Ala Ile
165 170 175
Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr Tyr Asn Ser Asp Asn
180 185 190
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
195 200 205
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
210 215 220
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
225 230 235 240
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
245 250 255
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
260 265 270
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
275 280 285
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
290 295 300
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
305 310 315 320
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
325 330 335
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
340 345 350
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
355 360 365
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
370 375 380
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
385 390 395 400
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
405 410 415
Ile Gly Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
420 425 430
Asn Lys Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu
435 440 445
Gln Glu Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr
450 455 460
Ala Gln Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 40
<211> 472
<212> PRT
<213> Artificial Sequence
<400> 40
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Asn Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Tyr Asn Ser Asp Asn Val
85 90 95
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys
100 105 110
Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr
115 120 125
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn
130 135 140
His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys
145 150 155 160
Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr
165 170 175
Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr
180 185 190
Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val
195 200 205
Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu
210 215 220
Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys
225 230 235 240
Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu
245 250 255
Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln
260 265 270
His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg
275 280 285
Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
290 295 300
Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
305 310 315 320
Gly Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn
325 330 335
Ser Ala Val Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp
340 345 350
Leu Lys Gly Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg
355 360 365
Tyr Leu Gln Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser
370 375 380
Tyr Leu Asn Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val
385 390 395 400
Phe Gly Asp Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp
405 410 415
Tyr Ala Ile Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr Gly Lys
420 425 430
Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu
435 440 445
Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln
450 455 460
Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 41
<211> 472
<212> PRT
<213> Artificial Sequence
<400> 41
Ala Ser Val Ser Ala Arg Thr Leu His Phe Gly Thr Ser Ala Thr Tyr
1 5 10 15
Ala Pro Tyr Glu Phe Val Asp Ala Asp Asn Lys Ile Val Gly Phe Asp
20 25 30
Ile Asp Val Ala Asn Ala Val Cys Lys Glu Met Gln Ala Glu Cys Ser
35 40 45
Phe Thr Asn Gln Ser Phe Asp Ser Leu Ile Pro Ser Leu Arg Phe Lys
50 55 60
Lys Phe Asp Ala Val Ile Ala Gly Met Asp Met Thr Pro Lys Arg Glu
65 70 75 80
Gln Gln Val Ser Phe Ser Gln Pro Tyr Tyr Tyr Asn Ser Asp Asn Val
85 90 95
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys
100 105 110
Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr
115 120 125
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn
130 135 140
His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys
145 150 155 160
Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr
165 170 175
Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr
180 185 190
Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val
195 200 205
Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu
210 215 220
Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys
225 230 235 240
Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu
245 250 255
Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln
260 265 270
His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg
275 280 285
Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
290 295 300
Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
305 310 315 320
Gly Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn
325 330 335
Ser Ala Val Val Val Thr Arg Lys Gly Ala Tyr His Thr Phe Ala Asp
340 345 350
Leu Lys Gly Lys Lys Val Gly Leu Glu Asn Gly Thr Thr His Gln Arg
355 360 365
Tyr Leu Gln Asp Lys Gln Gln Ala Ile Thr Pro Val Ala Tyr Asp Ser
370 375 380
Tyr Leu Asn Ala Phe Thr Asp Leu Lys Asn Asn Arg Leu Glu Gly Val
385 390 395 400
Phe Gly Asn Val Ala Ala Ile Gly Lys Trp Leu Lys Asn Asn Pro Asp
405 410 415
Tyr Ala Ile Met Asp Glu Arg Ala Ser Asp Pro Asp Tyr Tyr Gly Lys
420 425 430
Gly Leu Gly Ile Ala Val Arg Lys Asp Asn Asp Ala Leu Leu Gln Glu
435 440 445
Ile Asn Ala Ala Leu Asp Lys Val Lys Ala Ser Pro Glu Tyr Ala Gln
450 455 460
Met Gln Glu Lys Trp Phe Thr Gln
465 470
<210> 42
<211> 1416
<212> DNA
<213> Artificial Sequence
<400> 42
gccagcgtga gcgcccgtac cctgcatttt ggcaccaatg ccacctacgc gccgtatgag 60
ttcgtggacg cggataacaa gatcgtgggt tttgacattg atgtggcgaa cgccgtgtgc 120
aaagagatgc aggctgaatg cagcttcacc aaccagagct ttgacagcct gatcccgagc 180
ctgcgtttca agaaatttga cgctgtgatt gccggcatgg atatgacccc gaagcgcgag 240
cagcaggtga gcttcagcca gccgtactat tacaacagcg acaacgtcta tatcatggcc 300
gacaagcaga agaacggcat caaggccaac ttcaagatcc gccacaacgt cgaggacggc 360
agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 420
ctgcccgaca accactacct gagcttccag tccgtcctga gcaaagaccc caacgagaag 480
cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 540
gagctgtaca acgtggatgg cggtagcggt ggcaccggca gcaagggcga ggagctgttc 600
accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc 660
gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gctgatctgc 720
accaccggca agctgcccgt gccctggccc accctcgtga ccaccctcgg ctacggcctg 780
aagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg 840
cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc 900
cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 960
ggcttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacag cgccgtggtg 1020
gtgacccgta agggtgccta ccacaccttt gcggacctga aaggcaagaa ggtgggcctg 1080
gagaatggca ccacccacca gcgttacctg caagataagc agcaggctat caccccggtg 1140
gcctacgaca gctatctgaa cgcgttcacc gatctgaaaa acaaccgtct ggagggcgtg 1200
ttcggcgacg tggcggccat tggcaagtgg ctgaaaaaca acccggacta cgccattatg 1260
gatgaacgtg cgagcgaccc ggattactat ggcaagggtc tgggcatcgc tgtgcgcaaa 1320
gacaacgatg ccctgctgca agaaattaac gctgccctgg ataaggtgaa agcgagcccg 1380
gagtatgctc agatgcagga aaaatggttt acccag 1416
Claims (9)
1. 一种光学探针，包含精氨酸敏感多肽和光学活性多肽，其中光学活性多肽位于精氨酸敏感多肽的序列内，所述精氨酸敏感多肽如SEQ ID NO: 1所示，所述光学活性多肽选自以下任一种：cpYFP、cpGFP、cpBFP或cpmApple，其中，
所述光学活性多肽是cpYFP，其位于精氨酸敏感多肽的选自以下一个或多个位点：103/104，104/108，104/109，104/110，104/111，105/106，105/107，105/108， 105/110，105/111，106/108，106/109，107/108，107/109，107/110，107/111， 108/111，109/110，110/111，197/199，197/200，197/201，197/202，197/203，197/204，197/205，198/201，198/202，198/203，199/200，199/201，199/202，199/203，199/204，199/205，199/206，200/203，200/204，200/205，200/206，201/202，201/203，201/204，201/205，201/206，202/203，202/204，202/205，202/206，203/204，203/205，203/206，203/207，204/205，204/206，204/207，205/206，206/207，207/208或208/209；
所述光学活性多肽是cpGFP，其位于精氨酸敏感多肽的选自以下一个或多个位点：103/104，104/108，104/109，104/110，104/111，105/106，105/107，105/108，105/110，105/111，106/108，106/109，107/108，107/109，107/110，108/111，110/111，197/199，197/200，197/201，197/202，197/203，197/204，198/201，198/202，198/203，199/200，199/201，199/202，199/204，199/205，199/206，200/202，200/204，200/205，200/206，201/202，201/203，201/204，201/205，201/206，202/203，202/204，202/205，202/206，203/205，203/206，203/207，204/205，204/206或205/206；
所述光学活性多肽是cpBFP，其位于精氨酸敏感多肽的选自以下一个或多个位点：103/104，104/108，104/109，104/110，104/111，105/106，105/107，105/108，105/110，105/111，106/108，106/109，107/109，107/110，107/111，108/111，110/111，197/199，197/201，197/203，197/204，198/201，198/202，198/203，199/200，199/201，199/202，199/203，199/204，199/205，200/203，200/204，200/205，200/206，201/202，201/203，201/204，201/205，201/206，202/203，202/204，202/205，202/206，203/205，203/206，203/207，204/205，204/206，205/206，206/207或207/208；或
所述光学活性多肽是cpBFP，其位于精氨酸敏感多肽的选自以下一个或多个位点：103/104，104/108，104/109，104/110，104/111，105/106，105/107，105/108，105/110，105/111，106/108，106/109，107/108，107/109，107/110，107/111，108/111，109/110，110/111，197/199，197/201，197/203，197/204，197/205，198/201，198/202，198/203，199/200，199/201，199/202，199/203，199/204，199/205，199/206，200/203，200/204，200/205，200/206，201/202，201/203，201/204，201/205，201/206，202/203，202/205，202/206，203/204，203/206，203/207，204/205，204/206，205/206，206/207或207/208。
2.如权利要求1所述的光学探针，其特征在于，所述光学活性多肽位于精氨酸敏感多肽的107/111位点，并且所述精氨酸敏感多肽还包含选自下述的突变：（1）S30N，（2）D177N，和（3）R96M或R96K。
3. 一种核酸分子，包含选自以下的序列：
（1）编码权利要求1或2所述的光学探针的多核苷酸序列，和
（2）（1）的互补序列。
4.一种核酸构建物，其包含权利要求3所述的核酸分子。
5.如权利要求4所述的核酸构建物，其特征在于，所述核酸构建物是表达载体。
6.一种宿主细胞，所述宿主细胞：
（1）表达权利要求1或2所述的光学探针；
（2）包含权利要求3所述的核酸分子；或
（3）包含权利要求4或5所述的核酸构建物。
7.一种制备权利要求1或2所述的光学探针的方法，包括培养权利要求6所述的宿主细胞，和由培养物分离所述光学探针。
8.权利要求1或2所述的光学探针、权利要求3所述的核酸分子或权利要求4或5所述的核酸构建物在制备试剂盒中的应用。
9.一种检测试剂盒，其包含
（1）权利要求1或2所述的光学探针或权利要求7所述方法制备的光学探针；
（2）权利要求3所述的核酸分子；
（3）权利要求4或5所述的核酸构建物；或
（4）权利要求6所述的宿主细胞；和
利用光学探针检测精氨酸所需的其他试剂。
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| CN108395484A (zh) * | 2018-03-15 | 2018-08-14 | 华东理工大学 | 支链氨基酸荧光探针及其应用 |
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