CN114423871A

CN114423871A - 使用多(a)和多(u)聚合酶无模板酶促合成多核苷酸

Info

Publication number: CN114423871A
Application number: CN202080055871.7A
Authority: CN
Inventors: 蒂尔曼·海尼施; 伊莉斯·钱皮恩; 埃洛迪·苏尼; 米哈伊尔·索斯金
Original assignee: DNA Script SAS
Current assignee: DNA Script SAS
Priority date: 2019-07-30
Filing date: 2020-07-28
Publication date: 2022-04-29
Also published as: US20220403434A1; AU2020319829A1; KR20220052937A; JP2022543569A; CA3145783A1; EP4004222A1; WO2021018919A1; IL290127A

Abstract

本发明涉及使用多(A)和多(U)聚合酶从3’‑O‑可逆封闭的核苷三磷酸无模板酶促合成具有预定序列的多核糖核苷酸的方法和组合物。

Description

使用多(A)和多(U)聚合酶无模板酶促合成多核苷酸

背景

人们对通过使用无模板聚合酶(诸如末端脱氧核苷酸转移酶(TdT))的基于酶促的方法来补充或替代基于化学的合成方法产生了兴趣，因为这样的酶已被证明有效，并且具有反应条件温和无毒的益处，例如Ybert等人，国际专利公布WO2015/159023；Hiatt等人，美国专利5763594；Jensen等人,Biochemistry，57：1821-1832(2018)；等等。大多数基于酶的合成方法限于DNA合成并且需要使用可逆封闭的核苷三磷酸，以便在多核苷酸产物中获得期望的序列。遗憾地是，与未修饰的核苷三磷酸相比，天然TdT掺入这样的修饰的核苷三磷酸的效率降低。因此，已经有大量研究涉及开发新的对修饰的核苷三磷酸具有更好的掺入效率的TdT变体以合成DNA，例如Champion等人，美国专利公布US2019/0211315；Ybert等人，国际专利公布WO2017/216472等。

鉴于以上情况，如果新的无模板聚合酶可用于合成DNA和RNA并改进可逆封闭的核苷三磷酸的掺入，那么基于无模板酶促的多核苷酸合成领域将得到发展。

发明概述

本发明涉及使用多(A)和多(U)聚合酶及其变体进行多核苷酸的无模板酶促合成的方法、试剂盒和组合物。在一些实施方案中，采用多(A)和多(U)聚合酶的方法用于合成具有预定序列的RNA产物。在其他实施方案中，采用多(A)和多(U)聚合酶的方法用于合成具有预定序列的DNA产物。

在一些实施方案中，本发明涉及合成具有预定序列的多核苷酸的方法，该方法包括以下步骤：a)提供3’-末端核苷酸具有游离3’-羟基的起始子；以及b)重复以下的循环直至形成所述多核苷酸：(i)在延长条件下使具有游离3’-羟基的起始子或延长片段与3’-O-封闭的-核苷三磷酸和无模板聚合酶接触，使得起始子或延长片段通过3’-O-封闭的-核苷三磷酸的掺入被延长以形成3’-O-封闭的-延长片段，和(ii)使延长片段去封闭以形成具有游离3’-羟基的延长片段；其中无模板聚合酶是多(A)聚合酶(PAP)或多(U)聚合酶。在另外的实施方案中，起始子可以通过5’端附接至支持物。在另外的实施方案中，支持物可以是固体支持物。

在一些实施方案中，上述方法可以包括从起始子裂解多核苷酸的步骤。

在一些实施方案中，多核苷酸是多-2’-脱氧核糖核苷酸并且3’-O-封闭的-核苷三磷酸是3’-O-封闭的-2’-脱氧核糖核苷三磷酸。在另外的实施方案中，3’-O-封闭的-2’-脱氧核糖核苷酸三磷酸是3’-O-叠氮甲基-2’-脱氧核糖核苷三磷酸或3’-O-氨基-2’-脱氧核糖核苷三磷酸。在另外的实施方案中，多核苷酸是多核糖核苷酸并且所述3’-O-封闭的-核苷三磷酸是3’-O-封闭的-核糖核苷三磷酸。在一些实施方案中，3’-O-封闭的-核糖核苷三磷酸是3’-O-叠氮甲基-核糖核苷三磷酸。在另外的实施方案中，3’-叠氮甲基-O-核糖核苷三磷酸选自由以下组成的组：3’-叠氮甲基-O-腺苷三磷酸、3’-叠氮甲基-O-鸟苷三磷酸和3’-叠氮甲基-O-胞苷三磷酸、3’-叠氮甲基-O-尿苷三磷酸。

在一些实施方案中，本发明的方法中采用的多(A)聚合酶是这样的多(A)聚合酶变体，该多(A)聚合酶变体包含与选自SEQ ID NO:1、2、3、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26或27的氨基酸序列至少90％相同的氨基酸序列和以下位置处的甲硫氨酸的取代：关于SEQ ID NO:1的位置310处，关于SEQ ID NO:2的位置318处，关于SEQID NO:3的位置318处，关于SEQ ID NO:8的位置316处，关于SEQ ID NO:9的位置309处，关于SEQ ID NO:10的位置316处，关于SEQ ID NO:11的位置272处，关于SEQ ID NO:12的位置316处，关于SEQ ID NO:13的位置307处，关于SEQ ID NO:14的位置313处，关于SEQ ID NO:15的位置312处，关于SEQ ID NO:16的位置317处，关于SEQ ID NO:17的位置316处，关于SEQ IDNO:18的位置316处，关于SEQ ID NO:19的位置312处，关于SEQ ID NO:20的位置310处，关于SEQ ID NO:21的位置309处，关于SEQ ID NO:22的位置317处，关于SEQ ID NO:23的位置314处，关于SEQ ID NO:24的位置307处，关于SEQ ID NO:25的位置315处，关于SEQ ID NO:26的位置316处和关于SEQ ID NO:27的位置311处。特别地，所述位置处的所述甲硫氨酸的所述取代可以选自F、Y、V、E或T。选自所述SEQ ID NO:1、2、3、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26或27的所述氨基酸序列还可以包含以下位置处的缬氨酸的取代：关于SEQ ID NO:1的位置234处，关于SEQ ID NO:2的位置240处，关于SEQ ID NO:3的位置240处，关于SEQ ID NO:9的位置232处，关于SEQ ID NO:10的位置240处，关于SEQ IDNO:11的位置196处，关于SEQ ID NO:12的位置240处，关于SEQ ID NO:13的位置229处，关于SEQ ID NO:14的位置236处，关于SEQ ID NO:15的位置236处，关于SEQ ID NO:16的位置241处，关于SEQ ID NO:17的位置233处，关于SEQ ID NO:18的位置240处，关于SEQ ID NO:19的位置240处，关于SEQ ID NO:20的位置234处，关于SEQ ID NO:21的位置233处，关于SEQ IDNO:22的位置237处，关于SEQ ID NO:23的位置238处，关于SEQ ID NO:24的位置231处，关于SEQ ID NO:25的位置239处，关于SEQ ID NO:26的位置240处和关于SEQ ID NO:27的位置235处。特别地，所述位置处的所述缬氨酸的所述取代可以是丙氨酸或甘氨酸。

在上述组合物的另外的实施方案中，与SEQ ID NO:2或3至少90％相同的氨基酸序列还包含关于SEQ ID NO:2或3的位置410处的丙氨酸的取代。在一些实施方案中，位置410处的丙氨酸的取代是缬氨酸。

在一方面，本发明涉及用于合成具有预定序列的多核糖核苷酸的方法和试剂盒，包括以下步骤：a)提供3’-末端核苷酸具有游离3’-羟基的起始子；以及b)重复以下的循环：(i)在延长条件下使具有游离3’-O-羟基的起始子或延长片段与3’-O-封闭的核糖核苷三磷酸和多(A)聚合酶接触，使得起始子或延长片段通过3’-O-封闭的核糖核苷三磷酸的掺入被延长以形成3’-O-封闭的延长片段，和(ii)使延长片段去封闭以形成具有游离3’-羟基的延长片段，直至形成所述多核苷酸。在一些实施方案中，本发明的方法还包括从起始子裂解多核苷酸的步骤。在一些实施方案中，3’-O-封闭的核糖核苷三磷酸是3’-O-叠氮甲基-核糖核苷三磷酸。

在另一个方面，本发明涉及用于进行具有预定序列的多核糖核苷酸的无模板合成的试剂盒，该试剂盒包含多(A)聚合酶、附接至固体支持物的起始子和3’-O-封闭的核糖核苷三磷酸单体。所述3’-O-保护的核糖核苷三磷酸单体可以包括3’-O-叠氮甲基-核糖腺苷三磷酸、3’-O-叠氮甲基-核糖鸟苷三磷酸、3’-O-叠氮甲基-核糖胞苷三磷酸、3’-O-叠氮甲基-核糖胸苷三磷酸和3’-O-氨基-核糖尿苷三磷酸中的一种或更多种。所述多(A)聚合酶可以包括这样的多(A)聚合酶变体，该多(A)聚合酶变体具有与SEQ ID NO:1至少90％相同的氨基酸序列并在M310处具有取代，或具有与SEQ ID NO:3至少90％相同的氨基酸序列并在M318处具有取代；其中多(A)聚合酶变体能够(a)在没有模板的情况下合成核糖核酸片段以及(b)将所述3’-O-叠氮甲基-核糖核苷三磷酸掺入到核糖核酸片段上。

在另一个方面，本发明涉及这样的多(A)聚合酶变体，该多(A)聚合酶变体包含与选自SEQ ID NO:1、2、3、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26或27的氨基酸序列至少90％相同的氨基酸序列和以下位置处的甲硫氨酸的取代：关于SEQ IDNO:1的位置310处，关于SEQ ID NO:2的位置318处，关于SEQ ID NO:3的位置318处，关于SEQID NO:8的位置316处，关于SEQ ID NO:9的位置309处，关于SEQ ID NO:10的位置316处，关于SEQ ID NO:11的位置272处，关于SEQ ID NO:12的位置316处，关于SEQ ID NO:13的位置307处，关于SEQ ID NO:14的位置313处，关于SEQ ID NO:15的位置312处，关于SEQ ID NO:16的位置317处，关于SEQ ID NO:17的位置316处，关于SEQ ID NO:18的位置316处，关于SEQID NO:19的位置312处，关于SEQ ID NO:20的位置310处，关于SEQ ID NO:21的位置309处，关于SEQ ID NO:22的位置317处，关于SEQ ID NO:23的位置314处，关于SEQ ID NO:24的位置307处，关于SEQ ID NO:25的位置315处，关于SEQ ID NO:26的位置316处和关于SEQ IDNO:27的位置311处，其中PAP变体能够(a)在没有模板的情况下合成核糖核酸片段以及(b)将3’-O-叠氮甲基-核糖核苷三磷酸掺入到核糖核酸片段上或将3’-O-叠氮甲基-2’-脱氧核糖核苷三磷酸掺入到脱氧核糖核酸片段中。特别地，所述位置处的所述甲硫氨酸的所述取代可以选自F、Y、V、E或T。多(A)聚合酶变体还可以包含以下位置处的缬氨酸的取代：关于SEQ ID NO:1的位置234处，关于SEQ ID NO:2的位置240处，关于SEQ ID NO:3的位置240处，关于SEQ ID NO:9的位置232处，关于SEQ ID NO:10的位置240处，关于SEQ ID NO:11的位置196处，关于SEQ ID NO:12的位置240处，关于SEQ ID NO:13的位置229处，关于SEQ ID NO:14的位置236处，关于SEQ ID NO:15的位置236处，关于SEQ ID NO:16的位置241处，关于SEQID NO:17的位置233处，关于SEQ ID NO:18的位置240处，关于SEQ ID NO:19的位置240处，关于SEQ ID NO:20的位置234处，关于SEQ ID NO:21的位置233处，关于SEQ ID NO:22的位置237处，关于SEQ ID NO:23的位置238处，关于SEQ ID NO:24的位置231处，关于SEQ IDNO:25的位置239处，关于SEQ ID NO:26的位置240处和关于SEQ ID NO:27的位置235处。特别地，所述位置处的缬氨酸的所述取代可以是丙氨酸或甘氨酸。

在另一个方面，本发明涉及多(U)聚合酶(PUP)变体，每个PUP变体包含选自SEQ IDNO:4、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46或47的氨基酸序列至少90％相同的氨基酸序列和以下位置处的酪氨酸的取代：关于SEQ ID NO:4的位置212处，关于SEQ ID NO:28的位置189处，关于SEQ ID NO:29的位置184处，关于SEQ ID NO:30的位置227处，关于SEQ ID NO:31的位置478处，关于SEQ ID NO:32的位置192处，关于SEQ IDNO:33的位置186处，关于SEQ ID NO:34的位置243处，关于SEQ ID NO:35的位置196处，关于SEQ ID NO:36的位置253处，关于SEQ ID NO:37的位置284处，关于SEQ ID NO:38的位置182处，关于SEQ ID NO:39的位置187处，关于SEQ ID NO:40的位置203处，关于SEQ ID NO:41的位置224处，关于SEQ ID NO:42的位置204处，关于SEQ ID NO:43的位置337处，关于SEQ IDNO:44的位置296处，关于SEQ ID NO:45的位置291处，关于SEQ ID NO:46的位置218处和关于SEQ ID NO:47的位置366处，其中PUP变体能够(a)在没有模板的情况下合成核糖核酸片段以及(b)将3’-O-叠氮甲基-核糖核苷三磷酸掺入到核糖核酸片段上或将3’-O-叠氮甲基-2’-脱氧核糖核苷三磷酸掺入到脱氧核糖核酸片段中。特别地，所述位置处的所述酪氨酸的所述取代可以是丙氨酸或甘氨酸。

本发明通过提供无模板聚合酶和3’-O-修饰的核糖核苷三磷酸来有利地克服酶促多核苷酸合成领域中的上述问题，这提供了核糖核苷酸掺入生长的RNA片段中的更高比率。

附图简述

图1说明本发明的方法的实施方案的步骤。

图2A-图2C显示根据本发明的方法延伸1-5个核糖核苷酸的数据。

图3A-图3B显示在存在突变M310F/Y/Q/A/V/E/T的情况下使用单个3’-O-保护的-rATP的延伸(3A)和使用rGTP的多鸟苷酸化(3B)的数据。

图4是由粟酒裂殖酵母(schizosaccharomyces pombe)PUP产生的掺入产物的电泳图。“无延伸”意指，所示的列显示对应于引物的条带而没有任何延伸。

发明详述

这里更详细地公开了本发明的一般原理，特别地通过实例的方式，诸如在附图中示出并详细描述的那些。然而，应该理解的是，意图并不是将本发明限制于所描述的特定实施方案。本发明适用于各种修改和替代形式，其细节在若干实施方案中示出。意图是覆盖落入本发明的原理和范围内的所有修改、等同物和替代物。

除非另外指出，本发明的实践可以采用有机化学、分子生物学(包含重组技术)、细胞生物学和生物化学的常规技术和描述，这些都在本领域的技术内。这样的常规技术可以包括但不限于制备和使用合成肽、合成多核苷酸、单克隆抗体，核酸克隆、扩增、测序和分析以及相关技术。这样的常规技术的方案可在制造商的产品文献和标准实验室手册中找到，诸如Genome Analysis:A Laboratory Manual Series(第I-IV卷)；PCR Primer:ALaboratory Manual；和Molecular Cloning:A Laboratory Manual(全部来自ColdSpring Harbor Laboratory Press)；Lutz和Bornscheuer，编辑，Protein EngineeringHandbook(Wiley-VCH,2009)；Hermanson,Bioconjugate Techniques,第二版(AcademicPress,2008)；以及类似的参考文献。

本发明涉及用于在同一合成中使用多(A)聚合酶(PAP)、多(U)聚合酶(PUP)或PAP和PUP二者来合成多核苷酸(多核糖核酸或多脱氧核糖核酸)的方法和组合物。在一些实施方案中，PAP和/或PUP用于使用3’-O-可逆保护的-rNTP前体合成多核糖核酸，其中单一的PUP或PAP变体可用于偶联所有核糖核苷三磷酸单体，或在可选的实施方案中，其中不同的PUP和PAP可用于在特定RNA的合成中偶联不同种类的核糖核苷三磷酸单体。同样，在其他实施方案中，PAP和/或PUP可用于使用3’-O-可逆保护的-dNTP前体合成多脱氧核糖核酸，其中单一的PUP或PAP用于偶联所有脱氧核糖核苷三磷酸(dNTP)单体，或在可选的实施方案中，其中不同的PUP和PAP聚合酶可用于偶联不同种类的脱氧核糖核苷三磷酸单体。在一些实施方案中，本发明的方法采用已通过遗传工程修饰的PAP和/或PUP变体以改进合成中3’-O-封闭的-核糖核苷三磷酸和3’-O-封闭的-2’-脱氧核糖核苷三磷酸偶联到生长的多核苷酸链的效率。

本发明的方法，无论是用于合成RNA还是DNA，都包括诸如图1所示的重复的步骤循环，其中在每个循环中添加用于RNA合成的预定核糖核苷酸单体(或用于DNA合成的2’-脱氧核糖核苷酸单体)。提供了例如附接至固体支持物(102)的起始子多核苷酸(100)，其具有游离3’-羟基基团(103)。对于合成RNA，起始子通常是多核糖核苷酸，而对于合成DNA，起始子通常是多脱氧核糖核苷酸。在有效地将3’-O-保护的-rNTP(或3’-O-保护的-dNTP)酶促掺入到起始子多核苷酸(100)(或延长的起始子多核苷酸)的3’端上的条件(104)下，向起始子多核苷酸(100)(或后续循环中延长的起始子多核苷酸)中添加3’-O-可逆保护的-rNTP(或在DNA合成的情况中，3’-O-可逆保护的-dNTP)和PAP或PUP。该反应产生其3’-羟基被保护的延长的起始子多核苷酸(106)。如果延长的起始子多核苷酸含有完整的序列，那么3’-O-保护基团被去除，或者去保护，并且期望的序列从原始起始子多核苷酸裂解。这样的裂解可以使用多种单链裂解技术中的任一种来进行，例如，通过在原始起始子多核苷酸内的预定位置处插入可裂解核苷酸来进行。示例性可裂解核苷酸可以是被尿嘧啶DNA糖基化酶裂解的尿嘧啶核苷酸。此外，多种可裂解的连键(cleavable linkages)或可裂解的核苷酸可用于此目的。在一些实施方案中，裂解期望的多核苷酸在裂解链上留下天然的游离5’-羟基；然而，在可选的实施方案中，裂解步骤可以留下可以在后续步骤中去除(例如通过磷酸酶处理)的部分(moiety)，例如5’-磷酸。裂解步骤可以通过化学、热、酶或光化学方法进行。在一些实施方案中，可裂解核苷酸可以是核苷酸类似物，诸如脱氧尿苷或8-氧代-脱氧鸟苷，它们被特定的糖基化酶(例如尿嘧啶脱氧糖基化酶，随后分别是核酸内切酶VIII和8-氧代鸟嘌呤DNA糖基化酶)识别。在一些实施方案中，裂解可以通过以下完成：为起始子提供脱氧肌苷作为倒数第二个3’核苷酸，脱氧肌苷可以在起始子的3’端被内切核酸酶V裂解，在释放的多核苷酸上留下5’-磷酸。用于裂解单链多核苷酸的另外的方法在以下参考文献中公开，这些参考文献通过引用并入：美国专利第5,739,386号、第5,700,642号和第5,830,655号；和美国专利公布第2003/0186226号和第2004/0106728号；和在Urdea和Horn，美国专利5367066中。

如果延长的起始子多核苷酸不含有完整的序列，那么3’-O-保护基团被去除以暴露游离3’-羟基(103)，并且使延长的起始子多核苷酸经历另一个循环的核糖核苷酸添加和去保护。

在一些实施方案中，合成多核糖核苷酸的方法包括以下步骤：(a)提供具有游离3’-羟基的起始子；(b)在延伸条件下使具有游离3’-羟基的起始子或延伸中间体(即延长的起始子多核苷酸)与PAP在存在3’-O-封闭的核糖核苷三磷酸的情况下反应，以产生3’-O-封闭的延伸中间体；(c)使延伸中间体去封闭以产生具有游离3’-羟基的延伸中间体；以及(d)重复步骤(b)和(c)，直至合成所述多核糖核苷酸。(术语“延伸中间体”在本文中也可以称为“延长片段”或“延长的起始子多核苷酸”)。在一些实施方案中，如以上所述，起始子以例如通过其5’端附接至固体支持物的寡核苷酸提供。上述方法还可以包括在反应步骤或延伸步骤之后以及在去封闭步骤之后的洗涤步骤。例如，反应步骤可以包括在预定孵育时间段或反应时间之后去除未掺入的核糖核苷三磷酸(通过例如洗涤)的子步骤。这样的预定孵育时间段或反应时间可以是几秒钟(a few seconds)，例如30秒，至数分钟(several minutes)，例如30分钟。

在一些实施方案中，合成多核糖核苷酸的方法包括以下步骤：(a)提供具有游离3’-羟基的起始子；(b)在延伸条件下使具有游离3’-羟基的起始子或延伸中间体(即延长的起始子多核苷酸)与PAP或PUP在存在3’-O-封闭的核糖核苷三磷酸的情况下反应，以产生3’-O-封闭的延伸中间体；(c)使延伸中间体去封闭以产生具有游离3’-羟基的延伸中间体；以及(d)重复步骤(b)和(c)，直至合成所述多核糖核苷酸。(术语“延伸中间体”在本文中也可以称为“延长片段”或“延长的起始子多核苷酸”)。在一些实施方案中，如以上所述，起始子以例如通过其5’端附接至固体支持物的寡核苷酸提供。上述方法还可以包括在反应步骤或延伸步骤之后以及在去封闭步骤之后的洗涤步骤。例如，反应步骤可以包括在预定孵育时间段或反应时间之后去除未掺入的核糖核苷三磷酸(通过例如洗涤)的子步骤。这样的预定孵育时间段或反应时间可以是几秒钟(a few seconds)，例如30秒，至数分钟(severalminutes)，例如30分钟。

在一些实施方案中，合成多核苷酸的方法包括以下步骤：(a)提供具有游离3’-羟基的起始子；(b)在延伸条件下使具有游离3’-羟基的起始子或延伸中间体(即延长的起始子多核苷酸)与PAP或PUP在存在3’-O-封闭的核苷三磷酸的情况下反应，以产生3’-O-封闭的延伸中间体；(c)使延伸中间体去封闭以产生具有游离3’-羟基的延伸中间体；以及(d)重复步骤(b)和(c)，直至合成所述多核苷酸。(术语“延伸中间体”在本文中也可以称为“延长片段”或“延长的起始子多核苷酸”)。在一些实施方案中，如以上所述，起始子以例如通过其5’端附接至固体支持物的寡核苷酸提供。上述方法还可以包括在反应步骤或延伸步骤之后以及在去封闭步骤之后的洗涤步骤。例如，反应步骤可以包括在预定孵育时间段或反应时间之后去除未掺入的核糖核苷三磷酸(通过例如洗涤)的子步骤。这样的预定孵育时间段或反应时间可以是几秒钟(a few seconds)，例如30秒，至数分钟(several minutes)，例如30分钟。

上述方法还可以包括在反应步骤或延伸步骤之后以及在去封闭步骤之后的加帽步骤以及洗涤步骤。如以上提及的，在一些实施方案中，可以包括加帽步骤，其中非延伸的游离3’-羟基与阻止加帽链任何进一步延伸的化合物反应。在一些实施方案中，这样的化合物可以是双脱氧核苷三磷酸。在其他实施方案中，具有游离3’-羟基的非延伸链可以通过将其用3’-外切核糖核酸酶活性，例如RNA酶R(Epicentre)处理来降解。同样，在一些实施方案中，未能去封闭的链可以被处理以去除该链或使其对进一步的延伸呈惰性。

在一些包括寡核糖核苷酸的系列合成的实施方案中，加帽步骤可能是不期望的，因为加帽可能会阻止多于一种寡核苷酸的等摩尔量的产生。在没有加帽的情况下，序列将具有均匀分布的缺失错误，但是多于一种寡核糖核苷酸中的每一种将以等摩尔量存在。在未延伸片段被加帽的情况下，情况将不会如此。

在一些实施方案中，使用PAP或PUP的延伸或延长步骤的反应条件可以包括以下：反应条件1(用于引物+AM-rATP)：250uM AM-rATP、0.1uM ATTO488-(rA)5、1uM PAP、1x ATP缓冲液(20mM Tris-HCl、0.6mM MnCl₂、0.02mM EDTA、0.1％BSA、10％甘油、100mM咪唑，pH7-8)，37℃，30分钟。反应条件2(用于引物+AM-rGTP)：250uM rGTP、0.1uM ATTO488-(rA)5、1uM PAP、1x GTP缓冲液(0.6mM MnCl₂、0.1％BSA、10mM咪唑、pH 6)，37℃，30分钟。在上文中，“AM-rNTP”是指3’-叠氮甲基-O-核糖核苷三磷酸。

根据具体应用，去封闭和/或裂解的步骤可以包括多种化学或物理条件，例如光、热、pH、特定试剂诸如能够裂解指定化学键的酶的存在。选择3’-O-封闭基团和相应的去封闭条件的指导可以在诸如Wuts,Green’s Protection Groups in Organic Chemistry,第五版(Wiley 2014)的参考文献中发现。在一些实施方案中，裂解剂(有时也称为去封闭试剂或去封闭剂)是化学裂解剂，诸如，例如，二硫苏糖醇(DTT)。在可选的实施方案中，裂解剂可以是酶促裂解剂，诸如，例如，可以裂解3’-磷酸封闭基团的磷酸酶。本领域技术人员将理解，去封闭剂的选择取决于所使用的3’-核苷酸封闭基团的类型、是使用一种还是多于一种封闭基团、起始子是否被附接至活细胞或生物体或固体支持物等，这些都需要温和的处理。例如，膦诸如三(2-羧乙基)膦(TCEP)可以用于裂解3’O-叠氮甲基基团，钯络合物可以用于裂解3’O-烯丙基基团和3’-O-炔丙基基团，或亚硝酸钠可以用于裂解3’O-氨基基团。

如以上所述，在一些实施方案中，期望采用可以使用正交去封闭条件去除的两种或更多种封闭基团。以下示例性的封闭基团对可用于平行合成实施方案中，其中两种或更多种序列在相同的反应混合物中合成。应当理解，其他的封闭基团对，或者包含多于两种封闭基团的组可以可用于在本发明的这些实施方案中使用。

如本文所用，“起始子”(或等同术语，诸如，“起始片段”、“起始子核酸”、“起始子寡核苷酸”等)是指具有游离3’-末端的短寡核苷酸序列，其可被无模板聚合酶(诸如PAP或PUP)进一步延长。在一种实施方案中，起始片段是DNA或RNA起始片段。在可选的实施方案中，起始片段是RNA起始片段。在一种实施方案中，起始片段具有3个和100个之间的核苷酸，特别地，具有3个和20个之间的核苷酸。在一种实施方案中，起始片段是单链的。在可选的实施方案中，起始片段是双链的。在特定的实施方案中，用5’-伯胺合成的起始子寡核苷酸可以使用制造商的方案共价连接至磁珠。同样，用3’-伯胺合成的起始子寡核苷酸可以使用制造商的方案共价连接至磁珠或琼脂糖珠。适用于本发明实施方案的各种其他附接化学是本领域中熟知的，例如Integrated DNA Technologies手册,“Strategies for AttachingOligonucleotides to Solid Supports,”v.6(2014)；Hermanson,BioconjugateTechniques,第二版(Academic Press,2008)；和类似的参考文献。

本发明中采用的3’-O-封闭的rNTP中的许多可以从商业供应商(例如JenaBioscience、MyChemLabs等)购买或使用公开的技术合成，例如美国专利7057026；国际专利公布WO2004/005667、WO91/06678；Canard等人,Gene(以上引用)；Metzker等人,NucleicAcids Research,22:4259-4267(1994)；Meng等人,J.Org.Chem.,14:3248-3252(3006)；美国专利公布2005/037991；Zavgorodny等人,Tetrahedron Letters,32(51):7593-7596(1991)。

在另外的特定实施方案中，3’-封闭的核苷酸三磷酸被3’-O-炔丙基、3’-O-叠氮甲基、3’-O-NH₂或3’-O-烯丙基基团封闭。

在仍其他实施方案中，本发明的3’-O-封闭基团包括3’-O-甲基、3’-O-(2-硝基苄基)、3’-O-烯丙基、3’-O-胺、3’-O-叠氮甲基、3’-O-叔丁氧基乙氧基、3’-O-(2-氰乙基)和3’-O-炔丙基。

多(A)聚合酶和有用的变体

多种PAP可用于本发明的方法，包括为了改进的特性而工程化的PAP变体，改进的特性诸如3’-O-保护的rNTP的更高掺入率(对于特定保护基团，包括诸如，3’-O-叠氮甲基)、更大的稳定性和保质期、热稳定性、溶解度等。特别地，相对于野生型PAP，具有在M310(SEQID NO:1)或其他PAP(诸如来自各种不同物种的PAP)中的功能等同的残基处的突变的酵母PAP显示出3’-O保护的rNTP的改进的掺入。在一些实施方案中，本发明的酵母PAP变体具有SEQ ID NO:1的氨基酸序列，除了在M310处的取代。在一些实施方案中，这样的取代选自M310F/Y/V/E/T。特别地，取代M310F/Y允许3’-O-氨基-rATP的掺入，并且取代M310V/E/T改进3’-O-保护的-rGTP的掺入率。在其他实施方案中，本发明的酵母PAP变体具有与SEQ IDNO:1有至少90％同一性的氨基酸序列，除了在M310处的取代。

用于本发明的PAP变体包括下表1中列出的那些。在一些实施方案中，本发明的PAP变体包含至少在表1中所示的第二位置处的取代。在其他实施方案中，本发明的PAP变体的实施方案包含至少在表1中所示的第一位置处的取代。

表1

PAP变体：取代的位置

在一些实施方案中，如表1中所示的第一位置处的取代是A或G(因此，例如，对于SEQ ID NO:1，取代可写作V234A/G)。在一些实施方案中，如表1中所示的第二位置处的取代是F、Y、V、E或T(因此，例如，对于SEQ ID NO:1，取代可写作M310F/Y/V/E/T)。

在一些实施方案中，本发明的PAP变体具有表1的取代中的一种或更多种和与所示的SEQ ID NO具有至少80％同一性的同一性百分比值；在一些实施方案中，上述同一性百分比值是与所示的SEQ ID NO至少90％同一性；在一些实施方案中，上述同一性百分比值是与所示的SEQ ID NO至少95％同一性；在一些实施方案中，上述同一性百分比值是至少97％同一性；在一些实施方案中，上述同一性百分比值是至少98％同一性；在一些实施方案中，上述同一性百分比值是至少99％同一性。

在一些实施方案中，采用热稳定的PAP使得该方法可以在减少或消除正在合成的RNA或DNA中的二级结构的形成的温度实践。在一些实施方案中，热稳定PAP出现最高掺入率的温度范围高于40℃。在一些实施方案中，热稳定PAP出现最高掺入率的温度范围高于50℃。在一些实施方案中，热稳定PAP出现最高掺入率的温度范围在40℃和85℃之间。在一些实施方案中，热稳定PAP出现最高掺入率的温度范围在50℃和85℃之间。

多(U)聚合酶和有用的变体

与PAP一样，多种PUP可用于本发明的方法，包括为了改进的特性而工程化的PUP变体，改进的特性诸如3’-O-保护的-rNTP的更高掺入率(对于特定保护基团，包括诸如3’-O-叠氮甲基)、更大的稳定性和保质期、热稳定性、溶解度等。用于本发明的PUP变体包括下表2中列出的那些。在一些实施方案中，本发明的PUP变体包含至少在表2中所示的第一位置处的取代。在其他实施方案中，本发明的PUP变体的实施方案包含至少在表2中所示的第二位置处的取代。

表2

PUP变体：取代的位置

在一些实施方案中，如表2中所示的第一位置处的取代是A或G(因此，例如，对于SEQ ID NO:4，取代可写作Y212A/G)。在一些实施方案中，如表2中所示的第二位置处的取代是F、Y、V、E或T(因此，例如，对于SEQ ID NO:4，取代可写作H336F/Y/V/E/T)。

在一些实施方案中，本发明的PUP变体具有表2中的取代中的一种或更多种和与所示的SEQ ID NO具有至少80％同一性的同一性百分比值；在一些实施方案中，上述同一性百分比值是与所示的SEQ ID NO至少90％同一性；在一些实施方案中，上述同一性百分比值是与所示的SEQ ID NO至少95％同一性；在一些实施方案中，上述同一性百分比值是至少97％同一性；在一些实施方案中，上述同一性百分比值是至少98％同一性；在一些实施方案中，上述同一性百分比值是至少99％同一性。

在一些实施方案中，采用热稳定的PUP使得该方法可以在减少或消除正在合成的RNA或DNA中的二级结构的形成的温度实践。在一些实施方案中，热稳定PUP出现最高掺入率的温度范围高于40℃。在一些实施方案中，热稳定PUP出现最高掺入率的温度范围高于50℃。在一些实施方案中，热稳定PUP出现最高掺入率的温度范围在40℃和85℃之间。在一些实施方案中，热稳定PUP出现最高掺入率的温度范围在50℃和85℃之间。

PAP和PUP变体的产生

本发明的变体可以通过将已知的参考或野生型PAP编码多核苷酸或PUP编码多核苷酸突变、然后使用常规分子生物学技术表达它来产生。例如，编码具有期望序列的多肽的期望基因或DNA片段可以使用常规分子生物学技术，例如使用由以下描述的方案从合成片段组装：Stemmer等人,Gene,164:49-53(1995)；Kodumal等人,Proc.Natl.Acad.Sci.,101:15573-15578(2004)；等，或者这样的基因或DNA片段可以使用常规方案从选定物种的细胞中直接克隆。

可将编码期望的PAP或PUP变体的分离基因插入表达载体中以给出表达载体，表达载体然后可用于使用常规方案制备和表达变体PAP或PUP蛋白。可以将具有正确序列的载体转化到大肠杆菌(E.coli)生产菌株中。

以下程序是针对PAP变体描述的，但本领域普通技术人员可以将类似的程序应用于PUP变体。使用常规技术培养转化的菌株，从沉淀物提取PAP蛋白。例如，将先前制备的沉淀物在30℃至37℃的水浴中解冻。完全解冻后，将沉淀物重悬于包含50mM tris-HCL(Sigma)pH 7.5、150mM NaCl(Sigma)、0.5mM巯基乙醇(Sigma)、5％甘油(Sigma)、20mM咪唑(Sigma)和1片(用于100mL)蛋白酶抑制剂混合物(Thermofisher)的裂解缓冲液中。仔细地进行重悬，以便避免过早裂解和残留聚集物。通过若干次弗氏压碎器循环将重悬的细胞裂解，直至获得完全的颜色均匀性。通常使用的压力是14,000psi。然后将裂解物以10,000rpm离心1h至1h 30min。将离心液通过0.2μm过滤器，以在柱纯化前去除所有残渣。

PAP蛋白可以通过一步亲和程序从离心液中纯化。例如，Ni-NTA亲和柱(GEHealthcare)可以用于结合PAP聚合酶。最初，将柱用15倍柱体积的50mM tris-HCL(Sigma)pH 7.5、150mM NaCl(Sigma)和20mM咪唑(Sigma)洗涤和平衡。平衡后PAP聚合酶与柱结合；然后，洗涤缓冲液，例如包含50mM tris-HCL(Sigma)pH 7.5、500mM NaCl(Sigma)和20mM咪唑(Sigma)，可以以15倍柱体积应用于柱。这样的洗涤后，用50mM tris-HCL(Sigma)pH 7.5、500mM NaCl(Sigma)和0.5M咪唑(Sigma)洗脱PAP聚合酶。收集对应于最高浓度的感兴趣的PAP聚合酶的级分并汇集成单个样品。将汇集的级分用透析缓冲液(20mM Tris-HCl pH6.8、200mM NaCl、50mM MgOAc、100mM[NH₄]₂SO₄)透析。透析液随后在浓缩过滤器(AmiconUltra-30,Merk Millipore)的帮助下进行浓缩。将浓缩的酶分成小的等分试样，添加最终50％的甘油，并且然后将这些等分试样冷冻于-20℃并长期储存。在SDS-PAGE凝胶中分析5μL的各种纯化的酶的级分。

在一些实施方案中，PAP变体可以可操作地连接到包括共价键或非共价键的接头部分；氨基酸标签(例如，多氨基酸标签、多His标签、6His标签等)；化学化合物(例如，聚乙二醇)；蛋白-蛋白结合对(例如，生物素-亲和素)；亲和偶联物(affinity coupling)；捕获探针或这些的任何组合。接头部分可以与PAP变体分开或作为PAP变体的一部分。用于本发明的PAP变体的示例性His-标签是MASSHHHHHHSSGSENLYFQTGSSG-(SEQ ID NO:5)。标签-接头部分不干扰PAP变体的核苷酸结合活性或催化活性。

上述方法或等同方法产生分离的PAP或PUP变体，其可能混合有多种试剂诸如盐、pH缓冲液、载体化合物等，这些试剂对于活性和/或保存是必需的或有用的。

核苷酸掺入活性的测量

本发明的变体的核苷酸掺入效率可以通过延伸或延长测定来测量，例如，如在Boule等人(下文引用)；Bentolila等人(下文引用)；和Hiatt等人，美国专利5808045中描述的，后者通过引用并入本文。简言之，在这样的测定的一种形式中，将荧光标记的具有游离3’-羟基的寡核苷酸在PAP延伸条件下与待测试的PAP变体在存在可逆封闭的核糖核苷三磷酸的情况下反应预定持续时间，之后停止延伸反应，并在通过凝胶电泳分离后定量延伸产物和未延伸起始子寡核苷酸的量。通过这样的测定，可以容易地将PAP变体的掺入效率与其他变体的效率或野生型或参考PAP或其他聚合酶的效率进行比较。在一些实施方案中，PAP变体效率的量度可以是使用PAP变体的延伸产物的量与在等同测定中使用野生型PAP的延伸产物的量的比率(以百分比给出)。将试剂从水开始然后按表3的顺序添加到管中。

表3

延伸活性测定试剂

测定的产物可以通过常规的聚丙烯酰胺凝胶电泳进行分析。例如，上述测定的产物可以在16％的聚丙烯酰胺变性凝胶(Bio-Rad)中进行分析。凝胶在即将分析前通过将聚丙烯酰胺倾入玻璃板中并使其聚合而制备。将玻璃板中的凝胶安装在填充了TBE缓冲液(Sigma)的合适的槽中，用于电泳步骤。将待分析的样品加载到凝胶的顶部。在室温，在凝胶的顶部和底部之间施加500V至2,000V的电压3h至6h。分离后，凝胶荧光使用例如Typhoon扫描仪(GE Life Sciences)来扫描。使用Image J软件(imagej.nih.gov/ij/)或其等同软件分析凝胶图像，以计算修饰核苷酸的掺入百分比。

试剂盒

本发明包括用于进行本发明的方法的试剂盒。在一些实施方案中，本发明的试剂盒包含多(A)聚合酶和腺苷、鸟苷、尿苷和胞苷中的一种或更多种的3’-O-封闭的核糖核苷三磷酸。在其他实施方案中，这样的试剂盒还可以包含多(U)聚合酶。在一些实施方案中，本发明的试剂盒包含多(A)聚合酶和脱氧腺苷、脱氧鸟苷、脱氧胸苷和脱氧胞苷中的一种或更多种的3’-O-封闭的-2’-脱氧核糖核苷三磷酸。在其他实施方案中，这样的试剂盒还可以包含多(U)聚合酶。在一些实施方案中，本发明的试剂盒可以包含通过5’端附接至支持物的具有游离3’-羟基的起始子。在一些实施方案中，这样的支持物是固体支持物。这样的固体支持物可以包括珠诸如磁珠或琼脂糖珠，平面固体诸如玻璃载玻片或膜等。在一些实施方案中，本发明的试剂盒还可以包含能够从掺入的3’-O-封闭的核苷酸中去除3’封闭基团的去封闭剂。在一些实施方案中，试剂盒可以包含酵母PAP(诸如SEQ ID NO:1的酵母PAP)的M310突变体，或来自不同物种的PAP中的与M310功能等同的残基的突变体。在一些实施方案中，试剂盒可以包含PAP变体，该变体包含与选自SEQ ID NO:1、2或3的氨基酸序列至少90％相同的氨基酸序列以及在关于SEQ ID NO:1的位置310处的甲硫氨酸或在关于SEQ ID NO:2和3的位置318处的甲硫氨酸的取代，其中该变体能够(a)在没有模板的情况下合成核糖核酸片段以及(b)将3’-O-叠氮甲基-核糖核苷三磷酸掺入到核糖核酸片段上或将3’-O-叠氮甲基-2’-脱氧核糖核苷三磷酸掺入到脱氧核糖核酸片段中。

在一些实施方案中，在前述试剂盒的PAP变体中，在SEQ ID NO:1的位置310处的甲硫氨酸或SEQ ID NO:2或3的位置318处的甲硫氨酸的取代选自M310F/Y/V/E/T。在一些实施方案中，在前述试剂盒的PAP变体中，具有关于SEQ ID NO:1的位置234处的缬氨酸和关于SEQ ID NO:2和3的位置240处的缬氨酸的取代。在一些实施方案中，在前述试剂盒的PAP变体中，在SEQ ID NO:1的位置234处的缬氨酸和关于SEQ ID NO:2和3的位置240处的缬氨酸的取代是丙氨酸。在一些实施方案中，在前述试剂盒的PAP变体中，具有关于在SEQ ID:NO 2或3的位置410处的丙氨酸的取代。

在一些实施方案中，本发明的试剂盒可以包含具有与选自表1的SEQ ID NO:1-3和8-27的序列至少80％相同的氨基酸序列的PAP变体，该变体具有在如表1中所示的这样的所选序列的第一位置的取代或在第二位置的取代或在这两个位置的取代。在一些实施方案中，所选序列的这样的同一性百分比是至少90％同一性。在一些实施方案中，本发明的试剂盒可以包含具有与选自表2的SEQ ID NO:4和28-47的序列至少80％相同的氨基酸序列的PUP变体，该变体具有在如表2中所示的这样的所选序列的第一位置的取代或在第二位置的取代或在这两个位置的取代。在一些实施方案中，所选序列的这样的同一性百分比是至少90％同一性。

实施例1

向固定在固体支持物上的起始子多核苷酸添加一至五个核糖核苷酸

在存在PAP(Thermo)的情况下，用3’-O-叠氮甲基-核糖核苷三磷酸(AM-NTP)对Cp-RNA引物的2-3个循环的延伸。实验程序：1)Cp-珠的制备：100uL Cp-珠(珠上Cp引物的浓度～1uM)+1uL 100uM SynRDA_DNA-SynRDA_RNA；在室温孵育30分钟，用3x200 uL结合缓冲液(BB)(200mM caco,LiCl)洗涤珠，将珠重悬于25uL BB中；2)+1添加反应：250uM 3’O-封端核苷酸、3.2uM Cp-SynRDA_DNA-SynRDA_RNA、1x PAP_酵母Thermo反应缓冲液、100U/uL PAP_酵母(Thermo)，20uL终体积；在Eppendorf管中；30分钟、37℃、热混合器、1500rpm；3)洗涤+1产物：3X 200uL BB；4)TCEP去保护：将珠重悬于50uL 200uM TCEP，pH 7.0(从新打开的安瓿中稀释)，37℃、15分钟、热混合器、1500rpm；5)洗涤去保护的+1产物：3x 200uL结合缓冲液；6)+2/去保护/+3添加：参见1)-5)；7)洗脱+凝胶：添加20uL B-blue、涡旋、洗脱、应用于凝胶上。所用的Cp-RNA珠按以下方式制备：i)用DNA寡核苷酸(TCATTTCACTCTCACA-NH2)(SEQ IDNO:6)对显示末端羧酸基团的商用Dynabead M-270(购自Thermo Fisher)进行共价官能化。这些珠被命名为Cp-珠。然后将Cp-珠与序列ATTO488-TGTGAGAGTGAAATGAGGrUrGrUrGrArGrArGrUr-GrArArAr UrGrArGrG(命名为SynRDA_DNA-SynRDA_RNA)(SEQ ID NO:7)的DNA-RNA杂种引物一起孵育。

结果如图2A-图2C中所示。该数据显示：i)对于3’-O-AM-rATP和3’-O-AM-rUTP，使用相同的核苷酸可以实现至少5个循环的添加/去保护；ii)使用两种不同的3’-O-AM-rNTP(A和U)可以实现至少2个循环的添加/去保护；和iii)可以实现对3’-O-AM-保护的RNA的至少>95％的去保护。

实施例2

PAPM310突变对AM-rATP延长和核糖鸟苷酸化活性的影响

在本实验中，检查了PAP位置M310的突变对RNA引物的AM-rATP延长(图3A)和RNA引物的核糖鸟苷酸化(3B)活性的影响。在存在延长AM-rATP缓冲液(20mM Tris-HCl、0.6mMMnCl₂、0.02mM EDTA、0.1％BSA、10％甘油、100mM咪唑，pH 7-8)中0.1uM ATTO488-(rA)₅引物、0.5uM PAP突变体、250uM AM-rATP的情况下，在37℃、20uL的总反应体积，进行RNA引物的AM-rATP延长(图3A)持续30分钟。在存在rGTP聚合缓冲液(0.6mM MnCl₂、0.1％BSA、10mM咪唑，pH 6.2)中0.1uM ATTO488-(rA)₅引物、0.5uM PAP突变体、250uM AM-rATP的情况下，在37℃、20uL的总反应体积，进行RNA引物的核糖鸟苷酸化(图3B)持续30分钟。

实施例3

梭孢壳属PAP变体对AM-rNTP掺入的影响

梭孢壳属PAP(SEQ ID NO:3)使用常规技术经工程化具有三个取代(V240A、M318T和A410V)，之后表达和纯化修饰的多肽。通过进行实施例2中描述的测定比较野生型梭孢壳属PAP和突变的PAP掺入AM-NTP的能力，但以下条件除外：0.1μM Atto488-(rA)₇、3.0μMPAP、250μM AM-NTP。反应结果在下表4中给出。

表4

数据显示，梭孢壳属PAP变体掺入AM-rGTP和AM-rCTP单体的效率比对应的野生型PAP大得多。

实施例4

通过PUP掺入AM-NTP

在本实验中，粟酒裂殖酵母PUP(SEQ ID NO:4)用于使用实施例3的实验方案将AM-NTP掺到起始子中。通过凝胶电泳分离延伸产物，以给出图4中所示的电泳图。

定义

氨基酸由它们的单字母或三字母代码根据以下命名法表示：A：丙氨酸(Ala)；C：半胱氨酸(Cys)；D：天冬氨酸(Asp)；E：谷氨酸(Glu)；F：苯丙氨酸(Phe)；G：甘氨酸(Gly)；H：组氨酸(His)；I：异亮氨酸(Ile)；K：赖氨酸(Lys)；L：亮氨酸(Leu)；M：甲硫氨酸(Met)；N：天冬酰胺(Asn)；P：脯氨酸(Pro)；Q：谷氨酰胺(Gln)；R：精氨酸(Arg)；S：丝氨酸(Ser)；T：苏氨酸(Thr)；V：缬氨酸(Val)；W：色氨酸(Trp)和Y：酪氨酸(Tyr)。

提及取代残基时的“功能等同”意指变体PAP的取代残基与另一个具有与SEQ IDNO:1同源的序列的PAP序列中的残基具有相同的功能作用。功能等同的残基可以通过使用序列比对，例如，使用Mutalin line比对软件(http://multalin.toulouse.inra.fr/multalin/multalin.html；1988,Nucl.Acids Res.,16(22),25 10881-10890)来鉴定。比对后，功能等同的残基位于所考虑的不同序列的同源位置处。序列比对和功能等同残基的鉴定可以在任何PAP和它们的天然变体之间(包括物种间)进行确定。

提及蛋白时的“分离的(isolated)”意指这样的化合物，所述化合物已经被鉴定并与其自然环境的组分分离和/或回收或从非均相反应混合物中分离和/或回收。自然环境或反应混合物中的污染物组分是会干扰蛋白功能的物质，并可以包括酶、激素和其他蛋白或非蛋白溶质。在一些实施方案中，本发明的蛋白被纯化(1)至按蛋白重量计大于95％，并且最优选地按重量计大于99％，如通过Lowry法确定的，(2)至通过使用旋转杯测序仪(spinning cup sequenator)足以获得至少15个N末端或内部氨基酸序列的残基的程度，或(3)至使用考马斯蓝或优选地银染的还原或非还原条件下的SDS-PAGE的均一性。当通过重组方法制备时，本发明的分离的蛋白可以包括在重组细胞内原位的本发明的蛋白，因为该蛋白的自然环境中的至少一种组分将不存在。通常，本发明的分离的蛋白通过至少一个纯化步骤来制备。

“试剂盒”是指用于递送用于实施本发明的方法的材料或试剂的任何递送系统。在反应测定的情况下，这样的递送系统包括允许反应试剂(例如，适当的容器中的PAP酶、受保护的rNTP单体等)和/或支持材料(例如，缓冲液，用于进行测定的书面说明等)储存、从一个位置到另一个位置的运输或递送的系统和/或化合物(诸如稀释剂、表面活性剂、载体等)。例如，试剂盒包含含有相关反应试剂和/或支持材料的一种或更多种外罩(例如盒子)。这样的内含物可以一起或单独地被递送到预期的接收者。例如，第一个容器可以含有酶，而第二个或更多个容器可以含有rNTP单体、缓冲液、具有起始子的固体支持物等。

可互换使用的“突变体”或“变体”是指这样的多肽：来源于SEQ ID NO:1并且在一个或更多个位置包含修饰或改变，即取代、插入和/或缺失，且兼有无模板聚合酶活性和掺入一种或更多种可逆封闭的核苷三磷酸前体的能力。变体可以通过本领域熟知的各种技术获得。特别地，用于改变编码野生型蛋白的DNA序列的技术的实例包括但不限于定点诱变、随机诱变和合成寡核苷酸构建。诱变活性包括在蛋白或者在本发明的情况下聚合酶的序列中缺失、插入或取代一个或数个(several)氨基酸。以下术语用于指定取代：L238A表示参考序列或野生型序列的位置238处的氨基酸残基(亮氨酸，L)被改变为丙氨酸(A)。A132V/I/M表示亲本序列的位置132处的氨基酸残基(丙氨酸，A)被以下氨基酸之一取代：缬氨酸(V)、异亮氨酸(I)或甲硫氨酸(M)。取代可以是保守的或非保守的取代。保守取代的实例是在以下组内的取代：碱性氨基酸(精氨酸、赖氨酸和组氨酸)、酸性氨基酸(谷氨酸和天冬氨酸)、极性氨基酸(谷氨酰胺、天冬酰胺和苏氨酸)、疏水性氨基酸(甲硫氨酸、亮氨酸、异亮氨酸、半胱氨酸和缬氨酸)、芳香族氨基酸(苯丙氨酸、色氨酸和酪氨酸)和小氨基酸(甘氨酸、丙氨酸和丝氨酸)。

“多核苷酸”或“寡核苷酸”被可互换地使用并且均意指核苷酸单体的线性聚合物或其类似物。构成多核苷酸和寡核苷酸的单体能够通过单体与单体相互作用的常规模式(诸如Watson-Crick类型的碱基配对、碱基堆积、Hoogsteen或反向Hoogsteen类型的碱基配对等)与天然多核苷酸特异性结合。这样的单体及其核苷间连键可以是天然存在的，或者可以是其类似物，例如天然存在的或非天然存在的类似物。非天然存在的类似物可以包括PNA、硫代磷酸核苷间连键、含有允许标记物(诸如荧光团或半抗原等)附接的连接基团的碱基。每当寡核苷酸或多核苷酸的使用要求酶促加工(例如通过聚合酶延伸、通过连接酶连接等)时，普通技术人员将理解，在那些实例中，寡核苷酸或多核苷酸会在任何或一些位置处不含有核苷间连键、糖部分或碱基的某些类似物。通常多核苷酸的尺寸范围为几个单体单元例如5-40(此时多核苷酸通常被称为“寡核苷酸”)至数千个单体单元。除非另外指定或从上下文明显的，每当多核苷酸或寡核苷酸以字母(大写或小写)序列诸如“ATGCCTG”表示时，应该理解的是该核苷酸从左至右是5’→3’的顺序，并且“A”表示脱氧腺苷，“C”表示脱氧胞苷，“G”表示脱氧鸟苷，并且“T”表示胸苷，“I”表示脱氧肌苷，“U”表示尿苷。相应的核糖核苷酸可以被指定为“rA”、“rC”、“rG”和“rT”。除非另外说明，否则术语和原子编号惯例将遵循在Strachan和Read，Human Molecular Genetics 2(Wiley-Liss,New York,1999)中公开的那些。通常多核苷酸包含由磷酸二酯键连接的四种天然核苷(例如，脱氧腺苷、脱氧胞苷、脱氧鸟苷、脱氧胸苷(对于DNA)或它们的核糖对应物(对于RNA))；然而，它们还可以包含非天然的核苷酸类似物，例如包含修饰的碱基、糖或核苷间连键。本领域技术人员清楚，在酶的活性要求特定的寡核苷酸或多核苷酸底物(例如单链DNA、RNA/DNA双链体等)的情况下，则选择寡核苷酸或多核苷酸底物的合适组成完全是在普通技术人员的知识范围内，特别是在来自专著，诸如Sambrook等人，Molecular Cloning，第二版(Cold Spring HarborLaboratory，New York，1989)以及类似的参考文献的指导下。同样，寡核苷酸和多核苷酸可以指单链形式或双链形式(即寡核苷酸或多核苷酸及其相应互补物的双链体)。根据术语使用的上下文，普通技术人员将清楚哪种形式是意图的或是否两种形式都是意图的。

“引物”意指能够在与多核苷酸模板形成双链体后充当核酸合成的起始点并且从其3’末端沿模板延伸，使得形成延伸的双链体的天然或合成的寡核苷酸。引物的延伸通常用核酸聚合酶例如DNA或RNA聚合酶进行。在延伸过程中添加的核苷酸的顺序由模板多核苷酸的序列决定。通常引物通过DNA聚合酶延伸。引物通常具有14个至40个核苷酸的范围内或者18个至36个核苷酸的范围内的长度。引物用于各种核酸扩增反应，例如使用单种引物的线性扩增反应，或采用两种或更多种引物的聚合酶链式反应。选择用于特定应用的引物的长度和序列的指导是本领域普通技术人员熟知的，如由以下参考文献表明的，这些参考文献通过引用并入本文：Dieffenbach,编辑,PCR Primer:A Laboratory Manual,第二版(Cold Spring Harbor Press,New York,2003)。

“序列同一性”是指两个序列(诸如两个多肽序列或两个多核苷酸序列)之间的匹配(例如相同的氨基酸残基)的数目(或分数，通常以百分比表示)。序列同一性可以通过将序列对齐使得重叠和同一性最大化同时使序列空位最小化时对序列进行比较来确定。特别地，序列同一性可以使用许多数学全局或局部比对算法中的任何一种来确定，这取决于两个序列的长度。相似长度的序列优选地使用全局比对算法(例如，Needleman和Wunsch算法；Needleman和Wunsch，1970)进行比对，该算法将序列在整个长度上最优地对齐，而具有明显不同长度的序列优选地使用局部比对算法(例如，Smith和Waterman算法(Smith和Waterman，1981)或Altschul算法(Altschul等人，1997；Altschul等人，2005))进行比对。以确定氨基酸序列同一性百分比为目的的比对可以以本领域技术范围内的各种方式，例如，使用可在互联网网站诸如http://blast.ncbi.nlm.nih.gov/或ttp://www.ebi.ac.uk/Tools/emboss/上可得的公开可得的计算机软件来实现。本领域技术人员可以确定用于测量比对的合适参数，包括在被比较序列的全长上实现最大对齐所需的任何算法。出于本文的目的，％氨基酸序列同一性值是指使用逐对序列比对程序EMBOSS Needle生成的值，该程序使用Needman-Wunsch算法产生两个序列的最佳全局比对，其中所有检索参数被设置为默认值，即评分矩阵＝BLOSUM62，空位开放＝10，空位延伸＝0.5，末端空位罚分＝假，末端空位开放＝10并且末端空位延伸＝0.5。

“取代”意指氨基酸残基被另一种氨基酸残基替代。优选地，术语“取代”是指氨基酸残基被选自以下的另一种氨基酸残基替代：天然存在的20种标准氨基酸残基、罕见的天然存在的氨基酸残基(例如羟脯氨酸、羟赖氨酸、别羟赖氨酸、6-N-甲基赖氨酸、N-乙基甘氨酸、N-甲基甘氨酸、N-乙基天冬酰胺、别异亮氨酸、N-甲基异亮氨酸、N-甲基缬氨酸、焦谷氨酰胺、氨基丁酸、鸟氨酸、正亮氨酸、正缬氨酸)和通常合成获得的非天然存在的氨基酸残基(例如环己基-丙氨酸)。优选地，术语“取代”是指氨基酸残基被选自天然存在的20种标准氨基酸残基的另一种氨基酸残基替代。符号“+”指示取代的组合。

在本文中氨基酸由它们的单字母或三字母代码根据以下命名法表示：A：丙氨酸(Ala)；C：半胱氨酸(Cys)；D：天冬氨酸(Asp)；E：谷氨酸(Glu)；F：苯丙氨酸(Phe)；G：甘氨酸(Gly)；H：组氨酸(His)；I：异亮氨酸(Ile)；K：赖氨酸(Lys)；L：亮氨酸(Leu)；M：甲硫氨酸(Met)；N：天冬酰胺(Asn)；P：脯氨酸(Pro)；Q：谷氨酰胺(Gln)；R：精氨酸(Arg)；S：丝氨酸(Ser)；T：苏氨酸(Thr)；V：缬氨酸(Val)；W：色氨酸(Trp)和Y：酪氨酸(Tyr)。在本文件中，以下术语用于指定取代：L238A表示亲本序列的位置238处的氨基酸残基(亮氨酸，L)被改变为丙氨酸(A)。A132V/I/M表示亲本序列的位置132处的氨基酸残基(丙氨酸，A)被以下氨基酸之一取代：缬氨酸(V)、异亮氨酸(I)或甲硫氨酸(M)。取代可以是保守的或非保守的取代。保守取代的实例是在以下组内的取代：碱性氨基酸(精氨酸、赖氨酸和组氨酸)、酸性氨基酸(谷氨酸和天冬氨酸)、极性氨基酸(谷氨酰胺、天冬酰胺和苏氨酸)、疏水性氨基酸(甲硫氨酸、亮氨酸、异亮氨酸、半胱氨酸和缬氨酸)、芳香族氨基酸(苯丙氨酸、色氨酸和酪氨酸)和小氨基酸(甘氨酸、丙氨酸和丝氨酸)。

本公开内容不意图限于所阐述的特定形式的范围，而是意图涵盖本文描述的变化形式的替代、修改和等同物。此外，本公开内容的范围完全包含鉴于本公开内容对本领域技术人员可以变得明显的其他变化形式。本发明的范围仅由所附权利要求书限定。

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Asp Gln Pro Gln Val Pro Leu Ser Leu Thr Leu Ser Asp Lys Asn Leu

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Ala Asn Ile Phe Gly Phe Pro Gly Gly Val Ala Trp Ala Met Leu Val

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Ala Arg Ile Cys Gln Leu Tyr Pro Asn Ala Cys Ser Ala Val Ile Leu

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Pro Ala Tyr Pro Ser Met Cys Ala Thr His Asn Ile Thr Glu Ser Thr

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Asp Ile Phe Ser Asn Lys Lys Ser Trp Ala Asn Leu Phe Glu Lys Asn

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Asp Phe Phe Phe Arg Tyr Lys Phe Tyr Leu Glu Ile Thr Ala Tyr Thr

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Arg Gly Ser Asp Glu Gln His Leu Lys Trp Ser Gly Leu Val Glu Ser

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Lys Val Arg Leu Leu Val Met Lys Leu Glu Val Leu Ala Gly Ile Lys

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Ile Ala His Pro Phe Thr Lys Pro Phe Glu Ser Ser Tyr Cys Cys Pro

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Pro Thr Glu Glu Glu Lys Arg Leu Asn Asn Ala Leu His Gln Glu Leu

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Arg Ala Gln Gly Thr Phe Glu Ser Pro Ala Glu Thr Glu Lys Arg Lys

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Glu Val Leu Arg Gln Leu Glu Lys Ile Thr Asn Val Phe Val Gln Arg

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Ala Ala Ala Glu Lys Glu Pro Lys Asn Thr Ile Leu Ile Arg Asp Ala

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Pro Pro Gly Ala Ile Thr Asp Leu Thr Pro Val Pro Glu Ala Phe Val

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Pro Ile Ile Lys Phe Glu Phe Ser Gly Ile Ser Ile Asp Leu Ile Phe

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Ala Trp Ala Met Leu Val Ala Arg Val Cys Gln Leu Tyr Pro Lys Ala

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Pro Trp Pro Leu Pro Val Leu Leu Lys Asp Ile Glu Tyr Gly Cys Pro

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Asn His Arg Met Pro Ile Ile Thr Pro Ser Tyr Pro Ser Met Cys Ala

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Thr His Asn Val Gly Arg Ser Ser Met Ala Val Ile Lys Asp Glu Leu

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Glu Lys Gly Val Gln Val Thr Glu Asp Ile Met Arg Gly Lys Arg Pro

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Trp Lys Asp Leu Phe Thr Lys His Thr Phe Phe Thr Ser Gly Phe Arg

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Tyr Tyr Leu Thr Val Ile Ser Ser Ser Arg Thr Lys Lys Ala Gln Asn

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Val Trp Ser Gly Phe Val Glu Ser Arg Val Arg Leu Leu Val Asn Lys

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Tyr Asp Arg Glu His Arg Cys Lys Asn Asn Ala Gln Leu Glu Glu Val

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Val Ser Leu Gly Ser Leu Ala Tyr Met Tyr Lys Pro Ala Thr Gly Glu

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Ser Gly Ala Ser Val Lys Asn Glu Ala Pro Glu Ala Lys Gly Glu Asp

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Pro Thr Glu Gln Glu Lys Ala Leu Asn Lys Ala Leu His Asp Glu Leu

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Arg Ala Gln Gly Thr Phe Glu Ser Arg Ala Glu Thr Glu Lys Arg Lys

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Ala Ala Arg Glu Lys His Gly Lys Asn Ala Ile Leu Ile Arg Asp Ala

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Ile Gly Arg Val Phe Thr Tyr Gly Ser Tyr Arg Leu Gly Val Tyr Gly

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Pro Pro Ala Ala Ile Thr Asp Leu Thr Pro Val Pro Glu Ala Phe Val

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Pro Ile Ile Lys Phe Glu Phe Ser Gly Ile Ser Ile Asp Leu Ile Phe

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Cys Ser Ile Gln Thr Leu Ile Gln Leu Pro Ala Asp Lys Ser Trp Ser

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Leu Ala Asp Asn Asn Leu Leu Arg Gly Leu Ser Glu Asn Ala Val Arg

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Ser Leu Asn Gly Thr Arg Val Thr Asp Glu Ile Leu His Leu Val Pro

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Glu Pro Ala Thr Phe Lys Leu Ala Leu Arg Ala Ile Lys Leu Trp Ala

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Gln Arg Lys Ala Ile Tyr Ala Asn Ile Met Gly Tyr Pro Gly Gly Val

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Ala Trp Ala Met Leu Val Ala Arg Val Cys Gln Leu Tyr Pro Lys Ala

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Thr Ser Ala Val Ile Val Asn Lys Phe Phe Asn Ile Met Leu Lys Trp

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Pro Trp Pro Leu Pro Val Leu Leu Lys Asp Ile Glu Tyr Asn Gly Pro

275 280 285

Val Thr Arg Val Pro Val Trp Asn Pro Lys Leu Tyr Ala Ser Asp Arg

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Asn His Lys Met Pro Ile Ile Thr Pro Ala Tyr Pro Ser Met Cys Ala

305 310 315 320

Thr His Asn Val Gly Arg Ser Ser Met Val Val Ile Gln Gln Glu Leu

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Lys Lys Gly Ala Glu Val Thr Glu Glu Ile Met Leu Gly Arg Arg Pro

340 345 350

Trp Lys Asp Leu Phe Thr Lys His Thr Phe Phe Thr Ser Gly Phe Lys

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Tyr Tyr Leu Thr Val Ile Ser Ser Ser Arg Thr Lys Lys Ala Gln Asn

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Val Trp Ser Gly Phe Ile Glu Ser Arg Val Arg Leu Leu Val Asn Lys

385 390 395 400

Ile Glu Met His Pro Ser Ile Ala Leu Ala Arg Pro Phe Asn Lys Gly

405 410 415

Tyr Asp Arg Met His Arg Cys Lys Asn Asp Ala Gln Val Glu Glu Val

420 425 430

Val Ser Ala Gly Ser Leu Ala Tyr Val Tyr Thr Pro Pro Ala Phe Gly

435 440 445

Asp Glu Lys Val Lys Ser Glu Thr Lys Ser Glu Val Lys Gln Glu Val

450 455 460

Lys Gln Glu Val Arg Gln Asp Asp Val Ile Gln Asp Gly Val Pro Val

465 470 475 480

Lys Gln Glu Lys Ala Glu Val Arg Ala Glu Asp Gly Val Arg Ile Lys

485 490 495

Arg Glu Leu Ser Glu Glu Val Gln Leu Pro Pro Pro Thr Asn Val Lys

500 505 510

Pro Glu Pro His Asp Asp Glu Ala Lys Val Lys Leu Glu Asp Ile Pro

515 520 525

Glu Lys Glu Ala Gln Pro Glu Asp Met Glu Ile Tyr Thr Thr Asn His

530 535 540

Tyr Ile Gly Leu Gln Leu Val Glu Gly Ala Lys Ser Leu Asp Leu Ser

545 550 555 560

Arg Glu Val Asn Asp Trp Lys Ala Met Cys Met Ser Asn Glu Leu Tyr

565 570 575

Glu Glu Gly Leu Met Phe Leu Ser Ile Gln His Leu Lys Asn Thr Ala

580 585 590

Leu Pro Asp Asp Val Phe Glu Pro Gly Glu Val Lys Pro Arg Pro Gly

595 600 605

Lys Lys Val Leu Lys Arg Gly Ala Ser Glu Glu Pro Ser Lys Gln Gln

610 615 620

Pro Pro Ala Lys Arg Gln Ala His Val Gln Pro Arg Ala Pro Ala Ala

625 630 635 640

Gln Gln Pro Ser Ser Thr Ala Thr Ala Ala Ala Gly Ser Ser Gly Glu

645 650 655

Asn Leu Tyr Phe Gln Gly Ser Ser Gly Ser His His His His His His

660 665 670

<210> 4

<211> 405

<212> PRT

<213> 粟酒裂殖酵母(schizosaccharomyces pombe)

<400> 4

Met Asn Ile Ser Ser Ala Gln Phe Ile Pro Gly Val His Thr Val Glu

1 5 10 15

Glu Ile Glu Ala Glu Ile His Lys Asn Leu His Ile Ser Lys Ser Cys

20 25 30

Ser Tyr Gln Lys Val Pro Asn Ser His Lys Glu Phe Thr Lys Phe Cys

35 40 45

Tyr Glu Val Tyr Asn Glu Ile Lys Ile Ser Asp Lys Glu Phe Lys Glu

50 55 60

Lys Arg Ala Ala Leu Asp Thr Leu Arg Leu Cys Leu Lys Arg Ile Ser

65 70 75 80

Pro Asp Ala Glu Leu Val Ala Phe Gly Ser Leu Glu Ser Gly Leu Ala

85 90 95

Leu Lys Asn Ser Asp Met Asp Leu Cys Val Leu Met Asp Ser Arg Val

100 105 110

Gln Ser Asp Thr Ile Ala Leu Gln Phe Tyr Glu Glu Leu Ile Ala Glu

115 120 125

Gly Phe Glu Gly Lys Phe Leu Gln Arg Ala Arg Ile Pro Ile Ile Lys

130 135 140

Leu Thr Ser Asp Thr Lys Asn Gly Phe Gly Ala Ser Phe Gln Cys Asp

145 150 155 160

Ile Gly Phe Asn Asn Arg Leu Ala Ile His Asn Thr Leu Leu Leu Ser

165 170 175

Ser Tyr Thr Lys Leu Asp Ala Arg Leu Lys Pro Met Val Leu Leu Val

180 185 190

Lys His Trp Ala Lys Arg Lys Gln Ile Asn Ser Pro Tyr Phe Gly Thr

195 200 205

Leu Ser Ser Tyr Gly Tyr Val Leu Met Val Leu Tyr Tyr Leu Ile His

210 215 220

Val Ile Lys Pro Pro Val Phe Pro Asn Leu Leu Leu Ser Pro Leu Lys

225 230 235 240

Gln Glu Lys Ile Val Asp Gly Phe Asp Val Gly Phe Asp Asp Lys Leu

245 250 255

Glu Asp Ile Pro Pro Ser Gln Asn Tyr Ser Ser Leu Gly Ser Leu Leu

260 265 270

His Gly Phe Phe Arg Phe Tyr Ala Tyr Lys Phe Glu Pro Arg Glu Lys

275 280 285

Val Val Thr Phe Arg Arg Pro Asp Gly Tyr Leu Thr Lys Gln Glu Lys

290 295 300

Gly Trp Thr Ser Ala Thr Glu His Thr Gly Ser Ala Asp Gln Ile Ile

305 310 315 320

Lys Asp Arg Tyr Ile Leu Ala Ile Glu Asp Pro Phe Glu Ile Ser His

325 330 335

Asn Val Gly Arg Thr Val Ser Ser Ser Gly Leu Tyr Arg Ile Arg Gly

340 345 350

Glu Phe Met Ala Ala Ser Arg Leu Leu Asn Ser Arg Ser Tyr Pro Ile

355 360 365

Pro Tyr Asp Ser Leu Phe Glu Glu Ala Pro Ile Pro Pro Arg Arg Gln

370 375 380

Lys Lys Thr Asp Glu Gln Ser Asn Lys Lys Leu Leu Asn Glu Thr Asp

385 390 395 400

Gly Asp Asn Ser Glu

405

<210> 5

<211> 25

<212> PRT

<213> 人工序列(Artificial Sequence)

<220>

<223> His纯化标签

<400> 5

Met Ala Ser Ser His His His His His His Ser Ser Gly Ser Glu Asn

1 5 10 15

Leu Tyr Phe Gln Thr Gly Ser Ser Gly

20 25

<210> 6

<211> 16

<212> DNA

<213> 人工序列(Artificial Sequence)

<220>

<223> 引物

<400> 6

tcatttcact ctcaca 16

<210> 7

<211> 36

<212> DNA

<213> 人工序列(Artificial Sequence)

<220>

<223> 引物

<400> 7

tgtgagagtg aaatgaggug ugagagugaa augagg 36

<210> 8

<211> 566

<212> PRT

<213> 烧土火丝菌(Pyronema omphalodes)

<400> 8

Met Ser Ala Pro Pro Lys Gln Tyr Gly Thr Thr Pro Pro Ile Asn Thr

1 5 10 15

Asn Pro Pro Thr Lys Glu Asp Met Glu Arg Asn Ser Glu Leu Val Gln

20 25 30

Glu Leu Lys Asn Gln Asn Cys Phe Glu Glu Lys Ala Glu Ser Asp Lys

35 40 45

Arg Val Lys Val Leu Glu Ile Leu Gln Thr Leu Gly Glu Ala Phe Val

50 55 60

Lys Lys Ala Cys Ala Ala Lys Gly Leu Pro Asp His Leu Val Asn Asn

65 70 75 80

Ser Gly Gly Lys Ile Phe Thr Phe Gly Ser Tyr Arg Leu Gly Ala Tyr

85 90 95

Gly Pro Gly Ser Asp Ile Asp Thr Leu Leu Val Val Pro Ala His Ile

100 105 110

Glu Arg Ala Asp Phe Phe Gln His Val Pro Lys Met Leu Ser Glu Leu

115 120 125

Asn Pro Pro Ala Glu Glu Val Ser Pro Val Pro Asp Ala Phe Val Pro

130 135 140

Ile Ile Lys Phe Glu Leu Gln Ser Ile Ser Ile Asp Leu Ile Phe Ala

145 150 155 160

Lys Val Pro Ser Val Asn Ser Ile Pro Arg Asp Met Thr Leu Glu Asn

165 170 175

Lys Asp Ile Leu Lys Gly Cys Asp Glu Ala Asn Leu Arg Gly Leu Asn

180 185 190

Gly Val Arg Leu Thr Asp Glu Leu Leu Gly Leu Val Pro His Pro Val

195 200 205

Asn Phe Arg Met Ala Leu Arg Ala Ile Lys Leu Trp Ala Lys Ser Arg

210 215 220

Ala Ile Tyr Ala Asn Val Met Gly Phe Pro Gly Gly Ile Ala Trp Ala

225 230 235 240

Met Leu Val Ala Lys Ile Cys Gln Leu Tyr Pro Met Ala Val Ser Ala

245 250 255

Val Ile Val Cys Lys Phe Phe Thr Ile Tyr Thr Lys Trp Lys Trp Pro

260 265 270

Gln Pro Val Leu Leu Lys Asp Ile Leu Asp Glu Gly Gly Gln Gly Ala

275 280 285

Gly Leu Arg Val Trp Asn Pro Lys Ile Tyr Ala Ser Asp Arg Gly His

290 295 300

Leu Met Pro Val Ile Thr Pro Asn Tyr Pro Cys Met Cys Ser Thr His

305 310 315 320

Asn Ile Thr Lys Ser Thr Lys Ala Val Ile Leu Arg Glu Met Asp Arg

325 330 335

Ser Leu Asn Ile Ile His Glu Ile Met Asp Gly Lys Ser Pro Trp Ser

340 345 350

Lys Leu Phe Ala Pro His Thr Phe Phe Thr Gln Asp Tyr Arg Tyr Tyr

355 360 365

Leu Arg Val Ile Ser Ala Ala Arg Thr Lys Asp Glu Gln Leu Leu Trp

370 375 380

Ser Gly Leu Val Glu Ser Lys Leu Arg His Leu Val Ser Lys Leu Glu

385 390 395 400

Met Leu Asp Asn Ile Gln Leu Ala His Pro Phe Asn Lys Gly Phe Glu

405 410 415

Tyr Glu Val Glu Cys Asn Asn Glu Asp Glu Val Met Arg Val Ile Arg

420 425 430

Gly Glu Asn Ile Asp Gln Ser Glu Ala Asn Gly Thr Ala Val Asp Ser

435 440 445

Asn Gly Lys Ser Ser Lys Val Lys Ala Tyr Thr Thr Ser Phe Tyr Val

450 455 460

Gly Leu Val Leu Asp Thr Ser Lys Ser Lys Gln Phe Asp Ile Ser Trp

465 470 475 480

Ala Cys His Glu Phe Tyr Asp Ile Cys Lys Thr Trp Asn Leu Tyr Asn

485 490 495

Asp Asp Met His Ser Ile His Val Val Asn Thr Arg Asn Phe Glu Leu

500 505 510

Pro Asp Asn Val Phe Lys Pro Gly Glu Thr Lys Pro Thr Lys Lys Thr

515 520 525

Lys Thr Ile Lys Arg Lys Ile Pro Asn Ala Ala Gly Lys Lys Arg Gly

530 535 540

Ala Asp Glu Ile Ser Ala Pro Asp Ser Lys Arg Ala His Ile Asn Glu

545 550 555 560

Val Pro Pro Asn Ala Asn

565

<210> 9

<211> 598

<212> PRT

<213> 印度腥黑粉菌(Tilletia indica)

<400> 9

Met Thr Lys His Leu Gly Val Thr Pro Ile Val Ser Asp Ala Ser Pro

1 5 10 15

Thr Ala His Glu Ile Ala Leu Glu Thr Gln Leu Val Thr Glu Leu Thr

20 25 30

Thr Gln Asn Cys Phe Glu Ser Pro Gln Asp Ser Arg Leu Arg Glu Val

35 40 45

Val Leu Gly Lys Ala Asp Lys Leu Val Lys Glu Phe Val Tyr Arg Ala

50 55 60

Ala Ile Ala Arg Gly Leu Pro Glu Ser Ala Ala Arg Glu Leu Gly Gly

65 70 75 80

Lys Ile Phe Thr Phe Gly Ser Tyr Arg Leu Gly Val His Gly Pro Gly

85 90 95

Ser Asp Ile Asp Thr Leu Cys Val Val Pro Lys His Val Gln Arg Glu

100 105 110

Asp Phe Phe Thr Ile Phe Glu Gln Met Leu Arg Gln Arg Glu Asp Val

115 120 125

Thr Asp Val Ala Pro Val Pro Glu Ala Tyr Val Pro Leu Ile Ala Val

130 135 140

Asn Phe Met Gly Ile Ala Ile Asp Phe Leu Phe Ala Arg Leu Ser Leu

145 150 155 160

Ser Arg Ile Asp Asp Ser Leu Asp Leu Ser Asp Asn Thr Ile Leu Lys

165 170 175

Asn Met Asp Asp Gln Asp Ile Arg Ser Val Gly Gly Ser Arg Val Thr

180 185 190

Asp Glu Ile Leu Arg Leu Val Pro Asn Val Glu Thr Phe His Thr Ala

195 200 205

Leu Arg Ala Ile Lys Leu Trp Ala Gln Arg Arg Ala Ile Tyr Lys Asn

210 215 220

Met Val Gly Phe Pro Gly Gly Val Ala Trp Ala Met Leu Val Ala Arg

225 230 235 240

Ile Cys Gln Leu Tyr Pro Asn Gly Asn Ala Ala Val Ile Ile Glu Arg

245 250 255

Phe Phe Ile Ile Met Phe Gln Trp Asn Trp Pro Gln Pro Val Met Leu

260 265 270

Lys His His Ile Tyr Glu Ala Asp Pro Ile Leu Lys Val Trp Asn Pro

275 280 285

Arg Leu Tyr Ala Ala Asp Arg Ser His Arg Met Pro Ile Ile Thr Pro

290 295 300

Ala Tyr Pro Gly Met Cys Ala Thr His Asn Ile Thr Ala Ser Thr Gln

305 310 315 320

Ala Val Leu Thr Leu Glu Phe Lys Arg Gly Ile Glu Ile Met Ser Ala

325 330 335

Met Ser Ala Ser Ile Thr Asn Pro Ala Leu Pro Gln Thr Thr Trp Ala

340 345 350

Thr Leu Phe Glu Ser Arg Arg Phe Phe Glu Glu Tyr Lys Tyr Tyr Leu

355 360 365

Gln Ile Ile Ala Ser Ser Gly Ser Ala Asp Leu Gln Leu Lys Trp Ala

370 375 380

Gly Thr Val Glu Ser Lys Leu Arg His Leu Val Leu Ser Thr Glu Ala

385 390 395 400

Gln Pro Gly Val Lys Ile Ala His Pro Tyr Thr Arg Glu Ile Asp Leu

405 410 415

Val Ser Glu Cys His Thr Asp Glu Glu Val Arg Arg Val Ala Thr Gly

420 425 430

Asp Val Pro Pro Glu Val Ala Ala Arg Ala Arg Thr Val Glu Gly Glu

435 440 445

Gly Val Leu Pro Glu Ser Lys Glu Val Asp Lys Leu Lys Glu Ile Glu

450 455 460

Ser Lys Glu Glu Glu Ala Arg Lys Lys Gly Arg Arg Thr Ile Trp Thr

465 470 475 480

Thr Thr Phe Tyr Ile Gly Leu Ala Leu His Asp Gly Ser Asp Asp Lys

485 490 495

Thr Gly Gln Ser Ala Pro Lys Arg Leu Asp Leu Val Gly Pro Ile Gln

500 505 510

Asp Phe Lys Gly Arg Cys Thr Glu Trp Ala Thr Tyr Asp Glu Ser Thr

515 520 525

Met Gly Val Val Val Arg His Val Lys Arg Ser Gln Leu Pro Asp Ser

530 535 540

Val Phe Pro Asp Gly Lys Arg Pro Ala Ala Pro Val Lys Lys Val Val

545 550 555 560

Lys Lys Arg Lys Arg Asn Ala Gly Met Ala Glu Ala Ala Ala Glu Thr

565 570 575

Lys Glu Glu Glu Gly Ala Asp Glu Gln Glu Ala Lys Arg Ala Arg Thr

580 585 590

Asn Gly Asp Ser Ala Ala

595

<210> 10

<211> 574

<212> PRT

<213> Clathrospora elynae

<400> 10

Met Asn Gly Pro Pro Lys Thr Gln Tyr Gly Val Thr Ser Ala Ile Ser

1 5 10 15

Glu Ala Ala Pro Thr Glu Glu Asp Arg Leu Leu Asn Asp Lys Leu Ile

20 25 30

Glu Thr Leu Lys Arg Glu Asn Val Phe Glu Thr Pro Glu Gly Asn Ala

35 40 45

Lys Arg Glu Glu Val Ile Leu His Leu Gln Lys Val Val Glu Glu Phe

50 55 60

Val Arg Arg Val Gly Lys Gln Lys Gly Val Pro Gln Ser Thr Ile Asp

65 70 75 80

Ala Ala Gly Gly Lys Val Phe Thr Phe Gly Ser Tyr Ala Leu Gly Val

85 90 95

His Gly Pro Ser Ser Asp Ile Asp Thr Leu Val Val Ala Pro Lys Phe

100 105 110

Val Thr Ile Asp Glu Phe Phe Gln Thr Phe Pro Pro Thr Phe Lys Glu

115 120 125

Met Ser Arg Val Glu Asp Ile Lys Glu Phe Val Pro Val Glu Asp Ala

130 135 140

Phe Val Pro Ile Ile Lys Met Glu Tyr Arg Gly Val Ser Ile Asp Leu

145 150 155 160

Leu Phe Ala Ser Leu Pro Arg Met Ala Ser Ile Pro Lys Asp Met Asp

165 170 175

Thr Ile Asp Lys Lys Asn Leu Glu Gly Leu Gly Glu Ser Ala Thr Arg

180 185 190

Ser Val Asn Gly Thr Arg Val Thr Lys Glu Leu Leu Ala Ala Val Pro

195 200 205

Gln Asn Asn Ser Phe Arg His Ala Leu Arg Ala Ile Lys Leu Trp Ser

210 215 220

Ser Arg Arg Gly Ile Tyr Gly Ala Val Phe Gly Tyr Pro Gly Gly Val

225 230 235 240

Ala Trp Ala Ile Met Val Ala Arg Ile Cys Gln Leu Tyr Pro Phe Ala

245 250 255

Asn Gly Ala Thr Ile Val Ser Lys Phe Phe Ser Leu Met Tyr Lys Trp

260 265 270

Thr Trp Pro Arg Pro Val Met Leu Lys His Ile Glu Glu Gly Ala Met

275 280 285

Gly Leu Arg Val Trp Asn Pro Gln Ile Tyr Gly Gly Asp Arg Ala His

290 295 300

Leu Met Pro Ile Ile Thr Pro Ala Phe Pro Ser Met Cys Ala Thr His

305 310 315 320

Thr Val Met Pro Ser Thr Leu Arg Ile Met Lys Glu Glu Phe Gly Arg

325 330 335

Ala Asp Lys Ile Leu Gln His Val Phe Ala Gly Thr Lys Lys Trp Asp

340 345 350

Ala Leu Phe Glu Arg His Ser Phe Phe Thr Lys Asp His Lys Tyr Tyr

355 360 365

Leu Ser Val Val Ala Ala Ser Arg Thr Lys Glu Ala Asn Ser Thr Phe

370 375 380

Ser Gly Leu Val Gln Ser Lys Ile Arg His Ile Val Lys Gly Ile Asp

385 390 395 400

Asp Gly Gln Thr Gly Val Asp Ile Ala Arg Pro Tyr Ile Asp Tyr Phe

405 410 415

Glu Arg Tyr His Arg Cys Lys Asp Glu Asp Gln Ile Phe Gln Val Cys

420 425 430

Gln Gly Lys Leu Asp His Met Ile Pro Ala Ser Glu Leu Pro Ala Glu

435 440 445

Gly Thr Ala Pro Ala Asn Gly Asp Ser His Ile Met Tyr Thr Thr Thr

450 455 460

Phe Tyr Ile Gly Leu Thr Leu Pro Leu Gly Thr Ala Thr Glu Gly Thr

465 470 475 480

Lys Ser Leu Asp Ile Ser Tyr Pro Val Ser Gln Phe Lys Asn Phe Ile

485 490 495

Thr Asp Ala Asp Lys Phe Asp Lys Glu Thr Met Ser Val Lys Val Val

500 505 510

His Thr Arg Ser Ser Ala Leu Pro Asp Asp Val Phe Val Gln Gly Glu

515 520 525

Thr Arg Pro Lys Lys Pro Val Lys Glu Lys Lys Lys Lys Glu Ala Lys

530 535 540

Ser Ala Lys Arg Gln Phe Ala Asp Thr Gly Leu Asp Val Arg Asp Ser

545 550 555 560

Arg Leu Thr Lys Pro Ser Gln Arg Ser Ser Ala Ser Arg Glu

565 570

<210> 11

<211> 546

<212> PRT

<213> 环捕德氏霉(Drechslerella brochopaga)

<400> 11

Met Pro Ala Ser Val Asp Val Leu Lys Arg Leu Gln Lys Ile Thr Glu

1 5 10 15

Glu Phe Val Arg Gln Val Tyr Thr Ser Lys Asn Gln Asn Glu Leu Thr

20 25 30

Thr Asn Ser Ala Gly Gly Lys Val Phe Thr Tyr Gly Ser Tyr Arg Leu

35 40 45

Gly Val Val Gly Pro Gly Ser Asp Ile Asp Thr Leu Val Val Ala Pro

50 55 60

Lys Leu Val Thr Arg Glu Asp Phe Phe Lys Phe Tyr Pro Pro Leu Leu

65 70 75 80

Lys Ala Leu Asn Lys Ala Asn Glu Pro Pro Val Ile Glu Glu Leu Ala

85 90 95

Pro Val Pro Asp Ala Phe Val Pro Ile Ile Lys Phe Val Met Ser Gly

100 105 110

Ile Ser Ile Asp Leu Ile Phe Cys Arg Leu Gly Val Ala Gln Val Pro

115 120 125

Ala Asp Met Thr Leu Glu Asp Lys Asn Leu Leu Arg Gly Leu Asp Glu

130 135 140

Lys Glu Leu Arg Ser Leu Asn Gly Thr Arg Val Thr Asp Glu Ile Leu

145 150 155 160

Thr Leu Val Pro Val Pro Ala Val Phe Lys His Ala Leu Arg Ala Ile

165 170 175

Lys Ile Trp Ala Lys Cys Arg Ala Ile Tyr Gly Asn Val Tyr Gly Phe

180 185 190

Pro Gly Gly Val Ala Trp Ala Met Leu Val Ala Arg Ile Cys Gln Leu

195 200 205

Tyr Pro Ser Ala Val Ser Ala Val Ile Val Ser Lys Phe Phe Arg Ile

210 215 220

Leu Gly Gln Trp Asn Trp Pro Gln Pro Val Leu Leu Lys Pro Ile Glu

225 230 235 240

Asp Gly Pro Leu Asn Val Arg Val Trp Asn Pro Lys Leu Tyr Pro Ser

245 250 255

Asp Arg Asn His Leu Met Pro Ile Ile Thr Pro Ala Tyr Pro Ser Met

260 265 270

Cys Ser Thr His Asn Ile Thr Pro Ser Thr Lys Ala Val Ile Leu Gly

275 280 285

Glu Met Ala Lys Ala Ala Asp Ile Val Asp Arg Ile Ile Thr Gly Asn

290 295 300

Gly Asn Trp Asn Gln Leu Phe Gln Arg His Thr Phe Phe Thr Lys Asp

305 310 315 320

Tyr Lys Tyr Tyr Leu Thr Val Lys Ala Ser Ala Arg Asp Gln Glu Gln

325 330 335

Ser Val Lys Trp Ser Gly Leu Val Glu Ser Lys Ile Arg His Leu Val

340 345 350

Met Lys Leu Glu Met Leu Ser Asp Val Ile Ala Ser Ala Arg Pro Tyr

355 360 365

Val Lys Pro Phe Glu Lys Val Gln Tyr Cys Arg Asn Gln Glu Glu Ala

370 375 380

His Lys Ile Ala Met Gly Gln His Val Pro Asp Ser Pro Pro Ala Glu

385 390 395 400

Ala Ala Asn Ala Ala Thr Gly Glu Thr Ala Gly Glu Val Ser Glu Gln

405 410 415

Glu Lys Glu Gly Gly Val Pro Val Trp Ile Thr Thr Phe His Ile Gly

420 425 430

Ile Glu Leu Thr Pro Gly Met Val Gly Gln Val Asn Ile Ser Trp Pro

435 440 445

Ala Gln Glu Phe Tyr Ile Met Cys His Asn Trp Glu Gly Tyr Glu Glu

450 455 460

Asn Met His Cys Val Lys Leu Glu Leu Leu Arg Asn Trp Asn Leu Pro

465 470 475 480

Asp Glu Cys Phe Asp Phe Ser Lys Gly Asp Ala Lys Pro Ser Arg Pro

485 490 495

Ile Val Lys Arg Lys Val Ile Arg Lys Val Val Ser Ser Ser Glu Val

500 505 510

Ser Gln Ser Gly Met Lys Arg Thr His Ala Glu Glu Asn Pro Asn Ala

515 520 525

Asp Glu Gly Asp Val Lys Arg Thr Lys Val Asn Ala Pro Val Thr Val

530 535 540

Gln Gly

545

<210> 12

<211> 632

<212> PRT

<213> 早熟禾巨座壳菌(Magnaporthiopsis poae)

<400> 12

Met Ala Glu Arg Gln Leu Gly Val Thr Pro Pro Val Ser Val Ser Leu

1 5 10 15

Pro Ser Glu Phe Glu Leu Gln Ala Thr Asp Ala Leu Leu Ala Glu Leu

20 25 30

Arg Ala Gln Asn Ser Phe Glu Ser Pro Leu Glu Thr Gln Lys Arg His

35 40 45

Arg Val Leu Ala Ser Leu Gln Thr Ile Ala Asp Ala Phe Val Lys Val

50 55 60

Val Ala Gln Glu Arg Glu Pro His Asn Ala Val Leu Ile Lys Asp Ala

65 70 75 80

Arg Ala Lys Val Phe Ala Tyr Gly Ser Leu Arg Leu Gly Val Trp Gly

85 90 95

Pro Gly Ser Asp Ile Asp Thr Leu Val Val Gly Pro Arg Tyr Val Thr

100 105 110

Arg Glu Asp Tyr Phe Lys His Phe Pro Asp Leu Leu Val Lys Met Ala

115 120 125

Pro Pro Gly Ala Ile Thr Asp Leu Ala Val Val Gln Glu Ala Phe Val

130 135 140

Pro Ile Ile Lys Phe Glu Tyr His Gly Ile Ser Ile Asp Leu Ile Phe

145 150 155 160

Ser Arg Ile Ala Thr Leu Lys Gln Leu Pro Ser Asp Pro Ala Trp Asp

165 170 175

Leu Lys Asp Asn Asn Leu Leu Arg Gly Leu Asp Glu Lys Glu Leu Arg

180 185 190

Ser Val Asn Gly Thr Arg Val Thr Asp Glu Ile Leu Ser Leu Val Pro

195 200 205

Glu Gln Asn Ile Phe Arg Thr Ala Leu Arg Ala Ile Lys Leu Trp Ala

210 215 220

Gln Arg Arg Ala Val Tyr Gly Asn Ile Met Gly Phe Pro Gly Gly Val

225 230 235 240

Ala Trp Ala Met Leu Val Ala Arg Val Cys Gln Leu Tyr Pro Lys Ala

245 250 255

Thr Ser Ser Val Ile Val Asn Lys Phe Tyr Ser Ile Leu Leu Gln Trp

260 265 270

Pro Trp Pro Gln Pro Val Leu Leu Lys Pro Ile Gly Asp Gly Pro Leu

275 280 285

Gln Val Arg Val Trp Asn Pro Arg Leu Tyr Lys Gly Asp Ser Tyr His

290 295 300

Leu Met Pro Val Ile Thr Pro Ala Tyr Pro Ser Met Cys Ala Thr Phe

305 310 315 320

Asn Val Thr Arg Ser Ser Met Thr Val Ile Tyr Arg Glu Leu Gln Leu

325 330 335

Ala Ala Glu Val Thr Asn Asn Ile Met Cys Ser Thr Lys Pro Trp Lys

340 345 350

Asp Leu Phe Thr Lys His Thr Phe Phe Thr Lys Asp Phe Lys Tyr Tyr

355 360 365

Met Gln Val Ile Ser Ala Ser Arg Asp Lys Glu Ala His Lys Ile Trp

370 375 380

Ser Gly Phe Val Glu Ser Lys Val Arg Met Leu Val Gln Ser Leu Glu

385 390 395 400

Arg His Asp Ser Ile Ala Val Ala Arg Pro Phe Ile Lys Gly Phe Glu

405 410 415

Arg Phe His Arg Tyr Arg Asn Glu Glu Gln Phe Ala Gln Ile Leu Asp

420 425 430

Gly Asn Leu Gln His Met Val Lys Ala Gly Gly Asp Ala Ser Pro Ser

435 440 445

Gln Asp Gly Ile Gln Ala Lys Lys Glu Asp Asn Ser Ser Val Lys Ala

450 455 460

Glu Glu Asp Ala Lys Val Lys Gln Glu Asn Gly Glu Thr Pro Val Lys

465 470 475 480

Gln Glu Asn Gly Val Lys Val Lys Gly Glu Glu Ser Glu Glu Ala Pro

485 490 495

Leu Ala Ser Ile Pro Glu His Ser Pro Val Asp Thr Lys Gly Asp Ser

500 505 510

Lys Ala Gly Asp Glu Ala Leu Glu Met Tyr Thr Thr Thr His Tyr Ile

515 520 525

Gly Leu Glu Leu Asn Pro Ala Ala Lys Ser Leu Asp Leu Ser Phe Gln

530 535 540

Val Asn Asp Phe Arg Ala Leu Cys His Asn Trp Glu Lys Tyr Lys Asp

545 550 555 560

Glu Leu Ser Asp Arg Cys Tyr Val Asn Ile Lys Asp Leu Arg Asn Phe

565 570 575

Ala Leu Pro Glu Asp Leu Phe Glu Pro Gly Glu Val Lys Pro Val Lys

580 585 590

Ser Val Lys Lys Arg Pro Ala Ala Gly Ala Asn Ala Thr Gly Asp Lys

595 600 605

Lys Arg Pro Ala Asn Glu Asp Asn Ser Asn Pro Ala Lys Arg Arg Gln

610 615 620

Ala Ala Ser Val Ala Ala Ala Gly

625 630

<210> 13

<211> 631

<212> PRT

<213> Cryptococcus depauperatus

<400> 13

Met Leu Gly Val Thr Pro Pro Ile Ser Ile Glu Ala Pro Lys Pro Gly

1 5 10 15

Asp Ile His Ser Ser Glu Ala Leu Met Thr Asp Leu Ile Ala Met Asn

20 25 30

Gln Phe Glu Ser Asp Ser Glu Arg Lys Ile Arg Glu Lys Leu Leu Ser

35 40 45

Asn Ile Ala Gln Leu Val Val Lys Phe Val His Asp Val Ser Leu Lys

50 55 60

Ile Gly Met Ser Glu Lys Met Ala Ser Glu Ser Gly Gly Arg Ile Tyr

65 70 75 80

Thr Ser Gly Ser Tyr Arg Leu Gly Val His Gly Pro Gly Ser Asp Ile

85 90 95

Asp Thr Ile Cys Val Cys Pro Arg His Ile Tyr Arg Glu His Phe Phe

100 105 110

Gly Glu Phe Gln Glu Met Leu Arg Ala Trp Pro Glu Val Thr Glu Ile

115 120 125

Thr Ala Val Thr Gly Ala Phe Val Pro Val Met Lys Thr Val Ile Ser

130 135 140

Gly Val Glu Val Asp Leu Leu Phe Ala Arg Val Asn Leu Pro Glu Ala

145 150 155 160

Gly Asp Ser Leu Asp Ile Glu Lys Asp Glu Ile Leu Arg Gly Val Asp

165 170 175

Asp Ala Ser Gln Arg Ser Leu Asn Gly Pro Arg Val Thr Asp Met Ile

180 185 190

Leu Asn Leu Val Pro Asp Val Thr Thr Phe Arg Thr Ala Leu Arg Ser

195 200 205

Ile Arg Leu Trp Ala Arg Arg Arg Gly Ile Tyr Ser Asn Val Leu Gly

210 215 220

Phe Pro Gly Gly Val Ala Trp Ala Leu Leu Thr Ala Arg Ile Cys Gln

225 230 235 240

Leu Tyr Pro Asn Ala Thr Pro Ser Ile Ile Val Gly Lys Phe Phe Pro

245 250 255

Ile Tyr Tyr Gln Trp Asn Trp Pro Gln Pro Val Ile Leu Lys Lys Ile

260 265 270

Glu Asn Gly Pro Pro Asn Met Gln His Ala Val Trp Asn Pro Lys Leu

275 280 285

Asp Arg Arg Asp Met Ala His Arg Met Pro Val Ile Thr Pro Ala Tyr

290 295 300

Pro Ser Met Cys Ser Thr His Asn Ile Thr Ala Ser Thr Met Ser Ile

305 310 315 320

Ile Ser Lys Glu Met Leu Arg Ala Met Gln Ile Thr Asp Asp Ile Leu

325 330 335

Arg Glu Pro Gly Ala Ser Trp Ala Pro Leu Phe Glu Lys Val Asp Phe

340 345 350

Phe Ser Met Tyr Lys Thr Tyr Val Gln Val Val Ala Ser Ala Ser Thr

355 360 365

Ala Glu Gly Ile Lys Asp Trp Ser Gly Thr Val Glu Ala Arg Ile Arg

370 375 380

Thr Leu Val Gln Asp Leu Glu Asn Thr Asp Ser Ile Leu Thr Ala His

385 390 395 400

Pro Leu Val Gly Gly Val Ser Arg Val Phe Tyr Cys Thr Thr Glu Glu

405 410 415

Glu Gln Ala Ala Ala Ser Gln Gly Glu Leu Thr Ser Glu Met Ile Asp

420 425 430

Arg Thr Glu Glu Glu Val Thr Asp Lys Glu His Arg Asn Ile Phe Thr

435 440 445

Lys Ser Phe Phe Ile Gly Leu Glu Ile Glu Lys Lys Thr Lys Glu Gly

450 455 460

Gly Gly Arg Val Leu Asn Leu Phe Tyr Pro Ser Lys Lys Phe Cys Ser

465 470 475 480

Met Cys Gln Ser Trp Asp Arg Tyr Asn Glu Met Glu Met Ser Val Ile

485 490 495

Leu Arg Pro Ala Lys Arg Ser Asp Leu Pro Ser Phe Val Phe Pro Asp

500 505 510

Gly Ile Pro Ile Ser Lys Lys Lys Ala Lys Arg Gln Gln Gln Asn Gly

515 520 525

Ser Ala Asp Ala Ser Leu Ser Asp Ser Val Glu Gly Gln Gly Pro Ser

530 535 540

Lys Arg Thr Lys Ala Ala Pro Pro Ser Pro Asn Gln Ala Ser Asn Pro

545 550 555 560

Arg Cys Asn Gly Leu Pro Val Gln Asn Gly Ser Asp Pro Val Ser Ser

565 570 575

Lys Ile Gly Glu Glu Val Asp Ile Lys Pro Pro Pro Gly Ile Glu Asn

580 585 590

Leu Pro Pro Leu Ser Asn Ala Ala Met Ser Ser Phe Ala Thr Ala Ala

595 600 605

Lys Gly Val Ala Thr Thr Gln Asp Phe Asn Lys Glu Gly Leu Val Val

610 615 620

Leu Asn Gln Gln Ser Thr Leu

625 630

<210> 14

<211> 571

<212> PRT

<213> 二孢高氏白粉菌(Golovinomyces cichoracearum)

<400> 14

Met Ala Glu Lys Ser Phe Gly Val Thr Ala Pro Leu Ser Val Thr Pro

1 5 10 15

Pro Thr Glu Ser Glu Asn Gln Ala Ser Ser Ala Leu Ile Glu Glu Leu

20 25 30

Lys Arg Gln Asn Asn Tyr Glu Asn Ala Ile Glu Thr Ala Asn Arg Gln

35 40 45

Lys Val Leu Asn Ser Leu Gln Leu Ile Thr Glu Glu Phe Val Arg Gln

50 55 60

Val Ser Arg Ala Gln Gly Leu Pro Ala Asn Leu Ile Lys Ser Ala Gly

65 70 75 80

Gly Met Val Val Thr Phe Gly Ser Tyr Lys Leu Gly Val Ile Gly Pro

85 90 95

Gly Ser Asp Ile Asp Thr Leu Ile Val Ala Pro Gln Asn Val Thr Lys

100 105 110

Glu Asp Phe Phe Ser Arg Phe Pro Asp Leu Leu Arg Ser Met Ala Thr

115 120 125

Glu Gly Asn Ile Thr Glu Leu Thr Ala Lys Pro Asp Ala Phe Ala Pro

130 135 140

Cys Ile Thr Leu Lys Tyr Ala Gly Ile Asp Ile Asp Leu Leu Phe Gly

145 150 155 160

Arg Val Lys Leu Ser Gln Val Pro Arg Asn Leu Ser Leu Leu Asn Gln

165 170 175

Asn Met Leu Arg Gly Leu Asn Asn Glu Glu Val Arg Ser Leu Asn Gly

180 185 190

Val Arg Val Ala Asp Glu Ile Leu Asn Leu Val Pro Glu Pro Ala Ile

195 200 205

Phe Arg Thr Ala Leu Arg Thr Ile Lys Leu Trp Gly Thr Arg Arg Ala

210 215 220

Ile Ala Gly Asn Ile Tyr Gly Phe Pro Gly Gly Val Thr Trp Ala Ile

225 230 235 240

Leu Val Ala Arg Ile Cys Gln Leu Tyr Pro Lys Ala Thr Ser Ser Thr

245 250 255

Ile Val Phe Lys Phe Phe Arg Ile Met Glu Lys Trp Arg Trp Pro Met

260 265 270

Pro Val Leu Leu Lys Glu Ile Asp Asn Leu Asn Ala Leu Gly Leu Lys

275 280 285

Val Trp Asn Pro Lys Ile Tyr Gly Ser Asp Lys Asn His Ile Met Pro

290 295 300

Ile Ile Thr Pro Ala Tyr Pro Glu Met Cys Thr Thr His Asn Phe Ser

305 310 315 320

Leu Ser Thr Lys Ala Ile Leu Glu Lys Glu Leu Lys Arg Gly Gly Asp

325 330 335

Ile Thr Asp Arg Val Met Ser Gly Lys Ala Ser Trp Lys Asp Leu Phe

340 345 350

Ala Lys His Thr Phe Phe Thr Asn Gly Tyr Lys Tyr Tyr Leu Ser Val

355 360 365

Val Ser Ala Ser Arg Asn Lys Glu Ser Gln Leu Phe Trp Ser Gly Phe

370 375 380

Val Glu Ser Lys Val Arg Leu Leu Val Asn Lys Leu Glu Tyr His Pro

385 390 395 400

Ser Ile Ala Leu Ala His Pro Phe Asn Lys Gly Phe Thr Arg Val His

405 410 415

Arg Cys Arg Thr Glu Glu Glu Val Asp Leu Val Lys Asn Gly Ser Leu

420 425 430

Lys Phe His Ala Thr Asp Ile Ala Thr Ser Thr Thr Gly His Gly Leu

435 440 445

Ser Val Glu Thr Thr Ser Lys Glu Lys Leu Asn Glu Thr Lys Asn Val

450 455 460

Glu Gln Asp Glu Asn Leu Ile Met Val Tyr Thr Ala Thr His Tyr Ile

465 470 475 480

Gly Leu Glu Leu Ala Lys Gly Ala Lys Ser Leu Asp Leu Ser Tyr Glu

485 490 495

Val Glu Glu Phe Lys Ser Ile Cys Thr Ser Ser Glu Ala Tyr Asn Gln

500 505 510

Glu Glu Asn Ser Leu Gly Val Ala His Thr Lys Asn Cys Asp Leu Pro

515 520 525

Asp Asp Val Phe Thr Glu Gly Glu Leu Lys Pro Thr Arg Pro Leu Lys

530 535 540

Gln Lys Lys Lys Arg Pro Ala Thr Glu Asp Ser Asn Pro Val Pro Ser

545 550 555 560

Lys Arg Gln Gln Thr Ser Leu Ile Ala Val Gly

565 570

<210> 15

<211> 600

<212> PRT

<213> 威尼克何德霉(Hortaea werneckii)

<400> 15

Met Ala Glu Lys Gln Tyr Gly Val Thr Pro Ala Phe Ser Leu Glu Pro

1 5 10 15

Pro Ser Pro Lys Asp Leu Lys Leu Asn Asp Ala Leu Leu Ala Glu Phe

20 25 30

Lys Ala Gln Asn Asn Phe Ala Pro Gln Ser Asp Thr Glu Lys Arg Glu

35 40 45

Ala Ile Leu Lys Lys Leu Glu Gly Leu Leu Gln Arg Met Val Gln Glu

50 55 60

Val Gly Arg Lys Lys Gly Leu Pro Gln Ser Ile Leu Glu Val Ala Gly

65 70 75 80

Gly Lys Val Phe Thr Tyr Gly Ser Tyr Arg Leu Gly Val Tyr Gly Pro

85 90 95

Asn Ser Asp Val Asp Thr Leu Met Val Gly Pro Lys His Val Thr Arg

100 105 110

Glu Asp Phe Phe Glu His Met Pro Pro Leu Ile Arg Ser Ala Trp Ala

115 120 125

Glu Asp Gln Ile Gly Gly Leu Val Pro Val Pro Gly Ile Gly Thr Pro

130 135 140

Ile Ile Lys Leu Glu Leu Glu Gly Val Asp Ile Asp Leu Ile Tyr Ser

145 150 155 160

Ser Leu Gln Leu Ser Ser Ile Pro Lys Asp Ile Glu Leu Lys Asp Asp

165 170 175

Asn Leu Leu Arg Gly Leu Asp Asp Thr Asp Arg Arg Cys Val Asn Gly

180 185 190

Thr Arg Val Thr Asn Arg Ile Leu Glu Leu Val Pro Gln Thr Lys Thr

195 200 205

Phe Arg Leu Ala Leu Arg Ala Ile Lys Leu Trp Ser Ser Gln Arg Ala

210 215 220

Ile Tyr Gly Asn Ile Val Gly Phe Pro Gly Gly Val Ala Trp Ala Ile

225 230 235 240

Leu Val Ala Arg Val Cys Gln Leu Tyr Pro Lys Ala Ala Ala Pro Leu

245 250 255

Leu Ile Ser Lys Phe Phe Phe Ile Met Lys Arg Trp Asn Trp Pro Lys

260 265 270

Pro Val Phe Leu Gln His Lys Glu Glu Thr Ser Leu Gln Leu Arg Glu

275 280 285

Trp Asp Pro Ile Gln Tyr Arg Gly Asp Gly Phe His Leu Met Pro Ile

290 295 300

Leu Thr Pro Ala Tyr Pro Ser Met Asn Thr Ala His Thr Val Gly Pro

305 310 315 320

Ser Thr Lys Met Ile Ile Met Arg Glu Leu Glu Arg Gly Glu Asn Ile

325 330 335

Val Asn Asp Ile Tyr Ala Asn Lys Arg Pro Trp Lys Asp Leu Phe Gln

340 345 350

Arg His Thr Phe Phe Thr Asn Ala Tyr Lys His Tyr Ile Cys Val Val

355 360 365

Val Ala Ala Lys Asn Lys Asp Ala His Asp Asn Trp Ser Gly Leu Val

370 375 380

Asn Ser Lys Leu Lys Phe Leu Val Lys Gly Ile Glu Asp Ser Gly Gly

385 390 395 400

Ser Ser Val Glu Leu Val Gln Pro Phe Asn Lys Gly Phe Ser Arg Val

405 410 415

His Glu Cys Gln Thr Asn Glu Gln Val Glu Lys Val Leu Asp Gly Ser

420 425 430

Leu Glu Cys Gln Val Lys Glu Thr Lys Thr Thr Glu Glu Gly Asn Asp

435 440 445

Thr Ala Ile Ala Asn Gly Val Ala Gln Thr Asp Thr Glu Gly Leu Glu

450 455 460

Val Pro Lys Ala Asp Ser Glu Ala Arg Ala Glu Gly Ser Gly Thr Thr

465 470 475 480

Lys Leu Trp Thr Thr Thr Phe Tyr Leu Gly Ile Gly Leu Lys Lys Gly

485 490 495

Ala Thr Asp Leu Asp Ile Ser Val Pro Val Arg Asn Leu Gln Gly Asp

500 505 510

Cys Thr Ala Trp Ala Asp Tyr Asn Pro Asp Leu His Ser Ile Lys Ile

515 520 525

Lys His Ile Arg Asn Phe Asn Leu Pro Asp Asp Val Phe Lys Glu Gly

530 535 540

Glu Val Lys Pro Gln Arg Pro Lys Lys Lys Thr Asn Ala Ala Lys Ala

545 550 555 560

Ala Glu Ala Thr Ser Asn Lys Arg Ser Phe Ser Asp Thr Gly Leu Asp

565 570 575

Pro Asn Ala Asp Pro Ala Lys Arg Arg Gln Ser Gly Asn Val Pro Thr

580 585 590

Pro Val Ala Asn Gly Ser Val Gly

595 600

<210> 16

<211> 589

<212> PRT

<213> 污黑腐皮壳菌(Valsa sordida)

<400> 16

Met Ala Glu Ile Gln Tyr Gly Met Thr Pro Pro Leu Ser Thr Glu Leu

1 5 10 15

Pro Lys Asp Pro Glu Lys Arg Ala Asn Asp Ala Leu Phe Ala Glu Leu

20 25 30

Lys Ala Gln Asn Thr Tyr Glu His Ala Thr Glu Thr Gln Lys Arg Glu

35 40 45

Lys Val Leu Lys Ser Leu Gln Ser Ile Ala Asp Val Phe Val Gln Lys

50 55 60

Val Ala Gln Arg Val His Lys Glu Ser Pro Ala Ile Gln Lys Ser Ala

65 70 75 80

Arg Gly Arg Val Phe Thr Tyr Gly Ser Tyr Arg Leu Gly Val Phe Gly

85 90 95

Pro Gly Ser Asp Ile Asp Thr Leu Val Val Ala Pro Lys Tyr Val Thr

100 105 110

Arg Glu Asp Phe Phe Glu Leu Phe Pro Gly Leu Leu Lys Glu Leu Ala

115 120 125

Pro Ala Gly Ser Ile Thr Asp Leu Thr Ala Val Thr Asp Ala Phe Val

130 135 140

Pro Ile Ile Lys Phe Glu Tyr Ser Asp Ile Ser Ile Asp Leu Ile Phe

145 150 155 160

Ser Arg Ile Ala Thr Leu Thr Glu Ile Pro Ala Leu Thr Thr Lys Trp

165 170 175

Asp Leu Leu Asp Asn Asn Leu Leu Arg Gly Leu Asp Asp Ala Glu Leu

180 185 190

Arg Ser Leu Asn Gly Thr Arg Val Thr Asp Asp Ile Leu Lys Leu Val

195 200 205

Pro Glu Gln Thr Ser Phe Arg Leu Ala Leu Arg Ala Ile Lys Leu Trp

210 215 220

Ala Gln Arg Arg Ala Ile Tyr Ala Asn Ile Met Gly Phe Pro Gly Gly

225 230 235 240

Val Ala Trp Ala Met Leu Val Ala Arg Ile Cys Gln Leu Tyr Pro Lys

245 250 255

Ala Asn Gly Ala Val Ile Val Asp Lys Phe Phe His Ile Ile Arg Arg

260 265 270

Trp Pro Trp Pro Gln Pro Val Leu Leu Lys Asn Pro Glu Asp Gly Pro

275 280 285

Leu Gln Val Arg Val Trp Asn Pro Lys Val Tyr Lys Gly Asp Ser Tyr

290 295 300

His Leu Met Pro Val Ile Thr Pro Ala Tyr Pro Ser Met Cys Ser Thr

305 310 315 320

Phe Asn Val Thr His Ser Asn Lys Ala Val Ile Gln Lys Glu Leu Asp

325 330 335

His Phe Ala Asp Val Val Gln Gln Ile Met Met Gly Lys Leu Pro Trp

340 345 350

Lys Ser Leu Phe Val Lys His Thr Phe Phe Thr Lys Asp Tyr Lys Tyr

355 360 365

Tyr Ile Ala Val Ile Ala Ala Ser Thr Thr Lys Glu Asn Ser Lys Ile

370 375 380

Trp Gly Gly Phe Val Glu Ser Lys Ile Arg Leu Leu Val Gln Gly Leu

385 390 395 400

Glu Arg His Ser Ser Ile Arg Leu Ala Arg Pro Phe Asn Lys Gly Tyr

405 410 415

Glu Arg Lys His Arg Thr Val Ser Gly Met Asn Glu Trp His Asp Ile

420 425 430

Leu Asp Gly Ser Leu Asp Tyr Val Val Lys Asp Glu Gln Val Ala Asp

435 440 445

Glu Lys Ala Lys Thr Glu Pro Gly Asn Asp Ala Val Lys Thr Glu Pro

450 455 460

Thr Ser Pro Ala Lys Val Lys Gln Glu Glu Gly Ile Glu Ser Leu Glu

465 470 475 480

Ile Lys Asp Asp Ala Lys Glu Thr Asp Gly Lys Asn Glu Gln Asp Glu

485 490 495

Glu Asp Ala Glu Val Lys Pro Glu Thr Glu Asp Pro Asp Ser Pro Lys

500 505 510

Asp Val Tyr Thr Ser Thr His Tyr Ile Gly Ile Glu Leu His Asp Glu

515 520 525

His Tyr Ser Trp Ser Leu Pro Asp Asp Val Phe Glu Ala Gly Glu Thr

530 535 540

Lys Pro Thr Arg Pro Leu Lys Lys Lys Ala Ala Gln Pro Pro Asn Gly

545 550 555 560

Ala Lys Arg Lys Thr Pro Thr Glu Gly Ala Leu Asn Ser Ala Ile Asp

565 570 575

Ala Ala Lys Arg Gln Lys Thr Ser Thr Val Ser Ala Gly

580 585

<210> 17

<211> 588

<212> PRT

<213> 梅里科拉壁菌(Wallemia mellicola)

<400> 17

Met Ala Glu Lys Gln Trp Gly Ile Thr Pro Pro Ile Ser Leu Ala Ser

1 5 10 15

Pro Thr Asp Leu Glu Lys Gln Val Asp Ala Gly Leu Ile Gln Glu Leu

20 25 30

Lys Asp Gln Asn Thr Ile Glu Ser Glu Gln Glu Ser Leu Asn Arg Glu

35 40 45

Lys Val Leu Ala Leu Val Gln Ser Leu Val Lys Glu Phe Val Lys Arg

50 55 60

Val Ser Ile Arg Asn Gly Met Ser Glu Ser Leu Ala Asp Gln Ala Gly

65 70 75 80

Gly Lys Ile Phe Thr Phe Gly Ser Tyr Arg Leu Gly Val Asn Gln Pro

85 90 95

Gly Ala Asp Ile Asp Thr Leu Cys Val Val Pro Lys His Val Ser Arg

100 105 110

Glu Asp Phe Phe Asp Val Phe Glu Pro Leu Leu Lys Thr Arg Glu Glu

115 120 125

Val Ser Val Cys Ala Gly Val Pro Asp Ala Tyr Val Pro Ile Ile Lys

130 135 140

Thr Thr Ile Ser Gly Ile Glu Ile Asp Phe Leu Val Ala Arg Leu Ala

145 150 155 160

Leu Ala Thr Ile Pro Asp Asp Leu Glu Leu Ala Asp Asp Asn Leu Leu

165 170 175

Lys Asn Leu Asp Glu Arg Cys Val Arg Ser Leu Asn Gly Ser Arg Val

180 185 190

Thr Asp Glu Ile Leu Arg Val Val Pro Asn Val Gln Val Phe Arg Glu

195 200 205

Ser Leu Arg Cys Ile Lys Leu Trp Ala Gln Arg Arg Ala Ile Tyr Ser

210 215 220

Asn Val Asn Gly Phe Leu Gly Gly Val Ala Trp Ala Met Leu Val Ala

225 230 235 240

Arg Ile Cys Gln Leu Tyr Pro Asn Glu Asn Ala Gly Ala Val Ile Ala

245 250 255

Lys Phe Phe Thr Ile Leu Tyr Gln Trp Lys Trp Pro His Pro Val Leu

260 265 270

Leu Lys Glu Ile Glu Asp Gly Pro Leu Lys Val Arg Val Trp Asn Pro

275 280 285

Lys Val Gly Ser Ser Thr Leu Leu Ile Tyr Pro Ser Asp Arg Ala His

290 295 300

Arg Met Pro Ile Ile Thr Pro Ala Tyr Pro Ser Met Cys Ser Thr His

305 310 315 320

Asn Val Gly Pro Ser Thr Gln Glu Val Met Arg Gln Glu Phe Lys Arg

325 330 335

Gly Met Asp Ile Leu Asp Gly Ile His Lys Gly Thr Thr Thr Trp Ser

340 345 350

Gln Leu Phe Asn Lys His Glu Phe Phe Ser Lys Tyr Lys His Tyr Leu

355 360 365

Gln Ile Ile Ala Ser Gly Gln Thr Ala Glu Ser Gln Lys Lys Trp Ser

370 375 380

Gly Ala Val Glu Ser Lys Val Arg Gln Leu Val Ser Lys Leu Glu Leu

385 390 395 400

Val Asp Gly Ile Glu Leu Ala His Pro Phe Val Lys Gly Phe Ser Glu

405 410 415

Val Phe Ile Cys Leu Asn Asp Glu Glu Val Gln His Ala Ala Glu Tyr

420 425 430

Asn Pro Ser Glu Glu Val Lys Glu Arg Ser Lys Lys Pro Glu Glu Tyr

435 440 445

Glu Asn Lys Glu Ala Glu Asp Gly Ile His Arg Val Tyr Ile Ser Thr

450 455 460

Phe Tyr Ile Gly Leu Ala Ile Gln Pro Arg Asn Pro Glu Thr Asn Glu

465 470 475 480

Lys Arg Thr Leu Asn Leu Thr Tyr Pro Thr Asn Glu Phe Met Lys Leu

485 490 495

Thr Lys Leu Trp Asp Gln Phe Val Glu Gly Glu Met Lys Ile Phe Val

500 505 510

Lys Asn Ile Lys Ala Ser Ala Leu Pro Asp Val Val Phe Glu Gly Gly

515 520 525

Ile Arg Thr Ser Ser Ser Lys Ser Ser Lys Lys Lys Ser Lys Arg Pro

530 535 540

Ser Asn Val Met Asp Glu Ser Asn Gly Glu Asn Glu Gln Asn Lys Arg

545 550 555 560

Leu Lys Pro Asp Asn Thr Ser Asn Asn Glu Gln Lys Ser Glu Ala Thr

565 570 575

Ala Leu Ala Pro Ser Ala Val Pro Thr Glu Ser Thr

580 585

<210> 18

<211> 587

<212> PRT

<213> 扇形炭角菌(Xylaria flabelliformis)

<400> 18

Met Ala Glu Lys Thr Trp Gly Ile Thr Lys Pro Ile Ser Ser Ala Pro

1 5 10 15

Pro Thr Pro Ala Glu Ser His Ala Thr Ala Leu Leu Leu Glu Glu Leu

20 25 30

Arg Arg Gln Asn Thr Phe Glu Thr Ala Ser Glu Ile Lys Lys Arg Glu

35 40 45

Lys Val Val Asn Asp Leu Gln Arg Ile Ala Asp Glu Phe Val Arg Lys

50 55 60

Val Ala Arg Ala Lys Glu Pro His Asn Glu Val Leu Ile Arg Asp Ala

65 70 75 80

Arg Gly Glu Val Phe Thr Tyr Gly Ser Tyr Cys Leu Gly Val Tyr Gly

85 90 95

Pro Gly Ser Asp Ile Asp Thr Leu Val Thr Ala Pro Arg Tyr Val Thr

100 105 110

Arg Asp Asp Tyr Phe Lys Tyr Phe Pro Asp Leu Leu Thr Glu Met Ala

115 120 125

Pro Pro Gly Ala Ile Thr Asn Leu Thr Ala Val Glu Asp Ala Phe Val

130 135 140

Pro Ile Ile Lys Phe Glu Tyr Trp Gly Ile Ser Ile Asp Leu Ile Phe

145 150 155 160

Ser Arg Ile Ala Thr Leu Thr Gln Phe Pro Pro His Lys Gln Leu Glu

165 170 175

Leu Thr Ser Asn Glu His Leu Arg Gly Leu Asp Asp Arg Glu Leu Arg

180 185 190

Ser Leu Asn Gly Thr Arg Val Thr Lys Glu Ile Leu Asn Leu Val Pro

195 200 205

Glu Gln Ser Thr Phe Arg Thr Ala Leu Arg Ala Ile Lys Leu Trp Ala

210 215 220

Gln Arg Arg Ala Ile Tyr Ala Asn Ile Ile Gly Phe Pro Gly Gly Val

225 230 235 240

Val Trp Ala Met Met Val Ala Arg Val Cys Gln Leu Tyr Pro Lys Ala

245 250 255

Thr Ser Ala Thr Ile Val Gly Lys Phe Phe Leu Val Met Lys Gly Trp

260 265 270

Pro Trp Pro Ile Pro Val Gln Leu Lys His Met Glu Asp Gly Pro Leu

275 280 285

Asn Val Arg Val Trp Asn Pro Lys Ile Tyr Lys Ser Asp Ser Phe His

290 295 300

Leu Met Pro Val Ile Thr Pro Ala Tyr Pro Gln Met Cys Ala Thr Phe

305 310 315 320

Asn Ile Thr Lys Ser Thr Lys Thr Ile Ile Gln Arg Glu Leu Glu Arg

325 330 335

Gly Val Glu Leu Thr Asp Lys Ile Leu Gly Ser Glu Arg Pro Trp Lys

340 345 350

Asp Leu Phe Val Lys His Thr Phe Phe Thr Gln Asp His Lys Tyr Tyr

355 360 365

Leu Ala Val Ile Ala Thr Ser Thr Thr Lys Glu Ala His Lys Ile Trp

370 375 380

Ser Gly Phe Val Glu Ser Lys Val Arg Ile Leu Val Gly Glu Leu Glu

385 390 395 400

Arg His Ser Ser Ile Ala Leu Ala Arg Pro Phe Asn Lys Gly Phe Glu

405 410 415

Arg Gln His Leu Thr Lys Thr Asp Gln Gln Ala Ala Glu Val Gln Ser

420 425 430

Gly Ser Leu Ala Tyr Val Thr Glu Asp Glu Asp Asn Ser Asp Asp Ala

435 440 445

Thr Val Thr Lys Ala Glu Asn Gly Thr Asp Gly Asn Ala Asp Ser Ser

450 455 460

Asp Gln Lys Val Thr Ala Glu Asn Thr Thr Gln Pro Arg Arg Thr Tyr

465 470 475 480

Thr Thr Thr His Tyr Ile Gly Leu Glu Leu Ala Glu Gly Ser Lys Ser

485 490 495

Leu Asp Leu Ser Tyr Gln Val Asn Ala Phe Lys Gln Leu Cys Ser Glu

500 505 510

Trp Glu Lys Tyr Ser Ala Glu Phe Asn Ser Leu Ser Val Gln His Val

515 520 525

Lys Asn Ile Asn Leu Pro Glu Asp Val Phe Glu Pro Gly Glu Thr Lys

530 535 540

Pro Ala Arg Pro Leu Lys Lys Pro Thr Ala Asn Gly Thr Ala Pro Thr

545 550 555 560

Thr Gln Arg Lys Arg Asn Ala Pro Thr Glu Gly His Pro Pro Pro Ala

565 570 575

Lys Arg Gln Gln Ser Ser Ala Ala Ala Ala Gly

580 585

<210> 19

<211> 592

<212> PRT

<213> 球毛壳菌(Chaetomium globosum)

<400> 19

Met Ser Glu Ile Ile Tyr Gly Val Thr Pro Pro Ile Ser Thr Thr Leu

1 5 10 15

Pro Thr Glu Pro Glu Lys Arg Leu Asn His Ala Leu His Gln Glu Leu

20 25 30

Arg Ala Gln Gly Thr Phe Glu Ser Pro Gln Glu Thr Glu Lys Arg Lys

35 40 45

Glu Val Leu Arg Gln Leu Glu Lys Ile Thr Thr Val Phe Val Gln Arg

50 55 60

Ala Ala Ala Gln Lys Glu Pro Lys Asn Thr Phe Met Ile Arg Asp Ala

65 70 75 80

Ile Gly Arg Val Phe Thr Tyr Gly Ser Tyr Arg Leu Gly Val Tyr Gly

85 90 95

Pro Gly Ser Asp Met Asp Thr Leu Val Val Ala Pro Lys Tyr Val Thr

100 105 110

Val Glu Gln Tyr Phe Glu Ile Phe Pro Glu Val Leu Val Glu Met Ala

115 120 125

Pro Pro Gly Ala Ile Thr Asp Leu Thr Pro Val Pro Glu Ala Phe Val

130 135 140

Pro Ile Ile Lys Phe Glu Phe Ser Gly Ile Ser Ile Asp Leu Ile Phe

145 150 155 160

Cys Ser Ile Gln Thr Leu Lys Gln Leu Pro Asp Glu Lys Asn Trp Ser

165 170 175

Leu Ala Asp Asn Asn Leu Leu Arg Gly Leu Ser Glu Asn Glu Val Arg

180 185 190

Ser Leu Asn Gly Thr Arg Val Thr Asp Asp Ile Leu His Leu Val Pro

195 200 205

Glu Pro Ala Thr Phe Lys Leu Ala Leu Arg Ala Ile Lys Leu Trp Ala

210 215 220

Gln Arg Lys Ala Ile Tyr Ala Asn Ile Met Gly Tyr Pro Gly Gly Val

225 230 235 240

Ala Trp Ala Met Leu Val Ala Arg Val Cys Gln Leu Tyr Pro Lys Ala

245 250 255

Thr Ser Ala Val Val Val Asn Lys Phe Phe His Ile Met Phe Lys Trp

260 265 270

Pro Trp Pro Leu Pro Val Leu Leu Lys Asp Ile Glu Tyr Gly Ser Pro

275 280 285

Val Thr Arg Val Pro Val Trp Asn Pro Lys Ser Ser Arg Arg Ala Gly

290 295 300

Pro Lys Arg His Arg Thr Ser Met Arg Ala Ser Ser Asn Arg Gly Cys

305 310 315 320

Gly Cys Trp Leu Thr Asp Leu Arg Gly Thr Ile Leu Leu Pro Trp Pro

325 330 335

Gly Pro Ser Thr Arg Val Thr Thr Glu Asn Thr Asp Ala Arg Thr Ile

340 345 350

Gly Ser Trp Glu Glu Val Val Ser Val Gly Ser Leu Ala Tyr Val Tyr

355 360 365

Lys Pro Pro Ala Asp Ser Glu Glu Lys Ala Lys Ala Glu Pro Lys Val

370 375 380

Glu Pro Lys Arg Glu Val Lys Gln Glu Ile Lys Ala Glu Asn Arg Glu

385 390 395 400

Pro Ala Ala Ala Glu Glu Lys Leu Glu Thr Arg Gly Glu Asp Gly Met

405 410 415

Arg Ile Lys Gln Glu His Ser Glu Ser Thr Gln Pro Pro Pro Pro Ser

420 425 430

Asn Val Lys Pro Glu Pro His His Asp Thr Asn Ser Glu Val Lys Leu

435 440 445

Glu Asp Ile Pro Gln Lys Lys Lys Asp Glu Pro Asp Glu Met Glu Ile

450 455 460

Tyr Thr Thr Asn His Tyr Ile Gly Leu Gln Leu Val Glu Gly Ala Lys

465 470 475 480

Ser Leu Asp Leu Ser Arg Glu Val Asn Asp Trp Lys Ser Met Cys Thr

485 490 495

Ser Asn Glu Leu Phe Asp Glu Gly Leu Met Phe Leu Ser Ile Gln His

500 505 510

Leu Lys Asn Thr Asn Leu Pro Asp Asp Val Phe Glu Pro Gly Glu Thr

515 520 525

Lys Pro Arg Pro Val Lys Lys Asn Leu Lys Arg Ala Ala Ser Glu Gly

530 535 540

Pro Gly Asn Met Gln Pro Gln Pro Pro Val Lys Arg His Ala Pro Gly

545 550 555 560

Lys Pro Ser Ser His Ser Ala Gln Gln Gln Gln Gln Gln Gln Gln Lys

565 570 575

Gln Arg Gln Gln Arg Pro Ala Ser Thr Ala Ala Ala Ala Val Ala Gly

580 585 590

<210> 20

<211> 591

<212> PRT

<213> Lachancea thermotolerans

<400> 20

Met Asn Gln Gln Lys Thr Tyr Gly Val Thr Gly Pro Ile Ser Thr Ala

1 5 10 15

Gly Pro Thr Ala Ala Glu Asn Gln Leu Asn Asp Ala Leu Ile Gln Glu

20 25 30

Leu Lys Lys Glu Lys Ser Phe Glu Ser Glu Glu Asp Thr Lys Lys Arg

35 40 45

Val Glu Val Leu Arg Ile Leu Gln Asn Leu Ala Gln Glu Phe Val Tyr

50 55 60

Gln Val Ser Lys Lys Arg Asn Met Ser Asp Gly Met Ala Lys Asp Ala

65 70 75 80

Gly Gly Lys Ile Phe Thr Tyr Gly Ser Tyr Arg Leu Gly Val His Gly

85 90 95

Pro Gly Ser Asp Ile Asp Thr Leu Val Val Val Pro Lys His Val Thr

100 105 110

Arg Glu Asp Phe Phe Thr Val Phe Asp Gln Ile Leu Arg Thr Arg Ser

115 120 125

Glu Leu Glu Glu Ile Ala Pro Val Pro Asp Ala Phe Val Pro Ile Ile

130 135 140

Lys Ile Lys Phe Ser Gly Ile Ser Ile Asp Leu Ile Cys Ala Arg Leu

145 150 155 160

Asp Ile Ala Gln Val Pro Val Asn Leu Thr Leu Ala Asp Lys Asn Leu

165 170 175

Leu Arg Asn Leu Asp Glu Lys Asp Leu Arg Ala Leu Asn Gly Thr Arg

180 185 190

Val Thr Asp Glu Ile Leu Gln Leu Val Pro Lys Pro Thr Ser Phe Lys

195 200 205

Ile Ala Leu Arg Ala Ile Lys Leu Trp Ala Gln Arg Arg Ala Val Tyr

210 215 220

Ala Asn Ile Phe Gly Phe Pro Gly Gly Val Ala Trp Ala Met Leu Val

225 230 235 240

Ala Arg Ile Cys Gln Leu Tyr Pro Asn Ala Cys Ser Ala Val Ile Leu

245 250 255

Thr Arg Phe Phe His Ile Leu Thr Lys Trp Asn Trp Pro Gln Pro Val

260 265 270

Leu Leu Lys Pro Ile Glu Asp Gly Pro Leu Gln Val Arg Val Trp Asn

275 280 285

Pro Arg Ile Tyr Ala Gln Asp Arg Ser His Lys Met Pro Val Ile Thr

290 295 300

Pro Ala Tyr Pro Ser Met Cys Ala Thr His Asn Ile Ser Glu Ser Thr

305 310 315 320

Lys Lys Val Ile Leu Ala Glu Leu Glu Arg Gly Ala Gln Ile Ser Ser

325 330 335

Glu Ile Phe Ser Asn Lys Lys Thr Trp Ser Asp Leu Phe Gln Lys His

340 345 350

Asp Phe Phe Tyr Lys Tyr Lys Phe Tyr Leu Thr Val Met Ala Ser Thr

355 360 365

Ser Gly Ser Ser Glu Gln His Leu Lys Trp Ser Gly Leu Val Glu Ser

370 375 380

Lys Leu Arg Leu Leu Val Gln Lys Leu Glu Thr Leu Gly Gly Ile Asn

385 390 395 400

Leu Ala His Pro Phe Thr Lys Pro Phe Glu Ala Ser Tyr Val Tyr Asp

405 410 415

Asn Glu Ala Gln Cys Lys Asp Ile Ile Asp Asn Tyr Gly Thr His Lys

420 425 430

Ala Gln Asp Ile Leu Ala Gln Tyr Thr Glu Val Thr Asp Asp Asn Lys

435 440 445

Asp Thr Asp Gly Val Lys Asp Lys Ala Gln Val His Ile Thr Thr Met

450 455 460

Tyr Ile Gly Leu Asp Val Ala Leu Asp Gly Lys Lys Gln Val Asp Ile

465 470 475 480

His Val Pro Cys Ser Asp Phe Phe Asn Leu Cys Arg Ser Phe Ser Glu

485 490 495

Tyr Asp Asp Thr Asp Met Phe Ser Leu Met Ile Lys Tyr Val Lys Leu

500 505 510

Tyr Asp Leu Pro Asp Asn Val Tyr Val Glu Gly Glu Glu Arg Pro Val

515 520 525

Lys His Ser Lys Arg Lys Lys Leu Gly Lys Asp Ser Lys Lys Ser Lys

530 535 540

Arg Pro Lys Ser Asn Ser Leu Lys Ala Glu Glu Pro Thr Ser Gly Thr

545 550 555 560

Thr Asn Ser Glu Lys Asn Asn Ser Gln Gly Pro Asn Lys Pro Val Lys

565 570 575

His Glu Pro Pro Met Gln Thr Ser Pro Asn Pro Leu Ala Gly Ser

580 585 590

<210> 21

<211> 558

<212> PRT

<213> 八孢裂殖酵母(Schizosaccharomyces octosporus)

<400> 21

Met Ser Val Lys Gln Trp Gly Ile Thr Pro Pro Ile Ser Thr Ala Pro

1 5 10 15

Ala Thr Glu Gln Glu Asn Ala Leu Asn Thr Ala Leu Ile Asp Glu Leu

20 25 30

Lys Arg Gln Asn Leu Phe Glu Ser Ser Ala Glu Ser Glu Arg Arg Ile

35 40 45

Lys Val Leu Asp Asp Leu Gln Arg Ile Ala Thr Glu Phe Val Lys Lys

50 55 60

Val Ser Leu Ala Lys His Met Asn Glu Lys Met Ala Asn Glu Ala Gly

65 70 75 80

Gly Lys Ile Phe Thr Tyr Gly Ser Tyr Arg Leu Gly Val Tyr Gly Pro

85 90 95

Gly Ser Asp Ile Asp Thr Leu Leu Val Val Pro Lys His Val Ser Arg

100 105 110

Glu Asn Phe Phe Gln Asp Leu Glu Pro Met Leu Arg Glu Arg Glu Glu

115 120 125

Ile Thr Glu Leu Ala Ser Val Pro Asp Ala Tyr Val Pro Ile Ile Lys

130 135 140

Phe Lys Tyr Phe Asp Ile Ser Ile Asp Leu Ile Phe Ala Arg Leu Ser

145 150 155 160

Val Pro Lys Val Pro Arg Thr Leu Glu Leu Ser Asp Asn Asn Leu Leu

165 170 175

Lys Gly Val Glu Glu Arg Cys Ile Leu Ser Leu Asn Gly Thr Arg Val

180 185 190

Thr Asp Gln Ile Leu Gln Leu Val Pro Asn Arg Ala Val Phe Lys His

195 200 205

Ala Leu Arg Ala Val Lys Phe Trp Ala Gln Arg Arg Ala Ile Tyr Ala

210 215 220

Asn Val Ile Gly Phe Pro Gly Gly Val Ala Trp Ala Met Met Val Ala

225 230 235 240

Arg Ile Cys Gln Leu Tyr Pro Asn Ala Val Ser Ser Val Ile Val Ala

245 250 255

Lys Phe Phe Arg Ile Leu His Gln Trp Asn Trp Pro Gln Pro Ile Leu

260 265 270

Leu Lys Pro Ile Glu Asp Gly Pro Leu Gln Val Arg Ile Trp Asn Pro

275 280 285

Lys Leu Tyr Pro Ser Asp Lys Ala His Arg Met Pro Ile Ile Thr Pro

290 295 300

Ala Tyr Pro Ser Met Cys Ala Thr His Asn Ile Thr Pro Ser Thr Gln

305 310 315 320

Ala Ile Ile Leu Ser Glu Met Val Arg Ala Gly Glu Ile Ala Asp Gln

325 330 335

Ile Met Val Gly Ser Val Gly Trp Ser Ala Leu Phe Gln Lys His Asp

340 345 350

Phe Phe His Arg Tyr Lys His Tyr Leu Met Ile Thr Ala Ala Ser Lys

355 360 365

Thr Ala Glu Gly Gln Leu Lys Trp Ser Gly Leu Val Glu Ser Lys Leu

370 375 380

Arg His Leu Val Thr Arg Leu Glu Leu Val Glu Ala Ile Ala Leu Ala

385 390 395 400

His Pro Phe Asn Lys Gly Phe Asp Lys Ile His Asn Cys Lys Ser Glu

405 410 415

Glu Glu Ala His Gln Leu Ala Ser Gly Ile Ser Leu Asp Val Ala Ser

420 425 430

Gln Thr Ala Asp Leu Glu Arg Leu Ala Ala Asp Ala Asn Gly Gln Asn

435 440 445

Glu Asn Glu Asp Glu Lys Glu Glu Lys Lys Ile Tyr Pro Val Tyr Thr

450 455 460

Thr Thr Phe Tyr Ile Gly Leu Glu Leu Glu Lys Lys Lys Gly Gln Pro

465 470 475 480

Ile Lys Lys Leu Asp Ile Ser Trp Pro Ala Gln Glu Phe Tyr Glu Leu

485 490 495

Cys Lys Lys Trp Asp Arg Tyr Asp Glu Thr Gln Val Asn Val Phe Ile

500 505 510

Lys Asn Thr Arg Asn Val Asn Leu Pro Asp Glu Val Phe Glu Pro Gly

515 520 525

Glu Glu Lys Pro Lys Ser Ser Lys Lys Arg Ser Tyr Asn Asp Ala Gly

530 535 540

Asn Asn Asn Glu Thr Met Lys Arg Gln Lys Ile Ser Thr Ala

545 550 555

<210> 22

<211> 608

<212> PRT

<213> 棘状外瓶霉(Exophiala spinifera)

<400> 22

Met Ala Glu Lys Gln Trp Gly Met Thr Pro Pro Met Ser Thr Ala Leu

1 5 10 15

Pro Glu Pro Ile Asp Thr Glu Lys Thr Ala Asp Leu Ile Glu Glu Leu

20 25 30

Lys Lys Glu Asn Asn Tyr Glu Pro Leu Glu Ala Thr Gln Lys Arg Met

35 40 45

Ala Thr Leu Gly Leu Leu Asn Arg Val Thr Gln Glu Phe Val Arg Glu

50 55 60

Val Ser Arg Arg Arg Arg Met Pro Pro Ser Gln Ile Glu Gln Phe Gly

65 70 75 80

Gly Lys Ile Phe Pro Tyr Gly Ser Tyr Arg Leu Gly Val Phe Gly Pro

85 90 95

Gly Ser Asp Ile Asp Thr Leu Ala Val Ala Pro Arg His Val Thr Arg

100 105 110

Glu Asp Phe Phe Glu Tyr Phe Pro Thr Val Leu Lys Arg Met Thr Ala

115 120 125

Glu Gly Asp Ile Ser Ser Leu Thr Pro Val Pro Asp Ser Tyr Val Pro

130 135 140

Ile Ile Lys Leu Val Leu Asn Asp Ile Glu Ile Asp Leu Ile Phe Ala

145 150 155 160

Ser Ile Ala Ser Leu Gln Thr Ile Pro Lys Asn Leu Thr Leu Asn Asp

165 170 175

Asn Asn Leu Leu Thr Gly Leu Asp Gln Ala Thr Ile Arg Ala Val Thr

180 185 190

Gly Pro Arg Val Thr Asp Glu Ile Leu Ser Leu Val Pro Glu Gln Lys

195 200 205

Thr Phe Arg Thr Ala Leu Arg Ala Ile Lys Leu Trp Ala Gln Arg Arg

210 215 220

Ala Val Tyr Ala Asn Ile Val Gly Tyr Pro Gly Gly Val Ala Trp Ala

225 230 235 240

Met Leu Val Ala Arg Val Cys Gln Leu Tyr Pro His Ala Val Gly Ala

245 250 255

Thr Leu Val Asp Lys Phe Phe Phe Val Met Lys Asn Trp Asp Trp Pro

260 265 270

Thr Pro Val Met Leu Lys Asp Ile Glu Gln Pro Lys Pro Ser Gln Ala

275 280 285

Asn Glu Phe Lys Val Trp Asn Pro Ala Leu Tyr Lys Gly Asp Lys Lys

290 295 300

Met Leu Met Pro Ile Ile Thr Pro Ala Phe Pro Ser Met Ser Ala Thr

305 310 315 320

Tyr Asn Ile Ser Lys Ser Gly Lys Thr Val Ile Leu Arg Glu Leu Glu

325 330 335

Arg Ala Ser Gln Ile Thr Asn Asn Ile Phe Ala Gly Lys Ala Arg Trp

340 345 350

Ser Asp Leu Phe Lys Lys His Ser Phe Phe Thr Ala Asp His Lys Tyr

355 360 365

Tyr Leu Gly Val Thr Ala Ser Thr Leu Asn Ala Asp Ser Ala Lys Gln

370 375 380

Trp Ala Gly Leu Val Glu Ser Lys Val Arg Ile Phe Val Met Leu Leu

385 390 395 400

Glu Gly Ile Pro Asp Ile Thr Leu Ala Arg Pro Phe Thr Lys Gly Phe

405 410 415

Lys Arg Val His Lys Cys Ala Asp Glu Thr Gln Ile Arg Glu Val Gln

420 425 430

Lys Gly Ser Met Lys Tyr Lys Phe Glu Glu Thr Lys Thr Val Glu Thr

435 440 445

Thr Asp Pro Glu Leu Val Thr Ser Asn Gly Asp Gly Ala Ala Val Pro

450 455 460

Ala Ala Asn Gly Ala Lys Pro Glu Thr Asp Gln Gly Ala His Thr Val

465 470 475 480

Tyr Thr Tyr Thr Phe Tyr Ile Gly Ile Asp Thr Thr Ala Lys Gly Ser

485 490 495

Leu Asn Ile Ala Pro Ser Phe Gln Asn Phe Lys Asp Ile Cys Glu Gly

500 505 510

Trp Ala Ser Phe Asn Arg Asp Ser His Phe Leu Thr Leu Ala Ser Val

515 520 525

Lys Cys Trp Asp Leu Pro Asp Asp Val Phe Asp Ala Lys Ala Gly Glu

530 535 540

Val Lys Pro Ser Arg Pro Val Lys Lys Val Ala Lys Gln Val Lys Ala

545 550 555 560

Glu Ala Lys Ser Gly Val Arg Arg Ser Ile Asn Glu Val Glu Ala Thr

565 570 575

Glu Thr Asn Gly Asp Ala Ala Lys Arg Gln Lys Leu Met Thr Pro Thr

580 585 590

Pro Thr Pro Thr Pro Thr Pro Ala Pro Lys Pro Thr Ala Ala Pro Ala

595 600 605

<210> 23

<211> 626

<212> PRT

<213> 尖端赛多孢子菌(Scedosporium apiospermum)

<400> 23

Met Ala Asp Arg Gln Leu Gly Val Thr Pro Pro Ile Ser Met Ala Leu

1 5 10 15

Pro Thr Pro Ala Glu Ile Glu Ala Asn Asn Ala Met Val Glu Glu Leu

20 25 30

Arg Lys Gln Gly Ile Phe Glu Ser Lys Glu Glu Thr Asp Lys Arg Asn

35 40 45

Leu Val Leu Glu Ser Leu Gln Lys Met Cys Asp Glu Phe Val Thr Arg

50 55 60

Val Ala Gln Glu Lys Asp Gln Gly Leu Ala Arg Asp Ala Arg Gly Lys

65 70 75 80

Ile Phe Thr Tyr Gly Gly Phe Arg Leu Gly Val Phe Gly Pro Gly Ser

85 90 95

Asp Ile Asp Thr Leu Val Val Ala Pro Lys Tyr Val Thr Arg Asp Asp

100 105 110

Tyr Phe Ala Ile Phe Pro Asp Leu Leu Leu Glu Met Ala Pro Lys Gly

115 120 125

Ala Ile Thr Gly Leu Ala Val Val Thr Asp Ala Phe Val Pro Ile Ile

130 135 140

Lys Phe Glu Tyr Trp Gly Ile Ser Ile Asp Met Ile Phe Ser Arg Ile

145 150 155 160

Ala Met Leu Lys Lys Leu Pro Thr Asn Thr Ser Gln Phe Asn Leu Thr

165 170 175

Asp Thr Ala Leu Leu Arg Gly Leu Asp Glu Thr Glu Ile Arg Ser Leu

180 185 190

Asn Gly Thr Arg Val Ala His Glu Ile Leu Asp Leu Val Pro Glu Gln

195 200 205

Ser Thr Phe Gln Met Ala Leu Arg Ala Ile Lys Leu Trp Ala Gln Arg

210 215 220

Arg Ala Ile Tyr Ala Asn Val Met Gly Tyr Pro Gly Gly Val Ala Trp

225 230 235 240

Ala Met Leu Val Ala Arg Val Cys Gln Leu Tyr Pro Arg Ala Thr Ala

245 250 255

Ala Thr Ile Val Ser Lys Phe Phe Cys Ile Met Arg Gln Trp Pro Trp

260 265 270

Pro Gln Pro Val Leu Leu Lys His Ile Glu Arg Ala Pro Leu Gly Tyr

275 280 285

Arg Ile Trp Asn Pro Val Val Tyr Pro Ser Asp Lys His His Leu Met

290 295 300

Pro Ile Ile Thr Pro Ala Tyr Pro Ser Met Asn Ala Ala Tyr Asn Ile

305 310 315 320

Asn Arg Ser Ser Met Ser Ile Ile Gln Thr Glu Leu Ser Arg Ala Asp

325 330 335

Gly Ile Thr Asp Gly Ile Val Val Gly Gln Arg Pro Trp Ser Asp Leu

340 345 350

Phe Glu Lys His Thr Phe Phe Thr Ala Asp Tyr Lys Tyr Tyr Leu Ala

355 360 365

Ile Val Ser Ser Gly Ile Thr Lys Asp Ala His Lys Lys Trp Ser Gly

370 375 380

Phe Val Glu Ser Lys Val Arg Met Leu Val Leu Ala Leu Asp Arg His

385 390 395 400

Asp Thr Ile Ala Leu Ala Gln Ala Phe Val Lys Gly Tyr Asp Arg Val

405 410 415

His Lys Cys Asn Ala Asp Ser Glu Ile Gln Lys Val Gln Gln Gly Asp

420 425 430

Leu Ala Tyr Met Ile Lys Glu Glu Asp Ala Pro Lys Glu Glu Gln Thr

435 440 445

Ser Pro Val Lys Ser Glu Thr Lys Pro Asp Pro Gly Ala Asn Glu Lys

450 455 460

Asn Ser Ala Gly Gly Ser Asn Ser Asn Gly Asp Val Ala Gly Ser Ala

465 470 475 480

Asp Ala Val Lys Asn Glu Glu Gly Ala Asn Gly Val Lys Ala Gly Asp

485 490 495

Glu Asn Val Tyr Ile Tyr Thr Thr Thr His Tyr Ile Gly Leu Ala Leu

500 505 510

Arg Pro Gly Gln Lys Ser Ile Asn Leu Ala Asn Glu Val Gly Glu Phe

515 520 525

Lys Lys Leu Cys Lys Gly Trp Glu Lys Phe Asp His Gln Leu Asn Ala

530 535 540

Ile Thr Val Gln His Leu Arg Asn Ser Asp Leu Pro Asp Asp Val Phe

545 550 555 560

Ala Glu Gly Glu Val Lys Pro Arg Lys Arg Val Val Arg Lys Ala Asn

565 570 575

Gly Thr Gly Pro Ser Pro Pro Val Asp Ala Lys Gln Ala Thr Asn Gly

580 585 590

Thr Thr Ser Gln Val Asp Ser Lys Lys Arg Ala Ala Ala Glu Thr Gly

595 600 605

Ala Ala Ala Pro Ala Ala Lys Arg Arg Gln Pro Ala Ser Val Val Ala

610 615 620

Ala Gly

625

<210> 24

<211> 613

<212> PRT

<213> 桔绿木霉(Trichoderma citrinoviride)

<400> 24

Met Ala Glu Gln Asn Tyr Gly Val Thr Pro Pro Ile Ser Val Thr Leu

1 5 10 15

Pro Thr Glu Ala Glu Asn Arg Ala Ser Asp Ala Leu Leu Glu Glu Leu

20 25 30

Arg Arg Gln Lys Thr Phe Glu Ser Pro Ser Asp Thr Glu Lys Arg His

35 40 45

Glu Ile Cys Asn Glu Phe Val Arg Lys Val Ala Arg Glu Arg Glu Pro

50 55 60

Lys Asn Glu Ile Leu Ile Lys Asn Ala Arg Gly Lys Val Phe Thr Tyr

65 70 75 80

Gly Ser Phe Arg Leu Gly Val Phe Gly Pro Gly Ser Asp Ile Asp Thr

85 90 95

Leu Ile Val Ala Pro Lys Tyr Val Thr Arg Glu Asp Tyr Phe Asn Tyr

100 105 110

Phe Pro Asp Leu Leu Val Ser Met Ala Pro Pro Gly Ala Ile Thr Asp

115 120 125

Leu Thr Val Val Lys Asp Ala Phe Val Pro Ile Ile Lys Phe Glu Tyr

130 135 140

Ser Gly Ile Ser Ile Asp Leu Ile Phe Ser Arg Ile Ile Gln Lys Gln

145 150 155 160

Ile Ala Pro Asp Phe His Ser Leu Lys Asp Ser Ser Leu Leu Arg Gly

165 170 175

Leu Asp Glu Ala Glu Leu Arg Ser Leu Asn Gly Thr Arg Val Thr Asp

180 185 190

Glu Ile Leu Glu Leu Val Pro Glu Lys Ser Thr Phe Lys Leu Ala Leu

195 200 205

Arg Ala Ile Lys Leu Trp Ala Gln Arg Arg Ala Val Tyr Ala Asn Ile

210 215 220

Met Gly Phe Pro Gly Gly Val Ala Trp Ala Met Leu Val Ala Arg Val

225 230 235 240

Cys Gln Leu Tyr Pro Lys Ala Thr Met Ser Val Ile Val Asn Lys Phe

245 250 255

Phe Leu Val Ile Gly Gln Trp Arg Trp Pro Gln Pro Val Leu Leu Lys

260 265 270

Pro Ile Glu Gly Gly Pro Leu Pro Val Arg Val Trp Asn Pro Lys Val

275 280 285

Tyr Lys Gly Asp Ser Phe His Leu Met Pro Val Ile Thr Pro Ala Tyr

290 295 300

Pro Ser Met Cys Ala Thr Phe Asn Ile Thr Arg Ser Ser Met Thr Ile

305 310 315 320

Ile Gln Arg Glu Leu Arg Arg Gly Leu Glu Ile Ser Glu Gln Ile Met

325 330 335

Val Gly Lys Arg Pro Trp Ser Asp Leu Phe Val Lys His Thr Phe Phe

340 345 350

Thr Ser Gly Tyr Arg Tyr Tyr Ile Ser Val Val Ser Ala Ser Lys Asp

355 360 365

Lys Glu Ala His Lys Val Trp Ser Gly Tyr Val Glu Ser Lys Ile Arg

370 375 380

Met Leu Val Gln Lys Leu Glu Gln His Pro Ser Ile Ala Leu Ala His

385 390 395 400

Ala Phe Asn Lys Gly Tyr Asp Arg Arg His Leu Cys Arg Asn Glu Gln

405 410 415

Glu Ile Gly Gln Val Gln Glu Gly Ser Leu Glu Phe Met Ile Lys Asp

420 425 430

Trp Asp Asp Asn Lys Leu Asn Gly Ala Ala Ile Lys Pro Glu Lys Val

435 440 445

Glu Gly Asp Ser Lys Pro Asn Gly Ala Asp Ile Ile Gln Pro Glu Lys

450 455 460

Val Glu Gly Glu Ser Lys Leu Ser Asp Ile Pro Val Lys Pro Glu Met

465 470 475 480

Asp Glu Ala Glu Ser Lys Leu Ser Glu Val Ala Ile Lys Thr Glu Thr

485 490 495

Lys Asp Gly Ser Thr Glu Ser Val Phe Pro Val Glu Val Tyr Thr Thr

500 505 510

Thr His Tyr Ile Gly Leu Glu Leu Glu Glu Gly Ala Lys Ser Leu Asp

515 520 525

Leu Ser Tyr Gln Val Asp Glu Phe Lys Val Leu Cys Thr Ser Trp Lys

530 535 540

Lys Tyr Gln Glu Asp Leu Glu His Leu Val Ser Leu Gly Val Gln His

545 550 555 560

Val Arg Asn Phe Asn Leu Pro Asp Asp Val Phe Glu Pro Gly Glu Gln

565 570 575

Lys Pro Gln Lys Lys Ser Ser Ala Lys Ser Leu Ala Asn Lys Lys Arg

580 585 590

Gly Ala Pro Glu Asp Asn Thr Pro Pro Ala Lys Arg Gln Gln Ala Ser

595 600 605

Val Ala Ala Ala Gly

610

<210> 25

<211> 598

<212> PRT

<213> Aspergillus thermomutatus

<400> 25

Met Ser Ala Pro Pro Val Arg Gln Trp Gly Val Thr Pro Pro Ile Ser

1 5 10 15

Thr Ala Leu Pro Thr Pro Asp Glu Leu Ala Ala Asn Asp Asp Leu Ile

20 25 30

Ser Glu Leu Lys Ala Gln Asn Asn Phe Glu Ser Pro Ala Glu Thr Glu

35 40 45

Arg Arg Lys Gln Val Leu Gln Leu Ile Gln Arg Val Thr His Glu Phe

50 55 60

Val Lys Val Val Ser Arg Lys Lys Gly Leu Ser Pro Ala Ala Val Glu

65 70 75 80

Ala Ala Gly Gly Lys Ile Phe Thr Tyr Gly Ser Tyr Arg Leu Gly Val

85 90 95

Tyr Gly Pro Gly Ser Asp Ile Asp Thr Leu Val Val Gly Pro Lys His

100 105 110

Val Leu Ile Asp Asp Phe Phe Ser Asp Phe Pro Pro Val Leu Glu Lys

115 120 125

Met Ala Pro Pro Gly Ala Ile Glu Lys Met Thr Pro Val Pro Asp Ala

130 135 140

Phe Val Pro Ile Ile Lys Leu Glu Leu Ser Gly Ile Ser Ile Asp Leu

145 150 155 160

Ile Phe Ala Arg Leu Ile Val Ser Ser Val Pro Leu Asn Leu Asp Leu

165 170 175

Lys Asn Asn Asp Tyr Leu Arg Gly Leu Asp Glu Lys Glu Val Arg Ser

180 185 190

Leu Asn Gly Thr Arg Val Thr Asp Glu Ile Leu Glu Leu Val Pro Gln

195 200 205

Gln Lys Thr Phe Arg Leu Ala Leu Arg Ala Ile Lys Leu Trp Ala Gln

210 215 220

Arg Arg Ala Ile Tyr Ser Asn Ile Val Gly Phe Pro Gly Gly Val Ala

225 230 235 240

Trp Ala Met Leu Val Ala Arg Val Cys Gln Leu Tyr Pro His Ala Thr

245 250 255

Gly Ser Val Ile Val Gly Lys Phe Phe Arg Ile Met Asn Lys Trp Ala

260 265 270

Trp Pro Gln Pro Val Leu Leu Lys Pro Ile Glu Asp Gly Pro Leu Gln

275 280 285

Ile Lys Val Trp Asn Pro Lys Ile Tyr His Gly Asp Arg Phe His Leu

290 295 300

Met Pro Ile Ile Thr Pro Ala Tyr Pro Ser Met Cys Ala Thr His Asn

305 310 315 320

Ile Ser Met Ser Thr Lys Ala Val Ile Leu Arg Glu Leu Gln Arg Gly

325 330 335

Gly Asp Ile Val Asp Lys Ile Phe Leu Lys Gln Leu Thr Trp Asn Asp

340 345 350

Leu Phe Ala Arg His Ser Phe Phe Thr His Asp Tyr Lys Tyr Tyr Leu

355 360 365

Ser Ile Thr Ala Ser Ser Arg Thr Lys Glu Ala Glu Ser Val Trp Ser

370 375 380

Gly Leu Val Glu Ser Lys Ile Arg His Leu Val Gly Ala Leu Asp Arg

385 390 395 400

Lys Pro Thr Ile Ala Val Ala His Pro Phe Pro Lys Gly Phe Glu Arg

405 410 415

Val His Ile Ile Ser Asn Glu Glu Glu Ala Glu Ala Val Lys Asn Gly

420 425 430

Ser Thr Lys Tyr Gln Asp Lys Gly Thr Lys Thr Glu Thr Thr Asp Glu

435 440 445

Thr Lys Asp Ala Ala His Gln Ala Ala Ala Gln Ser Gly Val Glu Asn

450 455 460

Ala Glu Val Glu Pro Val Gly Glu Asn Ala Asn Gly Asp Ser Arg Ile

465 470 475 480

Ile Tyr Thr Thr Thr Tyr Tyr Ile Gly Leu Glu Leu Lys Pro Leu Glu

485 490 495

Pro Gly Ala Ser Arg Ser Leu Asp Ile Ser Thr Asp Ala Gln Ile Phe

500 505 510

Lys Ser Thr Cys Thr Ser Trp Ala Gly Tyr Gln Pro Gly Ile Asn Asp

515 520 525

Leu Ser Ile Thr His Val Arg Asn Phe Asp Leu Pro Glu Asp Val Phe

530 535 540

Gln Pro Gly Glu Ala Arg Pro Thr Arg Pro Lys Lys Lys Val Ile Lys

545 550 555 560

Lys Pro Glu Ala Gly Ala Gln Lys Arg Gly Ile Asp Ser Leu Asp Asp

565 570 575

Ala Ser Leu Pro Ala Ala Lys Arg Gln Val Thr Ser Asn Gly Ile Ser

580 585 590

Ser Thr Pro Thr Pro Ala

595

<210> 26

<211> 579

<212> PRT

<213> Sodiomyces alkalinus

<400> 26

Met Gln Asp Arg Pro Leu Gly Val Thr Pro Pro Ile Ser Thr Ser Leu

1 5 10 15

Pro Thr Asp Ala Glu His Ala Val Asn Glu Ser Leu Leu Glu Glu Leu

20 25 30

Lys Arg Gln Asn Thr Phe Glu Ser Pro Ala Glu Thr Ala Lys Arg Glu

35 40 45

Lys Val Leu Glu Gln Leu Gln Leu Ile Cys Asn Glu Phe Val Arg Arg

50 55 60

Val Ala Arg Glu Arg Glu Ala Gly Ser Glu Ala Leu Ile Lys Glu Ala

65 70 75 80

Arg Gly Arg Ile Phe Thr Tyr Gly Ser Phe Arg Leu Gly Val Tyr Gly

85 90 95

Pro Gly Ser Asp Ile Asp Ala Leu Val Val Ala Pro Lys Tyr Val Thr

100 105 110

Arg Asp Asp Tyr Phe Lys Leu Phe Pro Gly Leu Leu Gln Glu Ile Pro

115 120 125

Pro Thr Gly Ala Ile Thr Asp Leu Ala Val Val Ser Asp Ala Phe Val

130 135 140

Pro Ile Ile Lys Phe Asp Phe Leu Gly Ile Ser Ile Asp Leu Ile Phe

145 150 155 160

Ser Arg Ile Ala Ser Leu Lys Gln Leu Pro Lys Asp Lys Asp Trp Asn

165 170 175

Leu Lys Asp Ser Asn Ile Leu Arg Gly Leu Asp Glu Ala Glu Leu Arg

180 185 190

Ser Leu Asn Gly Thr Arg Val Thr Asp Glu Ile Ile Ser Leu Val Pro

195 200 205

Glu Pro Ser Thr Phe Arg Leu Ala Leu Arg Ala Ile Lys Leu Trp Ala

210 215 220

Gln Arg Arg Ala Val Tyr Ala Asn Ile Met Gly Phe Pro Gly Gly Val

225 230 235 240

Ala Trp Ala Met Leu Val Ala Arg Val Cys Gln Leu Tyr Pro Lys Ala

245 250 255

Thr Ser Ser Val Val Val Asn Lys Phe Phe His Ile Met Arg Arg Trp

260 265 270

Pro Trp Pro Gln Pro Val Leu Leu Lys Ala Val Glu Ser Gly Pro Leu

275 280 285

Gln Val Arg Val Trp Asn Pro Lys Leu Tyr Lys Gly Asp Gln Phe His

290 295 300

Leu Met Pro Ile Ile Thr Pro Ala Tyr Pro Ser Met Cys Ala Thr Tyr

305 310 315 320

Asn Ile Thr Lys Ser Ala Met Thr Val Ile Gln Arg Glu Leu Gln Arg

325 330 335

Gly Cys Glu Ile Thr Asp Ser Val Met Met Ser Lys Gln Pro Trp Ser

340 345 350

Asp Leu Phe Val Lys His Ala Phe Phe Thr Ser Asp Tyr Lys His Tyr

355 360 365

Ile Ser Val Ile Thr Thr Ser Thr Thr Lys Glu Ala His Lys Ile Trp

370 375 380

Ser Gly Tyr Val Glu Ser Lys Val Arg Val Leu Val Gln Gly Leu Glu

385 390 395 400

Gln His Pro Ser Ile Ala Leu Ala His Ala Phe Asn Lys Gly Tyr Asp

405 410 415

Arg Arg His Lys Cys Ser Ser Glu Gln Glu Ile Asn Gln Val Gln Glu

420 425 430

Gly Ser Leu Asp Tyr Leu Leu Lys Ala Gly Asp Ser Ser Ala Gly Asp

435 440 445

Pro Ala Glu Gly Asn Lys Ser Pro Arg Arg Pro Ala Asp Gly Glu His

450 455 460

Pro Glu Ile Thr Pro Glu Ala Thr Glu Thr Ile Val Phe Thr Thr Thr

465 470 475 480

His Tyr Ile Gly Leu Glu Leu Val Glu Gly Ala Lys Ser Leu Asp Leu

485 490 495

Ser Tyr Gln Val Asp Ser Phe Lys Gln Leu Cys Gly Gln Trp Glu Lys

500 505 510

Phe Asn Ser Asn Leu Asn Tyr Leu Ser Val Gln His Val Arg Asn Val

515 520 525

Lys Leu Pro Asp Asp Val Phe Glu Pro Cys Glu Thr Arg Pro Gln Lys

530 535 540

Lys Ser Ala Thr Asn Gly Ser Leu Gln Lys Lys Lys Arg Gly Ala Ser

545 550 555 560

Glu Val Asp Met His Pro Pro Ala Lys Arg Gln Gln Ser Ser Val Thr

565 570 575

Ala Ala Gly

<210> 27

<211> 606

<212> PRT

<213> 嗜酸新何德霉(Neohortaea acidophila)

<400> 27

Met Asp Pro Lys Leu Gly Val Ser Gly Thr Met Ser Met Glu Pro Pro

1 5 10 15

Ser Pro Lys Asp Ile Lys Leu Asp Asp Ala Leu Leu Gln Glu Leu Lys

20 25 30

Ser Arg Asn Glu Phe Glu Ala Pro Glu Glu Thr Gln Arg Arg His Ser

35 40 45

Val Leu Asp Arg Leu Glu Ser Val Leu Lys Arg Leu Val Ala Met Val

50 55 60

Gly Lys Gln Gln Gly Leu Pro Pro Gly Ile Leu Asn Glu Ala Gly Gly

65 70 75 80

Lys Ile Phe Thr Phe Gly Ser Phe Glu Leu Gly Val Tyr Gly Pro Lys

85 90 95

Ser Asp Met Asp Thr Leu Met Ala Ala Pro Lys His Val Ser Arg Glu

100 105 110

Asp Phe Phe Gln Tyr Met Pro Asp Leu Leu Arg Lys Glu Phe Lys Pro

115 120 125

Glu Glu Ile Ala Glu Leu Thr Pro Val Pro Gly Ile Ser Val Pro Ile

130 135 140

Ile Lys Leu Glu Leu Cys Gly Val Ser Val Asp Leu Ile Phe Cys Arg

145 150 155 160

Leu His Leu Gln Ser Val Pro Lys Ser Gln Glu Leu Ser Asn Leu Asp

165 170 175

Leu Leu Arg Gly Leu Asp Asp Thr Asp Leu Lys Cys Val Asn Gly Thr

180 185 190

Arg Val Thr Arg Arg Ile Leu Glu Leu Val Pro Gln Thr Lys Val Phe

195 200 205

Arg Met Ala Leu Arg Ala Val Lys Leu Trp Ala Lys Gln Arg Ala Leu

210 215 220

Tyr Gly Asn Ile Val Gly Tyr Pro Gly Gly Val Ala Tyr Ala Met Met

225 230 235 240

Val Ala Arg Ile Cys Gln Leu Tyr Pro Arg Ala Ala Ala Pro Leu Val

245 250 255

Ile Trp Lys Phe Phe Tyr Leu Met Arg Lys Trp Asn Trp Pro Ser Pro

260 265 270

Val Leu Leu Gln Asn His Glu Glu Gly Ser Ile Asn Leu Arg Glu Trp

275 280 285

Asp Pro Ser Ile Tyr Pro Gly Asp Lys Arg His Leu Met Pro Ile Ile

290 295 300

Thr Pro Ala Phe Pro Arg Met Asn Ala Cys His Thr Ile Gly Pro Ser

305 310 315 320

Thr Lys Lys Val Leu Leu Gln Glu Met Glu Arg Ala Glu Gly Ile Val

325 330 335

Arg Ser Ile Tyr Glu Ser Gly Arg Pro Trp Arg Asp Leu Phe Gln Arg

340 345 350

His Ser Phe Phe Thr Asp Ala Tyr Arg His Tyr Ile Cys Val Ile Thr

355 360 365

Ala Gly Arg Thr Lys Glu Ala Gln Gln Ala Trp Ser Gly Leu Val Glu

370 375 380

Ser Lys Val Lys Trp Leu Ile Val Gly Ile Glu His Ser Asp Ala Lys

385 390 395 400

Ser Val Glu Leu Val Gln Pro Tyr Asn Lys Gly Phe Asn Arg Val His

405 410 415

Glu Cys Lys Gly Asp Ala Asp Ile Asp Lys Thr Leu Asp Gly Asn Leu

420 425 430

Asp Cys Gln Val Lys Glu Ile Lys Thr Val Thr Thr Glu Gln Ala Gly

435 440 445

Asp Val Gln Phe Gln Ala Ala Ala Gln Thr Asp Thr Asp Gly Gln Glu

450 455 460

Val Pro Ala Pro Asn Gly Glu Ala Glu Val Pro Pro Gln Thr Asp Gly

465 470 475 480

Pro Gln Thr Ile Trp Thr Thr Thr Phe Tyr Leu Gly Ile Gly Leu Thr

485 490 495

Lys Gly Ala Asn Ser Leu Asp Ile Ser Ser Pro Ile Arg Asp Phe Thr

500 505 510

Gln Gln Cys Lys Glu Trp Gln Asn Tyr Asp Glu Asn Leu His Ser Ile

515 520 525

Arg Val Lys His Met Arg Asn Tyr Asp Leu Pro Ala Asp Val Phe Ala

530 535 540

Glu Gly Glu Thr Arg Pro Thr Arg Thr Lys Lys Lys Ser Ala Pro Lys

545 550 555 560

Thr Thr Asp Pro Thr Ala Met Asp Ala Ala Asn Lys Lys Arg Ser Phe

565 570 575

Asn Asn Ser Gly Leu Asp Thr Leu Asp Asp Pro Ala Lys Arg Arg Ala

580 585 590

Ser Ala Asn Gly Thr Ala Thr Pro Asn Gly Val Pro Pro Arg

595 600 605

<210> 28

<211> 333

<212> PRT

<213> 布氏锥虫(T. brucei)

<400> 28

Met Pro Pro Ser Pro Ala Val Val Gly Arg Ser Leu Val Asn Ser Phe

1 5 10 15

Lys Gln Phe Val Ser Lys Asp Leu His Thr Arg His Val Asp Ala Thr

20 25 30

Tyr Arg Leu Val Leu Asp Cys Val Ala Ala Val Asp Pro Leu Met Arg

35 40 45

Leu Tyr Thr Phe Gly Ser Thr Val Val Tyr Gly Val His Glu Lys Gly

50 55 60

Ser Asp Val Asp Phe Val Val Leu Asn Lys Thr Asp Val Glu Asp Gly

65 70 75 80

Lys Gly Gly Asp Ala Ala Thr Gln Val Ala Lys Gly Leu Gln Ala Asp

85 90 95

Ile Leu Ala Lys Leu Ala Arg Val Ile Arg Gln Lys His Leu Ser Trp

100 105 110

Asn Val Glu Glu Val Arg Arg Thr Arg Val Pro Val Val Arg Val Lys

115 120 125

Gly Gly Gly Ala Val Asp Phe Asp Ile Thr Ala Tyr Arg Arg Asn Gly

130 135 140

Val Arg Asn Ser Ala Leu Leu Arg Ala Tyr Phe Glu Gln Asn Pro Pro

145 150 155 160

Cys Arg Trp Leu Ser Met Ser Ile Lys Arg Trp Ser Lys Gln Thr Gly

165 170 175

Leu Asn Ala Ser Val Ile Gly Gly Ser Ile Thr Ser Tyr Gly Phe Asn

180 185 190

Leu Met Val Val Tyr Tyr Leu Leu Gln Arg Asn His Leu Gln Phe Val

195 200 205

Pro Pro Ser Thr Ile Asp Val Ser Arg Val Glu Pro Leu Pro Pro His

210 215 220

Leu Pro Leu Glu Glu Pro Ala Asp Glu Gly Leu Glu Leu Gly Thr Gln

225 230 235 240

Val Leu Asp Phe Leu His Phe Phe Leu His Glu Phe Asp Ser Asp Lys

245 250 255

Gln Val Ile Ser Leu Asn Arg Pro Gly Ile Thr Thr Lys Glu Glu Leu

260 265 270

Asp Trp Thr Lys Ser Ala Glu Asp Phe Ala Arg Met Asn Gly Glu Lys

275 280 285

Val His Tyr Gln Trp Cys Ile Glu Asp Pro Tyr Glu Leu Asn Leu Asn

290 295 300

Val Gly Arg Asn Val Thr Pro Leu Lys Arg Asp Phe Leu Arg Arg His

305 310 315 320

Leu Glu Lys Ala Arg Asp Thr Ala Leu Leu Thr Ile Val

325 330

<210> 29

<211> 349

<212> PRT

<213> 粟酒裂殖酵母(S. pombe)

<400> 29

Gly Ser His Met Ser Tyr Gln Lys Val Pro Asn Ser His Lys Glu Phe

1 5 10 15

Thr Lys Phe Cys Tyr Glu Val Tyr Asn Glu Ile Lys Ile Ser Asp Lys

20 25 30

Glu Phe Lys Glu Lys Arg Ala Ala Leu Asp Thr Leu Arg Leu Cys Leu

35 40 45

Lys Arg Ile Ser Pro Asp Ala Glu Leu Val Ala Phe Gly Ser Leu Glu

50 55 60

Ser Gly Leu Ala Leu Lys Asn Ser Asp Met Asp Leu Cys Val Leu Met

65 70 75 80

Asp Ser Arg Val Gln Ser Asp Thr Ile Ala Leu Gln Phe Tyr Glu Glu

85 90 95

Leu Ile Ala Glu Gly Phe Glu Gly Lys Phe Leu Gln Arg Ala Arg Ile

100 105 110

Pro Ile Ile Lys Leu Thr Ser Asp Thr Lys Asn Gly Phe Gly Ala Ser

115 120 125

Phe Gln Cys Asp Ile Gly Phe Asn Asn Arg Leu Ala Ile His Asn Thr

130 135 140

Leu Leu Leu Ser Ser Tyr Thr Lys Leu Asp Ala Arg Leu Lys Pro Met

145 150 155 160

Val Leu Leu Val Lys His Trp Ala Lys Arg Lys Gln Ile Asn Ser Pro

165 170 175

Tyr Phe Gly Thr Leu Ser Ser Tyr Gly Tyr Val Leu Met Val Leu Tyr

180 185 190

Tyr Leu Ile His Val Ile Lys Pro Pro Val Phe Pro Asn Leu Leu Leu

195 200 205

Ser Pro Leu Lys Gln Glu Lys Ile Val Asp Gly Phe Asp Val Gly Phe

210 215 220

Asp Asp Lys Leu Glu Asp Ile Pro Pro Ser Gln Asn Tyr Ser Ser Leu

225 230 235 240

Gly Ser Leu Leu His Gly Phe Phe Arg Phe Tyr Ala Tyr Lys Phe Glu

245 250 255

Pro Arg Glu Lys Val Val Thr Phe Arg Arg Pro Asp Gly Tyr Leu Thr

260 265 270

Lys Gln Glu Lys Gly Trp Thr Ser Ala Thr Glu His Thr Gly Ser Ala

275 280 285

Asp Gln Ile Ile Lys Asp Arg Tyr Ile Leu Ala Ile Glu Asp Pro Phe

290 295 300

Glu Ile Ser His Asn Val Gly Arg Thr Val Ser Ser Ser Gly Leu Tyr

305 310 315 320

Arg Ile Arg Gly Glu Phe Met Ala Ala Ser Arg Leu Leu Asn Ser Arg

325 330 335

Ser Tyr Pro Ile Pro Tyr Asp Ser Leu Phe Glu Glu Ala

340 345

<210> 30

<211> 480

<212> PRT

<213> T. boudieri

<400> 30

Met Ser Gly Pro Pro Ser Gly Pro Pro Ser Gly Pro Arg Tyr Arg Gly

1 5 10 15

Gly His Arg Asn Phe Gly Pro Arg His Leu Gln Ser Gln Glu Arg Gln

20 25 30

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro Tyr Gln Pro

35 40 45

Ser Pro Gln Glu Leu Ala Val Leu Ala Glu Val Val Ile Glu Gln Ile

50 55 60

Ala Leu Ala Thr Pro Ser Ala Glu Glu Leu Glu Arg Lys Thr Lys Leu

65 70 75 80

Arg Glu Arg Leu Arg Lys Leu Cys Thr Asn Val Ser Pro Thr Ala Glu

85 90 95

Leu Glu Ala Phe Gly Ser Leu Val Ser Gly Phe Ala Thr Val Gly Ser

100 105 110

Asp Leu Asp Leu Val Leu Val Asp Lys Gly Gly Ser Thr Asn Arg Pro

115 120 125

Phe Phe Glu Val Pro Leu Leu Leu Glu Gln Glu Leu Lys Asp Asn Gly

130 135 140

Leu Glu Ala Gln Leu Leu Ala Lys Thr Arg Val Pro Ile Leu Lys Ile

145 150 155 160

Lys Gln Pro Ala Thr Glu Glu Tyr Pro Lys Glu Val Ala Ala Asp Ile

165 170 175

Gly Phe Ala Asn Pro Leu Ala Ile Arg Asn Thr Asp Met Leu Ser Met

180 185 190

Tyr Ser Lys Cys Asp Pro Arg Val Ile Asp Met Val Arg Phe Ile Lys

195 200 205

Arg Trp Ala Lys Arg Arg Lys Ile Asn Asn Pro Tyr Lys Gly Thr Leu

210 215 220

Ser Ser Tyr Gly Tyr Val Leu Met Val Leu His Tyr Leu Ile Asn Ile

225 230 235 240

Val Glu Pro Pro Val Leu Pro Asn Leu Gln Leu Tyr Pro Ile Pro Ala

245 250 255

Glu Thr Pro Lys Asp Glu Ile Thr Thr Glu Glu Gly His Asn Val Trp

260 265 270

Tyr Tyr Lys Asp Val Ala Glu Ile Gln Arg Arg Val Ala Glu Gly Thr

275 280 285

Met Thr Thr Asn Thr Met Asp Leu Pro Leu Leu Leu Leu Gly Phe Phe

290 295 300

Glu Phe Tyr Ala Tyr Arg Phe Gly Trp Val Lys Asp Ile Ile Ser Ile

305 310 315 320

Arg Thr Lys Gly Gly Leu Ile Ser Lys Val Asp Lys Gly Trp Thr Val

325 330 335

Val Ala Val Arg Val Gly Lys Cys Glu Ala Glu Tyr Lys Asp Arg Tyr

340 345 350

Leu Phe Ala Ile Glu Asp Pro Phe Glu Thr Asn His Asn Val Ser Arg

355 360 365

Thr Cys Asn Ile Tyr Gly Val Arg Lys Ile Arg Asp Glu Phe Lys Arg

370 375 380

Ala Asn Arg Ile Met Arg Met Arg Glu Gly Val Gly Ala Leu Arg Tyr

385 390 395 400

Lys Leu Phe Glu Glu Ala Pro Glu Asp Val Arg Pro Gln His His Gln

405 410 415

Lys Gln Ser Ile Ala Gly Glu Glu His Val Asn Gly Ala Pro Glu Ser

420 425 430

Gly Arg Glu Asp Arg Glu Gly Asn Gln Leu Val Glu Glu Leu Glu Gly

435 440 445

His Ser Asn Gly Asn Gly Ile His Pro Ser Glu Gly Tyr Pro Gly Pro

450 455 460

Gly Leu Ala Ala Val Val Glu Gly Val Gln Arg Leu Ser Val Asp Ala

465 470 475 480

<210> 31

<211> 761

<212> PRT

<213> D. stenobrocha

<400> 31

Met Glu Leu Thr Thr Gly Asp Phe Gly Gly Pro Pro Gly Ala Ser Asn

1 5 10 15

Ala Thr Gly Pro Gly Gln Lys Pro Glu Asp Pro Arg Thr Met Asp Pro

20 25 30

Val Gly Glu Gln Thr Leu Glu Ser Arg Leu Arg Gly Met Leu Leu His

35 40 45

Gly Gly Pro Pro Pro Val Asp Pro Arg Thr Gln Leu Arg His Pro Ser

50 55 60

Gly Leu Ala Gln Gln Ala Gln Gln Ala Gln Gln Gln Pro Ile Gly Ile

65 70 75 80

Asp Pro Ala Val Pro Gly His Leu His Gln Leu Gln Thr Ala Ala Ala

85 90 95

Pro Pro Gln Gln Ser Arg Leu Pro Pro Ser Pro Asn Ile His His Gln

100 105 110

His Leu Gln Pro Arg Leu Leu Gln Arg Ser Asn Thr Leu Pro Pro Pro

115 120 125

Gly Gly Leu Thr Gln Pro Pro Pro Gly Leu Glu Arg Ala Gly Thr Phe

130 135 140

His Gly Pro Val Pro Val Ala Gln Phe Gly Tyr His Gly Ser Tyr Pro

145 150 155 160

Thr Ala His Ser Gln Arg Gly Arg Tyr Gly Asn Arg Arg Asn Gly Phe

165 170 175

Gln Asn Ala Pro Val Pro Gln Met Asn Asn Asn Leu His Phe Pro Pro

180 185 190

Leu Gly Thr Val Pro Ser Gln Pro Pro Pro Pro Leu Ser Glu Arg Ser

195 200 205

Ser Leu Ser Asn Met Asn Asn Pro Arg Arg His Ser Gly Pro Gly His

210 215 220

Tyr His Thr Asp Asn Asn His Ser Pro Pro Tyr Lys Arg Tyr Glu Ala

225 230 235 240

Phe His Gly Ala Pro Gly Ser Pro Gln Gly Tyr Ser Gly Ser Pro Pro

245 250 255

Gln Asn Arg Gln Gly Arg Pro Tyr His Ser Ser Arg Gly Ser His Gly

260 265 270

Gly Arg Asn Asn Arg Asn Phe Trp Pro Leu Asp Tyr Asp Gly Leu Thr

275 280 285

Asn Tyr Ala Lys Tyr Ile Val Glu Ser Val Ser Pro Thr Pro Glu Glu

290 295 300

Ile Ala Met Lys Asp Asn Met Leu Arg Arg Ile Ser Glu Ile Cys Asp

305 310 315 320

Lys Leu Val Pro Gly Ser Arg Ile Ile Pro Phe Gly Ser Leu Val Ser

325 330 335

Gly Phe Ala Thr Lys Gly Ala Asp Met Asp Val Ile Phe Ala His Asp

340 345 350

Thr Ile Asp Pro Ala Pro Ser Ser His Glu Ser Asn Ile Pro Val Arg

355 360 365

Leu Ala Asn Glu Phe Leu Lys Arg Gly Phe Glu Val Asp Leu Leu Ile

370 375 380

Lys Thr Arg Val Pro Ile Leu Lys Leu Lys Thr Pro Gly Glu Pro Val

385 390 395 400

Ser Arg Pro Gly Ser Pro Val Ser Glu Gly Asp Gly Asp Ser Ser Glu

405 410 415

Glu Pro Trp Pro Glu Asn Val Ser Cys Asp Ile Gly Phe Lys Ala His

420 425 430

Leu Gly Ile Thr Asn Ser His Phe Phe Arg Thr Tyr Ser His Cys Asp

435 440 445

His Arg Phe Arg Glu Met Val Leu Phe Val Lys Gln Trp Ser Lys Asn

450 455 460

Arg Asp Leu Asn Ser Pro Tyr Phe Gly Thr Leu Ser Ser Tyr Gly Tyr

465 470 475 480

Val Leu Met Val Ala His Phe Leu Ile Asn Val Val Gln Pro Pro Val

485 490 495

Leu Pro Asn Leu Gln Leu Ile Pro Pro Ala Ala Glu Thr Ala Glu Ser

500 505 510

Glu Leu Met Gln Glu Gly Phe Asn Ile Trp Tyr Phe Lys Asp Leu Glu

515 520 525

Arg Ile Ala Ser Gly Glu Phe Leu Pro Gly Gly Arg Asn Glu Met Ser

530 535 540

Leu Gly Gln Leu Val His Glu Phe Phe Gln Tyr Tyr Thr Thr Asn Phe

545 550 555 560

Asn Phe Val Ser Glu Cys Val Thr Ile Arg Thr Pro Gly Gly Val Met

565 570 575

Tyr Lys Gln Glu Lys Gly Trp Thr Ser Ala Arg Glu Arg Val Gly Glu

580 585 590

Met Asn Asn Thr Tyr Gln Asp Arg Tyr Leu Leu Ala Leu Glu Asp Pro

595 600 605

Phe Glu Val Ser His Asn Val Gly Arg Thr Cys Gly Gly Ala Gly Val

610 615 620

Arg Arg Ile Arg Ala Glu Met Gln Arg Ala Ala His Ile Ile Arg Lys

625 630 635 640

Val Ser Ala Lys Glu Gly Gly Thr Pro Arg Ser Ala Gly Trp Glu Met

645 650 655

Pro Leu Val Val Glu Asp Leu Met Thr Thr Val Arg Glu Thr Asn Arg

660 665 670

Gly Phe Arg Asn Arg Arg Val Asn Gln Lys Asn Leu Met Glu Trp Ile

675 680 685

Arg Asn Asp Trp Thr Val Gly Lys Cys Leu Val Gln Ile Asp Ala Ala

690 695 700

Ala Glu Glu Leu Arg Lys Lys Arg Glu Leu Gly Leu Pro Asp Asp Ala

705 710 715 720

Glu Leu Pro Pro Glu Ser Glu Glu Asp Ser Glu Gly Asp Glu Asp Ala

725 730 735

Ser Glu Glu Glu Glu Glu Ser Ser Ser Asp Asp Gly Ala Val Arg Val

740 745 750

Val Gly Gly Gly Ser Arg Gly Gly Val

755 760

<210> 32

<211> 337

<212> PRT

<213> 植物单胞菌属(Phytomonas sp.)

<400> 32

Met Thr Ser Leu Ser Ala Val Asn Val Gly Lys Leu Val Cys Asp Ser

1 5 10 15

Phe Gly Lys Leu Leu Ser Lys Asn Leu Asp Ile Lys Pro Ile Val Gln

20 25 30

Thr His Glu Ile Val Ser Gln Ser Met Gln Thr Val Asp Pro Ser Met

35 40 45

Ala Leu Tyr Val Phe Gly Ser Thr Ala Val Tyr Gly Phe His Glu Ser

50 55 60

Gly Cys Asp Val Asp Phe Val Ala Leu Asn Gln Lys Asp Val Ser Asp

65 70 75 80

Gly Lys Ala Ala Asp Pro Ser Ser Glu Ile Ala Lys Gly Leu Gln Val

85 90 95

Asp Phe Leu Ser Arg Leu Glu Ala Ser Leu Arg Glu Met His Asn Leu

100 105 110

Ala Trp Lys Met Asp Leu Val Arg Arg Thr Arg Val Pro Val Leu Arg

115 120 125

Val Lys Gly Asp Pro Cys Gly Ile Asp Phe Asp Val Thr Ala Arg Arg

130 135 140

Arg Asn Gly Val Arg Asn Ser Ala Leu Leu Ser Ala Tyr Phe Lys Gln

145 150 155 160

Lys Pro Glu Thr Arg Trp Leu Ser Met Ala Ile Lys Gln Trp Ser Lys

165 170 175

Arg Ala Gly Phe Asn Met Ser Val Asp Gly Gly Cys Leu Thr Ser Tyr

180 185 190

Gly Cys Asn Leu Met Val Val Tyr Tyr Leu Leu Gln Arg Gln Leu Val

195 200 205

Lys Phe Val Asp Pro Glu Arg Cys Asp Val Ala His Ile Pro Ser Leu

210 215 220

Pro Ser Tyr Leu Pro Leu Glu His Pro Ala Gln Asn Gly Ser Glu Leu

225 230 235 240

Gly Asp Met Val Leu Asp Phe Leu Asn Tyr Tyr Leu His Glu Phe Asn

245 250 255

Pro Glu Thr Glu Val Ile Ser Leu Ser Arg Ala Asp Lys Thr Thr Lys

260 265 270

Glu Met Ile Tyr Trp Thr Lys Gln Ala Glu Asp Met Ala Arg Ile Ser

275 280 285

Gly Glu Lys Val Ser Tyr Arg Trp Cys Ile Glu Asp Pro Phe Glu His

290 295 300

Asn Leu Asn Val Gly Arg Tyr Val Thr Pro Phe Lys Leu Thr Leu Leu

305 310 315 320

Arg Lys His Met Glu Arg Ala Lys Glu Thr Ala Leu Leu Leu Ser Ile

325 330 335

Ser

<210> 33

<211> 355

<212> PRT

<213> 舞行波豆虫(B. saltans)

<400> 33

Met Leu Ser Pro Ala Ala Ile Gly Lys Ala Ile Leu Asp His His Ala

1 5 10 15

Ser Leu Ile Gly Ala Asp Leu Ser Val Lys Arg Val His Asp Ala His

20 25 30

Ser Leu Val Lys Asn Ser Leu Asp Ser Val Ala Pro Asp Leu Lys Leu

35 40 45

Tyr Thr Phe Gly Ser Ser Thr Val Phe Gly Phe His Glu Pro Lys Ser

50 55 60

Asp Val Asp Phe Val Ala Leu Arg Gln Glu Asp Ile Val Asp Gly Lys

65 70 75 80

Gly Gly Asp Ser Thr Ser Gln Leu Ala Lys Gly Leu Gln Thr Gln Val

85 90 95

Leu Ala Lys Leu Ala Ala Ser Val Arg Gln Lys Asn Val Gln Trp Ala

100 105 110

Val Glu Glu Val Arg Arg Ala Arg Val Pro Val Val Lys Val Lys Ala

115 120 125

Pro His Ile Asp Phe Asp Ile Thr Ala His Arg Arg Asn Gly Val Arg

130 135 140

Asn Ser Ala Leu Leu Arg His Tyr Leu Thr Gln Val Pro Glu Asn Arg

145 150 155 160

Trp Leu Ser Ile Ala Ile Lys Ser Trp Ser Lys Arg Val Gly Met Asn

165 170 175

Gly Pro Val Gly Gly Tyr Leu Thr Ser Tyr Gly Phe Asn Ile Leu Val

180 185 190

Val Tyr Tyr Leu Leu His Arg Arg Arg His Glu Gln Pro Glu Val Glu

195 200 205

Gln Lys Glu Gly Gln Asp Gly Ala Ala Ser Ala Ser Val Asp Gly Lys

210 215 220

Gln Gln Lys Glu Leu Thr Phe Ile Asp Lys Asp Thr Leu Asp Val Ser

225 230 235 240

Leu Ile Pro Pro Ile Pro Glu Tyr Leu Ala Leu Glu Pro Pro Asn Pro

245 250 255

Glu Thr Leu Gly Glu Gln Val Leu Asp Phe Phe Asp Phe Tyr Leu Ser

260 265 270

Arg Phe Pro Met Glu Ser His Val Ile Ser Leu Ser His Lys Glu Pro

275 280 285

Ile Thr Lys Gln Ser Leu Asn Trp Thr Lys Thr Ala Glu Asp Met Lys

290 295 300

Asn Asn Thr Ser Met Glu Lys Val Phe Tyr Arg Leu Cys Ile Glu Asp

305 310 315 320

Pro Tyr Glu Val Asn Leu Asn Val Gly Arg Asn Val Ser Pro Phe Lys

325 330 335

Phe Asp Leu Met Lys Lys His Phe Val Lys Gly Arg Ala Thr Ala Leu

340 345 350

Gly Leu Leu

355

<210> 34

<211> 425

<212> PRT

<213> A. deanei

<400> 34

Met Pro Pro Asn Pro Arg Gln Leu Gly Ala Ala Met Ile Glu Gln Phe

1 5 10 15

Arg Pro Leu Leu Gln Ala Pro Gln Gln Ser Gln Leu Thr Val Leu Ser

20 25 30

Glu Ala Pro Leu Phe Gln Arg Ile Gln Arg Glu Val His Glu Leu Asp

35 40 45

Pro Gly Ala Arg Val Tyr Ile Phe Gly Ser Thr Arg Val Tyr Gly Phe

50 55 60

His Asp Ser Gly Ala Tyr Gln Gln Gln Ser Ala Gln Val Pro Pro Ser

65 70 75 80

Asp Val Asp Met Ala Val Leu Arg Arg Glu Asp Leu Leu Asp Gly Ser

85 90 95

Gly Val Asp Pro Ser Ser Glu Leu Thr Arg Asn Val Gln Ala Glu Phe

100 105 110

Leu Glu Lys Leu Leu Asn Arg Leu Gln Gln Ser Glu Gly Ser Ala Ser

115 120 125

Ile Arg Ser Asn Cys Ser Ser Leu Val Lys Arg Thr Arg Val Pro Val

130 135 140

Leu Arg Val Lys His Leu Val Ser Asn His Ser Pro Pro His Asp Lys

145 150 155 160

Glu Asp Asn Ile Ser Val Gln Val Pro Phe Asp Phe Asp Ile Thr Cys

165 170 175

Gly Arg Arg Cys Gly Ile Arg Asn Ser Ala Leu Phe Phe His Tyr Val

180 185 190

Gln Gln Met Pro Met Ile Arg Phe Leu Leu Leu Ser Val Lys Lys Trp

195 200 205

Ser Lys Gln Thr Gly Leu Asn Ser Ala Ile Lys Ala Pro Tyr Ser Asn

210 215 220

Asn Asn Asn Gln Asn His Asn Leu Thr Ser Val Met Gly Gly Ser Leu

225 230 235 240

Thr Ser Tyr Gly Phe His Val Leu Leu Leu Tyr Tyr Leu Leu Arg Arg

245 250 255

Gly Val Val Gln Tyr Ile Pro Met Thr Val Ser Ser Ala Ala Gly Ser

260 265 270

Thr Asp Arg Ile Pro Asn Pro Gln Leu Asp Val Asn Ala Ile Pro Pro

275 280 285

Val Pro Thr Phe Leu Pro Leu Leu Asp Pro Ala Leu Glu Pro His Gly

290 295 300

Pro Gln Gly Asp Pro Val Arg Tyr Leu Gly Glu Leu Ala Leu Asp Trp

305 310 315 320

Cys His Phe Tyr Leu His Glu Phe Asp Val Glu Lys Glu Val Val Ser

325 330 335

Leu Ser Arg Phe Asp Asp Tyr Phe Ala Asp Ser Pro Pro Ser Gly Pro

340 345 350

Phe Pro Val Ile Thr Lys Asp Val Leu Gly Trp Thr Lys Gln Arg Glu

355 360 365

Asp Gln Gly Arg Leu Arg Gly Glu Lys Val His Tyr Thr Leu Cys Ile

370 375 380

Glu Asp Pro Tyr Glu Val Asn Leu Asn Val Gly Arg Asn Val Thr Ser

385 390 395 400

Leu Lys Trp Met Leu Phe Arg Lys His Leu Glu Lys Ala Ile Gln Thr

405 410 415

Gly Leu Leu Leu Cys Pro Pro Ser Lys

420 425

<210> 35

<211> 432

<212> PRT

<213> P. lactucaedebilis

<400> 35

Met Pro Ser Ser Ser Pro Leu Gly Ala Thr Leu Pro Asn Ser Ser Ser

1 5 10 15

Leu Ala Val Ser Ala Ala Ala Ala Val Val Pro Asp Pro Leu Phe Ala

20 25 30

Phe Thr Gln Phe Ala Val Asp Ala Val Arg Asn Asn Thr Val Ser His

35 40 45

Ala Glu Leu Val Glu Lys Glu Arg Leu Arg Gln Glu Leu Glu Ala Val

50 55 60

Leu Gln Ala Leu Gln Pro Thr Ala Arg Leu Val Val Tyr Gly Ser Leu

65 70 75 80

Ala Asn Asn Leu Ala Ile Ala Asn Cys Asp Met Asp Leu Met Asp Ala

85 90 95

Leu His Asp Val Ser Val Asp Leu Pro Ala Leu Tyr Thr Glu Ala Leu

100 105 110

Asn Ser Gln Gly Tyr Ser Val Lys Leu Leu Ser Lys Thr Arg Thr Pro

115 120 125

Ile Val Lys Val Ser Leu Glu His Met Ala Val Pro Tyr Cys Val Asp

130 135 140

Ile Ser Phe Asp Asn Ala Leu Ala Leu His Asn Thr Lys Leu Ile Ala

145 150 155 160

Thr Tyr Ala Ala Cys Asp Ser Arg Val Pro Ile Leu Leu Met Phe Val

165 170 175

Lys Leu Trp Thr Ala Val Arg Arg Ile Asn Asp Pro His Ser Gly Thr

180 185 190

Leu Ser Ser Tyr Gly Tyr Ala Leu Leu Leu Ile Phe Tyr Leu Gln Asn

195 200 205

Arg Cys Asn Ser Pro Pro Val Leu Pro Asn Leu Gln Leu Ile Ser Ala

210 215 220

Met Gly Thr Arg Ser Ala Asp Glu Leu Glu Cys Ser Gly Tyr Asp Ile

225 230 235 240

Trp Phe Phe Lys Asp Val Glu Lys Ile Gln Gln Ala Gln Val Val Lys

245 250 255

Asn Thr Ser Ser Ile Gly Thr Leu Leu Glu Gly Phe Phe Ser Tyr Phe

260 265 270

Ala Tyr Glu Phe Asp Phe Arg Asp Leu Cys Ile Ser Ile Arg Thr Pro

275 280 285

Gly Gly Ile Val Thr Lys Thr Ala Lys Thr Trp Thr Gln Met Val Glu

290 295 300

His Leu Asn Asp Arg Gly Asp Ala Lys Val Lys Asp Arg Tyr Ile Leu

305 310 315 320

Ser Ile Glu Asp Pro Phe Glu Ile Val His Asn Val Gly Arg Ser Val

325 330 335

Thr Arg Ala Gly Leu Tyr Glu Ile Arg Gly Glu Phe Met Ala Ala Thr

340 345 350

Arg Tyr Val Arg Gly Asn Lys Leu Asp Glu Ile Leu Asn Pro His Arg

355 360 365

Arg Glu Ile Val Ala Pro Thr Ser Ser Thr Ala Met His Ser Pro Gln

370 375 380

Gln Gln His Gly Thr Gln Gln Val Ser Pro Gly Glu Gly Ser Ser Pro

385 390 395 400

Thr Pro Pro Ala Val His Ala Glu Ala Arg Pro Ser Thr Gly Thr Ala

405 410 415

Thr Ser Thr Lys Thr Ser Gln Gln Ala Glu Thr Val Val Lys Ala Glu

420 425 430

<210> 36

<211> 437

<212> PRT

<213> S. culicis

<400> 36

Met Pro Pro Pro Thr Pro Met Gln Leu Gly Arg Ala Ile Leu Arg His

1 5 10 15

Tyr Ala Pro Ser Ile Val Val Pro Thr Ala Ala Thr Ser Gly Pro Ile

20 25 30

Ser Ser Ala Ala Ala Ser Thr Ala Ser Leu Ala Ser Ile His Pro Leu

35 40 45

Phe Glu Phe Ile Gln Gly Cys Ala Arg Arg Val Asp Pro Gln Thr His

50 55 60

Leu Tyr Val Phe Gly Ser Thr Arg Val Tyr Gly Phe Ser Glu Val Ser

65 70 75 80

Ala Pro Thr Ser Val Gly Pro Pro Ala Ala Leu Ala Arg Pro Val Lys

85 90 95

Asn Asp Val Asp Ile Ala Ala Leu Ser Ala Ala Asp Val Ala Thr Thr

100 105 110

Pro Ser Pro His Ser Ala Asp Ala Val Val Asp Lys Gly Ser Glu Leu

115 120 125

Ala Lys Ser Leu Gln Ile Asp Phe Leu Glu Lys Leu Lys Met Gln Leu

130 135 140

Gln Thr Gln Arg Gln His Gln Gly Asp Gly Ala Pro Pro Ala Ala Ser

145 150 155 160

Leu Ser Trp Glu Met Glu Val Val Lys Arg Ala Arg Val Pro Val Leu

165 170 175

Arg Leu Gln Pro Arg Pro Ala Tyr Thr Thr Ser Ala Ala Gly Gly Val

180 185 190

Pro Thr Tyr Asn Val Asp Val Thr Tyr Gly Arg Arg Cys Gly Val Leu

195 200 205

Asn Ser Ala Leu Leu Arg Arg Tyr Met Asp Gln Gln Pro Asp Leu Arg

210 215 220

Tyr Leu Cys Leu Ala Val Lys Arg Trp Ser Lys Leu Thr Gly Leu Asn

225 230 235 240

Thr Ala Thr Ser Pro Asp Gly Gly Gly Leu Thr Ser Tyr Gly Phe His

245 250 255

Leu Leu Leu Val Tyr Tyr Ala Leu Arg Arg Arg Leu Val Ala Tyr Val

260 265 270

Ala Pro Glu Asn Ile Gln Trp Gly Asp Ile Ala Pro Val Pro Ala Ala

275 280 285

Leu Pro Leu Arg Phe Pro Gly Asp Ala Asp Gly Ala Asp Ala Trp Arg

290 295 300

Gly Arg Ser Leu Gly Glu Arg Ile Ala Gln Asp Asp Ala Ala Ala Ala

305 310 315 320

Arg Val Gly Glu Trp Ala Leu Asp Phe Val Arg Phe Tyr Leu His Glu

325 330 335

Phe Asp Tyr Glu Arg Asp Val Ala Ser Leu Ser Arg Gly Ala Ala Ala

340 345 350

Ala Gly Leu Val Thr Thr Glu Ala Leu Gln Trp Thr Arg Gln Glu Glu

355 360 365

Tyr Ala Ala Arg Gly Arg Gly Glu Tyr Leu Phe Tyr Arg Phe Cys Ile

370 375 380

Glu Asp Pro Tyr Glu Ile Asn Leu Asn Val Gly Arg His Met Ser Ser

385 390 395 400

Val Lys Leu Met Ile Phe Lys Lys His Leu Glu Lys Ala Leu Glu Thr

405 410 415

Gly Leu Ala Phe Ile Pro Ala Asp Glu Lys Ala Lys Gln Arg Val Lys

420 425 430

Pro Lys Gly Lys Glu

435

<210> 37

<211> 467

<212> PRT

<213> 裂孢蛙粪霉(B. meristosporus)

<400> 37

Met Ser Leu Glu Thr Asp Arg Ile Val Ser Glu Phe Lys Asp Leu Ala

1 5 10 15

Ile Ser Gln Thr Ser Glu Thr Asn Gln Pro Val Pro Asp Val Lys Val

20 25 30

Ser Ser Val Glu Asp Ser Gln His His Ser Lys Ala His Pro Pro Lys

35 40 45

Asn Glu Asn Gly Asn Asn Gln Gly His Arg His Gly Lys Lys Pro Phe

50 55 60

His Arg Gly Ser Thr Phe Gly Glu Gln Gly Lys Ser Asp Arg Ser Tyr

65 70 75 80

Gly Ala Arg Ser Phe His Pro Asn Arg Gly Thr Gly Gln Tyr Glu Gly

85 90 95

Lys Arg Val Leu Asp Lys Ser Asp Pro Arg Tyr Leu Lys Ala Arg Ala

100 105 110

Arg Phe Ile Ser Val Leu Thr Glu Gln Val Ser Glu Leu Tyr Leu Lys

115 120 125

Leu Thr Pro Ser Val Glu Glu Val Ser Arg Arg Glu Gln Leu Tyr Phe

130 135 140

Arg Ile His Ser Ile Cys Glu Glu Leu Phe Pro Asp Ala Gln Leu Phe

145 150 155 160

Gln Phe Gly Ser Thr Ala Asn Gly Phe Gly Leu Lys Asn Ala Asp Val

165 170 175

Asp Phe Cys Leu Cys Thr Ala Glu Asn Ser Ser Leu Thr Ala Val Ala

180 185 190

Phe Val Glu Gln Leu Gly Ser Val Leu Lys Glu Arg Gly Met Glu Asn

195 200 205

Val Met Leu Leu Thr Arg Thr Arg Ile Pro Ile Ile Lys Leu Lys Asp

210 215 220

Pro Val Ser Glu Leu Asn Cys Asp Ile Gly Phe Asn Asn Arg Leu Ala

225 230 235 240

Val Tyr Asn Thr Arg Leu Leu Arg Met Tyr Ala Glu Val Asp Ser Arg

245 250 255

Val Lys Glu Ile Val Ala Ile Val Lys His Trp Ala Lys Ser Arg Gln

260 265 270

Ile Asn Glu Pro Tyr Leu Gly Thr Leu Ser Ser Tyr Ala Tyr Val Leu

275 280 285

Met Ile Ile Phe Val Leu Gln Gln Arg Gly Val Leu Pro Cys Leu Gln

290 295 300

Gln Ile Cys Glu Gly Glu Lys Lys Glu Val Leu Val Asp Asn Tyr Asp

305 310 315 320

Ala Tyr Phe Phe Asp Asn Ile Thr Asp Leu Pro Lys Tyr Trp Lys Ser

325 330 335

Gln Asn Asn Glu Ser Leu Gly Glu Leu Leu Tyr Glu Phe Phe Arg Phe

340 345 350

Tyr Ala Ser Asp Phe Ser Tyr Tyr Gly Ser Val Val Ser Val Arg Thr

355 360 365

Gly Gln Val Met Ser Lys Glu Glu Lys Gly Trp Thr Ala Glu Ala Thr

370 375 380

Arg Glu Ser Gly Ala Ser His Val Gln Asp Arg Tyr Trp Val Cys Ile

385 390 395 400

Glu Asp Pro Phe Glu Val Thr His Asn Leu Gly Arg Pro Val Asn Lys

405 410 415

Asn Ser Leu Tyr Thr Ile Arg Gly Glu Phe Met Arg Ala Ala Asn Ile

420 425 430

Leu Gly Gly Thr Arg Thr Ser Thr Ile Leu Glu Arg Leu Cys Gln Val

435 440 445

Tyr Val Pro Glu Glu Glu Pro Pro Arg Ala Pro Pro Ala Pro His Glu

450 455 460

Arg Ser Asn

465

<210> 38

<211> 354

<212> PRT

<213> 加利福尼亚新美鞭菌(N. californiae)

<400> 38

Met Lys Thr Thr Glu Arg Thr Cys Thr Phe Glu Phe Tyr Asp Glu Leu

1 5 10 15

Thr Glu Glu Ile Asn Lys Ile Val Asn Lys Ile Lys Ala Lys Pro Asn

20 25 30

Asp Leu Lys Lys Arg Glu Asn Leu Phe Asn Tyr Ile Lys Leu Val Ala

35 40 45

Lys Lys Ile Phe Pro Asn Ser Lys Ala Tyr Lys Tyr Gly Ser Ile Glu

50 55 60

Asn Gly Phe Ser Leu Thr Asn Gly Asp Ile Asp Ile Cys Ile Leu Asn

65 70 75 80

Asn Lys Glu Phe Asn Asn Thr Pro Ala Glu Cys Val Glu Ile Leu Gly

85 90 95

Glu Lys Leu Lys Glu Asp Gly Ile Asn Asp Ile Lys Leu Leu Ile Arg

100 105 110

Ala Arg Val Pro Ile Ile Lys Met Lys Asp Asn Lys Ser Gly Ile Ser

115 120 125

Cys Asp Ile Gly Phe Gln Asn Lys Leu Ala Ile Gln Asn Thr Lys Leu

130 135 140

Ile Asn Ala Tyr Ser Lys Ile Asp Asp Arg Phe Lys Gln Met Val Phe

145 150 155 160

Ile Ile Lys Tyr Trp Ser Lys Met Lys Asn Ile Asn Glu Pro Tyr Met

165 170 175

Gly Thr Leu Ser Ser Tyr Cys Phe Ile Leu Met Ile Ile His Phe Leu

180 185 190

Gln Ile Lys Glu Pro Pro Val Leu Pro Asn Leu Gln Lys Ile Tyr Leu

195 200 205

Asp Asn Phe Ile Glu Tyr Glu Tyr Ile Asp Asp Phe Asn Val Ser Phe

210 215 220

Phe Glu Asn Ile Asp Glu Leu His Lys Tyr Trp Asn Ser Lys Asn Asn

225 230 235 240

Glu Ser Leu Gly Glu Leu Leu Val Glu Phe Phe Lys Tyr Tyr Ala Asn

245 250 255

Asp Phe Pro Tyr Ile Thr Gly Val Ala Ser Ile Arg Val Gly Asn Ile

260 265 270

Ile Thr Lys Glu Glu Lys Lys Trp Thr Arg Glu His Gln Phe Glu Ile

275 280 285

Asn Lys Ser Asn Ser Val Lys Asp Arg Tyr Trp Phe Cys Val Glu Asp

290 295 300

Pro Phe Glu Ile Thr His Asn Leu Gly Arg Pro Val Asp Arg Lys Ser

305 310 315 320

Leu Phe Arg Ile Arg Gly Glu Phe Leu Lys Ala Val Lys Ile Ile Asn

325 330 335

Asn Lys Asn Leu Thr Ala Leu Gln Val Leu Glu Lys Leu Leu Glu Lys

340 345 350

Asn Ile

<210> 39

<211> 344

<212> PRT

<213> Perkinsela sp.

<400> 39

Met Leu Asp Pro Ser Glu Thr Cys Met Gly Arg Ile Lys Asn Ile His

1 5 10 15

Ala Ile Ile Thr Ser Lys Leu Met Gly Leu Leu Val Glu Lys Asn Ala

20 25 30

Gln Ile Tyr Cys Phe Gly Ser Ser Met Val Asn Gly Val Val Glu Lys

35 40 45

Lys Ser Asp Val Asp Val Val Tyr Leu Thr Arg Glu Asp Leu Gln Lys

50 55 60

Pro Leu Ser Ile Gly Asp Val Cys Asn Pro Gln Ser Arg Ser Glu Gln

65 70 75 80

Thr Ser Ile Leu Ser Ala Ile Ser Lys Ile Leu Met Lys Asp Ser Glu

85 90 95

Leu Phe Ser Thr Val Gln Glu Lys Pro Arg Ala Arg Val Pro Tyr Val

100 105 110

Arg Gly Val Leu Lys Asn Gly Leu Glu Ile Asp Ile Ser Ala His Arg

115 120 125

Arg Asn Gly Val Arg Asn Ser Leu Leu Leu Arg Ser Tyr Phe Ser Gln

130 135 140

Ile Pro Pro Thr Arg Pro Ser Leu Pro Asn Ala Thr Thr Ser Val Tyr

145 150 155 160

Arg Met Leu Ser Leu Ala Leu Lys Phe Trp Gly Lys Arg Thr Gly Leu

165 170 175

Val Asp Pro Val Gln Thr Phe Leu Thr Ser Tyr Ala Phe Asn Val Leu

180 185 190

Ile Cys Tyr Tyr Leu Gln Gln Arg Gly Gly Met Asp Phe Ile His Pro

195 200 205

Glu Ser Ile Leu Ile Pro Lys Gly His Pro Thr Val Pro Asp Tyr Arg

210 215 220

Glu Ile Ala Leu Ala Gly Thr Gln Gly His Thr Cys Leu Gly Trp Tyr

225 230 235 240

Met Arg Asp Phe Leu Lys Phe Tyr Asn His Glu Phe Asp Tyr Asn Asn

245 250 255

Thr Val Val Ser Leu Ser Arg Lys Gly Ile Thr Thr Lys Glu Tyr Leu

260 265 270

Gly Trp Gly Leu Arg Asp Glu Glu Arg Met His Gly Thr Asp Gly Asn

275 280 285

Ala Phe Phe Tyr Arg Phe Cys Val Glu Asp Pro Tyr Glu Asn Arg Leu

290 295 300

Asn Leu Gly Arg Phe Val Thr Pro Leu Arg Tyr Ser Met Phe Arg Met

305 310 315 320

Ala Leu His Gln Ala Gln Leu Asn Gly Phe Gly Tyr Leu Asn Leu Lys

325 330 335

Asn Tyr Gly Ala Lys Ile Val Asp

340

<210> 40

<211> 352

<212> PRT

<213> S. complicate

<400> 40

Met Met Ala His Pro Ser Gln Gly Gly Ala His Pro Leu Ser Ala Gly

1 5 10 15

Pro Thr Asn Pro Gln Pro Tyr Gln Asp Ala Tyr Ser Gly Phe Thr Glu

20 25 30

Tyr Val Leu Ser Ile Asn Arg Pro Gln Tyr Pro Ser Ala Asn Glu Met

35 40 45

Tyr Leu Lys Glu Gln Leu Arg Ser Leu Ile Asp Val Glu Ile Val Gln

50 55 60

Arg Ile Gln Pro Ser Ala Arg Val Ile Pro Phe Gly Ser Leu Val Asn

65 70 75 80

Gly Phe Ala Thr Ala Asn Ser Asp Leu Asp Leu Cys Ile Leu Asp Asp

85 90 95

Ser Pro Asn Pro Arg Tyr Lys Ile Lys Thr Glu Leu Pro Glu Val Leu

100 105 110

Ala Ala Glu Phe Glu Arg Tyr Gly Phe Glu Val Lys Leu Leu Thr Lys

115 120 125

Thr Arg Ile Pro Ile Ile Lys Leu Val Gln Gly Pro Thr Ala Gln Phe

130 135 140

Pro Leu Gly Leu Ala Met Asp Ile Gly Phe Glu Asn Arg Leu Ala Leu

145 150 155 160

His Asn Thr Arg Leu Leu Ala Thr Tyr Ser Arg Ile Asp Ser Arg Leu

165 170 175

Arg Glu Met Val Leu Phe Val Lys His Trp Ala Lys Val Arg Gly Ile

180 185 190

Asn Ser Ser Tyr His Gly Thr Leu Ser Ser Tyr Gly Tyr Val Leu Thr

195 200 205

Ile Leu His Phe Leu Ile Asn Val Ala Ser Pro Ser Cys Leu Pro Asn

210 215 220

Leu Gln His Ile Gly Ala Gln Val Pro Val Pro Phe Glu Glu Leu Glu

225 230 235 240

Cys Glu Gly Tyr Asn Ile Trp Phe Phe Lys Asp Leu Thr Asn Ile Pro

245 250 255

Pro Ser Leu Asn Arg Arg Ser Ile Gly Glu Leu Met Ala Glu Tyr Phe

260 265 270

Ser Tyr Tyr Ala Gln Phe Asp Phe Arg Asn Met Val Ile Ser Ile Arg

275 280 285

Thr Pro Gly Gly Leu Leu Thr Lys Thr Ser Lys Gly Trp Thr Gln Ala

290 295 300

Leu Asp Arg Val Gly Pro Glu Asp Gln Lys Val Arg Ser Arg Asn Phe

305 310 315 320

Leu Cys Ile Glu Asp Pro Phe Glu Ile Thr His Asn Val Gly Arg Thr

325 330 335

Val Gly Lys Asn Gly Leu Tyr Asp Val Trp Pro Phe Val Ser Phe Ala

340 345 350

<210> 41

<211> 388

<212> PRT

<213> 赭黄齿耳(S. ochraceum)

<400> 41

Met Ala Thr Val Phe Ala Gln Ser His Ser Leu Leu Asn Thr Arg Gln

1 5 10 15

Pro His Gln His Val Gln Gln Pro Ala Gln Gln Pro Pro Gln Gln Gln

20 25 30

Gln Gln Gln Gln Ser Thr Arg Pro Lys Gln Leu Ser Lys Pro Arg Phe

35 40 45

Tyr Ala Glu Phe Ser Gln Cys Leu Phe Asp Phe Val Ile Gln Leu Leu

50 55 60

Pro Thr Pro Glu Glu Leu Ala Ile Lys Glu Asp Val Arg Lys Leu Leu

65 70 75 80

Glu Arg Leu Ile Arg Thr Leu Glu Pro Asp Ser Arg Leu Leu Ser Phe

85 90 95

Gly Ser Thr Ala Asn Gly Phe Ser Leu Arg Asn Ser Asp Met Asp Leu

100 105 110

Cys Cys Leu Ile Asp Ser Asp Asp Arg Leu Ala Ala Ser Asp Leu Val

115 120 125

Thr Met Leu Gly Asp Leu Leu Glu Arg Glu Thr Lys Phe His Val Lys

130 135 140

Pro Leu Pro Tyr Ala Arg Ile Pro Ile Val Lys Leu Ser Leu Asp Pro

145 150 155 160

Ser Pro Gly Leu Pro Phe Gly Ile Ala Cys Asp Ile Gly Phe Glu Asn

165 170 175

Arg Leu Ala Leu Glu Asn Thr Arg Leu Leu Met Cys Tyr Ala Met Ile

180 185 190

Asp Pro Ala Arg Val Arg Thr Met Val Leu Phe Leu Lys Val Trp Cys

195 200 205

Lys Arg Arg Lys Ile Asn Ser Pro Tyr Lys Gly Thr Leu Ser Ser Tyr

210 215 220

Gly Tyr Val Leu Leu Val Ile Phe Phe Leu Val His Val Lys Asn Pro

225 230 235 240

Pro Val Leu Pro Asn Leu Gln Ser Met Pro Pro Leu Arg Pro Met Asp

245 250 255

Lys Glu Glu Ser Thr Leu Asn Gly His Asn Val Trp Phe Phe Asp Asp

260 265 270

Ile Asn Leu Leu Arg Gln Arg Trp Gln Ser Thr Asn Thr Glu Ser Val

275 280 285

Ala Glu Leu Leu Val Asp Phe Phe Lys Phe Tyr Ser Arg Glu Phe Pro

290 295 300

Tyr Asn Ser Gly Val Val Ser Ile Arg Ala Gly Gly Ile Lys Lys Asp

305 310 315 320

Ser Lys Gly Trp Phe Ser Glu Ala Glu Arg Gly Ser Ala Arg Glu Arg

325 330 335

Asn Arg Leu Cys Ile Glu Asp Pro Phe Glu Thr Asp Phe Asn Val Ala

340 345 350

Arg Cys Val Thr Arg Asp Gly Leu Tyr Thr Ile Arg Gly Glu Leu Met

355 360 365

Arg Ala Ser Arg Ile Leu Ala Ala Arg Pro Glu Arg Pro Ile Val Ala

370 375 380

Leu Ala Gln Leu

385

<210> 42

<211> 421

<212> PRT

<213> 点地梅裸柄伞(G. androsaceus)

<400> 42

Met Ala Ala Tyr Ser Pro Thr Met Ser Ser Asn Gln His Tyr His Glu

1 5 10 15

Leu Asn Ser Ala Ser Pro Pro Ala Arg Arg Lys Trp Leu Ala Asp Phe

20 25 30

Ser Glu Cys Leu Phe Ser Phe Val Ile Gln Leu Leu Pro Thr Gln Glu

35 40 45

Glu Leu Ala Val Lys Glu Asp Val Arg Lys Leu Leu Glu Arg Leu Ile

50 55 60

Arg Thr Ile Glu Pro Glu Ser Arg Leu Leu Ser Phe Gly Ser Thr Ala

65 70 75 80

Asn Gly Tyr Gly Leu Arg Asn Ser Asp Met Asp Leu Cys Cys Leu Ile

85 90 95

Asp Ser Glu Glu Arg Leu Ala Ala Ser Asp Leu Val Thr Met Leu Gly

100 105 110

Asp Leu Leu Glu Arg Glu Thr Lys Phe His Val Lys Pro Leu Pro His

115 120 125

Ala Arg Ile Pro Ile Val Lys Leu Ser Leu Asp Pro Ser Pro Gly Leu

130 135 140

Pro Leu Gly Ile Ala Cys Asp Ile Gly Phe Glu Asn Arg Leu Ala Leu

145 150 155 160

Glu Asn Thr Arg Leu Leu Met Cys Tyr Ala Met Ile Asp Pro Thr Arg

165 170 175

Val Arg Thr Met Val Leu Phe Leu Lys Val Trp Ser Lys Arg Arg Lys

180 185 190

Ile Asn Ser Pro Tyr Lys Gly Thr Leu Ser Ser Tyr Gly Tyr Val Leu

195 200 205

Leu Val Ile Tyr Phe Leu Val His Val Lys Asn Pro Pro Val Leu Pro

210 215 220

Asn Leu Gln Gln Met Pro Pro Leu Arg Pro Ile Pro Lys Glu Glu Thr

225 230 235 240

Tyr Leu Gly Gly Phe Asn Thr Trp Phe Phe Asp Asp Ile Glu Leu Leu

245 250 255

Arg Gln Arg Trp His Ser Glu Asn Thr Glu Thr Val Ala Glu Leu Leu

260 265 270

Ile Asp Phe Phe Arg Tyr Phe Ser Arg Asp Phe Pro Tyr Gly Val Gly

275 280 285

Val Ala Ser Ile Arg Ala Gly Leu Leu Lys Lys Asp Ser Lys Gly Trp

290 295 300

Gln Asn Asp Leu Ser Ala Ser Arg Tyr Asn Asp Ala Arg Glu Arg Asn

305 310 315 320

Arg Phe Cys Ile Glu Asp Pro Phe Glu Thr Asp Tyr Asn Val Ala Arg

325 330 335

Cys Val Thr Lys Asp Gly Leu Tyr Thr Ile Arg Gly Glu Phe Met Arg

340 345 350

Ala Ser Arg Val Leu Ala Ala Arg Pro Glu Arg Ala Ile Leu Ala Leu

355 360 365

Ala Asp Leu Cys Asp Glu Arg Lys Asp Glu Asp Pro Cys Arg Arg Ala

370 375 380

Ser Leu Tyr Ser Phe Ser Ser Ser Ala Pro Thr Asn Thr Val Phe Gly

385 390 395 400

Gly Lys Ser Ile Asn Ala Phe Gln Arg Asn Thr Asp Tyr Ser Leu Ser

405 410 415

Phe Asp Gln Thr Pro

420

<210> 43

<211> 512

<212> PRT

<213> 马媾疫锥虫(T. equiperdum)

<400> 43

Met Gly Val Arg Leu Tyr Ser Cys Asp Ala Cys Pro His Ala Val Phe

1 5 10 15

Thr Thr His Ala Ala Leu Leu Ala His Ala Glu Glu His His Ala Asp

20 25 30

Leu Leu Pro Asp His Ala Arg Leu Arg Arg Ile Ala Gln Lys Leu Asn

35 40 45

Pro Val Trp Asn Arg Ala Leu Asn Ala Arg Arg Asn Thr Ile Thr Ser

50 55 60

Trp Gly Lys Lys Ile Phe His Val Ala Ala Gln Arg Asp Ala Gly Glu

65 70 75 80

Ser Lys Met Gln Glu Ala His Arg Ala Arg Ala Gln Leu Glu Cys Val

85 90 95

Val Arg Arg Trp His Asp Lys Ala Arg Val Phe Ile Phe Gly Ser Ser

100 105 110

Val Ala Met Gly Val Trp Asp Gly Thr Ala Asp Ile Asp Phe Ala Val

115 120 125

Val Asp Val Asp Ala Met Glu Arg Gly Ser Trp Pro Pro Leu Glu Lys

130 135 140

Asn Ala Val Arg Ser Ile Thr Glu Leu Leu Arg Arg Val Gly Phe Ser

145 150 155 160

Phe Val Asn Leu Glu Pro Ile Ser His Ala Arg Val Pro Ile Ile Lys

165 170 175

His His Ala Ser Ser Pro Ile Leu Thr Val Ala Arg Arg Asp Ala Glu

180 185 190

Asp Val Val Ala Arg Ser Ile Arg Phe Ile Leu Asn Gly Pro Ala Thr

195 200 205

Arg Glu Asp Arg Leu Leu Leu Glu Gly Ser Val Arg Asp Ala Val Gly

210 215 220

Pro Thr Gly Val Gln Gln Val Trp Trp Asn Arg Thr Ser Asp Met Met

225 230 235 240

Ser Ala Thr Leu Glu Ser Thr Thr Ala Ala Val Arg Ala Ala Met Cys

245 250 255

Ser Pro Ala Leu Ala Ser Ala Ser Leu Arg Thr Lys Val Gln Pro Ala

260 265 270

His Asp Glu Cys Arg Pro Glu Leu Tyr Asn Ile Asp Phe Ala Leu Ser

275 280 285

Phe Arg Ala Phe Gly Ile Arg Asn Ser Thr Leu Leu Arg Lys Tyr Leu

290 295 300

Leu Ser His Pro Cys Ala Arg Pro Gly Ala Ile Val Leu Lys Asp Trp

305 310 315 320

Ser Lys Thr Ser Gly Val Asn Asn Ser Val Asn Gly Tyr Phe Thr Ser

325 330 335

Tyr Ala Ile Asn Ile Met Trp Ile Tyr Tyr Leu Val Gln Lys Gly Tyr

340 345 350

Val Pro Tyr Val Asp Pro Leu Glu Ile Pro Glu Ser Leu Val Asn Tyr

355 360 365

Thr Asp Phe Asp Pro Arg Tyr Thr Pro Met Ile Asp Pro Glu Ile Thr

370 375 380

Asn Thr Glu Arg Glu Glu Leu Tyr Lys Ala Ala Gly Asp Met Leu Val

385 390 395 400

Gly Phe Phe Tyr Phe Tyr Ser Phe Glu Phe Asp Trp Gly His Asn Val

405 410 415

Ile Ser Leu Asn Arg Pro Gly Ile Thr Thr Lys Arg Met Leu Gly Trp

420 425 430

His Val Glu Asp Val Val Pro Val Ala Ser Thr Ser Val Ser Ser Gly

435 440 445

Gly Gly Gly Ser Asn Val Lys Arg His Pro Thr Arg Tyr Glu Leu Cys

450 455 460

Ile Glu Asp Pro Tyr Glu Glu Asn Leu Asn Leu Gly Arg His Ile Gly

465 470 475 480

Val Thr Lys Ser Leu Arg Val Arg Thr Glu Leu Tyr Arg Gly Leu Leu

485 490 495

Ser Leu Leu Lys Glu Gly Glu Thr Arg Ser Cys Val Phe Ala Ala Ala

500 505 510

<210> 44

<211> 531

<212> PRT

<213> 尖顶羊肚菌(M. conica)

<400> 44

Met Thr Ala Gly Arg Asp Ile Arg Ser Pro Pro Arg Gly Gln Arg Gly

1 5 10 15

Gln Gly Phe Asn Lys Ser Gln Asn Ser Pro Arg Ala Leu Pro Leu Asn

20 25 30

Ala Leu Asp His Phe Pro Pro Leu Gly Thr Gln Pro Thr Lys Ala Lys

35 40 45

Thr Pro Pro Pro Asn Tyr Ser Gln Gln Gln Gln Ser Thr Thr Ala Lys

50 55 60

Pro Gln Gln Arg Thr Gln Tyr Gln Pro Leu Pro Ile Asp Thr Arg Asn

65 70 75 80

Thr Asn Pro His His Ala Arg Gly Glu Val His Gly Arg Arg Asn Phe

85 90 95

Arg Gly Asp Tyr Asn Gln Arg Gln Leu Arg Arg Asp Ile Gly Pro Glu

100 105 110

Val Cys Gln Tyr Leu Ser Glu Leu Ser Lys Lys Val Ile Ala Glu Ala

115 120 125

Ala Pro Pro Glu Ser Glu Ile Leu Val Lys Arg Ala Leu Leu Glu Arg

130 135 140

Leu Glu Ala Ile Ser Lys Thr Ile Val Pro Asp Ala Lys Leu Ile Ala

145 150 155 160

Phe Gly Ser Leu Val Thr Gly Phe Ala Thr Ala Asn Ser Asp Leu Asp

165 170 175

Val Ile Phe Thr Gly Gly Asp Arg Val Asp Ile Leu Asn Glu Ser Ser

180 185 190

Asp Asp Pro Ser Ser Asn Phe Arg Ile Pro Met Leu Leu Glu Ser Lys

195 200 205

Leu Gln Glu Glu Gly Phe Gly Thr Thr Leu Leu Thr Lys Thr Arg Val

210 215 220

Pro Ile Leu Lys Leu Val Gln Lys Ala Thr Glu Gln Ser Pro Tyr Glu

225 230 235 240

Leu Gln Cys Asp Ile Gly Phe Ser Asn His Leu Ala Leu Tyr Asn Thr

245 250 255

Gln Met Leu Leu Thr Tyr Ser Lys Cys Asp Pro Arg Val Lys Glu Met

260 265 270

Met Ile Phe Ile Lys Trp Trp Ala Lys Arg Arg His Ile Asn Asn Pro

275 280 285

Tyr Arg Gly Thr Leu Ser Ser Tyr Gly Tyr Ala Leu Ile Val Leu His

290 295 300

Tyr Leu Ile Asn Val Val Lys Pro Pro Val Leu Pro Asn Leu Gln Thr

305 310 315 320

Phe Pro Val Pro Asp Ser Ala Pro Thr Asn Glu Ile Ile Phe Glu Gly

325 330 335

Asp Ser Asp Asp Thr Phe Glu Ile Trp Phe Tyr Lys Asp Ile Glu Lys

340 345 350

Leu Pro Lys Ser Asp Asn Ala Met Asp Ile Gly Glu Leu Leu Lys Gly

355 360 365

Phe Phe Glu Tyr Tyr Ala His Asn Phe Gln Trp Gly Arg Glu Val Val

370 375 380

Ser Ile Arg Thr Lys Gly Gly Leu Met Thr Lys Gln Glu Lys Gly Trp

385 390 395 400

Val Ala Ala Val Ile Lys Pro Gly Arg Thr Glu Asn Ser Glu Val Lys

405 410 415

Asn Arg Tyr Leu Phe Ala Val Glu Asp Pro Phe Glu Thr Glu His Asn

420 425 430

Val Ser Arg Thr Cys Asn Gly Pro Gly Val Asn Arg Ile Lys Asp Glu

435 440 445

Phe Lys Arg Ala Val Trp Leu Ile Arg Val Arg Asp Gly Gly Lys Thr

450 455 460

Leu Phe Gln Asn Leu Cys Met Glu Ala Pro Pro Glu Arg Val Trp Val

465 470 475 480

Arg Arg Glu Asp Arg Asp Arg Arg Gly Ile Glu Glu Ala Gly Gly His

485 490 495

Gly Ser Lys Asp His Gln Gly Ala Glu Gly Glu Ala Gly Asp Gly Val

500 505 510

Lys His Glu Ser Val Val Leu Ser Glu Asp Gly Arg Asn Ala Leu Glu

515 520 525

Ser Leu Val

530

<210> 45

<211> 474

<212> PRT

<213> P. murina

<400> 45

Met Asp Ser Val Lys Gly Phe Glu Gly Val Glu Ser Val Val Phe Gln

1 5 10 15

Lys Glu Met Lys Lys Leu Thr Lys Ile Ser Asp Phe Ser Gly Lys Asn

20 25 30

Tyr Gly Ser Gly Asn Glu Ile Glu Asp Leu Lys Lys Ser Leu Glu Lys

35 40 45

Met Met Leu Glu Ser Val Asn Ile Gln Glu Lys Gly Val Phe Leu Arg

50 55 60

Glu Lys Gly Ser Glu Gly Ser Tyr Glu Ala Asn Leu Asn Asp Lys Asp

65 70 75 80

Val Leu Lys Asn Asn Lys Lys Asp Ser Asn Phe Arg Asn Ile Lys Tyr

85 90 95

Tyr Glu Pro Lys Asp Tyr Gly Leu Tyr Val Asn Gly Lys Ala Val Trp

100 105 110

Arg Lys Asn Tyr Asp Glu Tyr Phe Val Leu Ser Thr Phe Ile Ser His

115 120 125

Thr Leu Lys His Ile Met Pro Thr Asn Glu Glu Ile Ser Gln Lys Glu

130 135 140

Arg Phe Arg Phe Phe Leu Ser Glu Ile Leu Asn Thr Cys Arg Pro Ser

145 150 155 160

Ala Lys Leu Val Leu Phe Gly Ser Val Ala Ser Gly Leu Ala Ile Val

165 170 175

Asn Ser Asp Met Asp Phe Cys Val Met Asp Asp Ser Leu Asp Leu His

180 185 190

Thr Asp Glu Phe Leu Lys Ile Phe Ser Glu Glu Ile Lys Lys Tyr Gly

195 200 205

Met Glu Thr Thr Leu Leu Phe Arg Thr Arg Val Ser Ile Ile Lys Val

210 215 220

Asn Ser Lys Gly Ser Phe Asn Phe Pro Gln Gly Ile Ser Cys Asp Ile

225 230 235 240

Gly Phe Asn Asn Lys Leu Ala Ile Tyr Asn Thr Lys Leu Leu Ala Thr

245 250 255

Tyr Ser Lys Cys Asp His Arg Val Arg Lys Ile Ile Leu Phe Val Lys

260 265 270

Tyr Trp Ala Lys Arg Arg Lys Ile Asn Asp Pro Tyr His Gly Thr Leu

275 280 285

Ser Ser Tyr Gly Tyr Val Leu Leu Ile Leu His Tyr Leu Ile Asn Ile

290 295 300

Val Pro Ile Pro Leu Leu Pro Asn Leu Gln Gln Met Lys Val Ala Ser

305 310 315 320

Trp Arg Ser Ile Pro Gln Ser Glu Ile Glu Cys Asp Gly Tyr Asn Val

325 330 335

Trp Phe Tyr Lys Asp Ile Asp Ile Ser Cys Ser Phe Ser Asn Asn Thr

340 345 350

Asp Ser Leu Gly Lys Leu Val Tyr Gly Phe Phe Tyr Tyr Tyr Ala Tyr

355 360 365

Gln Phe Asn Trp Lys Asp His Val Val Ser Ile Arg Thr Gln Thr Gly

370 375 380

Leu Leu Thr Lys Gln Glu Lys Gly Trp Thr Gln Ala Met Glu Arg Val

385 390 395 400

Phe Val Ser Asp Lys Ile Phe Lys Asp Arg Tyr Ile Leu Ala Ile Glu

405 410 415

Asp Pro Phe Glu Ile Thr His Asn Val Gly Arg Thr Val Asn Lys Gln

420 425 430

Ser Ser His Ile Ile Arg Gly Glu Phe Phe Arg Ala Ser Lys Leu Ser

435 440 445

Ser Ser Lys Leu Arg Lys Lys Ile Phe Asn Glu Leu Cys Glu Glu Arg

450 455 460

Ile Ile Ile Asn Asn Phe Ser Asn Val Ile

465 470

<210> 46

<211> 445

<212> PRT

<213> 日本裂殖酵母(S. japonicus)

<400> 46

Met Ser Gly Asn Gly Asn Gly His Phe Ile Pro Gly Val His Thr Val

1 5 10 15

Glu Glu Ile Glu Arg Gln Phe Val Asn Leu Ala Leu Gln Lys Arg Asn

20 25 30

Gln Ala Ala Ala Ala Ala Ala Ala Ala Ala Glu Arg Glu Leu Asp Pro

35 40 45

Val Thr Cys Phe Leu Leu Ser Thr Tyr Asp Asp Val Arg Val Ser Asp

50 55 60

Asp Glu Leu Arg Glu Lys Asp Ala Ile Met Asn Leu Leu Lys His Val

65 70 75 80

Val His Ser Val Arg Pro Glu Ala Asp Ile Val Ala Phe Gly Ser Ile

85 90 95

Gln Ser Gly Leu Ala Leu Lys Asn Ser Asp Ile Asp Ala Cys Ile Leu

100 105 110

Leu Pro Asp Ile Gly Glu Glu Met Glu Glu Phe Ala Ser Glu Cys Phe

115 120 125

Glu Arg Phe Thr Ala Leu Gly Phe Glu Gly Lys Tyr Leu Arg Lys Ala

130 135 140

Arg Ile Pro Ile Ile Lys Leu Leu Ser Asp Thr Lys Asn Arg Tyr Tyr

145 150 155 160

Tyr Gly Phe Gln Cys Asp Ile Gly Phe Asn Asn Gln Leu Ala Ile Tyr

165 170 175

Asn Thr Ser Leu Leu His Gln Tyr Ser Leu Ile Asp Pro Arg Cys Lys

180 185 190

Gln Leu Ala Ile Leu Val Lys Tyr Trp Ala Lys Gln Lys Arg Ile Asn

195 200 205

Ser Pro Tyr Tyr Gly Thr Leu Ser Ser Tyr Gly Tyr Val Leu Met Val

210 215 220

Leu Phe Tyr Leu Ile His Val Val Arg Pro Ala Val Leu Pro Asn Leu

225 230 235 240

Gln Asp Ser Pro His Lys Gln Asp Leu Tyr Val Glu Gly Phe Asn Val

245 250 255

Gly Phe Val Arg Gly Thr Thr Val Ala Arg Arg Asn Thr Glu Ser Leu

260 265 270

Pro Gln Leu Leu Ala Gly Phe Tyr Gly Phe Phe Ala His Glu Phe Asn

275 280 285

Tyr Arg Glu Ser Val Ile Ser Ile Arg Gln Pro Gly Gly Leu Leu Lys

290 295 300

Lys Val Asp Lys Asp Trp Thr Leu Ala Lys Glu His Thr Gly Ser Ala

305 310 315 320

Asp Gln Val Ile Lys Asp Arg Tyr Val Leu Ala Ile Glu Asp Pro Phe

325 330 335

Glu Ile Thr His Asn Val Gly Arg Thr Val Ser Lys Ala Gly Leu Phe

340 345 350

Glu Ile Arg Gly Glu Phe Met Gln Ala Thr Arg Leu Leu Asn Ala Ala

355 360 365

Thr Leu His Ser Thr Ser Ala Ile Arg Lys Leu Arg Ala Ser Leu Phe

370 375 380

Lys Glu Arg Leu Glu Pro Lys Ser His Leu Lys Tyr Gln Lys Ala Leu

385 390 395 400

Arg Gln Lys Arg Met Gly Thr Glu Gly Lys Ala Ala Ala Glu Gly Lys

405 410 415

Thr Asn Asn Pro Thr Ala Ala Ser Gly Ser Asp Ser Ala Pro Ser Glu

420 425 430

Ser Ala Ser Arg Thr Ala Thr Val Glu Ser Arg Glu Pro

435 440 445

<210> 47

<211> 620

<212> PRT

<213> A. nigricans

<400> 47

Met Asn Pro Pro Gln Gln Thr Thr Gly Leu Glu Asp His Leu Arg Ser

1 5 10 15

Leu Ile Ile Asn Asn Gln Thr Pro Pro Asn His Ser Ile Ser Pro Ala

20 25 30

Ala Lys Ile Pro His Lys His His Pro Arg Thr Ser Thr Asp Ser Arg

35 40 45

Gly Phe Val Arg Ser Gln Thr Ile His Arg Pro Gly Pro Ala Ala Ser

50 55 60

Gln Pro Pro Ser Asn Gly Pro Asn Arg Arg Phe Pro Gly Glu Ala Gln

65 70 75 80

Arg Pro Asp His Arg Pro Pro His Leu Arg Gly Pro Ser Gln Asn Arg

85 90 95

Asn Tyr Trp His Gly Ala Pro Pro Pro Pro Arg Gln Val Val Pro Gln

100 105 110

Gly Leu Ser Pro Pro Ala Ala Thr Asn Leu His His Phe Pro Pro Leu

115 120 125

Gly Ala Lys Val Ala Pro Pro Pro Pro Ser Leu Pro Ser Tyr His Ser

130 135 140

Val Pro Thr Ala Pro Cys His Arg Pro Glu Asn Gln Arg Pro Val Tyr

145 150 155 160

Gln Asn Asp Asn Arg Asp Ser Tyr His Tyr Lys Gln Arg Gly Leu Tyr

165 170 175

Asp Gly Tyr Leu Ala Glu Gln Trp Arg Glu Leu Asp Lys Met Ala Lys

180 185 190

Glu Val Leu Leu Thr Ala Thr Pro Val Asp Gly Glu Lys Glu Ala Lys

195 200 205

Glu Lys Phe Met Gln Asp Leu Glu Ala Val Cys Gln Lys Val Glu Pro

210 215 220

Thr Ala Lys Leu Ile Pro Phe Gly Ser Ile Val Thr Gly Phe Ala Thr

225 230 235 240

Arg Gly Ser Asp Ile Asp Cys Val Phe Thr Ser Asn Thr Asp Ser Ala

245 250 255

Leu Gln Asn Asp Lys Ile Thr Glu Pro Glu Thr Pro Phe Asp Arg His

260 265 270

Asp Glu Leu Val Thr Gln Leu Gln Glu Ser Gly Tyr Asn Ala Gln Leu

275 280 285

Leu Thr Arg Thr Arg Val Pro Ile Ile Lys Leu Val Arg Pro Ala Thr

290 295 300

Pro Asp Val Pro Asp Ser Val Ser Cys Asp Ile Gly Phe Asn Asn Phe

305 310 315 320

Leu Ala Ile His Asn Thr Arg Leu Leu Arg Thr Tyr Ala Ala Cys Asp

325 330 335

Glu Arg Leu Val Gln Met Val Leu Phe Ile Lys Trp Trp Ala Lys Arg

340 345 350

Arg His Ile Asn Ser Pro Tyr Arg Gly Thr Leu Ser Ser Tyr Gly Tyr

355 360 365

Ala Leu Leu Ile Val His Tyr Leu Ile Asn Ile Ala Gln Pro Pro Val

370 375 380

Leu Pro Asn Leu Gln Leu Phe His Pro Pro Ala Thr Gln Thr Ser Thr

385 390 395 400

Glu Leu Gln Tyr Glu His Asn Gly Asn Val Cys Asn Ile Trp Tyr Leu

405 410 415

Lys Asp Thr Ser Thr Leu Pro Arg Ser Ala Asn Lys Ala Ser Ile Gly

420 425 430

Glu Leu Leu Arg Gly Phe Phe Glu Tyr Tyr Ala Phe Thr Phe Arg Trp

435 440 445

Gln Asn Asp Val Val Ser Ile Arg Thr Ala Gly Gly Ile Leu Thr Lys

450 455 460

Met Glu Lys Asn Trp Cys Ala Ala Arg Ser Arg Pro Gly Gly Arg Glu

465 470 475 480

Glu Gly Gln Val Trp Glu Val Lys Asp Arg Val Asp Val Gln His Arg

485 490 495

Tyr Leu Leu Ala Ile Glu Asp Pro Phe Glu Thr Thr His Asn Val Ala

500 505 510

Arg Thr Cys Thr Phe Asn Gly Val Gly Arg Ile Lys Ser Glu Leu Lys

515 520 525

Arg Ala Met Tyr Leu Ile Lys Ser Arg Asp Pro Gly His His Arg Leu

530 535 540

Arg Thr Glu Leu Phe Glu Glu Ala Pro Pro Glu Arg Pro Phe Val Arg

545 550 555 560

Asn Gly Lys Glu Val Val Asp Lys Leu Glu Lys Val Glu Ile Val Glu

565 570 575

Glu Arg Ala Gly Val Ala Val Glu Ile Val Glu Glu Arg Ala Gly Val

580 585 590

Ala Val Glu Ile Val Glu Glu Arg Ala Gly Val Ala Leu Glu Val Val

595 600 605

Glu Glu Arg Ala Gly Ala Ala Val Glu Val Asp Val

610 615 620

Claims

1.一种合成具有预定序列的多核苷酸的方法，所述方法包括以下步骤：

a)提供3’-末端核苷酸具有游离3’-羟基的起始子；以及

b)重复以下的循环直至形成所述多核苷酸：(i)在延长条件下使具有游离3’-羟基的所述起始子或延长片段与3’-O-封闭的核苷三磷酸和无模板聚合酶接触，使得所述起始子或延长片段通过3’-O-封闭的-核苷三磷酸的掺入被延长以形成3’-O-封闭的-延长片段，和(ii)使所述延长片段去封闭以形成具有游离3’-羟基的延长片段；

其中所述无模板聚合酶是多(A)聚合酶(PAP)或多(U)聚合酶。

2.根据权利要求1所述的方法，其中所述起始子通过5’端附接至支持物。

3.根据权利要求2所述的方法，其中所述支持物是固体支持物。

4.根据权利要求1至3中任一项所述的方法，所述方法还包括从所述起始子裂解所述多核苷酸的步骤。

5.根据权利要求1至4中任一项所述的方法，其中所述多核苷酸是多-2’-脱氧核糖核苷酸并且所述3’-O-封闭的-核苷三磷酸是3’-O-封闭的-2’-脱氧核糖核苷三磷酸。

6.根据权利要求5所述的方法，其中所述3’-O-封闭的-2’-脱氧核糖核苷酸三磷酸是3’-O-叠氮甲基-2’-脱氧核糖核苷三磷酸或3’-O-氨基-2’-脱氧核糖核苷三磷酸。

7.根据权利要求1至4中任一项所述的方法，其中所述多核苷酸是多核糖核苷酸并且所述3’-O-封闭的-核苷三磷酸是3’-O-封闭的-核糖核苷三磷酸。

8.根据权利要求1至7中任一项所述的方法，其中所述多(A)聚合酶是这样的多(A)聚合酶变体，所述多(A)聚合酶变体包含与选自SEQ ID NO:1、2、3、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26或27的氨基酸序列至少90％相同的氨基酸序列和以下位置处的甲硫氨酸的取代：

关于SEQ ID NO:1的位置310处，

关于SEQ ID NO:2的位置318处，

关于SEQ ID NO:3的位置318处，

关于SEQ ID NO:8的位置316处，

关于SEQ ID NO:9的位置309处，

关于SEQ ID NO:10的位置316处，

关于SEQ ID NO:11的位置272处，

关于SEQ ID NO:12的位置316处，

关于SEQ ID NO:13的位置307处，

关于SEQ ID NO:14的位置313处，

关于SEQ ID NO:15的位置312处，

关于SEQ ID NO:16的位置317处，

关于SEQ ID NO:17的位置316处，

关于SEQ ID NO:18的位置316处，

关于SEQ ID NO:19的位置312处，

关于SEQ ID NO:20的位置310处，

关于SEQ ID NO:21的位置309处，

关于SEQ ID NO:22的位置317处，

关于SEQ ID NO:23的位置314处，

关于SEQ ID NO:24的位置307处，

关于SEQ ID NO:25的位置315处，

关于SEQ ID NO:26的位置316处，和

关于SEQ ID NO:27的位置311处。

9.根据权利要求8所述的方法，其中所述位置处的所述甲硫氨酸的所述取代选自F、Y、V、E或T。

10.根据权利要求8或9所述的方法，其中选自所述SEQ ID NO:1、2、3、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26或27的所述氨基酸序列还包含以下位置处的缬氨酸的取代：

关于SEQ ID NO:1的位置234处，

关于SEQ ID NO:2的位置240处，

关于SEQ ID NO:3的位置240处，

关于SEQ ID NO:9的位置232处，

关于SEQ ID NO:10的位置240处，

关于SEQ ID NO:11的位置196处，

关于SEQ ID NO:12的位置240处，

关于SEQ ID NO:13的位置229处，

关于SEQ ID NO:14的位置236处，

关于SEQ ID NO:15的位置236处，

关于SEQ ID NO:16的位置241处，

关于SEQ ID NO:17的位置233处，

关于SEQ ID NO:18的位置240处，

关于SEQ ID NO:19的位置240处，

关于SEQ ID NO:20的位置234处，

关于SEQ ID NO:21的位置233处，

关于SEQ ID NO:22的位置237处，

关于SEQ ID NO:23的位置238处，

关于SEQ ID NO:24的位置231处，

关于SEQ ID NO:25的位置239处，

关于SEQ ID NO:26的位置240处，和

关于SEQ ID NO:27的位置235处。

11.根据权利要求10所述的方法，其中所述位置处的所述缬氨酸的所述取代是丙氨酸或甘氨酸。

12.根据权利要求7至11中任一项所述的方法，其中所述3’-封闭的-O-核糖核苷三磷酸是3’-叠氮甲基-O-核糖核苷三磷酸。

13.根据权利要求12所述的方法，其中所述3’-叠氮甲基-O-核糖核苷三磷酸选自由以下组成的组：3’-叠氮甲基-O-腺苷三磷酸、3’-叠氮甲基-O-鸟苷三磷酸和3’-叠氮甲基-O-胞苷三磷酸以及3’-叠氮甲基-O-尿苷三磷酸。

14.一种试剂盒，所述试剂盒用于进行具有预定序列的多核糖核苷酸的无模板合成，所述试剂盒包含多(A)聚合酶、附接至固体支持物的起始子和3’-O-封闭的核糖核苷三磷酸单体。

15.根据权利要求14所述的试剂盒，其中所述3’-O-保护的核糖核苷三磷酸单体包括3’-O-叠氮甲基-核糖腺苷三磷酸、3’-O-叠氮甲基-核糖鸟苷三磷酸、3’-O-叠氮甲基-核糖胞苷三磷酸、3’-O-叠氮甲基-核糖胸苷三磷酸和3’-O-氨基-核糖尿苷三磷酸中的一种或更多种。

16.根据权利要求14或15所述的试剂盒，其中所述多(A)聚合酶包括这样的多(A)聚合酶变体，所述多(A)聚合酶变体具有与SEQ ID NO:1至少90％相同的氨基酸序列并在M310处具有取代，或具有与SEQ ID NO:3至少90％相同的氨基酸序列并在M318处具有取代；其中所述多(A)聚合酶变体能够(a)在没有模板的情况下合成核糖核酸片段以及(b)将所述3’-O-叠氮甲基-核糖核苷三磷酸掺入到核糖核酸片段上。

17.一种多(A)聚合酶(PAP)变体，所述PAP变体包含与选自SEQ ID NO:1、2、3、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26或27的氨基酸序列至少90％相同的氨基酸序列，和以下位置处的甲硫氨酸的取代：

关于SEQ ID NO:1的位置310处，

关于SEQ ID NO:2的位置318处，

关于SEQ ID NO:3的位置318处，

关于SEQ ID NO:8的位置316处，

关于SEQ ID NO:9的位置309处，

关于SEQ ID NO:10的位置316处，

关于SEQ ID NO:11的位置272处，

关于SEQ ID NO:12的位置316处，

关于SEQ ID NO:13的位置307处，

关于SEQ ID NO:14的位置313处，

关于SEQ ID NO:15的位置312处，

关于SEQ ID NO:16的位置317处，

关于SEQ ID NO:17的位置316处，

关于SEQ ID NO:18的位置316处，

关于SEQ ID NO:19的位置312处，

关于SEQ ID NO:20的位置310处，

关于SEQ ID NO:21的位置309处，

关于SEQ ID NO:22的位置317处，

关于SEQ ID NO:23的位置314处，

关于SEQ ID NO:24的位置307处，

关于SEQ ID NO:25的位置315处，

关于SEQ ID NO:26的位置316处，和

关于SEQ ID NO:27的位置311处，

其中所述PAP变体能够(a)在没有模板的情况下合成核糖核酸片段以及(b)将3’-O-叠氮甲基-核糖核苷三磷酸掺入到核糖核酸片段上或将3’-O-叠氮甲基-2’-脱氧核糖核苷三磷酸掺入到脱氧核糖核酸片段中。

18.根据权利要求17所述的PAP变体，其中所述位置处的所述甲硫氨酸的所述取代选自F、Y、V、E或T。

19.根据权利要求17或18所述的PAP变体，所述PAP变体还包含以下位置处的缬氨酸的取代：

关于SEQ ID NO:1的位置234处，

关于SEQ ID NO:2的位置240处，

关于SEQ ID NO:3的位置240处，

关于SEQ ID NO:9的位置232处，

关于SEQ ID NO:10的位置240处，

关于SEQ ID NO:11的位置196处，

关于SEQ ID NO:12的位置240处，

关于SEQ ID NO:13的位置229处，

关于SEQ ID NO:14的位置236处，

关于SEQ ID NO:15的位置236处，

关于SEQ ID NO:16的位置241处，

关于SEQ ID NO:17的位置233处，

关于SEQ ID NO:18的位置240处，

关于SEQ ID NO:19的位置240处，

关于SEQ ID NO:20的位置234处，

关于SEQ ID NO:21的位置233处，

关于SEQ ID NO:22的位置237处，

关于SEQ ID NO:23的位置238处，

关于SEQ ID NO:24的位置231处，

关于SEQ ID NO:25的位置239处，

关于SEQ ID NO:26的位置240处，和

关于SEQ ID NO:27的位置235处。

20.根据权利要求19所述的PAP变体，其中在所述位置的缬氨酸的所述取代是丙氨酸或甘氨酸。

21.一种多(U)聚合酶(PUP)变体，所述PUP变体包含与选自SEQ ID NO:4、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46或47的氨基酸序列至少90％相同的氨基酸序列和以下位置处的酪氨酸的取代：

关于SEQ ID NO:4的位置212处，

关于SEQ ID NO:28的位置189处，

关于SEQ ID NO:29的位置184处，

关于SEQ ID NO:30的位置227处，

关于SEQ ID NO:31的位置478处，

关于SEQ ID NO:32的位置192处，

关于SEQ ID NO:33的位置186处，

关于SEQ ID NO:34的位置243处，

关于SEQ ID NO:35的位置196处，

关于SEQ ID NO:36的位置253处，

关于SEQ ID NO:37的位置284处，

关于SEQ ID NO:38的位置182处，

关于SEQ ID NO:39的位置187处，

关于SEQ ID NO:40的位置203处，

关于SEQ ID NO:41的位置224处，

关于SEQ ID NO:42的位置204处，

关于SEQ ID NO:43的位置337处，

关于SEQ ID NO:44的位置296处，

关于SEQ ID NO:45的位置291处，

关于SEQ ID NO:46的位置218处，和

关于SEQ ID NO:47的位置366处，

其中所述PUP变体能够(a)在没有模板的情况下合成核糖核酸片段以及(b)将3’-O-叠氮甲基-核糖核苷三磷酸掺入到核糖核酸片段上或将3’-O-叠氮甲基-2’-脱氧核糖核苷三磷酸掺入到脱氧核糖核酸片段中。

22.根据权利要求21所述的PUP变体，其中所述位置处的所述酪氨酸的所述取代是丙氨酸或甘氨酸。