CN113004420B - 乳酸光学探针及其制备方法和应用 - Google Patents

乳酸光学探针及其制备方法和应用 Download PDF

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CN113004420B
CN113004420B CN202011516287.5A CN202011516287A CN113004420B CN 113004420 B CN113004420 B CN 113004420B CN 202011516287 A CN202011516287 A CN 202011516287A CN 113004420 B CN113004420 B CN 113004420B
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杨弋
赵玉政
李写
陈念
张则一
张秀泽
黄立
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East China University of Science and Technology
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Abstract

本发明涉及一种乳酸光学探针及其制备方法和应用。一方面，本发明涉及一种光学探针，包含乳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乳酸敏感多肽或其功能变体的序列内。本发明也涉及上述探针的制备方法及其在检测乳酸中的应用。

Description

乳酸光学探针及其制备方法和应用
技术领域
本发明涉及光学探针技术领域，尤其涉及一种乳酸光学探针及其制备方法和应用。
背景技术
乳酸是一种α-羟基酸，在水中能电离出一个氢离子H+，而形成CH3CH(OH)COO-乳酸根离子。乳酸是手性分子，由两种对映异构体组成，分别是D-(-)-乳酸和L-(+)-乳酸。在人体中乳酸可能以两种不同的构型存在，其中L-乳酸占绝大多数。当血浆中L-乳酸浓度大于5mM，血pH值低于7.35时，可能为乳酸性酸中毒。乳酸性酸中毒的原因包括线粒体内丙酮酸运输网络缺乏、丙酮酸脱氢酶复合体缺陷、柠檬酸循环障碍、先天线粒体呼吸链障碍、组织缺氧等原因。乳酸稳态与糖代谢有关，因此糖尿病与乳酸代谢紊乱有关。与健康对照组相比，糖尿病患者的基础全身葡萄糖氧化率降低，而非氧化性糖酵解的基础全身比率增加，导致过量乳酸的形成。血液中L-乳酸浓度上升曾经一度被认为是在缺氧的条件下骨骼肌通过糖酵解途径提供能量而生成的废物，现在我们知道在完全有氧条件下机体内各种不同细胞产生L-乳酸，并通过外周循环被几乎所有的组织用作燃料，这一点现在已被广泛接受。乳酸是机体内主要的能量来源，在肺肿瘤细胞生长过程中乳酸在三羧酸循环的过程中的贡献超过葡萄糖。由于肿瘤细胞大量摄取葡萄糖，在有氧条件下利用糖酵解途径为肿瘤细胞的生长增殖提供能量，同时产生大量的代谢物乳酸。乳酸是一种有效的抑制免疫细胞功能和生存的抑制剂，导致肿瘤细胞逃脱免疫反应，说明肿瘤来源的乳酸与肿瘤环境中的免疫细胞之间的重要相互作用。
参与乳酸代谢的代谢途径对于了解运动的生理反应以及糖尿病、癌症等常见疾病的发病机制具有重要意义。不论是乳酸的产生还是消除都依赖由乳酸脱氢酶(LDH)催化的可逆的氧化还原反应。动物在正常的代谢和运动过程中，葡萄糖通过糖酵解产生丙酮酸，丙酮酸通过乳酸脱氢酶(LDH)不断产生L-乳酸，但是乳酸的浓度并不会上升，直到乳酸的生成速率超过其消除速率。在剧烈的运动过程中，血液中的葡萄糖通过无氧呼吸快速合成ATP以供机体需要，同时生成大量的丙酮酸，随后立即转化成乳酸，此时乳酸生成速率高于机体清除乳酸的速率，从而导致乳酸的浓度上升。机体内的乳酸有两条去路，一是重新生成丙酮酸进入有氧呼吸链氧化供能；二是在肝脏通过糖异生转变成葡萄糖进入外周循环。乳酸是最主要的糖异生前体物质，在激烈运动时，肌细胞内的糖原通过糖酵解形成丙酮酸，在肌肉内无6-磷酸葡萄糖酸酶，所以无法催化6-磷酸葡萄糖生成葡萄糖。所以乳酸通过细胞膜弥散进入血液后，再入肝，在肝脏乳酸脱氢酶作用下变成丙酮酸，接着通过糖异生途径生成葡萄糖，又回到血液中以供肌肉和大脑对葡萄糖的需要，这个循环称作Cori-cycle，又称乳酸循环。乳酸上调缺氧诱导因子1α(HIF-1α)和血管内皮生长因子(VEGF)，在血管生成中具有不可或缺的作用。鉴于乳酸新陈代谢中的核心作用，乳酸现在被认为是原发性和转移性癌症代谢的组成部分，一些研究表明乳酸在肿瘤生长和转移性疾病进展中起着关键作用。
正是由于乳酸具有上述重要的作用，因此乳酸含量的检测也尤为重要。乳酸的常规检测方法包括NaOH溶液滴定法，高效液相色谱法(HPLC)(白冬梅，等，分析化学，2001，29(4)：413-415)，此外由于乳酸是手性分子，具有旋光性特点，也可采用旋光仪检测；紫外线(UV)-酶法(Immonen,K.,等,Meat Sci,2000.54(2):p.163-7.)；酶电极分析法(Tanner,R.K.等,Eur J Appl Physiol,2010.109(3):p.551-9.)。但是这些方法并不适用于活细胞研究，存在很多缺陷：需要经过耗时的样品处理过程，例如细胞破碎、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、高通量检测乳酸的方法。
发明内容
本发明的目的在于提供在细胞内、外实时原位、高通量、定量检测乳酸的探针和方法。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供一种乳酸光学探针，包含乳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乳酸敏感多肽或其功能变体的序列内。乳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。
本发明提供了一种乳酸光学探针，包括乳酸敏感多肽B和光学活性多肽A，其中光学活性多肽A位于乳酸敏感多肽B的序列内，将乳酸敏感多肽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，乳酸敏感多肽包括乳酸结合蛋白的乳酸结合域。在一个实施方案中，乳酸敏感多肽源自大肠杆菌。在一个实施方案中，乳酸敏感多肽是乳酸结合蛋白或其功能片段。在一个或多个实施方案中，所述乳酸结合蛋白是LldR蛋白。在一个实施方案中，乳酸敏感多肽具有SEQ ID NO:1所示的序列，或与其有至少35％、40％、50％、60％、70％、80％、85％、90％、95％、99％序列相同性并保留乳酸结合功能的序列。在一个实施方案中，乳酸敏感多肽具有SEQ ID NO:1所示的序列第80-258位氨基酸，或与其有至少35％、40％、50％、60％、70％、80％、85％、90％、95％、99％序列相同性并保留乳酸结合功能的序列。
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)、绿色荧光蛋白(如SEQID 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。本发明所述接头可以是任何长度的任何氨基酸序列。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：乳酸敏感多肽的第一部分B1-Y-光学活性多肽A-乳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，光学活性多肽位于乳酸敏感多肽的选自以下的位置中：残基93-97、119-121、137-141、158-161、185-191、208-210和/或230-232，所述乳酸敏感多肽是SEQ ID NO:1第80-258位氨基酸所示的乳酸结合蛋白功能片段，编号对应于乳酸结合蛋白的全长。在一个实施方案中，光学活性多肽置换乳酸敏感多肽的选自以下的位置中的一个或多个氨基酸：残基93-97、119-121、137-141、158-161、185-191、208-210和/或230-232，所述乳酸敏感多肽是SEQ ID NO:1第80-258位氨基酸所示的乳酸结合蛋白功能片段，编号对应于乳酸结合蛋白的全长。
在一个实施方案中，光学活性多肽位于乳酸敏感多肽的选自以下的一个或多个位点：93/94，93/95，93/96，93/97，94/95，94/96，94/97，95/96，95/97，96/97，119/120，119/121，120/121，137/138，137/139，137/140，137/141，138/139，138/140，138/141，139/140，139/141，140/141，158/159，158/160，158/161，159/160，159/161，160/161，185/186，185/187，185/188，185/189，185/190，185/191，186/187，186/188，186/189，186/190，186/191，187/188，187/189，187/190，187/191，188/189，188/190，188/191，189/190，189/191，190/191，208/209，208/210，209/210，230/231，230/232和/或231/232。优选地，光学活性多肽位于乳酸敏感多肽的选自以下的一个或多个位点：185/186，185/187，185/188，185/189，185/190，185/191，186/187，186/188，186/189，186/190，186/191，187/188，187/189，187/190，187/191，188/189，188/190，188/191，189/190，189/191或190/191。在一个或多个实施方案中，本发明B1-A-B2式光学探针可为当cpYFP位于乳酸结合蛋白或其功能片段的185/186，185/187，185/188，185/189，185/190，185/191，186/187，186/188，186/189，186/190，186/191，187/188，187/189，187/190，187/191，188/189，188/190，188/191，189/190，189/191或190/191处的探针。在示例性实施方案中，本发明B1-A-B2式光学探针可为当cpYFP位于乳酸结合蛋白或其功能片段的185/186，185/187，185/188，185/189，185/190，186/187，186/188，186/189，186/190，187/189，189/191和190/191处的探针。在一个或多个实施方案中，所述乳酸结合蛋白的功能片段如SEQ ID NO:1第80-258位所示。在一个实施方案中，本发明光学探针具有SEQ ID NO:6-17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乳酸敏感多肽的突变体。在一个实施方案中，所述突变位于乳酸结合蛋白或其功能片段的第185、第189和/或第190位。
在一个或多个实施方案中，所述突变包含：P189R和P190D、P189R和P190A、P189R和P190I、P189R和P190Q、P189R和P190N、P189D和P190D、P189D和P190E、P189D和P190V、P189D和P190L、P189D和P190F、P189D和P190I、P189D和P190Q、P189D和P190N、P189D和P190G、P189D和P190Y、P189D和P190W、P189E和P190R、P189E和P190A、P189E和P190V、P189E和P190Q、P189A和P190L、P189A和P190F、P189A和P190M、P189A、P189A和P190N、P189A和P190G、P189A和P190H、P189A和P190T、P189V和P190D、P189V和P190E、P189V和P190A、P189V、P189V和P190N、P189V和P190H、P189V和P190Y、P189L和P190V、P189L和P190F、P189L和P190M、P189L和P190G、P189L和P190H、P189F和P190D、P189F和P190L、P189F和P190F、P189F和P190I、P189F和P190N、P189F和P190H、P189F和P190Y、P189F和P190K、P189F和P190T、P189F和P190W、P189I和P190R、P189I和P190D、P189I和P190A、P189I和P190V、P189I和P190M、P189I和P190Q、P189I和P190G、P189I和P190Y、P189I和P190S、P189I和P190T、P189M和P190R、P189M和P190D、P189M和P190E、P189M和P190F、P189M和P190G、P189M和P190S、P189M和P190W、P189C和P190D、P189C和P190E、P189C和P190F、P189C和P190I、P189C和P190M、P189C和P190C、P189C、P189C和P190H、P189C和P190Y、P189C和P190S、P189C和P190W、P190L、P190F、P190I、P190Q、P190N、P190K、P190T、P189Q和P190E、P189Q和P190A、P189Q和P190V、P189Q和P190M、P189Q和P190C、P189Q和P190Q、P189Q和P190H、P189Q和P190S、P189N和P190R、P189N和P190D、P189N和P190L、P189N和P190F、P189N和P190C、P189N、P189N和P190N、P189N和P190G、P189N和P190H、P189N和P190Y、P189N和P190T、P189G和P190V、P189G和P190F、P189G和P190M、P189G和P190C、P189G和P190G、P189G和P190H、P189G和P190K、P189G和P190W、P189H和P190R、P189H和P190D、P189H和P190E、P189H和P190L、P189H和P190S、P189Y和P190R、P189Y和P190L、P189Y和P190N、P189Y和P190H、P189Y和P190S、P189Y和P190T、P189K和P190D、P189K和P190E、P189K和P190V、P189K和P190L、P189K和P190F、P189K和P190I、P189K和P190M、P189K、P189K和P190Q、P189K和P190N、P189K和P190Y、P189K和P190K、P189K和P190T、P189S和P190E、P189S和P190A、P189S和P190L、P189S和P190F、P189S和P190M、P189S和P190C、P189S、P189S和P190Q、189S和P190Y、P189S和P190K、P189S和P190S、P189T和P190R、P189T和P190D、P189T和P190M、P189T和P190C、P189T、P189T和P190Q、P189T和P190N、P189T和P190H、P189T和P190Y、P189T和P190K、P189T和P190W、P189W和P190A、P189W和P190V、P189W和P190F、P189W、P189W和P190Q、P189W和P190H、P189W和P190S、P189W和P190T、P189W和P190W。在一个或多个实施方案中，所述突变还包含M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185P、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R、M185E或M185K；优选地，所述突变还包含M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R或M185K。
在一个或多个实施方案中，所述突变包含P189R和P190A，P189D和P190D，P189D和P190E，P189D和P190Q，P189D和P190Y，P189A和P190N，P189A和P190G，P189V和P190H，P189F和P190I，P189F和P190N，P189F和P190K，P189I和P190D，P189I和P190A，P189I和P190V，P189I和P190M，P189M和P190R，P189M和P190E，P189M和P190F，P189M和P190G，P189M和P190S，P189C和P190E，P190Q，P189Q和P190M，P189Q和P190C，P189N和P190N，P189G和P190F，P189H和P190L，P189H和P190S，P189Y和P190L，P189K和P190V，P189K和P190T，P189S和P190A，P189S和P190M，P189S和P190Q，P189S和P190K，P189S和P190S，P189T和P190D，P189W和P190A，P189W和P190T，P189C和P190D，P189C和P190Y，P189N和P190Y，P189R和P190I，P189M和P190D，P189H和P190R，P189N，P189F和P190D，P189F和P190H，P189N和P190F，P189C和P190F，P189H和P190D，或P189S。在一个或多个实施方案中，所述突变还包含M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185P、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R、M185E或M185K；优选地，所述突变还包含M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R或M185K。
在一个或多个实施方案中，所述突变包含P189S，P189C和P190D，P189C和P190Y，P189N和P190Y，P189R和P190I，P189M和P190D，P189H和P190R，P189N，P189F和P190D，P189F和P190H，P189N和P190F，P189C和P190F，或P189H和P190D。在一个或多个实施方案中，所述突变还包含M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185P、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R、M185E或M185K；优选地，所述突变还包含M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R或M185K。
在一个或多个实施方案中，所述突变包含：(1)P189C和P190D、P189M和P190D、P189F和P190D，或P189H和P190D，和任选的(2)M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185P、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R、M185E或M185K。
在一个或多个实施方案中，所述突变包含：(1)P189C和P190D、P189M和P190D、或P189H和P190D，和任选的(2)M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185P、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R、M185E或M185K；或者，所述突变包含：(1)P189F和P190D，和任选的(2)M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R或M185K。
本发明光学探针中的乳酸敏感多肽可为上述具有一个或多个突变的乳酸敏感多肽。在一个或多个实施方案中，包含突变的乳酸敏感多肽的光学探针对乳酸的响应高于或低于未突变的对应物。
在示例性实施方案中，本发明光学探针可为乳酸结合蛋白功能片段的185/189位点插入有cpYFP并且具有选自以下的一个或多个突变的探针：P189R和P190D、P189R和P190A、P189R和P190I、P189R和P190Q、P189R和P190N、P189D和P190D、P189D和P190E、P189D和P190V、P189D和P190L、P189D和P190F、P189D和P190I、P189D和P190Q、P189D和P190N、P189D和P190G、P189D和P190Y、P189D和P190W、P189E和P190R、P189E和P190A、P189E和P190V、P189E和P190Q、P189A和P190L、P189A和P190F、P189A和P190M、P189A、P189A和P190N、P189A和P190G、P189A和P190H、P189A和P190T、P189V和P190D、P189V和P190E、P189V和P190A、P189V、P189V和P190N、P189V和P190H、P189V和P190Y、P189L和P190V、P189L和P190F、P189L和P190M、P189L和P190G、P189L和P190H、P189F和P190D、P189F和P190L、P189F和P190F、P189F和P190I、P189F和P190N、P189F和P190H、P189F和P190Y、P189F和P190K、P189F和P190T、P189F和P190W、P189I和P190R、P189I和P190D、P189I和P190A、P189I和P190V、P189I和P190M、P189I和P190Q、P189I和P190G、P189I和P190Y、P189I和P190S、P189I和P190T、P189M和P190R、P189M和P190D、P189M和P190E、P189M和P190F、P189M和P190G、P189M和P190S、P189M和P190W、P189C和P190D、P189C和P190E、P189C和P190F、P189C和P190I、P189C和P190M、P189C和P190C、P189C、P189C和P190H、P189C和P190Y、P189C和P190S、P189C和P190W、P190L、P190F、P190I、P190Q、P190N、P190K、P190T、P189Q和P190E、P189Q和P190A、P189Q和P190V、P189Q和P190M、P189Q和P190C、P189Q和P190Q、P189Q和P190H、P189Q和P190S、P189N和P190R、P189N和P190D、P189N和P190L、P189N和P190F、P189N和P190C、P189N、P189N和P190N、P189N和P190G、P189N和P190H、P189N和P190Y、P189N和P190T、P189G和P190V、P189G和P190F、P189G和P190M、P189G和P190C、P189G和P190G、P189G和P190H、P189G和P190K、P189G和P190W、P189H和P190R、P189H和P190D、P189H和P190E、P189H和P190L、P189H和P190S、P189Y和P190R、P189Y和P190L、P189Y和P190N、P189Y和P190H、P189Y和P190S、P189Y和P190T、P189K和P190D、P189K和P190E、P189K和P190V、P189K和P190L、P189K和P190F、P189K和P190I、P189K和P190M、P189K、P189K和P190Q、P189K和P190N、P189K和P190Y、P189K和P190K、P189K和P190T、P189S和P190E、P189S和P190A、P189S和P190L、P189S和P190F、P189S和P190M、P189S和P190C、P189S、P189S和P190Q、189S和P190Y、P189S和P190K、P189S和P190S、P189T和P190R、P189T和P190D、P189T和P190M、P189T和P190C、P189T、P189T和P190Q、P189T和P190N、P189T和P190H、P189T和P190Y、P189T和P190K、P189T和P190W、P189W和P190A、P189W和P190V、P189W和P190F、P189W、P189W和P190Q、P189W和P190H、P189W和P190S、P189W和P190T、P189W和P190W。在进一步的实施方案中，所述突变还包含M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185P、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R、M185E或M185K；优选地，所述突变还包含M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R或M185K。
在示例性实施方案中，本发明光学探针可为乳酸结合蛋白功能片段的185/189位点插入有cpYFP并且具有选自以下的一个或多个突变的探针：P189S，P189C和P190D，P189C和P190Y，P189N和P190Y，P189R和P190I，P189M和P190D，P189H和P190R，P189N，P189F和P190D，P189F和P190H，P189N和P190F，P189C和P190F，或P189H和P190D。在示例性实施方案中，所述乳酸结合蛋白功能片段是SEQ ID NO:1的第80-258位，所述突变是P189N，P189S，P189C和P190F，P189N和P190F，P189N和P190Y，P189H和P190R，P189R和P190I，P189F和P190H，P189C和P190Y，P189C和P190D，P189M和P190D，P189H和P190D，或P189F和P190D。在进一步的实施方案中，所述突变还包含M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185P、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R、M185E或M185K；优选地，所述突变还包含M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R或M185K。
在一些具体实施方案中，本发明光学探针可为乳酸结合蛋白功能片段的185/189位点插入有cpYFP并且具有突变的探针，所述乳酸结合蛋白功能片段是SEQ ID NO:1的第80-258位，所述突变包含：(1)P189C和P190D、P189M和P190D、P189F和P190D，或P189H和P190D，和任选的(2)M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185P、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R、M185E或M185K。优选地，所述突变包含：(1)P189C和P190D、P189M和P190D、或P189H和P190D，和任选的(2)M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185P、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R、M185E或M185K；或者，所述突变包含：(1)P189F和P190D，和任选的(2)M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R或M185K。
在一个实施方案中，本发明光学探针具有SEQ ID NO:18-30、34-40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:6-30、34-40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:6-30、34-40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。在优选实施方案中，本发明提供的光学探针由SEQID NO:6-30、34-40中任一所示序列组成。在更优选的实施方案中，本发明提供的光学探针包含SEQ ID NO:30、34-40或由其组成。
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。实施方案在一些实施方案中，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:6-30、34-40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SEQID NO:34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核酸构建物的细胞。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。
本发明还提供了上述乳酸光学探针的制备方法，包括以下步骤：1)将编码本文所述乳酸光学探针的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)分离乳酸光学探针。
本发明还提供检测样品中乳酸的方法，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乳酸的方法，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乳酸。
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乳酸光学探针或融合多肽或如本文所述方法制备的乳酸光学探针或融合多肽在乳酸细胞内/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：本发明提供的乳酸光学探针易于成熟，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，可在细胞内外实时原位、高通量、定量检测乳酸，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乳酸光学探针对乳酸的最高响应达到对照的13倍以上，并且可以在细胞浆、线粒体、细胞核、内质网、溶酶体和高尔基体等亚细胞结构中对细胞进行定位、定性、定量检测，并且可以进行高通量的化合物筛选以及血液中乳酸定量检测。
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1所述的示例性乳酸光学探针的SDS-PAGE图；
图2为实施例2所述的示例性的包含cpYFP和乳酸结合蛋白的乳酸光学探针对乳酸响应变化图；
图3为实施例3所述的示例性的包含cpGFP和乳酸结合蛋白的乳酸光学探针对乳酸响应变化图；
图4为实施例4所述的示例性的包含cpBFP和乳酸结合蛋白的乳酸光学探针对乳酸响应变化图；
图5为实施例5所述的示例性的包含cpmApple和乳酸结合蛋白的乳酸光学探针对乳酸响应变化图；
图6为实施例6所述的示例性的在乳酸结合蛋白的位点185/189插入cpYFP的乳酸光学探针的突变体对乳酸的响应情况；
图7A为实施例7所述的示例性的在乳酸结合蛋白的位点185/189插入有cpYFP的乳酸光学探针的突变体对不同浓度乳酸的滴定曲线；图7B为实施例8所述的乳酸光学探针的突变体对不同浓度乳酸的滴定曲线；
图8为实施例9所述的示例性乳酸光学探针的荧光光谱性质图；
图9为实施例9所述的示例性乳酸光学探针的特异性检测的柱状图；
图10为实施例10所述的示例性乳酸光学探针在哺乳动物细胞中的亚细胞器定位图片；
图11为实施例11所述的对示例性乳酸光学探针在哺乳动物细胞的乳酸跨膜运输进行动态监测的示意图；
图12为实施例12所述的示例性乳酸光学探针在活细胞水平进行高通量化合物筛选的点图；
图13为实施例13所述的示例性乳酸光学探针对小鼠和人血液中的乳酸进行定量的柱状图。
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“乳酸敏感多肽”或“乳酸响应多肽”指对乳酸产生响应的多肽，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。本文所述乳酸敏感多肽还可包括其功能变体。乳酸敏感多肽的功能变体包括但不限于可以与乳酸相互作用从而发生与亲本乳酸敏感多肽相同或相似变化的变体。
本发明所述乳酸敏感多肽包括但不限于乳酸结合蛋白LldR或与其有90％以上同源性的变体。本发明所述示例性乳酸结合蛋白LldR来源于大肠杆菌JM109。LldR是细菌转录因子由乳酸结合/调节域和DNA结合域组成。示例性LldR蛋白如SEQ ID NO:1所示。在一个或多个实施方案中，乳酸敏感多肽包含乳酸结合蛋白的功能片段(乳酸结合域)，即80-258位氨基酸。当描述本发明光学探针或乳酸结合蛋白时(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。
本文所用术语“光学探针”是指与光学活性多肽融合的乳酸敏感多肽。发明人发现，乳酸敏感多肽例如乳酸结合蛋白专一性地对生理浓度的乳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。借助不同乳酸浓度下测定的荧光蛋白的荧光绘制标准曲线，可以检测并分析乳酸的存在和/或水平。
在本发明的光学探针中，光学活性多肽(例如荧光蛋白)可操作地插入乳酸敏感多肽中。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包括其功能变体。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，本发明中乳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。
本发明所述的乳酸光学探针包括乳酸敏感多肽B，例如乳酸结合蛋白或其乳酸结合域(第80-258位)或变体，和光学活性多肽A，例如荧光蛋白。光学活性多肽A插入到乳酸敏感多肽B中，将B分为B1和B2两个部分，形成B1-A-B2式的探针结构；乳酸敏感多肽B和乳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乳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方向的乳酸敏感多肽的任何位置。具体地，光学活性多肽位于乳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乳酸结合蛋白中的插入位点所在区域，如氨基酸残基93-97、119-121、137-141、158-161、185-191、208-210和/或230-232区域。示例性地，光学活性多肽位于乳酸结合蛋白或其功能片段的选自以下的一个或多个位点：93/94，93/95，93/96，93/97，94/95，94/96，94/97，95/96，95/97，96/97，119/120，119/121，120/121，137/138，137/139，137/140，137/141，138/139，138/140，138/141，139/140，139/141，140/141，158/159，158/160，158/161，159/160，159/161，160/161，185/186，185/187，185/188，185/189，185/190，185/191，186/187，186/188，186/189，186/190，186/191，187/188，187/189，187/190，187/191，188/189，188/190，188/191，189/190，189/191，190/191，208/209，208/210，209/210，230/231，230/232或231/232处。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乳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乳酸敏感多肽的氨基酸94-96。在一个实施方案中，光学活性多肽位于乳酸结合蛋白或其功能片段的选自以下的一个或多个位点：185/186，185/187，185/188，185/189，185/190，185/191，186/187，186/188，186/189，186/190，186/191，187/188，187/189，187/190，187/191，188/189，188/190，188/191，189/190，189/191或190/191。在一个实施方案中，光学活性多肽位于乳酸结合蛋白或其功能片段的选自以下的一个或多个位点：185/186，185/187，185/188，185/189，185/190，186/187，186/188，186/189，186/190，187/189，189/191和190/191。在一个实施方案中，光学活性多肽位于乳酸结合蛋白或其功能片段的选自以下的一个或多个位点：185/189，186/189或187/189。优选地，本发明光学探针可为当cpYFP位于乳酸结合蛋白或其功能片段的185/186，185/187，185/188，185/189，185/190，185/191，186/187，186/188，186/189，186/190，186/191，187/188，187/189，187/190，187/191，188/189，188/190，188/191，189/190，189/191或190/191处的探针。在示例性实施方案中，本发明光学探针可为当cpYFP位于乳酸结合蛋白或其功能片段的185/186，185/187，185/188，185/189，185/190，186/187，186/188，186/189，186/190，187/189，189/191和190/191处的探针。在一个或多个实施方案中，所述乳酸结合蛋白的功能片段如SEQ ID NO:1第80-258位所示。在一个实施方案中，本发明光学探针具有SEQID NO:6-17所示序列或由其组成。
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。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。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、乳酸、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乳酸敏感多肽。所述突变可位于乳酸结合蛋白或其功能片段的P189和/或P190位点和任选的M185位点。
示例性地，在一个或多个实施方案中，所述突变选自P189R和P190D、P189R和P190A、P189R和P190I、P189R和P190Q、P189R和P190N、P189D和P190D、P189D和P190E、P189D和P190V、P189D和P190L、P189D和P190F、P189D和P190I、P189D和P190Q、P189D和P190N、P189D和P190G、P189D和P190Y、P189D和P190W、P189E和P190R、P189E和P190A、P189E和P190V、P189E和P190Q、P189A和P190L、P189A和P190F、P189A和P190M、P189A、P189A和P190N、P189A和P190G、P189A和P190H、P189A和P190T、P189V和P190D、P189V和P190E、P189V和P190A、P189V、P189V和P190N、P189V和P190H、P189V和P190Y、P189L和P190V、P189L和P190F、P189L和P190M、P189L和P190G、P189L和P190H、P189F和P190D、P189F和P190L、P189F和P190F、P189F和P190I、P189F和P190N、P189F和P190H、P189F和P190Y、P189F和P190K、P189F和P190T、P189F和P190W、P189I和P190R、P189I和P190D、P189I和P190A、P189I和P190V、P189I和P190M、P189I和P190Q、P189I和P190G、P189I和P190Y、P189I和P190S、P189I和P190T、P189M和P190R、P189M和P190D、P189M和P190E、P189M和P190F、P189M和P190G、P189M和P190S、P189M和P190W、P189C和P190D、P189C和P190E、P189C和P190F、P189C和P190I、P189C和P190M、P189C和P190C、P189C、P189C和P190H、P189C和P190Y、P189C和P190S、P189C和P190W、P190L、P190F、P190I、P190Q、P190N、P190K、P190T、P189Q和P190E、P189Q和P190A、P189Q和P190V、P189Q和P190M、P189Q和P190C、P189Q和P190Q、P189Q和P190H、P189Q和P190S、P189N和P190R、P189N和P190D、P189N和P190L、P189N和P190F、P189N和P190C、P189N、P189N和P190N、P189N和P190G、P189N和P190H、P189N和P190Y、P189N和P190T、P189G和P190V、P189G和P190F、P189G和P190M、P189G和P190C、P189G和P190G、P189G和P190H、P189G和P190K、P189G和P190W、P189H和P190R、P189H和P190D、P189H和P190E、P189H和P190L、P189H和P190S、P189Y和P190R、P189Y和P190L、P189Y和P190N、P189Y和P190H、P189Y和P190S、P189Y和P190T、P189K和P190D、P189K和P190E、P189K和P190V、P189K和P190L、P189K和P190F、P189K和P190I、P189K和P190M、P189K、P189K和P190Q、P189K和P190N、P189K和P190Y、P189K和P190K、P189K和P190T、P189S和P190E、P189S和P190A、P189S和P190L、P189S和P190F、P189S和P190M、P189S和P190C、P189S、P189S和P190Q、189S和P190Y、P189S和P190K、P189S和P190S、P189T和P190R、P189T和P190D、P189T和P190M、P189T和P190C、P189T、P189T和P190Q、P189T和P190N、P189T和P190H、P189T和P190Y、P189T和P190K、P189T和P190W、P189W和P190A、P189W和P190V、P189W和P190F、P189W、P189W和P190Q、P189W和P190H、P189W和P190S、P189W和P190T、P189W和P190W。在一些具体实施方案中，所述突变选自P189R和P190A，P189D和P190D，P189D和P190E，P189D和P190Q，P189D和P190Y，P189A和P190N，P189A和P190G，P189V和P190H，P189F和P190I，P189F和P190N，P189F和P190K，P189I和P190D，P189I和P190A，P189I和P190V，P189I和P190M，P189M和P190R，P189M和P190E，P189M和P190F，P189M和P190G，P189M和P190S，P189C和P190E，P190Q，P189Q和P190M，P189Q和P190C，P189N和P190N，P189G和P190F，P189H和P190L，P189H和P190S，P189Y和P190L，P189K和P190V，P189K和P190T，P189S和P190A，P189S和P190M，P189S和P190Q，P189S和P190K，P189S和P190S，P189T和P190D，P189W和P190A，P189W和P190T，或P189S，P189C和P190D，P189C和P190Y，P189N和P190Y，P189R和P190I，P189M和P190D，P189H和P190R，P189N，P189F和P190D，P189F和P190H，P189N和P190F，P189C和P190F，或P189H和P190D。包含上述突变的光学探针对乳酸的响应超过对照的1.5倍。在一些实施方式中，所述突变选自以下的一个或多个：P189S，P189C和P190D，P189C和P190Y，P189N和P190Y，P189R和P190I，P189M和P190D，P189H和P190R，P189N，P189F和P190D，P189F和P190H，P189N和P190F，P189C和P190F，或P189H和P190D。包含上述突变的光学探针对乳酸的响应超过对照的2倍。在优选实施方案中，所述突变是P189C和P190D，P189M和P190D，P189H和P190D或P189F和P190D。
在进一步的实施方案中，所述突变还包含M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185P、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R、M185E或M185K。在进一步优选的实施方案中，所述突变还包含M185F、M185Y、M185L、M185I、M185Q、M185G、M185H、M185A、M185N、M185C、M185W、M185S、M185V、M185D、M185T、M185R或M185K。
本文所用术语“功能变体”、“衍生物”和“类似物”是指基本上保持与原始多肽或蛋白(例如乳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(例如乳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，本发明的乳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。在示例性实施方式中，核酸序列如SEQ ID NO:31所示，其表示乳酸结合蛋白功能片段的185/189位点插入有cpYFP并具有P189F/P190D突变的探针的编码序列。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。该试剂盒还任选包含利用光学探针检测乳酸所需的其他试剂。本领域知晓常规的所述其他试剂。
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。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，分离得到乳酸光学探针。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。本发明对分离所述乳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乳酸光学探针在乳酸实时定位、定量检测以及高通量化合物筛选中的应用。在一个方面，所述的乳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，进行乳酸的实时定位；通过乳酸标准滴加曲线进行相应乳酸的定量检测。本发明所述的乳酸标准滴加曲线是根据乳酸光学探针在不同浓度乳酸的情况下的荧光信号绘制而成。本发明所述乳酸光学探针直接转入细胞中，在乳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。本发明乳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，测定乳酸含量的变化，从而筛选出对乳酸含量变化有影响的化合物。在本发明中所述的乳酸光学探针在乳酸实时定位、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乳酸光学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pCDFDuet-cpYFP，pCDFDuet-乳酸结合蛋白质粒由华东理工大学蛋白质实验室构建，pCDFDuet质粒载体购自Novagen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。乳酸等化合物均购自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，硫酸链霉素和嘌呤霉素购自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包括：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.3DNA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)、1mL10×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.将表达载体转化到JM109(DE3)细胞中，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24-36小时。
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.纯化的蛋白经过SDS-PAGE鉴定后，使用测定缓冲液(100mM HEPES，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将乳酸配制成终浓度为1M的储液。
5.取100μl 5μM的蛋白溶液，37℃温育5分钟，分别加入乳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的蛋白溶液，37℃温育5分钟，加入乳酸滴定，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的蛋白溶液，37℃温育5分钟，加入乳酸，测定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pCDNA3.1+为基础的乳酸光学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，乳酸结合蛋白质粒
通过PCR扩增大肠杆菌基因中的LldR(80-258)基因，PCR产物凝胶电泳后回收后用BamHI和EcoRI酶切，同时对pCDFDuet1载体进行相应的双酶切。用T4 DNA连接酶连接后，用产物转化MachI，转化的MachI涂布于LB平板(硫酸链霉素50ug/mL)，置于37℃培养过夜。将生长MachI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pCDFDuet-LldR(80-258)为基础根据乳酸结合蛋白晶体结构选择了下述位点插入cpYFP，得到相应pCDFDuet-LldR(80-258)-cpYFP质粒：93/94，93/95，93/96，93/97，94/95，94/96，94/97，95/96，95/97，96/97，119/120，119/121，120/121，137/138，137/139，137/140，137/141，138/139，138/140，138/141，139/140，139/141，140/141，158/159，158/160，158/161，159/160，159/161，160/161，185/186，185/187，185/188，185/189，185/190，185/191，186/187，186/188，186/189，186/190，186/191，187/188，187/189，187/190，187/191，188/189，188/190，188/191，189/190，189/191，190/191，208/209，208/210，209/210，230/231，230/232或231/232。示例性的光学探针的氨基酸序列如表1所示。
表1，光学探针的序列
Figure BDA0002848026110000251
Figure BDA0002848026110000261
利用PCR产生cpYFP的DNA片段，同时通过反向PCR扩增产生含有不同断裂位点的pCDFDuet-LldR(80-258)线性化载体，将线性化的pCDFDuet-LldR(80-258)和cpYFP片段在同源重组酶的作用下连接产生重组质粒，通过菌落PCR，选取阳性克隆，由上海杰李生物技术有限公司完成测序。
经过测序正确后，将重组质粒转化到BL21(DE3)中诱导表达，并纯化蛋白质，通过SDS-PAGE电泳大小在48Kda附近。该大小符合pCDFDuet-LldR(80-258)-cpYFP表达出的含His-tag纯化标签的LldR(80-258)-cpYFP融合蛋白质的大小。结果如图1所示。
将纯化的LldR(80-258)-cpYFP融合蛋白质和对照蛋白cpYFP进行乳酸响应检测，将含有10mM乳酸的融合荧光蛋白质的检测信号除以无乳酸的融合荧光蛋白质的检测信号。结果如图2所示，检测结果显示对乳酸响应超过1.5倍的光学探针有在185/186，185/187，185/188，185/189，185/190，186/187，186/188，186/189，186/190，187/189，189/191和190/191位点或者其家族蛋白的对应氨基酸位点实施插入的光学探针。
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2中的方法将cpYFP替换为cpGFP，构建乳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。如图3所示，检测结果显示对乳酸响应超过1.5倍的光学探针有在188/190，和189/190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2中的方法将cpYFP替换为cpBFP，构建乳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。如图4所示，检测结果显示对乳酸响应超过1.5倍的光学探针有在187/190，和187/191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2中的方法将cpYFP替换为cpmApple，构建乳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。如图5所示，检测结果显示对乳酸响应超过1.5倍的光学探针有在185/190，185/191，186/190，186/191，187/189和188/191位点。
实施例6，光学探针双位点突变体的表达和检测
在LldR(80-258)-185/189-cpYFP的基础上构建189和190两个位点的突变体。通过反向PCR线性化质粒pCDFDuet-LldR(80-258)-185/189-cpYFP，引物中引入突变位点的突变序列，对得到的PCR产物在PNK、T4 DNA连接酶和PEG4000的作用下加磷连接，转化BL21(DE3)，然后进行筛选。将融合蛋白和对照蛋白cpYFP含有10mM乳酸的检测信号除以无乳酸的检测信号，各个突变体对乳酸响应情况如表2所示。其中对乳酸响应超过2倍的样品为P189/P190(WT)，P189S(12H4)，P189C/P190D(14B5)，P189C/P190Y(14D2)，P189N/P190Y(3D1)，P189R/P190I(14H5)，P189M/P190D(10F4)，P189H/P190R(13H6)，P189N(2F5)，P189F/P190D(3C2)，P189F/P190H(2G3)，P189N/P190F(9B6)，P189C/P190F(2A5)和P189H/P190D(1A3)。示例性的光学探针突变体的序列如表3所示。示例性的核酸序列如SEQ ID NO:31(LldR(80-258)-185/189-P189F/P190D-cpYFP)所示。
表2，189与190突变体对乳酸的响应
Figure BDA0002848026110000271
表3
Figure BDA0002848026110000272
Figure BDA0002848026110000281
实施例7，双位点突变体的光学探针的滴定曲线
对于实施例6中所得的对乳酸响应超过2倍的光学探针，即P189/P190(WT)，P189N(2F5)，P189C/P190F(2A5)，P189N/P190F(9B6)，P189N/P190Y(3D1)，P189H/P190R(13H6)，P189R/P190I(14H5)，P189F/P190H(2G3)，P189S(12H4)，P189C/P190Y(14D2)，P189C/P190D(14B5)，P189M/P190D(10F4)，P189H/P190D(1A3)和P189F/P190D(3C2)进行浓度梯度的乳酸检测，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。野生型WT及突变体2F5，2A5，9B6，3D1，13H6，14H5，2G3，12H4，14D2，14B5，10F4，1A3和3C2的Kd(结合常数)分别为112μM、67μM、114μM、46μM、95μM、194μM、70μM、152μM、88μM、41μM、57μM、234μM、100μM、和95μM，变化幅度分别为3.4倍、2.1倍、2.3倍、2.5倍、3.1倍、3.2倍、3.5倍、3.7倍、3.8倍、4.3倍、5.3倍、7.0倍、8.9倍和13.4倍，结果如图7A所示。
实施例8，光学探针三位点突变体的表达和检测
对于实施例7中基于189和190两个位点突变体中，对乳酸响应超过5倍的光学探针为P189C/P190D(14B5)、P189M/P190D(10F4)、P189F/P190D(3C2)和P189H/P190D(1A3)。在这四个突变体的基础上对M185位点进行饱和突变，全部突变体对乳酸响应情况如表4所示。其中对乳酸响应超过2倍的样品为M185F/P189F/P190D、M185I/P189F/P190D、M185G/P189F/P190D、M185H/P189F/P190D、M185A/P189F/P190D、M185S/P189F/P190D、M185V/P189F/P190D、M185F/P189H/P190D、M185Y/P189H/P190D、M185L/P189H/P190D、M185I/P189H/P190D、M185G/P189H/P190D、M185Q/P189H/P190D、M185N/P189H/P190D、M185C/P189H/P190D、M185W/P189H/P190D、M185S/P189H/P190D、M185V/P189H/P190D、M185D/P189H/P190D、M185T/P189H/P190D、M185E/P189H/P190D、M185F/P189M/P190D、M185Y/P189M/P190D、M185L/P189M/P190D、M185I/P189M/P190D、M185G/P189M/P190D、M185Q/P189M/P190D、M185H/P189M/P190D、M185A/P189M/P190D、M185C/P189M/P190D、M185W/P189M/P190D、M185S/P189M/P190D、M185V/P189M/P190D、M185T/P189M/P190D、M185E/P189M/P190D、M185F/P189C/P190D、M185Y/P189C/P190D、M185L/P189C/P190D、M185I/P189C/P190D、M185G/P189C/P190D、M185Q/P189C/P190D、M185H/P189C/P190D、M185A/P189C/P190D、M185P/P189C/P190D、M185N/P189C/P190D、M185C/P189C/P190D、M185W/P189C/P190D、M185S/P189C/P190D、M185V/P189C/P190D、M185D/P189C/P190D、M185T/P189C/P190D、M185E/P189C/P190D、M185K/P189C/P190D。对乳酸响应超过5倍的样品为：M185F/P189H/P190D、M185L/P189H/P190D、M185I/P189H/P190D、M185S/P189H/P190D、M185V/P189H/P190D、M185L/P189M/P190D、M185A/P189M/P190D。
示例性的光学探针突变体的序列如表5所示，对这9个样品进行浓度梯度的乳酸检测，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。M185F/P189H/P190D、M185L/P189H/P190D(G9)、M185I/P189H/P190D、M185S/P189H/P190D、M185V/P189H/P190D、M185L/P189M/P190D、M185A/P189M/P190D(G25)的Kd(结合常数)分别为61μM、154μM、42μM、660μM、210μM、62μM、1000μM，变化幅度分别为8倍、15倍、5.6倍、7倍、6.2倍、5.5倍、8.4倍，结果如图7B所示。
表4，三位点突变体对乳酸的响应
Figure BDA0002848026110000291
表5
Figure BDA0002848026110000292
Figure BDA0002848026110000301
实施例9，光学探针的光谱性能和特异性
示例性地，将纯化的乳酸光学探针3C2、10F4和G9分别进行0mM和10mM乳酸处理10分钟后，使用荧光分光光度计进行荧光谱的检测。
对激发光谱的测定：以350nm至515nm的激发范围和530nm的发射波长记录激发光谱，每5nm读取一次。结果显示，探针3C2、10F4和G9在约420和500nm处有两个激发峰，如图8A、8D和8G所示。
对发射光谱的测定：固定激发波长分别为420nm和490nm，记录500-600nm的发射光谱，每5nm读取一次。结果显示，探针3C2在添加10mM乳酸后，在420nm激发下荧光强度降低为添加0mM乳酸的0.31倍；在490nm激发下荧光强度增强为添加0mM乳酸的4.2倍，如图8B和8C所示。探针10F4在420nm激发下荧光强度降低为添加0mM乳酸的0.37倍；在490nm激发下荧光强度增强为添加0mM乳酸的3倍，如图8E和8F所示。探针G9在添加10mM乳酸后，在420nm激发下荧光强度降低为添加0mM乳酸的0.18倍；在490nm激发下荧光强度增强为添加0mM乳酸的2.8倍，如图8H和8I所示。
对纯化的乳酸光学探针3C2、10F4、G9和G25测定其特异性，结果表明探针具有很好的特异性，如图9所示。
实施例10，光学探针的亚细胞器定位
本实施例中，使用不同的定位信号肽与光学探针融合，将光学探针定位到不同的细胞器中。
用融合不同定位信号肽的光学探针质粒转染HeLa细胞36小时后，使用PBS冲洗，置于HBSS溶液中使用倒置荧光显微镜进行FITC通道下荧光检测。结果如图10所示。乳酸光学探针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、细胞外膜、细胞核、内质网、线粒体、核排阻等亚细胞器中。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11，乳酸跨膜运输的动态监测
用胞浆表达的光学探针质粒转染HeLa细胞36小时后，使用PBS冲洗，置于HBSS溶液中，然后添加10mM乳酸，检测30min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，添加乳酸的样品的485/420逐渐增加，3C2和10F4最高可以分别达到4.8和3.1倍，而不添加乳酸的对照组的485/420为1保持不变。
实施例12，在活细胞中基于光学探针进行高通量化合物筛选
本实施例中，我们使用胞浆表达乳酸探针3C2的HeLa细胞进行高通量化合物筛选。
经转染的HeLa细胞使用PBS冲洗，置于HBSS溶液中(无乳酸)处理1小时，然后使用10μM的化合物处理1小时。各样品中分别滴加乳酸。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作为对照进行标准化。结果如图12所示。在使用的2000种化合物中，绝大部分的化合物对乳酸进入细胞影响极小。有12种化合物能够提高细胞对乳酸的摄取能力，另外有6种化合物能够明显降低细胞对乳酸的摄取。
实施例13，光学探针定量检测血液中的乳酸
在本实施例中，我们使用纯化的乳酸探针3C2对小鼠和人的血液上清中的乳酸进行分析。
将乳酸探针3C2与稀释的血液上清混合处理10分钟后，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，小鼠血液中的乳酸含量在2.4mM左右，人血液中的乳酸含量在1.7mM左右。
由以上实施例可知，本发明提供的乳酸光学探针，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、定量检测乳酸；并且能够进行高通量的化合物筛选。
其它实施方案
本说明书描述了许多实施方案。然而应理解，本领域技术人员通过阅读本说明书获知的不背离本发明的构思和范围的各种改进，也应包括在所附权利要求书的范围内。
序列表
<110> 华东理工大学
<120> 乳酸光学探针及其制备方法和应用
<130> 20A578
<141> 2020-12-21
<150> 201911318003.9
<151> 2019-12-19
<160> 40
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Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Leu Val Pro Pro Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala
355 360 365
Val Ile Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala
370 375 380
Met Met Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp
385 390 395 400
Glu Asp Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His
405 410 415
Asn Glu His Ser Arg Glu Lys Asn Ala
420 425
<210> 8
<211> 424
<212> PRT
<213> Artificial Sequence
<400> 8
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Val Pro Pro Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val
355 360 365
Ile Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met
370 375 380
Met Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu
385 390 395 400
Asp Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn
405 410 415
Glu His Ser Arg Glu Lys Asn Ala
420
<210> 9
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 9
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Pro Pro Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 10
<211> 422
<212> PRT
<213> Artificial Sequence
<400> 10
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Pro Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile Asp
355 360 365
Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met Ala
370 375 380
His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp Gln
385 390 395 400
Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu His
405 410 415
Ser Arg Glu Lys Asn Ala
420
<210> 11
<211> 426
<212> PRT
<213> Artificial Sequence
<400> 11
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Tyr Asn Ser Asp
100 105 110
Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn
115 120 125
Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp
130 135 140
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
145 150 155 160
Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn
165 170 175
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
180 185 190
Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly
195 200 205
Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile
210 215 220
Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser
225 230 235 240
Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu
245 250 255
Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
260 265 270
Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met
275 280 285
Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln
290 295 300
Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala
305 310 315 320
Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys
325 330 335
Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
340 345 350
Tyr Asn Leu Val Pro Pro Val Phe Ser Gln Leu Thr Glu Gln His Gln
355 360 365
Ala Val Ile Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys
370 375 380
Ala Met Met Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe
385 390 395 400
Asp Glu Asp Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu
405 410 415
His Asn Glu His Ser Arg Glu Lys Asn Ala
420 425
<210> 12
<211> 425
<212> PRT
<213> Artificial Sequence
<400> 12
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Tyr Asn Ser Asp
100 105 110
Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn
115 120 125
Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp
130 135 140
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
145 150 155 160
Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn
165 170 175
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
180 185 190
Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly
195 200 205
Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile
210 215 220
Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser
225 230 235 240
Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu
245 250 255
Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
260 265 270
Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met
275 280 285
Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln
290 295 300
Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala
305 310 315 320
Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys
325 330 335
Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
340 345 350
Tyr Asn Val Pro Pro Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala
355 360 365
Val Ile Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala
370 375 380
Met Met Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp
385 390 395 400
Glu Asp Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His
405 410 415
Asn Glu His Ser Arg Glu Lys Asn Ala
420 425
<210> 13
<211> 424
<212> PRT
<213> Artificial Sequence
<400> 13
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Tyr Asn Ser Asp
100 105 110
Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn
115 120 125
Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp
130 135 140
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
145 150 155 160
Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn
165 170 175
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
180 185 190
Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly
195 200 205
Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile
210 215 220
Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser
225 230 235 240
Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu
245 250 255
Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
260 265 270
Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met
275 280 285
Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln
290 295 300
Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala
305 310 315 320
Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys
325 330 335
Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
340 345 350
Tyr Asn Pro Pro Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val
355 360 365
Ile Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met
370 375 380
Met Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu
385 390 395 400
Asp Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn
405 410 415
Glu His Ser Arg Glu Lys Asn Ala
420
<210> 14
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 14
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Tyr Asn Ser Asp
100 105 110
Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn
115 120 125
Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp
130 135 140
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
145 150 155 160
Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn
165 170 175
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
180 185 190
Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly
195 200 205
Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile
210 215 220
Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser
225 230 235 240
Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu
245 250 255
Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
260 265 270
Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met
275 280 285
Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln
290 295 300
Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala
305 310 315 320
Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys
325 330 335
Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
340 345 350
Tyr Asn Pro Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 15
<211> 425
<212> PRT
<213> Artificial Sequence
<400> 15
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Leu Tyr Asn Ser
100 105 110
Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala
115 120 125
Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala
130 135 140
Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu
145 150 155 160
Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro
165 170 175
Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala
180 185 190
Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser
195 200 205
Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro
210 215 220
Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val
225 230 235 240
Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys
245 250 255
Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val
260 265 270
Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His
275 280 285
Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val
290 295 300
Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg
305 310 315 320
Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu
325 330 335
Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu
340 345 350
Glu Tyr Asn Pro Pro Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala
355 360 365
Val Ile Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala
370 375 380
Met Met Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp
385 390 395 400
Glu Asp Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His
405 410 415
Asn Glu His Ser Arg Glu Lys Asn Ala
420 425
<210> 16
<211> 425
<212> PRT
<213> Artificial Sequence
<400> 16
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Leu Val Pro Tyr
100 105 110
Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile
115 120 125
Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln
130 135 140
Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val
145 150 155 160
Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys
165 170 175
Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr
180 185 190
Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly
195 200 205
Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val
210 215 220
Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe
225 230 235 240
Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr
245 250 255
Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr
260 265 270
Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro
275 280 285
Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly
290 295 300
Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys
305 310 315 320
Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile
325 330 335
Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His
340 345 350
Lys Leu Glu Tyr Asn Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala
355 360 365
Val Ile Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala
370 375 380
Met Met Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp
385 390 395 400
Glu Asp Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His
405 410 415
Asn Glu His Ser Arg Glu Lys Asn Ala
420 425
<210> 17
<211> 426
<212> PRT
<213> Artificial Sequence
<400> 17
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Leu Val Pro Pro
100 105 110
Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly
115 120 125
Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val
130 135 140
Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
145 150 155 160
Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser
165 170 175
Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val
180 185 190
Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp
195 200 205
Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly
210 215 220
Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys
225 230 235 240
Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu
245 250 255
Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
260 265 270
Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr
275 280 285
Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu
290 295 300
Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr
305 310 315 320
Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg
325 330 335
Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly
340 345 350
His Lys Leu Glu Tyr Asn Val Phe Ser Gln Leu Thr Glu Gln His Gln
355 360 365
Ala Val Ile Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys
370 375 380
Ala Met Met Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe
385 390 395 400
Asp Glu Asp Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu
405 410 415
His Asn Glu His Ser Arg Glu Lys Asn Ala
420 425
<210> 18
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 18
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Asn Pro Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 19
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 19
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Ser Pro Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 20
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 20
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Cys Phe Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 21
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 21
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Asn Phe Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 22
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 22
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Asn Tyr Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 23
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 23
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn His Arg Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 24
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 24
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Arg Ile Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 25
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 25
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Phe His Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 26
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 26
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Cys Tyr Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 27
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 27
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Cys Asp Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 28
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 28
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Met Asp Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 29
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 29
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn His Asp Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 30
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 30
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Met Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Phe Asp Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 31
<211> 1272
<212> DNA
<213> Artificial Sequence
<400> 31
atggagcaaa acatcgtcca gccgctaaaa acactgatgg ccgatgatcc ggattacagt 60
ttcgatattc tggaagcccg ctacgccatt gaagccagca ccgcatggca tgcggcaatg 120
cgcgccacac ctggcgacaa agaaaagatt cagctttgct ttgaagcaac gctaagtgaa 180
gacccggata tcgcctcaca agcggacgtt cgttttcatc tggcgattgc cgaagcctca 240
cataacatcg tgctgctgca aaccatgcgc ggtttcttcg atgtcctgca atcctcagtg 300
aagcatagcc gtcagcggat gtacaacagc gacaacgtct atatcatggc cgacaagcag 360
aagaacggca tcaaggccaa cttcaagatc cgccacaacg tcgaggacgg cagcgtgcag 420
ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 480
aaccactacc tgagcttcca gtccgtcctg agcaaagacc ccaacgagaa gcgcgatcac 540
atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac 600
aacgtggatg gcggtagcgg tggcaccggc agcaagggcg aggagctgtt caccggggtg 660
gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 720
gagggcgagg gcgatgccac ctacggcaag ctgaccctga agctgatctg caccaccggc 780
aagctgcccg tgccctggcc caccctcgtg accaccctcg gctacggcct gaagtgcttc 840
gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 900
tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 960
gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 1020
gaggacggca acatcctggg gcacaagctg gagtacaact ttgatgtttt ttcacaactg 1080
accgaacaac atcaggctgt cattgacgcc atttttgccg gtgatgctga cggggcgcgt 1140
aaagcaatga tggcgcacct tagttttgtt cacaccacca tgaaacgatt cgatgaagat 1200
caggctcgcc acgcacggat tacccgcctg cccggtgagc ataatgagca ttcgagggag 1260
aaaaacgcat aa 1272
<210> 32
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 32
nnknnkgttt tttcacaact gaccgaac 28
<210> 33
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 33
gttgtactcc agcttgtgcc ccag 24
<210> 34
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 34
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Phe Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn His Asp Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 35
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 35
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Leu Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn His Asp Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 36
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 36
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Ile Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn His Asp Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 37
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 37
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Ser Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn His Asp Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 38
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 38
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Val Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn His Asp Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 39
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 39
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Leu Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Met Asp Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420
<210> 40
<211> 423
<212> PRT
<213> Artificial Sequence
<400> 40
Met Glu Gln Asn Ile Val Gln Pro Leu Lys Thr Leu Met Ala Asp Asp
1 5 10 15
Pro Asp Tyr Ser Phe Asp Ile Leu Glu Ala Arg Tyr Ala Ile Glu Ala
20 25 30
Ser Thr Ala Trp His Ala Ala Met Arg Ala Thr Pro Gly Asp Lys Glu
35 40 45
Lys Ile Gln Leu Cys Phe Glu Ala Thr Leu Ser Glu Asp Pro Asp Ile
50 55 60
Ala Ser Gln Ala Asp Val Arg Phe His Leu Ala Ile Ala Glu Ala Ser
65 70 75 80
His Asn Ile Val Leu Leu Gln Thr Met Arg Gly Phe Phe Asp Val Leu
85 90 95
Gln Ser Ser Val Lys His Ser Arg Gln Arg Ala Tyr Asn Ser Asp Asn
100 105 110
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
115 120 125
Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His
130 135 140
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
145 150 155 160
Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu
165 170 175
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
180 185 190
Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly
195 200 205
Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
210 215 220
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
225 230 235 240
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
245 250 255
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
260 265 270
Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys
275 280 285
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
290 295 300
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
305 310 315 320
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
325 330 335
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
340 345 350
Asn Met Asp Val Phe Ser Gln Leu Thr Glu Gln His Gln Ala Val Ile
355 360 365
Asp Ala Ile Phe Ala Gly Asp Ala Asp Gly Ala Arg Lys Ala Met Met
370 375 380
Ala His Leu Ser Phe Val His Thr Thr Met Lys Arg Phe Asp Glu Asp
385 390 395 400
Gln Ala Arg His Ala Arg Ile Thr Arg Leu Pro Gly Glu His Asn Glu
405 410 415
His Ser Arg Glu Lys Asn Ala
420

Claims (11)

1.一种光学探针，包含乳酸敏感多肽或其突变体和光学活性多肽，其中光学活性多肽位于乳酸敏感多肽或其突变体的序列内，其中，
乳酸敏感多肽如SEQ ID NO:1的第80-258位氨基酸所示或如SEQ ID NO:1所示，
所述乳酸敏感多肽突变体的突变为：P189R和P190D、P189R和P190A、P189R和P190I、P189R和P190Q、P189R和P190N、P189D和P190D、P189D和P190E、P189D和P190V、P189D和P190L、P189D和P190F、P189D和P190I、P189D和P190Q、P189D和P190N、P189D和P190G、P189D和P190Y、P189D和P190W、P189E和P190R、P189E和P190A、P189E和P190V、P189E和P190Q、P189A和P190L、P189A和P190F、P189A和P190M、P189A、P189A和P190N、P189A和P190G、P189A和P190H、P189A和P190T、P189V和P190D、P189V和P190E、P189V和P190A、P189V、P189V和P190N、P189V和P190H、P189V和P190Y、P189L和P190V、P189L和P190F、P189L和P190M、P189L和P190G、P189L和P190H、P189F和P190D、P189F和P190L、P189F和P190F、P189F和P190I、P189F和P190N、P189F和P190H、P189F和P190Y、P189F和P190K、P189F和P190T、P189F和P190W、P189I和P190R、P189I和P190D、P189I和P190A、P189I和P190V、P189I和P190M、P189I和P190Q、P189I和P190G、P189I和P190Y、P189I和P190S、P189I和P190T、P189M和P190R、P189M和P190D、P189M和P190E、P189M和P190F、P189M和P190G、P189M和P190S、P189M和P190W、P189C和P190D、P189C和P190E、P189C和P190F、P189C和P190I、P189C和P190M、P189C和P190C、P189C、P189C和P190H、P189C和P190Y、P189C和P190S、P189C和P190W、P190L、P190F、P190I、P190Q、P190N、P190K、P190T、P189Q和P190E、P189Q和P190A、P189Q和P190V、P189Q和P190M、P189Q和P190C、P189Q和P190Q、P189Q和P190H、P189Q和P190S、P189N和P190R、P189N和P190D、P189N和P190L、P189N和P190F、P189N和P190C、P189N、P189N和P190N、P189N和P190G、P189N和P190H、P189N和P190Y、P189N和P190T、P189G和P190V、P189G和P190F、P189G和P190M、P189G和P190C、P189G和P190G、P189G和P190H、P189G和P190K、P189G和P190W、P189H和P190R、P189H和P190D、P189H和P190E、P189H和P190L、P189H和P190S、P189Y和P190R、P189Y和P190L、P189Y和P190N、P189Y和P190H、P189Y和P190S、P189Y和P190T、P189K和P190D、P189K和P190E、P189K和P190V、P189K和P190L、P189K和P190F、P189K和P190I、P189K和P190M、P189K、P189K和P190Q、P189K和P190N、P189K和P190Y、P189K和P190K、P189K和P190T、P189S和P190E、P189S和P190A、P189S和P190L、P189S和P190F、P189S和P190M、P189S和P190C、P189S、P189S和P190Q、P189S和P190Y、P189S和P190K、P189S和P190S、P189T和P190R、P189T和P190D、P189T和P190M、P189T和P190C、P189T、P189T和P190Q、P189T和P190N、P189T和P190H、P189T和P190Y、P189T和P190K、P189T和P190W、P189W和P190A、P189W和P190V、P189W和P190F、P189W、P189W和P190Q、P189W和P190H、P189W和P190S、P189W和P190T、P189W和P190W，M185F和P189F和P190D、M185Y和P189F和P190D、M185L和P189F和P190D、M185I和P189F和P190D、M185G和P189F和P190D、M185Q和P189F和P190D、M185H和P189F和P190D、M185A和P189F和P190D、M185P和P189F和P190D、M185N和P189F和P190D、M185C和P189F和P190D、M185W和P189F和P190D、M185S和P189F和P190D、M185V和P189F和P190D、M185D和P189F和P190D、M185T和P189F和P190D、M185R和P189F和P190D、M185K和P189F和P190D、M185F和P189H和P190D、M185Y和P189H和P190D、M185L和P189H和P190D、M185I和P189H和P190D、M185G和P189H和P190D、M185Q和P189H和P190D、M185H和P189H和P190D、M185A和P189H和P190D、M185P和P189H和P190D、M185N和P189H和P190D、M185C和P189H和P190D、M185W和P189H和P190D、M185S和P189H和P190D、M185V和P189H和P190D、M185D和P189H和P190D、M185T和P189H和P190D、M185R和P189H和P190D、M185E和P189H和P190D、M185K和P189H和P190D、M185F和P189M和P190D、M185Y和P189M和P190D、M185L和P189M和P190D、M185I和P189M和P190D、M185G和P189M和P190D、M185Q和P189M和P190D、M185H和P189M和P190D、M185A和P189M和P190D、M185P和P189M和P190D、M185N和P189M和P190D、M185C和P189M和P190D、M185W和P189M和P190D、M185S和P189M和P190D、M185V和P189M和P190D、M185D和P189M和P190D、M185T和P189M和P190D、M185R和P189M和P190D、M185E和P189M和P190D、M185K和P189M和P190D、M185F和P189C和P190D、M185Y和P189C和P190D、M185L和P189C和P190D、M185I和P189C和P190D、M185G和P189C和P190D、M185Q和P189C和P190D、M185H和P189C和P190D、M185A和P189C和P190D、M185P和P189C和P190D、M185N和P189C和P190D、M185C和P189C和P190D、M185W和P189C和P190D、M185S和P189C和P190D、M185V和P189C和P190D、M185D和P189C和P190D、M185T和P189C和P190D、M185R和P189C和P190D、M185E和P189C和P190D、或M185K和P189C和P190D，
光学活性多肽位于乳酸敏感多肽的选自以下的一个或多个位点：185/186，185/187，185/188，185/189，185/190，185/191，186/187，186/188，186/189，186/190，186/191，187/189，187/190，187/191，188/190，188/191，189/190，189/191和190/191。
2.如权利要求1所述的光学探针，其中，所述光学探针的氨基酸序列如SEQ ID NO:6-30、34-40中任一项所示。
3.一种核酸分子，其：
（1）编码权利要求1或2所述的光学探针；或
（2）是（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、权利要求5所述的核酸构建物在制备检测样品中的乳酸的试剂盒中的应用。
9.如权利要求8所述的应用，其特征在于，所述检测是乳酸定位或定量检测。
10.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、权利要求4或5所述的核酸构建物在筛选化合物中的应用，所述化合物为对乳酸含量的变化有影响的化合物。
11.一种检测试剂盒，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；或
（2）权利要求3所述的核酸分子；或
（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
利用光学探针检测乳酸所需的其他试剂。
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