CN115135770A - 使用无歧化作用末端脱氧核苷酸转移酶变体的多核苷酸无模板酶学合成 - Google Patents

使用无歧化作用末端脱氧核苷酸转移酶变体的多核苷酸无模板酶学合成 Download PDF

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CN115135770A
CN115135770A CN202080096423.1A CN202080096423A CN115135770A CN 115135770 A CN115135770 A CN 115135770A CN 202080096423 A CN202080096423 A CN 202080096423A CN 115135770 A CN115135770 A CN 115135770A
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托马斯·伊贝尔特
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Abstract

本发明涉及缺少歧化作用活性的末端脱氧核苷酸转移酶(TdT)变体用于任何预定序列的多核苷酸的无模板酶学合成的用途。这种TdT变体实现正确序列多核苷酸的更高产率。

Description

使用无歧化作用末端脱氧核苷酸转移酶变体的多核苷酸无模 板酶学合成
背景技术
使用末端脱氧核苷酸转移酶(TdT)的不依赖模板的多核苷酸酶学合成已经引起更多的兴趣，因为这类酶的经证实的效率和温和无毒反应条件的益处，例如Ybert等，国际专利公开WO2015/159023；Hiatt等，美国专利5763594；Jensen等，Biochemistry,57:1821-1832(2018)。遗憾的是，在TdT天然形式中，TdT具有所谓的歧化酶活性，包括在自由焦磷酸存在下，现有的多核苷酸n聚体底物的焦磷酸分解以产生(n-1)聚体底物和自由脱氧核糖核苷三磷酸单体，例如Anderson等，Nucleic Acids Research,27(15):3190-3196(1999)。如果天然TdT用于多核苷酸合成，这种不希望的反应必然严重降低全长正确序列产物的产率。
考虑到上述情况，如果可获得不具有这种歧化酶活性并且可用于多核苷酸合成的新的无模板TdT变体，则无模板的多核苷酸酶学合成领域被推进。
发明概述
本发明涉及缺少歧化酶活性的末端脱氧核苷酸转移酶(TdT)变体在合成预定序列的多核苷酸中的应用。在一个方面，本发明涉及使用TdT变体来无模板地合成预定序列的多核苷酸的方法，该方法不发生由歧化酶活性引起的产率降低，所述TdT变体在其FMR基序中的蛋氨酸或功能等同氨基酸以及GGFRR基序中的第二精氨酸或功能等同氨基酸处具有取代。在一些实施方案中，本发明涉及在氨基酸位置M192和R336(其中编号是相对于天然小鼠TdT，SEQ ID NO:1)具有取代的TdT在不使用模板的情况下用于酶学合成任何给定序列的多核苷酸的用途。本发明包括任何具有这种取代或者在其它TdT中的功能等同的残基上的取代的TdT的用途，例如没有BRCT样片段的TdT、非小鼠物种的TdT、嵌合TdT、与其它肽或蛋白质融合的TdT等。在一些实施方案中，这种TdT变体可以通过在其它氨基酸位置处的突变被进一步修饰，以改善多种特性，包括稳定性，可制造性和将3’-O-可逆终止的脱氧核苷三磷酸并入生长中的多核苷酸链的效率。
本发明部分地基于认识和理解到对M192和R336(或功能等同位置)的取代抑制TdT中的歧化作用活性，从而提高最终合成产物中全长正确序列多核苷酸的产率。
在一些实施方案中，本发明涉及使用末端脱氧核苷酸转移酶(TdT)变体无模板地酶学合成预定多核苷酸的方法，所述TdT变体包含与选自SEQ ID NO：2、3、4、5、6、7、8、9、10、11、12、13、14、15、26、27、28、29、35、36、37、38、39、40或41的氨基酸序列具有至少60％同一性的氨基酸序列；所述TdT变体在以下残基具有取代：相对于SEQ ID NO：2、3、4、6、7、8、11和13的第63位的蛋氨酸，或相对于SEQ ID NO:27的第48位的亮氨酸，或相对于SEQ ID NO：26、28或29的第61位的亮氨酸，或相对于SEQ ID NO:5的第62位的亮氨酸，或相对于SEQ ID NO:12的第63位的亮氨酸，或相对于SEQ ID NO:9的第64位的蛋氨酸，或相对于SEQ ID NO:15的第47位的蛋氨酸，或相对于SEQ ID NO:35和40的第48位的蛋氨酸，或相对于SEQ ID NO:9的第64位的蛋氨酸，或相对于SEQ ID NO：10、36、37、38、39和41的第61位的蛋氨酸，或相对于SEQ ID NO:14的第66位的蛋氨酸；并且所述TdT变体在以下残基具有取代：相对于SEQ IDNO:2、3、4、6、7、8、9、11和12的第207位的第一精氨酸，或相对于SEQ ID NO:5的第206位的第一精氨酸，或相对于SEQ ID NO:9的第208位的第一精氨酸，或相对于SEQ ID NO:10、28、29、36、37、38、39和41的第205位的第一精氨酸，或相对于SEQ ID NO:15的第190位的第一精氨酸，或相对于SEQ ID NO:27、35和40的第192位的第一精氨酸，或相对于SEQ ID NO:26的第204位的第一精氨酸或相对于SEQ ID NO:13的第216位的第一精氨酸，或相对于SEQ ID NO:14的第210位的第一精氨酸，其中TdT变体(i)能够在无模板的情况下合成核酸片段，和(ii)能够将3’-O-修饰的核苷酸并入核酸片段的自由3’-羟基上。
在一些实施方案中，上述同一性百分比值与所指示的SEQ ID NO具有至少80％的同一性；在一些实施方案中，上述百分比同一性值与所指示的SEQ ID NO具有至少90％的同一性；在一些实施方案中，上述百分比同一性值与所指示的SEQ ID NO具有至少95％的同一性；在一些实施方案中，上述同一性百分比值为至少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％的同一性；在一些实施方案中，上述同一性百分比值是至少99％的同一性。关于(ii)，这种3’-O-修饰的核苷酸可以包括3’-O-NH2-三磷酸核苷，3’-O-叠氮基甲基-三磷酸核苷，3’-O-烯丙基-三磷酸核苷，3’-O-(2-硝基苄基)-三磷酸核苷，或3’-O-炔丙基-三磷酸核苷。在一些实施方案中，FMR基序中的蛋氨酸或其功能等同氨基酸的氨基酸取代选自R、Q、G、A、V、D、N、H或E。(例如，SEQ ID NO：1的M192R/Q/G/A/V/D/N/H/E，或SEQ ID NO：2的M63R/Q/G/A/V/D/N/H/E)。在一些实施方案中，所述蛋氨酸(或其功能等同残基)的取代是R或Q。在一些实施方案中，GGFRR基序的第二精氨酸的取代选自N、L、K、H、G、D、A或P。在一些实施方案中，所述取代是L或N。
附图简要说明
图1展示了使用本发明的TdT变体的无模板酶学核酸合成方法的步骤。
图2显示在M192和R336(或功能等同的残基)处具有和不具有取代的TdT的引物延伸反应产物的电泳图。
发明详细描述
本发明的一般原理在此更详细地公开，特别是通过实例，例如在附图中所示和详细描述的那些实例。然而，应当理解，本发明不应局限于所描述的具体实施方案。本发明可以进行各种修改和替换形式，下文对于几个实施方案示出了这些修改和替换形式的细节。本申请旨在涵盖落入本发明的原理和范围内的所有修改方案、等同方案和替换方案。
除非另有说明，本发明的实践可以采用有机化学、分子生物学(包括重组技术)，细胞生物学和生物化学的常规技术和描述，它们在本领域的技术范围内。这种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。用于这种常规技术的操作指南可以在来自制造商的产品文献和标准实验室手册中找到，例如Genome Analysis:A Laboratory Manual Series(Vols.I-IV)；PCRPrimer:A Laboratory Manual；以及Molecular Cloning:A Laboratory Manual(均来自冷泉港实验室出版社)；Lutz and Bornscheuer编,Protein Engineering Handbook(Wiley-VCH,2009)；Hermanson,Bioconjugate Techniques,第二版(Academic Press,2008)等。
每个天然TdT具有序列基序的顺序(例如从N末端到C末端：FMR、VSC、GGFRR、GHD、TGSR、FER等)。在一些实施方案中，FMR基序是位于185-200位氨基酸区段(参考SEQ ID NO:1)中或附近的3氨基酸区段，其选自FMR、FLR、FGR、FRR和YMR。
本发明部分地基于这样的发现：M192和R336残基对于TdT中的歧化作用活性是必需的，并且该活性可以通过取代M192处的蛋氨酸和R336处的精氨酸(或者在非小鼠物种中相应的氨基酸)来消除。这些残基相对于天然小鼠TdT(SEQ ID NO:1)编号，但是对于其它TdT中的功能等同残基同样适用，其它TdT包括但不限于，具有SEQ ID NO 2、3、4、5、6、7、8、9、10、11、12、13、14、15、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40和41的氨基酸序列的TdT。歧化作用活性的效果显示在图2中，图2显示在存在(+)和不存在(-)3’-O-NH2-dGTP单体的情况下，由编号为1、2、4、5、6、7、8、9和10的各种TdT变体延伸的引物的电泳数据(对TdT变体11没有进行负性单体对照)。TdT变体5(200)是唯一具有M192R和R336L取代的变体。结果显示，在没有任何3’-O-保护-dNTP的情况下，具有歧化作用活性的TdT变体催化末端磷酸键的焦磷酸分解(例如TdT变体6的n-1点(202))，以及释放的dNTP与引物的偶联(例如TdT变体6的n+1点(204))。相反，在存在或不存在3’-O-保护-dNTP的情况下，TdT变体5没有假的n-1或n+1点。因此，使用根据本发明的具有M192和R336取代的TdT变体的酶学多核苷酸合成允许以更高产率产生全长正确序列多核苷酸的产物。
无模板酶学合成
多核苷酸的无模板酶学合成可以通过多种已知方案、使用无模板聚合酶进行，例如末端脱氧核苷酸转移酶(TdT)，包括经改造以具有改善的特性的TdT变体，改善的特性为例如更高的温度稳定性或在并入3’-O-封闭的三磷酸脱氧核苷(3’-O-封闭的dNTP)中更高的效率，例如Ybert等人的国际专利公开WO/2015/159023；Ybert等，国际专利公开WO/2017/216472；Hyman，美国专利5436143；Hiatt等，美国专利5763594；Jensen等人，Biochemistry,57:1821-1832(2018)；Mathews等人，Organic&Biomolecular Chemistry,DOI:0.1039/c60ob01371f(2016)；Schmitz等人，Organic Lett.,1(11):1729-1731(1999)。
在一些实施方案中，酶学DNA合成方法包括重复的步骤循环，例如图1所示，其中在每个循环中加入预定的核苷酸。提供了具有自由3’-羟基(103)的引发多核苷酸(100)，例如附接于固体支持物(102)。在能有效地将3’-O-保护的-dNTP酶学并入到引发多核苷酸(100)(或延伸的引发多核苷酸)的3’末端的条件(104)下，向引发多核苷酸(100)(或后续循环中的延伸的引发多核苷酸)中加入3’-O-保护的dNTP和TdT变体。该反应产生3’-羟基被保护的延伸的引发多核苷酸(106)。如果延伸的引发多核苷酸含有完整的序列，则3’-O-保护基团被去除或脱保护，并且期望的序列被从原始引发多核苷酸切割。这种切割可以使用多种单链切割技术中的任一种进行，例如，通过在原始引发多核苷酸内的预定位置插入可切割的核苷酸或可切割的接头。示例性的可切割核苷酸或接头包括但不限于(i)被尿嘧啶DNA糖基化酶切割的尿嘧啶核苷酸；(ii)可光切割的基团，例如硝基苄基接头，如美国专利5,739,386中所述；或者被内切核酸酶V切割的肌苷。在一些实施方案中，切割的多核苷酸可以具有自由的5'-羟基；在其它实施方案中，切割的多核苷酸可具有5'-磷酸化末端。如果延伸的引发多核苷酸不包含完整的序列，则除去3’-O-保护基团以暴露自由的3’-羟基(103)，并且延伸的引发多核苷酸经历另一循环的核苷酸添加和脱保护。
如本文所用，关于指定基团(例如核苷酸或核苷的3’-羟基)的术语“保护的”和“封闭的”可互换使用，并且旨在表示某种部分共价连接至该指定基团，防止在化学或酶学过程期间基团的化学变化。当指定基团是三磷酸核苷或其中已并入3’-保护的(或封闭的)-三磷酸核苷的延伸片段(或“延伸中间体”)的3’-羟基时，被阻止的化学变化是延伸片段(或“延伸中间体”)通过酶偶联反应的进一步或随后的延伸。
在一些实施方案中，在每个合成步骤中，在3’-O-可逆性封闭的dNTP存在下，使用TdT将有序序列的核苷酸偶联至引发核酸。在一些实施方案中，合成寡核苷酸的方法包括以下步骤：(a)提供具有自由3’-羟基的引发物；(b)在延伸条件下，在3’-O-封闭的三磷酸核苷存在下，使所述引发物或具有自由3’-羟基的延伸中间体与TdT反应，以产生3’-O-封闭的延伸中间体；(c)解封闭所述延伸中间体，以产生具有自由3’-羟基的延伸中间体；和(d)重复步骤(b)和(c)，直至合成多核苷酸。(有时“延伸中间体”也称为“延伸片段”。)在一些实施方案中，引发物作为例如通过其5'末端连接到固体支持物上的寡核苷酸提供。上述方法还可以包括在反应或延伸步骤之后以及在解封闭步骤之后的洗涤步骤。例如，反应步骤可以包括在预定的孵育期或反应时间之后，例如通过洗涤除去未并入的三磷酸核苷的子步骤。这种预定的孵育时间或反应时间可以是数秒(例如30秒)至数分钟(例如30分钟)。
上述方法还可以包括封端步骤，以及在反应或延伸步骤之后以及在解封闭步骤之后的洗涤步骤。如上所述，在一些实施方案中，可以包括封端步骤，其中非延伸的自由3’-羟基与防止封端链的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二三磷酸脱氧核苷。在其它实施方案中，具有自由3’-羟基的非延伸链可通过用3’外切核酸酶活性(例如Exo I)处理而降解。例如，参见Hyman，美国专利5436143。同样，在一些实施方案中，未被解封闭的链可以被处理，以除去这样的链或使这样的链对进一步的延伸是惰性的。
在包括寡核苷酸的连续合成的一些实施方案中，封端步骤可能是不希望的，因为封端可能阻止产生等摩尔量的多种寡核苷酸。在没有封端的情况下，序列将具有均匀分布的缺失错误，但是多种寡核苷酸中的每一种将以相等的摩尔量存在。在非延伸片段被封端时则不是这样的情形。
在一些实施方案中，用于延伸或延伸步骤的反应条件可包括以下：2.0μM纯化的TdT；125-600μM 3’O-封闭的dNTP(例如3’O-NH2-封闭的dNTP)；约10至约500mM的二甲胂酸钾缓冲液(pH为6.5-7.5)和约0.01至约10mM的二价阳离子(例如COCl2或MnCl2)，其中延伸反应可以在50μL反应体积中，在室温至45℃范围内的温度下进行3分钟。在3’O-封闭的dNTP是3’O-NH2-封闭的dNTP的实施方案中，用于解封闭步骤的反应条件可以包括以下：700mMNaNO2；1M乙酸钠(用乙酸调节至pH4.8-6.5)，其中解封闭反应可以在50μL体积中在室温至45℃的范围内的温度下进行30秒至几分钟。
根据具体应用，解封闭和/或切割的步骤可以包括各种化学或物理条件，例如光，热，pH，能够切割指定化学键的具体试剂(如酶)的存在。选择3’-O-封闭基团和相应的解封闭条件方面的指导可以在以下参考文献中找到，将这些文献通过援引的方式并入本文：美国专利5808045；美国专利8808988；国际专利公开WO91/06678；以及以下引用的参考文献。在一些实施方案中，切割剂(有时也称为解封闭试剂或解封闭剂)是化学切割剂，例如二硫苏糖醇(DTT)。在其他实施方案中，切割剂可以是酶切割剂，例如磷酸酶，其可以切割3’-磷酸封闭基团。本领域技术人员将理解，解封闭剂的选择取决于所用的3’-核苷酸封闭基团的类型，是否使用一个或多个封闭基团，引发物是否连接到活细胞或生物体或固体支持物等，这种情况则需要温和的处理。例如，膦(如三(2-羧乙基)膦(TCEP))可用于切割3’-O-叠氮基甲基，钯络合物可用于切割3’O-烯丙基，或亚硝酸钠可用于切割3’-O-氨基。在具体的实施方案中，切割反应涉及TCEP、钯络合物或亚硝酸钠。
如上所述，在一些实施方案中，期望使用两个或更多个可以使用正交解封闭条件除去的封闭基团。以下示例性封闭基团对可用于并行合成实施方案，例如上文所述的那些。应理解，其它封闭基团对或含有多于两个的基团可用于本发明的这些实施方案中。
3’-O-NH2 3’-O-叠氮基甲基
3’-O-NH2 3’-O-烯丙基
3’-O-NH2 3’-O-磷酸
3’-O-叠氮基甲基 3’-O-烯丙基
3’-O-叠氮基甲基 3’-O-磷酸
3’-O-烯丙基 3’-O-磷酸
在活细胞上合成寡核苷酸需要温和的解封闭或脱保护条件，即，不破坏细胞膜、不变性蛋白质、不干扰关键细胞功能等的条件。在一些实施方案中，脱保护条件在与细胞存活相容的生理条件的范围内。在这样的实施方案中，酶学脱保护是期望的，因为酶学脱保护可以在生理条件下进行。在一些实施方案中，特定的可酶学去除的封闭基团与用于去除所述封闭基团的特定的酶相关联。例如，基于酯或酰基的封闭基团可以用酯酶(如乙酰酯酶或类似的酶)除去，并且可以用3’-磷酸酶(如T4多核苷酸激酶)除去磷酸封闭基团。例如，3’-O-磷酸可以通过用100mM Tris-HCl(pH6.5)、10mM MgCl2、5mM 2-巯基乙醇和一个单位T4多核苷酸激酶的溶液处理来除去。反应在37℃的温度下进行1分钟。
“3’-磷酸封闭的”核苷酸或“3’-磷酸保护的”核苷酸是指其中3’位的羟基被含有磷酸的部分的存在封闭的核苷酸。本发明的3’-磷酸封闭的核苷酸的实例是核苷酸-3’磷酸单酯/核苷酸-2’,3’-环磷酸酯，核苷酸-2’-磷酸单酯和核苷酸-2’或3’-烷基磷酸酯二酯，以及核苷酸-2’或3’-焦磷酸酯。还可以使用硫代磷酸或此类化合物的其它类似物，只要该取代不能防止由磷酸酶导致自由3’OH的脱磷酸化。
3’-O-酯保护的dNTP或3’-O-磷酸保护的dNTP的合成和酶学脱保护的其它实例描述于以下参考文献中：Canard et al,Proc.Natl.Acad.Sci.,92:10859-10863(1995)；Canard et al,Gene,148:1-6(1994)；Cameron et al,Biochemistry,16(23):5120-5126(1977)；Rasolonjatovo et al,Nucleosides&Nucleotides,18(4&5):1021-1022(1999)；Ferrero et al,Monatshefte fur Chemie,131:585-616(2000)；Taunton-Rigby et al,J.Org.Chem.,38(5):977-985(1973)；Uemura et al,Tetrahedron Lett.,30(29):3819-3820(1989)；Becker et al,J.Biol.Chem.,242(5):936-950(1967)；Tsien,国际专利公开WO1991/006678。
本文所用“引发物”(或等同术语，如“引发片段”,“引发核酸”,“引发寡核苷酸”等)是指具有自由3’-末端的短寡核苷酸序列，其可通过无模板聚合酶(如TdT)进一步延伸。在一个实施方案中，引发片段是DNA引发片段。在另一个实施方案中，引发片段是RNA引发片段。在一个实施方案中，引发片段具有3-100个核苷酸，特别是3-20个核苷酸。在一个实施方案中，引发片段是单链的。在另一个实施方案中，引发片段是双链的。在一个具体的实施方案中，用5'-伯胺合成的引发寡核苷酸可以使用制造商的方案共价连接于磁珠。同样，用3’伯胺合成的引发寡核苷酸可以使用制造商的方案共价连接到磁珠。适合与本发明的实施方案一起使用的各种其它附接化学方案在本领域中是熟知的，例如Integrated DNATechnologies brochure,“Strategies for Attaching Oligonucleotides to SolidSupports,”v.6(2014)；Hermanson,Bioconjugate Techniques,Second Edition(AcademicPress,2008)和类似文献。
在本发明中使用的许多3’-O-封闭的dNTP可以从商业销售商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，Gene(上文引用)；Metzker等人，Nucleic Acids Research,22:4259-4267(1994)；Meng等，J.Org.Chem.,14:3248-3252(3006)；美国专利公开2005/037991。在一些实施方案中，经修饰的核苷酸包含经修饰的核苷酸或核苷分子，所述经修饰的核苷酸或核苷分子包含嘌呤或嘧啶碱基和核糖或脱氧核糖糖部分，所述核糖或脱氧核糖糖部分具有与其共价连接的可移除的3’-OH封闭基团，使得所述3’碳原子连有以下结构的基团：
-O-Z
其中-Z是-C(R’)2-O-R”、-C(R’)2-N(R”)2、-C(R’)2-N(H)R”、-C(R’)2-S-R”和-C(R’)2-F中的任一个，其中每个R”是可移除保护基团或可移除保护基团的一部分；每个R’独立地为氢原子，烷基，取代的烷基，芳基烷基，烯基，炔基，芳基，杂芳基，杂环基，酰基，氰基，烷氧基，芳氧基，杂芳氧基或酰胺基，或通过连接基团连接的可检测标记；条件是在一些实施方案中，这样的取代基具有多至10个碳原子和/或多至5个氧或氮杂原子；或(R’)2表示式＝C(R”’)2的基团，其中每个R”’可以相同或不同，并且选自包含氢原子和卤素原子以及烷基的组，条件是在一些实施方案中，每个R”’的烷基具有1至3个碳原子；并且其中所述分子可以反应以产生中间体，其中每个R”交换为H，或者其中Z是-(R')2-F，F交换为OH、SH或NH2，优选OH，所述中间体在水性条件下离解，以提供具有自由3’OH的分子；条件是当Z是-C(R’)2-S-R”时，两个R’基团不都是H。在某些实施方式中，修饰的核苷酸或核苷的R’是烷基或取代的烷基，条件是这样的烷基或取代的烷基具有1至10个碳原子和0至4个氧或氮杂原子。在某些实施方案中，经修饰核苷酸或核苷的-Z具有式-C(R’)2-N3。在某些实施方案中，Z是叠氮基甲基。
在一些实施方案中，Z是可切割有机部分，具有或不具有杂原子，分子量为200或更小。在其它实施方案中，Z是可切割有机部分，具有或不具有杂原子，分子量为100或更小。在其它实施方案中，Z是可切割有机部分，具有或不具有杂原子，分子量为50或更小。在一些实施方案中，Z是可酶学切割有机部分，具有或不具有杂原子，分子量为200或更小。在其它实施方案中，Z是可酶学切割有机部分，具有或不具有杂原子，分子量为100或更小。在其它实施方案中，Z是可酶学切割有机部分，具有或不具有杂原子，分子量为50或更小。在其它实施方案中，Z是分子量为200或更小的可酶学切割的酯基。在其它实施方案中，Z是可被3’磷酸酶除去的磷酸基团。在一些实施方案中，以下3’磷酸酶中的一种或多种可与制造商推荐的方案一起使用：T4多核苷酸激酶，小牛肠碱性磷酸酶，重组虾碱性磷酸酶(例如可得自NewEngland Biolabs,Beverly,MA)。
在进一步的具体实施方案中，3’-封闭的三磷酸核苷被3’-O-叠氮基甲基，3’-O-NH2或3’-O-烯丙基封闭。在其它实施方案中，3’-封闭的三磷酸核苷被3’-O-叠氮基甲基，3’-O-NH2封闭。在其它实施方案中，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-氨基、3’-O-叠氮基甲基、3’-O-叔丁氧基乙氧基、3’-O-(2-氰基乙基)和3’-O-炔丙基。
在一些实施方案中，上述TdT变体在表1所示的半胱氨酸、第二精氨酸和谷氨酸处具有取代。
表1
Figure BDA0003792871540000131
本发明的具体TdT变体DS1001至DS1018在表2中列出。TdT变体DS1001至DS1018中的每一个包含与SEQ ID NO 2具有至少60％同一性的氨基酸序列，并且在指定位置包含所述取代。在一些实施方案中，TdT变体DS1001至DS1018包含与SEQ ID NO 2具有至少80％同一性的氨基酸序列，并且在指定位置包含所述取代。在一些实施方案中，TdT变体DS1001至DS1018包含与SEQ ID NO 2具有至少90％同一性的氨基酸序列，并且在指定位置包含所述取代。在一些实施方案中，TdT变体DS1001至DS1018包含与SEQ ID NO 2具有至少95％同一性的氨基酸序列，并且在指定位置包含所述取代。在一些实施方案中，TdT变体DS1001至DS1018包含与SEQ ID NO 2具有至少97％同一性的氨基酸序列，并且在指定位置包含所述取代。在一些实施方案中，TdT变体DS1001至DS1018包含与SEQ ID NO 2具有至少98％同一性的氨基酸序列，并且在指定位置包含所述取代。在一些实施方案中，TdT变体DS1001至DS1018包含与SEQ ID NO 2具有至少99％同一性的氨基酸序列，并且在指定位置包含所述取代。
表2.用于本发明方法的具体TdT变体
Figure BDA0003792871540000141
Figure BDA0003792871540000151
如上所述的本发明的TdT变体各自包含与指定的SEQ ID NO具有一定序列同一性百分比的氨基酸序列，其条件是存在指定的取代。在一些实施方案中，以这种方式描述的本发明的嵌合TdT变体与指定的SEQ ID NO之间的序列差异的数目和类型可以是由于取代、缺失和/或插入，并且被取代、缺失和/或插入的氨基酸可能包括任何氨基酸。在一些实施方案中，这样的缺失、取代和/或插入仅包括天然存在的氨基酸。在一些实施方案中，取代仅包括保守型或同义型(synonymous)氨基酸改变，如Grantham,Science,185:862-864(1974)中所述。也就是说，氨基酸的取代只能在其同义型氨基酸组的成员中发生。在一些实施方案中，可以使用的同义型氨基酸组列于表3A中。
表3A：氨基酸同义组I
Figure BDA0003792871540000161
Figure BDA0003792871540000171
在一些实施方案中，可以使用的同义氨基酸组列于表3B中。
表3B：氨基酸同义组II
Figure BDA0003792871540000172
Figure BDA0003792871540000181
核苷酸并入活性的测量
本发明变体的核苷酸并入效率可通过延伸或延长测定来测量，例如如Boule等(下文引用)，Bentolila等(下文引用)，和Hiatt等人的美国专利5808045中所述，后者通过援引加入的方式并入到本文中。简言之，在这种测定的一种形式中，具有自由3’-羟基的荧光标记的寡核苷酸在TdT延伸条件下与待测试的变体TdT在可逆封闭的三磷酸核苷存在下反应预定的持续时间，之后终止延伸反应，并且在通过凝胶电泳分离后，定量延伸产物和未延伸的引发寡核苷酸的量。通过这种测定，变体TdT的并入效率可以容易地与其它变体或野生型或参考TdT或其它聚合酶的效率比较。在一些实施方案中，变体TdT效率的量度可以是在同样测定中使用变体TdT的延伸产物的量与使用野生型TdT的延伸产物的量的比率(以百分比给出)。
在一些实施方案中，以下具体延伸测定可用于测量TdT的并入效率：所用引物如下：
5'-AAAAAAAAAAAAAAGGGG-3'(SEQ ID NO:20)
所述引物在5’末端还具有ATTO荧光染料。使用的代表性修饰核苷酸(在表4中记为dNTP)包括3’-O-氨基-2’,3’-二脱氧核苷酸-5’-三磷酸(ONH2,Firebird Biosciences)，例如3’-O-氨基-2’,3’-二脱氧腺苷-5’-三磷酸。对于每个测试的不同变体，使用一个管进行反应。将试剂加入管中，从水开始，然后按表4的顺序加入。在37℃下孵育30分钟后，通过添加甲酰胺(Sigma)终止反应。
表4：延伸活性测定试剂
Figure BDA0003792871540000191
活性缓冲液包含例如补充有CoCl2的TdT反应缓冲液(可获自New EnglandBiolabs)。
通过常规聚丙烯酰胺凝胶电泳分析测定的产物。例如，上述测定的产物可以在16％聚丙烯酰胺变性凝胶(Bio-Rad)中分析。在分析即将开始前制备凝胶，通过将聚丙烯酰胺倾倒在玻璃平板内并使其聚合。将玻璃平板内的凝胶安装在填充用于电泳步骤的TBE缓冲液(Sigma)的改造罐中。将待分析的样品加载在凝胶的顶部。在室温下，在凝胶的顶部和底部之间施加500至2000V的电压，持续3至6h。分离后，使用例如Typhoon扫描仪(GE LifeSciences)扫描凝胶荧光。使用ImageJ软件(imagej.nih.gov/ij/)或其同类软件分析凝胶图像，以计算修饰核苷酸的并入百分比。
发夹完成测定。在一个方面，本发明包括测量聚合酶(例如TdT变体)将dNTP并入多核苷酸(即“测试多核苷酸”)的3’末端的能力的方法。一种这样的方法包括在反应条件下提供具有自由3’羟基的测试多核苷酸，其中所述测试多核苷酸基本上仅是单链的，但在用聚合酶(如TdT变体)延伸时形成包含单链环和双链茎的稳定发夹结构，从而允许通过双链多核苷酸的存在检测3’末端的延伸。双链结构可以以多种方式检测，包括但不限于，插入双链结构时优先发荧光的荧光染料，延伸多核苷酸上的受体(或供体)和寡核苷酸上的供体(或受体)之间的荧光共振能量转移(FRET)，所述寡核苷酸与新形成的发夹茎形成三链体，FRET受体和供体均连接到测试多核苷酸并且在形成发夹或类似结构等时，被带入FRET附近。在一些实施方案中，在延伸单个核苷酸后，测试多核苷酸的茎部的长度在4至6个碱基对的范围内；在其它实施方案中，这样的茎部的长度为4至5个碱基对；在其它实施方案中，这种茎部的长度为4个碱基对。在一些实施方案中，测试多核苷酸的长度在10至20个核苷酸的范围内；在其它实施方案中，测试多核苷酸具有12-15个核苷酸的长度。在一些实施方案中，用这样的核苷酸延伸测试多核苷酸是有利的或方便的，所述核苷酸使不具有延伸的茎和具有延伸的茎的熔解温度之间的差异最大化；因此，在一些实施方案中，用dC或dG延伸测试多核苷酸(因此选择具有用于茎形成的合适互补核苷酸的测试多核苷酸)。
用于发夹完成测定的示例性测试多核苷酸包括p875(5'-CAGTTAAAAACT)(SEQ IDNO:16)，其通过用dGTP延伸而完成；p876(5'-GAGTTAAAACT)(SEQ ID NO:17)，其通过用dCTP延伸而完成；和p877(5'-CAGCAAGGCT)(SEQ ID NO:18)，其通过用dGTP延伸而完成。用于这些测试多核苷酸的示例性反应条件可以包括：2.5-5μM的测试多核苷酸，
Figure BDA0003792871540000201
的1∶4000稀释液(来自Biotium,Inc.,Fremont,CA的插入染料)，200mM卡可基酸盐KOH pH6.8，1mMCoCl2，0-20％DMSO和3’ONH2 dGTP和所需浓度的TdT。使用常规荧光计(例如TECAN读数器)在28-38℃的反应温度下，使用设置为360nm的激发过滤器和设置为635nm的发射过滤器，可以通过
Figure BDA0003792871540000211
染料的荧光增加来监测发夹的完成。
在本发明该方面的一些实施方案中，可以通过以下步骤测试TdT变体对无模板三磷酸核苷并入的能力：(a)在一定条件下，将具有自由3’羟基的测试多核苷酸、TdT变体和三磷酸核苷组合，其中测试多核苷酸是单链的，但并入三磷酸核苷后形成具有双链茎区的发夹；和(b)检测形成的双链茎区的量，作为TdT变体并入三磷酸核苷的能力的量度。在一些实施方案中，三磷酸核苷是3’-O-封闭的三磷酸核苷。
变体TdT的产生
本发明的变体的产生可以通过突变已知的参考或野生型TdT编码多核苷酸，然后使用常规分子生物学技术将其表达。例如，编码小鼠TdT(SEQ ID NO:2)的核酸构建体可以使用常规分子生物学技术(例如使用Stemmer et al,Gene,164:49-53(1995)；Kodumal etal,Proc.Natl.Acad.Sci.,101:15573-15578(2004)等文献描述的方案)，从合成片段组装，或者可以使用Boule et al,Mol.Biotechnology,10:199-208(1998),or Bentolila etal,EMBO J.,14:4221-4229(1995)等文献描述的方案从小鼠细胞直接克隆。
例如，可以将分离的TdT基因插入表达载体，如pET32(Novagen)，得到载体pCTdT，然后可以使用常规方法制备和表达变体TdT蛋白。具有正确序列的载体可以转化入大肠杆菌生产菌株中。
使用常规技术培养转化的菌株成为沉淀物，从沉淀物中提取TdT蛋白。例如，将预先制备的沉淀物在30至37℃水浴中解冻。一旦完全解冻，将沉淀物重悬于裂解缓冲液中，裂解缓冲液包含50mM tris-HCL(Sigma)pH7.5，150mM NaCl(Sigma)，0.5mM巯基乙醇(Sigma)，5％甘油(Sigma)，20mM咪唑(Sigma)和1tab 100mL蛋白酶混合物抑制剂(Thermofisher)。小心地进行再悬浮，以避免裂解不足和剩余聚集体。重悬的细胞通过几个弗氏压碎器循环裂解，直到获得完全的颜色均匀性。所用的通常压力为14000psi。然后将裂解物以10000rpm离心1小时至1小时30分钟。在柱纯化之前，使离心物通过0.2μm过滤器以除去任何碎片。
TdT蛋白可以在一步法亲和操作中从离心物中纯化。例如，Ni-NTA亲和柱(GEHealthcare)用于结合聚合酶。初始，用15柱体积的50mM tris-HCL(Sigma)pH7.5、150mMNaCl(Sigma)和20mM咪唑(Sigma)洗涤和平衡柱。平衡后，将聚合酶结合到柱上。然后，将15倍柱体积的包含50mM tris-HCL(Sigma)pH7.5、500mM NaCl(Sigma)和20mM咪唑(Sigma)的洗涤缓冲液加到柱上15倍柱体积。洗涤后，用50mM tris-HCL(Sigma)pH7.5、500mM NaCl(Sigma)和0.5M咪唑(Sigma)洗脱聚合酶。收集对应于最高浓度的目标聚合酶的级分，并将其汇集在单个样品中。合并的级分用透析缓冲液(20mM Tris-HCl pH6.8，200mM NaCl,50mMMgOAc,100mM[NH4]2SO4)透析。随后在浓缩过滤器(Amicon Ultra-30,Merk Millipore)的帮助下浓缩透析液。将浓缩的酶分成小份，最后加入50％甘油，然后将这些小份在-20℃冷冻并长期保存。在SDSPAGE凝胶中分析5μL纯化酶的不同级分。
在一些实施方案中，TdT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，聚氨基酸标签、聚His标签、6His标签等)，化合物(例如，聚乙二醇)，蛋白-蛋白结合对(例如，生物素-抗生物素蛋白)，亲和偶联，捕获探针，或这些的任意组合。接头部分可以与TdT变体分开或作为TdT变体的一部分。用于本发明的TdT变体的示例性His标签是MASSHHHHHSSGSENLYFQTGSSG-(SEQ ID NO:19))。标签-接头部分不干扰核苷酸结合活性或TdT变体的催化活性。
上述方法或等同方法产生分离的TdT变体，其可以与对于活性和/或保存是必需的或有用的各种试剂混合，所述试剂例如盐，pH缓冲剂，载体化合物等。
用于实施本发明方法的试剂盒
本发明包括用于实施本发明方法的多种试剂盒。在一个方面，本发明的试剂盒包含本发明的TdT变体，所述TdT变体配制在适于进行如本文所述的无模板酶学多核苷酸合成的制剂中。这样的试剂盒还可以包括合成缓冲液，其提供用于优化3’-O-保护的dNTP向生长链的无模板添加或并入的反应条件。在一些实施方案中，本发明的试剂盒还包括3’-O-可逆保护的dNTP。在这样的实施方案中，3’-O-可逆保护的dNTP可以包含3’-O-氨基-dNTP或3’-O-叠氮基甲基-dNTP。在进一步的实施方案中，试剂盒可以包括以下组分中的一种或多种(单独包括或与上述组分一起)：(i)用于进行如本文所述的脱保护步骤的脱保护剂，(ii)附接有引发物的固体载体，(iii)用于从固体载体释放完成的多核苷酸的切割剂，(iv)用于在酶学添加或偶联步骤结束时除去未反应的3’-O-保护的dNTP的洗涤试剂或缓冲液以及，(v)合成后的处理试剂，如净化柱，脱盐试剂，洗脱试剂等。
关于以上组分(ii)和(iii)，某些引发物和切割剂一起存在。例如，包含肌苷可切割核苷酸的引发物可以与内切核酸酶V切割剂一起存在；包含硝基苄基可光切割接头的引发物可以与合适的光源一起，用于切割可光切割接头；包含尿嘧啶的引发物可以与尿嘧啶DNA糖基化酶切割剂一起存在等。
定义
氨基酸由根据以下命名法的单字母或三字母代码表示：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：蛋氨酸(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)。
关于两个或多个不同TdT中的氨基酸位置的“功能等同”是指(i)在各自位置的氨基酸在TdT中起相同的功能作用，和(ii)氨基酸存在于分别TdT的氨基酸序列中的同源氨基酸位置处。如本文所用的术语，可以使用常规序列比对工具和/或常规蛋白质建模工具来确定两种或更多种不同TdT的氨基酸序列中的功能等同氨基酸残基。在一些实施方案中，可以使用比对工具Multalin鉴定功能等同氨基酸(Mitchell,Bioinformatics,9(5):614-615(1993))。
关于蛋白质，“分离的”是指这样的化合物，其已经从其天然环境的组分或从非均相反应混合物中鉴定和分离和/或回收。天然环境或反应混合物的污染物组分是会干扰蛋白质功能的物质，并且可以包括酶，激素和其它蛋白质或非蛋白质溶质。在一些实施方案中，本发明的蛋白质被纯化为(1)通过Lowry方法测定，大于95重量％的蛋白质，最优选大于99重量％的蛋白质，(2)通过使用旋转杯定序器足以获得N末端或内部氨基酸序列的至少15个残基的程度，或(3)通过SDS-PAGE、在还原或非还原条件下、使用考马斯蓝或优选银染验证达到同质性。当通过重组方法制备时，本发明的分离的蛋白质可以在重组细胞内原位包括本发明的蛋白质，因为蛋白质的天然环境的至少一个组分将不存在。通常，通过至少一个纯化步骤制备本发明的分离的蛋白质。
“试剂盒”是指用于递送用于实施本发明方法的材料或试剂的任何递送系统。在反应测定的上下文中，这样的递送系统包括允许从一个位置到另一个位置储存、运输或递送反应试剂(例如，在适当容器中的一种或多种TdT变体，反应缓冲液，3’-O-保护的-dNTP，脱保护试剂，附接有引发物的固体支持物等)和/或支持材料(例如，用于进行测定的书面说明、缓冲液等)的系统和/或化合物(例如稀释剂，表面活性剂，载体等)。例如，试剂盒包括含有相关反应试剂和/或支持材料的一个或多个外壳(例如盒子)。这种内容物可以一起或分开递送到预期的接受者。例如，第一容器可含有一种或多种用于合成方法的TdT变体，第二或另外的容器可含有脱保护剂，具有引发物的固体支持物，3’-O-保护的dNTP等。N末端
本文所用的关于TdT变体的氨基酸序列的“基序”是指在具体位置的TdT氨基酸序列的亚序列或片段，其在不同物种、特别是进化相关物种的TdT的氨基酸序列中是保守的(但可能不是相同的)。这种保守序列基序的实例描述于Motea et al,Biochim.Biophys.Acta.1804(5):1151-1166(2010)；Delarue et al,EMBO J.,21:427-439(2002)和类似的参考文献。示例性TdT基序包括(从N末端到C末端排序)：FMR、VSC、GGFRR、KMT、GHD、TGSR、FER等。如本文所提及的，序列基序通过最常见的序列发生或共有序列来鉴定，例如“TGSR”，但是这样的命名并不意味着排除使用常规比对工具，如Multalin(Mitchell,Bioinformatics,9(5):614-615(1993))与TGSR比对的其它子序列。例如，提及“TGSR基序”包括与小鼠的片段“TGSR”比对的负鼠TdT的片段“SGSR”。在一些实施方案中，序列基序被定义为序列表达集合。例如，在一些实施方案中，FMR基序由FMR、FLR、FGR、FRR和YMR组成的组中的序列组成。
“突变体”或“变体”可互换使用，并且指衍生自本文所述的天然或参考TdT多肽并且在一个或多个位置包含修饰或改变(即，取代、插入和/或缺失)的多肽。变体可以通过本领域熟知的各种技术获得。具体而言，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、序列重排和合成寡核苷酸构建。诱变活性在于蛋白质序列(在本发明中为聚合酶)中缺失、插入或取代一个或多个氨基酸。以下术语用于表示取代：L238A表示参考序列或野生型序列的第238位氨基酸残基(亮氨酸，L)被改变为丙氨酸(A)。A132V/I/M表示母体序列的第132位的氨基酸残基(丙氨酸，A)被下列氨基酸之一取代：缬氨酸(V)，异亮氨酸(I)或蛋氨酸(M)。取代可以是保守的或非保守的取代。保守取代的实例为碱性氨基酸(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(蛋氨酸，亮氨酸，异亮氨酸，半胱氨酸和缬氨酸)，芳族氨基酸(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)的组内取代。
“序列同一性”是指两个序列(例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，通常表示为百分比)。通过比较比对时的序列来确定序列同一性，以便最大化重叠和同一性，同时最小化序列空位。特别地，根据两个序列的长度，序列同一性可以使用多种数学全局或局部比对算法中的任何一种来确定。相似长度的序列的比对优选使用全局比对算法(例如Needleman和Wunsch算法；Needleman和Wunsch,1970)，其在整个长度上最优地比对序列，而明显不同长度的序列的比对优选使用局部比对算法(例如Smith和Waterman算法(Smith和Waterman,1981)或Altschul算法(Altschul等,1997；Altschul等,2005))。用于确定氨基酸序列同一性百分比的比对可以以本领域技术人员所熟知的各种方式实现，例如，使用可在互联网网站上提供的可公开获得的计算机软件，如http://blast.ncbi.nlm.nih.gov/或http://www.ebi.ac.uk/tools/emboss/。本领域技术人员可以确定用于测量比对的适当参数，包括在所比较的序列的整个长度上实现最大比对所需的任何算法。出于本文的目的，氨基酸序列同一性％值是指使用成对序列比对程序EMBOSS Needle产生的值，其使用Needleman-Wunsch算法创建两个序列的最佳全局比对，其中所有搜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＝false，末端空位开放＝10和末端空位延伸＝0.5。如本文所用，用于比较参考序列与变体序列的同一性百分比值不包括明确指定的包含变体序列的取代的氨基酸位置。也就是说，同一性百分比关系是在参考蛋白的序列和包含变体中取代的明确指定位置之外的变体蛋白的序列之间。因此，例如，如果参考序列和变体序列各自包含100个氨基酸，并且变体序列在25和81位具有突变，那么同源性百分比将是关于1-24、26-80和82-100位序列。
“多核苷酸”或“寡核苷酸”可互换使用，并且各自表示核苷酸单体或其类似物的线性聚合物。构成多核苷酸和寡核苷酸的单体能够通过单体-单体相互作用的规则模式(例如Watson-Crick型碱基配对，碱基堆叠，Hoogsteen或反向Hoogsteen型碱基配对等)与天然多核苷酸特异性结合。这样的单体和它们的核苷间键可以是天然存在的或可以是类似物，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包括PNA，硫代磷酸核苷间键，含有允许连接标记的连接基团的碱基，例如荧光团或半抗原等。当使用寡核苷酸或多核苷酸需要酶学加工(例如通过聚合酶延伸，通过连接酶连接等)时，本领域普通技术人员将理解，在那些情况下，寡核苷酸或多核苷酸不会在任何位置或某些位置含有核苷间键、糖部分或碱基的某些类似物。多核苷酸的大小通常在数个单体单位，例如5-40个(多核苷酸通常被称为“寡核苷酸”时)，至几千个单体单位的范围内。当多核苷酸或寡核苷酸由字母序列(大写或小写)表示时，例如“ATGCCTG”，应当理解，核苷酸从左至右为5'→3’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，“T”表示胸苷，“I”表示脱氧肌苷，“U”表示尿苷，除非另有说明或从上下文能清楚判断出另外的情形。除非另有说明，术语和原子编号惯例遵循Strachan and Read,Human Molecular Genetics 2(Wiley-Liss,New York,1999)中公开的那些。通常，多核苷酸包含通过磷酸二酯键连接的四种天然核苷(例如对于DNA的脱氧腺苷、脱氧胞苷、脱氧鸟苷、脱氧胸苷或对于RNA的它们的核糖对应物)，然而多核苷酸也可以包含非天然核苷酸类似物，例如包括修饰的碱基、糖或核苷间键。本领域技术人员清楚的是，当酶具有对活性的特定寡核苷酸或多核苷酸底物要求时，例如单链DNA、RNA/DNA双链体等，那么选择用于寡核苷酸或多核苷酸底物的合适的组分完全在普通技术人员的知识范围内，特别是在来自文献的指导下，例如Sambrook等，Molecular Cloning,SecondEdition(Cold Spring HarborLaboratory,，New York，1989)等参考文献。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的双链体及其相应的互补物)。对于本领域的普通技术人员来说，从术语使用的上下文中可以清楚意指哪种形式或者指这两种形式。
“引物”是指天然的或合成的寡核苷酸，其在与多核苷酸模板形成双链体时能够充当核酸合成的起始点，并从其3’末端沿着模板延伸，从而形成延伸的双链体。引物的延伸通常用核酸聚合酶进行，例如DNA或RNA聚合酶。在延伸过程中加入的核苷酸序列由模板多核苷酸的序列决定。通常引物由DNA聚合酶延伸。引物通常具有14-40个核苷酸的长度，或18-36个核苷酸的长度。引物用于多种核酸扩增反应，例如使用单一引物的线性扩增反应，或使用两种或多种引物的聚合酶链反应。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，如以下参考文献所证明的：编者Dieffenbach，PCR Primer:ALaboratory Manual，第2版(Cold Spring Harbor Press,New York,2003)。
“取代”是指氨基酸残基被另一个氨基酸残基替代。优选地，术语“取代”是指将一个氨基酸残基替换为另一个氨基酸残基，另一个氨基酸残基选自天然存在的标准20个氨基酸残基、稀有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)和非天然存在的氨基酸残基，非天然存在的氨基酸残基经常是合成制备(例如，环己基-丙氨酸)。优选地，术语“取代”是指将一个氨基酸残基替换为另一个氨基酸残基，另一个氨基酸残基选自天然存在的标准20个氨基酸残基。符号“+”表示取代的组合。
氨基酸在本文中由根据以下命名法的单字母或三字母代码表示：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：蛋氨酸(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)。
在本文中，使用以下术语表示取代：L238A表示母体序列的第238位的氨基酸残基(亮氨酸，L)被改变为丙氨酸(A)。A132V/I/M表示母体序列的第132位的氨基酸残基(丙氨酸，A)被下列氨基酸之一取代：缬氨酸(V)，异亮氨酸(I)或蛋氨酸(M)。取代可以是保守取代或非保守取代。保守取代的实例为以下各组内的取代：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(蛋氨酸，亮氨酸，异亮氨酸，半胱氨酸和缬氨酸)，芳族氨基酸(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)。
本申请不旨在局限于所阐述的特定形式的范围，而是旨在涵盖本文中描述的变体方案的替代、修改和等同方案。此外，本申请的范围完全涵盖鉴于本申请内容对于本领域技术人员而言可以清楚获得的其它变化。本发明的范围仅由所附权利要求限定。
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<110> DNA斯克瑞普特公司(DNA SCRIPT)
<120> 使用无歧化作用末端脱氧核苷酸转移酶变体的多核苷酸无模板酶学合成
<130> B3165PC00
<150> EP19216556
<151> 2019-12-16
<160> 49
<170> PatentIn version 3.5
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Arg Asp Leu Val Leu Phe Ile Leu Glu Lys Lys Met Gly Thr Thr Arg
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Arg Ala Phe Leu Met Glu Leu Ala Arg Arg Lys Gly Phe Arg Val Glu
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Asn Glu Leu Ser Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser
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Gly Ser Asp Val Leu Glu Trp Leu Gln Leu Gln Asn Ile Lys Ala Ser
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Ser Glu Leu Glu Leu Leu Asp Ile Ser Trp Leu Ile Glu Cys Met Gly
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Ala Gly Lys Pro Val Glu Met Met Gly Arg His Gln Leu Val Val Asn
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Arg Asn Ser Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Val Pro Ala
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Pro Ala Val Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr
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Glu Asn Asp Glu Leu Arg Glu Asn Glu Gly Ser Cys Leu Ala Phe Met
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Arg Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met
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Lys Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Ser Ile
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Ile Glu Gly Ile Ile Glu Asp Gly Glu Ser Ser Glu Ala Lys Ala Val
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Leu Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe
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Gly Val Gly Leu Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg
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Thr Leu Ser Lys Ile Gln Ser Asp Lys Ser Leu Arg Phe Thr Gln Met
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Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Asn
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Arg Pro Glu Ala Glu Ala Val Ser Met Leu Val Lys Glu Ala Val Val
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Thr Phe Leu Pro Asp Ala Leu Val Thr Met Thr Gly Gly Phe Arg Arg
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Gly Lys Met Thr Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu
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Ala Thr Glu Asp Glu Glu Gln Gln Leu Leu His Lys Val Thr Asp Phe
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Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys
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Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe Gly
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Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly
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Lys Lys Met Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser
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Gln Tyr Pro Thr Leu Lys Thr Pro Glu Ser Glu Val Ser Ser Phe Thr
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Ala Ser Val Leu Lys Ser Leu Pro Phe Pro Val Thr Arg Met Lys Asp
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Asp Glu Arg Tyr Lys Ser Phe Lys Glu Phe Thr Ser Val Phe Gly Val
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Gly Val Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Leu Arg Thr Val
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145 150 155 160
Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Ser Lys Ala
165 170 175
Glu Ala Asp Ala Val Ser Ser Ile Val Lys Asn Thr Val Cys Thr Phe
180 185 190
Leu Pro Asp Ala Leu Val Thr Ile Thr Gly Gly Phe Arg Arg Gly Lys
195 200 205
Lys Ile Gly His Asp Ile Asp Phe Leu Ile Thr Ser Pro Gly Gln Arg
210 215 220
Glu Asp Asp Glu Leu Leu His Lys Gly Leu Leu Leu Tyr Cys Asp Ile
225 230 235 240
Ile Glu Ser Thr Phe Val Lys Glu Gln Ile Pro Ser Arg His Val Asp
245 250 255
Ala Met Asp His Phe Gln Lys Cys Phe Ala Ile Leu Lys Leu Tyr Gln
260 265 270
Pro Arg Val Asp Asn Ser Ser Tyr Asn Met Ser Lys Lys Cys Asp Met
275 280 285
Ala Glu Val Lys Asp Trp Lys Ala Ile Arg Val Asp Leu Val Ile Thr
290 295 300
Pro Phe Glu Gln Tyr Ala Tyr Ala Leu Leu Gly Trp Thr Gly Ser Arg
305 310 315 320
Gln Phe Gly Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg Lys Met
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Met Leu Asp Asn His Ala Leu Tyr Asp Lys Arg Lys Arg Val Phe Leu
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Lys Ala Gly Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr
355 360 365
Val Glu Pro Trp Glu Arg Asn Ala
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<210> 6
<211> 387
<212> PRT
<213> 负鼠(截短型)
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Ser Ala Asn Pro Asp Pro Thr Ala Gly Thr Leu Asn Ile Leu Pro Pro
1 5 10 15
Thr Thr Lys Thr Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Ile
20 25 30
Asn Asn His Asn Gln Arg Phe Thr Asp Ala Phe Glu Ile Leu Ala Lys
35 40 45
Asn Tyr Glu Phe Lys Glu Asn Asp Asp Thr Cys Leu Thr Phe Met Arg
50 55 60
Ala Ile Ser Val Leu Lys Cys Leu Pro Phe Glu Val Val Ser Leu Lys
65 70 75 80
Asp Thr Glu Gly Leu Pro Trp Ile Gly Asp Glu Val Lys Gly Ile Met
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Glu Glu Ile Ile Glu Asp Gly Glu Ser Leu Glu Val Gln Ala Val Leu
100 105 110
Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly
115 120 125
Val Gly Leu Lys Thr Ala Asp Lys Trp Tyr Arg Met Gly Phe Arg Thr
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Leu Asn Lys Ile Arg Ser Asp Lys Thr Leu Lys Leu Thr Lys Met Gln
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Lys Ala Gly Leu Cys Tyr Tyr Glu Asp Leu Ile Asp Cys Val Ser Lys
165 170 175
Ala Glu Ala Asp Ala Val Ser Leu Leu Val Gln Asp Ala Val Trp Thr
180 185 190
Phe Leu Pro Asp Ala Leu Val Thr Ile Thr Gly Gly Phe Arg Arg Gly
195 200 205
Lys Glu Phe Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ala
210 215 220
Glu Lys Glu Gln Glu Asp Gln Leu Leu Gln Lys Val Thr Asn Leu Trp
225 230 235 240
Lys Lys Gln Gly Leu Leu Leu Tyr Cys Asp Leu Ile Glu Ser Thr Phe
245 250 255
Glu Asp Leu Lys Leu Pro Ser Arg Lys Ile Asp Ala Leu Asp His Phe
260 265 270
Gln Lys Cys Phe Leu Ile Leu Lys Leu Tyr His His Lys Glu Asp Lys
275 280 285
Arg Lys Trp Glu Met Pro Thr Gly Ser Asn Glu Ser Glu Ala Lys Ser
290 295 300
Trp Lys Ala Ile Arg Val Asp Leu Val Val Cys Pro Tyr Asp Arg Tyr
305 310 315 320
Ala Phe Ala Leu Leu Gly Trp Ser Gly Ser Arg Gln Phe Glu Arg Asp
325 330 335
Leu Arg Arg Tyr Ala Thr His Glu Lys Lys Met Met Leu Asp Asn His
340 345 350
Ala Leu Tyr Asp Lys Thr Lys Lys Ile Phe Leu Lys Ala Lys Ser Glu
355 360 365
Glu Glu Ile Phe Ala His Leu Gly Leu Glu Tyr Ile Gln Pro Ser Glu
370 375 380
Arg Asn Ala
385
<210> 7
<211> 381
<212> PRT
<213> 地鼠(截短型)
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Asp Cys Pro Ala Ser His Asp Ser Ser Pro Gln Lys Thr Glu Ser Ala
1 5 10 15
Ala Val Gln Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu
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Asn Asn His Asn His Ile Phe Thr Asp Ala Phe Glu Ile Leu Ala Glu
35 40 45
Asn Cys Glu Phe Arg Glu Asn Glu Gly Ser Tyr Val Thr Tyr Met Arg
50 55 60
Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Ser Ile Ile Ser Met Lys
65 70 75 80
Asp Thr Glu Gly Ile Pro Cys Leu Ala Asp Lys Val Lys Cys Val Ile
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Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu
100 105 110
Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe Gly
115 120 125
Val Gly Leu Lys Thr Ala Glu Lys Trp Phe Arg Leu Gly Phe Arg Thr
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Leu Ser Gly Ile Met Asn Asp Lys Thr Leu Lys Leu Thr His Met Gln
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Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg
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Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala
180 185 190
Phe Leu Pro Asp Ala Ile Val Thr Met Thr Gly Gly Phe Arg Arg Gly
195 200 205
Lys Lys Val Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu Ala
210 215 220
Thr Glu Glu Gln Glu Gln Gln Leu Leu His Lys Val Ile Thr Phe Trp
225 230 235 240
Glu Lys Glu Gly Leu Leu Leu Tyr Cys Asp Leu Tyr Glu Ser Thr Phe
245 250 255
Glu Lys Leu Lys Met Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe
260 265 270
Gln Lys Cys Phe Leu Ile Leu Lys Leu His Arg Glu Cys Val Asp Asp
275 280 285
Gly Thr Ser Ser Gln Leu Gln Gly Lys Thr Trp Lys Ala Ile Arg Val
290 295 300
Asp Leu Val Val Cys Pro Tyr Glu Cys Arg Ala Phe Ala Leu Leu Gly
305 310 315 320
Trp Thr Gly Ser Pro Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr
325 330 335
His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr
340 345 350
Lys Arg Lys Phe Leu Ser Ala Asp Ser Glu Glu Asp Ile Phe Ala His
355 360 365
Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375 380
<210> 8
<211> 381
<212> PRT
<213> 犬(截短型)
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Asp Tyr Thr Ala Ser Pro Asn Pro Glu Leu Gln Lys Thr Leu Pro Val
1 5 10 15
Ala Val Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu
20 25 30
Asn Asn Tyr Asn Asn Val Phe Thr Asp Ala Phe Glu Val Leu Ala Glu
35 40 45
Asn Tyr Glu Phe Arg Glu Asn Glu Val Phe Ser Leu Thr Phe Met Arg
50 55 60
Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys
65 70 75 80
Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Gln Val Lys Cys Ile Ile
85 90 95
Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu
100 105 110
Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly
115 120 125
Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Thr
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Leu Ser Lys Ile Lys Ser Asp Lys Ser Leu Lys Phe Thr Pro Met Gln
145 150 155 160
Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg
165 170 175
Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Gly Ala
180 185 190
Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly
195 200 205
Lys Lys Met Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser
210 215 220
Thr Asp Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp
225 230 235 240
Glu Arg Lys Gly Leu Leu Leu Tyr Cys Asp Leu Val Glu Ser Thr Phe
245 250 255
Glu Lys Leu Lys Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe
260 265 270
Gln Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Gly
275 280 285
Gly Lys Cys Ser Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val
290 295 300
Asp Leu Val Met Cys Pro Tyr Glu Arg Arg Ala Phe Ala Leu Leu Gly
305 310 315 320
Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Ser
325 330 335
His Glu Arg Lys Met Ile Leu Asp Asn His Ala Leu Tyr Asp Lys Thr
340 345 350
Lys Lys Ile Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His
355 360 365
Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375 380
<210> 9
<211> 382
<212> PRT
<213> 鼹鼠(截短型)
<400> 9
Gly Asp Cys Pro Ala Ser His Asp Ser Ser Pro Gln Lys Thr Glu Ser
1 5 10 15
Ala Ala Val Gln Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr
20 25 30
Leu Asn Asn His Asn His Ile Phe Thr Asp Ala Phe Glu Ile Leu Ala
35 40 45
Glu Asn Cys Glu Phe Arg Glu Asn Glu Gly Ser Tyr Val Thr Tyr Met
50 55 60
Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Ser Ile Ile Ser Met
65 70 75 80
Lys Asp Thr Glu Gly Ile Pro Cys Leu Ala Asp Lys Val Lys Cys Val
85 90 95
Ile Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val
100 105 110
Leu Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe
115 120 125
Gly Val Gly Leu Lys Thr Ala Glu Lys Trp Phe Arg Leu Gly Phe Arg
130 135 140
Thr Leu Ser Gly Ile Met Asn Asp Lys Thr Leu Lys Leu Thr His Met
145 150 155 160
Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr
165 170 175
Arg Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp
180 185 190
Ala Phe Leu Pro Asp Ala Ile Val Thr Met Thr Gly Gly Phe Arg Arg
195 200 205
Gly Lys Lys Val Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu
210 215 220
Ala Thr Glu Glu Gln Glu Gln Gln Leu Leu His Lys Val Ile Thr Phe
225 230 235 240
Trp Glu Lys Glu Gly Leu Leu Leu Tyr Cys Asp Leu Tyr Glu Ser Thr
245 250 255
Phe Glu Lys Leu Lys Met Pro Ser Arg Lys Val Asp Ala Leu Asp His
260 265 270
Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu His Arg Glu Cys Val Asp
275 280 285
Asp Gly Thr Ser Ser Gln Leu Gln Gly Lys Thr Trp Lys Ala Ile Arg
290 295 300
Val Asp Leu Val Val Cys Pro Tyr Glu Cys Arg Ala Phe Ala Leu Leu
305 310 315 320
Gly Trp Thr Gly Ser Pro Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala
325 330 335
Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys
340 345 350
Thr Lys Arg Lys Phe Leu Ser Ala Asp Ser Glu Glu Asp Ile Phe Ala
355 360 365
His Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375 380
<210> 10
<211> 379
<212> PRT
<213> 鼠兔(截短型)
<400> 10
Glu Tyr Ser Ala Asn Pro Ser Pro Gly Pro Gln Ala Thr Pro Ala Val
1 5 10 15
Tyr Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn
20 25 30
His Asn His Ile Phe Thr Asp Ala Phe Glu Ile Leu Ala Glu Asn Tyr
35 40 45
Glu Phe Lys Glu Asn Glu Gly Cys Tyr Val Thr Tyr Met Arg Ala Ala
50 55 60
Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Val Ser Met Lys Asp Thr
65 70 75 80
Glu Gly Ile Pro Cys Leu Glu Asp Lys Val Lys Ser Ile Met Glu Glu
85 90 95
Ile Ile Glu Glu Gly Glu Ser Ser Glu Val Lys Ala Val Leu Ser Asp
100 105 110
Glu Arg Tyr Gln Cys Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Ser Leu Ser
130 135 140
Asn Ile Arg Leu Asp Lys Ser Leu Lys Phe Thr Gln Met Gln Lys Ala
145 150 155 160
Gly Phe Arg Tyr Tyr Glu Asp Ile Val Ser Cys Val Thr Arg Ala Glu
165 170 175
Ala Glu Ala Val Asp Val Leu Val Asn Glu Ala Val Arg Ala Phe Leu
180 185 190
Pro Asp Ala Phe Ile Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys
195 200 205
Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu Leu Thr Glu
210 215 220
Glu Asp Glu Gln Gln Leu Leu His Lys Val Met Asn Leu Trp Glu Lys
225 230 235 240
Lys Gly Leu Leu Leu Tyr His Asp Leu Val Glu Ser Thr Phe Glu Lys
245 250 255
Leu Lys Gln Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe Gln Lys
260 265 270
Cys Phe Leu Ile Phe Lys Leu Tyr His Glu Arg Val Gly Gly Asp Arg
275 280 285
Cys Arg Gln Pro Glu Gly Lys Asp Trp Lys Ala Ile Arg Val Asp Leu
290 295 300
Val Met Cys Pro Tyr Glu Cys His Ala Phe Ala Leu Leu Gly Trp Thr
305 310 315 320
Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Ser His Glu
325 330 335
Arg Lys Met Ile Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg
340 345 350
Val Phe Leu Gln Ala Glu Asn Glu Glu Glu Ile Phe Ala His Leu Gly
355 360 365
Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375
<210> 11
<211> 384
<212> PRT
<213> 刺猬 (截短型)
<400> 11
Asp Ala Ser Phe Gly Ser Asn Pro Gly Ser Gln Asn Thr Pro Pro Leu
1 5 10 15
Ala Ile Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Ser Leu
20 25 30
Asn Asn Cys Asn His Ile Phe Thr Asp Ala Leu Asp Ile Leu Ala Glu
35 40 45
Asn His Glu Phe Arg Glu Asn Glu Val Ser Cys Val Ala Phe Met Arg
50 55 60
Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys
65 70 75 80
Asp Thr Lys Gly Ile Pro Cys Leu Gly Asp Lys Ala Lys Cys Val Ile
85 90 95
Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Ile Leu
100 105 110
Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly
115 120 125
Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Thr
130 135 140
Leu Asn Lys Ile Met Ser Asp Lys Thr Leu Lys Leu Thr Arg Met Gln
145 150 155 160
Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Ala Lys
165 170 175
Ala Glu Ala Asp Ala Val Ser Val Leu Val Gln Glu Ala Val Trp Ala
180 185 190
Phe Leu Pro Asp Ala Met Val Thr Met Thr Gly Gly Phe Arg Arg Gly
195 200 205
Lys Lys Leu Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ala
210 215 220
Thr Glu Glu Glu Glu Gln Gln Leu Leu Pro Lys Val Ile Asn Phe Trp
225 230 235 240
Glu Arg Lys Gly Leu Leu Leu Tyr His Asp Leu Val Glu Ser Thr Phe
245 250 255
Glu Lys Leu Lys Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe
260 265 270
Gln Lys Cys Phe Leu Ile Leu Lys Leu His Leu Gln His Val Asn Gly
275 280 285
Val Gly Asn Ser Lys Thr Gly Gln Gln Glu Gly Lys Asn Trp Lys Ala
290 295 300
Ile Arg Val Asp Leu Val Met Cys Pro Tyr Glu Arg Arg Ala Phe Ala
305 310 315 320
Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg
325 330 335
Phe Ala Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr
340 345 350
Asp Lys Thr Lys Arg Ile Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile
355 360 365
Phe Ala His Leu Gly Leu Asp Tyr Ile Asp Pro Trp Glu Arg Asn Ala
370 375 380
<210> 12
<211> 381
<212> PRT
<213> 树鼩(截短型)
<400> 12
Asp His Ser Thr Ser Pro Ser Pro Gly Pro Gln Lys Thr Pro Ala Leu
1 5 10 15
Ala Val Gln Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu
20 25 30
Asn Asn Cys Asn Arg Val Phe Thr Asp Ala Phe Glu Thr Leu Ala Glu
35 40 45
Asn Tyr Glu Phe Arg Glu Asn Glu Asp Ser Ser Val Ile Phe Leu Arg
50 55 60
Ala Ala Ser Val Leu Arg Ser Leu Pro Phe Thr Ile Thr Ser Met Arg
65 70 75 80
Asp Thr Glu Gly Leu Pro Cys Leu Gly Asp Lys Val Lys Cys Val Ile
85 90 95
Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Asn Ala Val Leu
100 105 110
Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe Gly
115 120 125
Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Thr
130 135 140
Leu Ser Arg Val Arg Ser Asp Lys Ser Leu His Leu Thr Arg Met Gln
145 150 155 160
Gln Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Ala Ser Cys Val Thr Arg
165 170 175
Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Gly Ala
180 185 190
Phe Leu Pro Asp Ala Leu Val Thr Ile Thr Gly Gly Phe Arg Arg Gly
195 200 205
Lys Lys Thr Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser
210 215 220
Thr Glu Glu Lys Glu Glu Glu Leu Leu Gln Lys Val Leu Asn Leu Trp
225 230 235 240
Glu Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe
245 250 255
Glu Lys Leu Lys Thr Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe
260 265 270
Pro Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Gly
275 280 285
Asp Lys Pro Ser Gln Gln Glu Gly Lys Ser Trp Lys Ala Ile Arg Val
290 295 300
Asp Leu Val Met Cys Pro Tyr Glu Arg His Ala Phe Ala Leu Leu Gly
305 310 315 320
Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr
325 330 335
His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr
340 345 350
Lys Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Asp Ile Phe Ala His
355 360 365
Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375 380
<210> 13
<211> 394
<212> PRT
<213> 鸭嘴兽(截短型)
<400> 13
Leu Thr Asn Ser Ala Pro Ile Asn Cys Met Thr Glu Thr Pro Ser Leu
1 5 10 15
Ala Thr Lys Gln Val Ser Gln Tyr Ala Cys Glu Arg Arg Thr Thr Leu
20 25 30
Asn Asn Cys Asn Gln Lys Phe Thr Asp Ala Phe Glu Ile Leu Ala Lys
35 40 45
Asp Phe Glu Phe Arg Glu Asn Glu Gly Ile Cys Leu Ala Phe Met Arg
50 55 60
Ala Ile Ser Val Leu Lys Cys Leu Pro Phe Thr Ile Val Arg Met Lys
65 70 75 80
Asp Ile Glu Gly Val Pro Trp Leu Gly Asp Gln Val Lys Ser Ile Ile
85 90 95
Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Ser Val Lys Ala Val Leu
100 105 110
Asn Asp Glu Arg Tyr Arg Ser Phe Gln Leu Phe Asn Ser Val Phe Glu
115 120 125
Val Gly Leu Thr Asp Asn Gly Glu Asn Gly Ile Ala Arg Gly Phe Gln
130 135 140
Thr Leu Asn Glu Val Ile Thr Asp Glu Asn Ile Ser Leu Thr Lys Thr
145 150 155 160
Thr Leu Ser Thr Ser Leu Trp Asn Tyr Leu Pro Gly Phe Leu Tyr Tyr
165 170 175
Glu Asp Leu Val Ser Cys Val Ala Lys Glu Glu Ala Asp Ala Val Tyr
180 185 190
Leu Ile Val Lys Glu Ala Val Arg Ala Phe Leu Pro Glu Ala Leu Val
195 200 205
Thr Leu Thr Gly Gly Phe Arg Arg Gly Lys Lys Ile Gly His Asp Val
210 215 220
Asp Phe Leu Ile Ser Asp Pro Glu Ser Gly Gln Asp Glu Gln Leu Leu
225 230 235 240
Pro Asn Ile Ile Lys Leu Trp Glu Lys Gln Glu Leu Leu Leu Tyr Tyr
245 250 255
Asp Leu Val Glu Ser Thr Phe Glu Lys Thr Lys Ile Pro Ser Arg Lys
260 265 270
Val Asp Ala Met Asp His Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu
275 280 285
His His Gln Lys Val Asp Ser Gly Arg Tyr Lys Pro Pro Pro Glu Ser
290 295 300
Lys Asn His Glu Ala Lys Asn Trp Lys Ala Ile Arg Val Asp Leu Val
305 310 315 320
Met Cys Pro Phe Glu Gln Tyr Ala Tyr Ala Leu Leu Gly Trp Thr Gly
325 330 335
Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Lys
340 345 350
Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Lys Ile
355 360 365
Phe Leu Lys Ala Glu Ser Glu Glu Asp Ile Phe Thr His Leu Gly Leu
370 375 380
Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
385 390
<210> 14
<211> 384
<212> PRT
<213> 跳鼠 (截短型)
<400> 14
Ser Ser Glu Leu Glu Leu Leu Asp Val Ser Trp Leu Ile Glu Cys Met
1 5 10 15
Gly Ala Gly Lys Pro Val Glu Met Thr Gly Arg His Gln Leu Val Lys
20 25 30
Gln Thr Phe Cys Leu Pro Gly Phe Ile Leu Gln Asp Ala Phe Asp Ile
35 40 45
Leu Ala Glu Asn Cys Glu Phe Arg Glu Asn Glu Ala Ser Cys Val Glu
50 55 60
Phe Met Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Ile
65 70 75 80
Ser Val Lys Asp Thr Glu Gly Ile Pro Trp Leu Gly Gly Lys Val Lys
85 90 95
Cys Val Ile Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys
100 105 110
Ala Leu Leu Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser
115 120 125
Val Phe Gly Val Gly Leu Lys Thr Ala Glu Arg Trp Phe Arg Met Gly
130 135 140
Phe Arg Thr Leu Ser Thr Val Lys Leu Asp Lys Ser Leu Thr Phe Thr
145 150 155 160
Arg Met Gln Lys Ala Gly Phe Leu His Tyr Glu Asp Leu Val Ser Cys
165 170 175
Val Thr Arg Ala Glu Ala Glu Ala Val Ser Val Leu Val Gln Gln Ala
180 185 190
Val Val Ala Phe Leu Pro Asp Ala Leu Val Ser Met Thr Gly Gly Phe
195 200 205
Arg Arg Gly Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser
210 215 220
Pro Glu Ala Thr Glu Glu Glu Glu Gln Gln Leu Leu His Lys Val Thr
225 230 235 240
Asn Phe Trp Glu Gln Lys Gly Leu Leu Leu Tyr Cys Asp His Val Glu
245 250 255
Ser Thr Phe Glu Lys Cys Lys Leu Pro Ser Arg Lys Val Asp Ala Leu
260 265 270
Asp His Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu Tyr Arg Glu Arg
275 280 285
Val Asp Ser Val Lys Ser Ser Gln Gln Glu Gly Lys Gly Trp Lys Ala
290 295 300
Ile Arg Val Asp Leu Val Met Cys Pro Tyr Glu Cys Arg Ala Phe Ala
305 310 315 320
Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg
325 330 335
Tyr Ala Thr His Glu Arg Lys Met Arg Leu Asp Asn His Ala Leu Tyr
340 345 350
Asp Lys Thr Lys Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile
355 360 365
Phe Ala His Leu Gly Leu Glu Tyr Ile Glu Pro Leu Glu Arg Asn Ala
370 375 380
<210> 15
<211> 364
<212> PRT
<213> 野生型美洲狮
<400> 15
Val Val Lys Lys Ile Pro Leu Tyr Ala Cys Gln Arg Arg Thr Thr Leu
1 5 10 15
Asn Asn Phe Asn His Ile Phe Thr Asp Ala Phe Glu Val Leu Ala Glu
20 25 30
Asn Tyr Glu Phe Lys Glu Asn Glu Ile Ser Ser Ala Thr Phe Met Arg
35 40 45
Ala Ala Ser Val Leu Lys Leu Pro Phe Thr Ile Ile Ser Met Lys Asp
50 55 60
Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Cys Val Ile Glu
65 70 75 80
Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn
85 90 95
Asp Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly Val
100 105 110
Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu
115 120 125
Ser Lys Ile Lys Ser Asp Lys Thr Leu Lys Phe Thr Gln Met Gln Lys
130 135 140
Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala
145 150 155 160
Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe
165 170 175
Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys
180 185 190
Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ile Pro Gly Ser Thr
195 200 205
Asp Glu Glu Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Gln
210 215 220
Arg Lys Glu Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu
225 230 235 240
Lys Leu Lys Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe Gln
245 250 255
Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Gly
260 265 270
Lys Cys Ser Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp
275 280 285
Leu Val Met Cys Pro Tyr Glu Arg Arg Ala Phe Ala Leu Leu Gly Trp
290 295 300
Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr His
305 310 315 320
Glu Arg Lys Met Ile Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys
325 330 335
Lys Ile Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu
340 345 350
Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
355 360
<210> 16
<211> 12
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 测试多核苷酸p875
<400> 16
cagttaaaaa ct 12
<210> 17
<211> 11
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 测试多核苷酸p876
<400> 17
gagttaaaac t 11
<210> 18
<211> 10
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 测试多核苷酸p877
<400> 18
cagcaaggct 10
<210> 19
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 示例性His标签
<400> 19
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> 20
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 引物序列
<400> 20
aaaaaaaaaa aaaagggg 18
<210> 21
<211> 381
<212> PRT
<213> 小鼠(截短型TdT)
<400> 21
Asn Ser Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Ala Pro Ala Pro
1 5 10 15
Val Val Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu
20 25 30
Asn Asn Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu
35 40 45
Asn Asp Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg
50 55 60
Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys
65 70 75 80
Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile
85 90 95
Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu
100 105 110
Asn Asp Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly
115 120 125
Val Gly Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr
130 135 140
Leu Glu Lys Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln
145 150 155 160
Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Ala Glu Arg Val Asn Arg
165 170 175
Pro Glu Ala Glu Ala Ile Arg Met Leu Val Lys Glu Ala Val Val Thr
180 185 190
Phe Leu Pro Asp Ala Leu Val Thr Met Thr Gly Gly Phe Arg Leu Gly
195 200 205
Lys Met Thr Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu Ala
210 215 220
Thr Glu Asp Glu Glu Gln Gln Leu Leu His Lys Val Thr Asp Phe Trp
225 230 235 240
Lys Gln Gln Gly Leu Leu Leu Tyr Cys Asp Ile Leu Glu Ser Thr Phe
245 250 255
Glu Lys Phe Lys Gln Pro Ser Arg Thr Val Asp Ala Leu Asp His Phe
260 265 270
Gln Lys Cys Phe Leu Ile Leu Lys Leu Asp His Pro Arg Val His Ser
275 280 285
Val Lys Ser Gly Gln Gln Glu Gly Lys Gly Trp Lys Ala Ile Arg Val
290 295 300
Asp Leu Val Met Cys Pro Tyr Asp Arg Arg Ala Phe Ala Leu Leu Gly
305 310 315 320
Trp Thr Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr
325 330 335
His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr
340 345 350
Lys Arg Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His
355 360 365
Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375 380
<210> 22
<211> 369
<212> PRT
<213> 牛
<400> 22
Lys Thr Pro Pro Leu Val Val Lys Lys Ile Ser Gln Tyr Ala Cys Gln
1 5 10 15
Arg Lys Thr Thr Leu Asn Asn Tyr Asn Glu Ile Phe Thr Arg Ala Phe
20 25 30
Glu Ile Leu Ala Glu Asn Ser Glu Phe Lys Glu Asn Glu Glu Ser Tyr
35 40 45
Val Thr Phe Arg Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr
50 55 60
Ile Ile Ser Met Lys Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys
65 70 75 80
Val Lys Arg Ile Ile Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu
85 90 95
Val Lys Ala Val Leu Asn Asp Glu Arg Tyr Gln Ala Phe Lys Leu Phe
100 105 110
Thr Ser Val Phe Gly Val Gly Arg Lys Thr Ser Glu Lys Trp Phe Arg
115 120 125
Met Gly Phe Arg Ser Leu Glu Lys Ile Arg Ser Asp Lys Thr Leu Lys
130 135 140
Phe Thr Lys Met Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val
145 150 155 160
Ser Gly Val Thr Arg Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys
165 170 175
Glu Ala Val Trp Ala Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly
180 185 190
Gly Phe Arg Leu Gly Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile
195 200 205
Thr Ser Pro Gly Ser Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val
210 215 220
Ile Asn Leu Trp Glu Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val
225 230 235 240
Glu Ser Thr Phe Glu Lys Phe Lys Leu Pro Ser Arg Gln Val Asp Thr
245 250 255
Leu Asp His Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu His His Gln
260 265 270
Arg Val Asp Ser Ser Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys
275 280 285
Ala Ile Arg Val Asp Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe
290 295 300
Ala Leu Leu Gly Trp Thr Gly Ser Ala Phe Phe Asn Arg Asp Ile Arg
305 310 315 320
Arg Tyr Ala Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu
325 330 335
Tyr Asp Lys Thr Lys Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Glu
340 345 350
Ile Phe Ala His Leu Gly Leu Asp Tyr Ile Glu Pro Arg Glu Arg Asn
355 360 365
Ala
<210> 23
<211> 378
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 没有His标签和接头的M57
<400> 23
Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Ala Pro Ala Pro Val Val
1 5 10 15
Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn
20 25 30
Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn Asp
35 40 45
Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala Ser
50 55 60
Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp Thr
65 70 75 80
Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu Glu
85 90 95
Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp
100 105 110
Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Glu
130 135 140
Lys Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys Ala
145 150 155 160
Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg Ala Glu
165 170 175
Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu
180 185 190
Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys Lys
195 200 205
Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu
210 215 220
Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys Lys
225 230 235 240
Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe
245 250 255
Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys
260 265 270
Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser
275 280 285
Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val
290 295 300
Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly
305 310 315 320
Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg
325 330 335
Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val
340 345 350
Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu
355 360 365
Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375
<210> 24
<211> 378
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 没有His标签和接头的M59
<400> 24
Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Ala Pro Ala Pro Val Val
1 5 10 15
Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn
20 25 30
Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn Asp
35 40 45
Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala Ser
50 55 60
Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp Thr
65 70 75 80
Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu Glu
85 90 95
Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp
100 105 110
Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Glu
130 135 140
Lys Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys Ala
145 150 155 160
Gly Phe Leu Tyr Tyr Glu Asp Leu Ala Glu Arg Val Asn Arg Pro Glu
165 170 175
Ala Glu Ala Ile Arg Met Leu Val Lys Glu Ala Val Val Thr Phe Leu
180 185 190
Pro Asp Ala Leu Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys Met
195 200 205
Thr Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu
210 215 220
Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys Lys
225 230 235 240
Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe
245 250 255
Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys
260 265 270
Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser
275 280 285
Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val
290 295 300
Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly
305 310 315 320
Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg
325 330 335
Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val
340 345 350
Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu
355 360 365
Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375
<210> 25
<211> 381
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> M27
<400> 25
Asn Ser Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Val Pro Ala Pro
1 5 10 15
Val Val Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu
20 25 30
Asn Asn Tyr Asn Gln Leu Phe Thr Asp Ala Leu Asp Ile Leu Ala Glu
35 40 45
Asn Asp Glu Phe Arg Glu Asn Glu Glu Ser Cys Leu Ala Phe Arg Arg
50 55 60
Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys
65 70 75 80
Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Ser Ile Ile
85 90 95
Glu Gly Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu
100 105 110
Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe Gly
115 120 125
Val Gly Leu Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr
130 135 140
Leu Ser Lys Ile Gln Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln
145 150 155 160
Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Asn Arg
165 170 175
Pro Glu Ala Glu Ala Val Ser Met Leu Val Lys Glu Ala Val Val Thr
180 185 190
Phe Leu Pro Asp Ala Leu Val Thr Met Thr Gly Gly Phe Arg Leu Gly
195 200 205
Lys Met Thr Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu Ala
210 215 220
Thr Glu Asp Glu Glu Gln Gln Leu Leu His Lys Val Thr Asp Phe Trp
225 230 235 240
Lys Gln Gln Gly Leu Leu Leu Tyr Cys Asp Ile Leu Glu Ser Thr Phe
245 250 255
Glu Lys Phe Lys Gln Pro Ser Arg Thr Val Asp Ala Leu Asp His Phe
260 265 270
Gln Lys Cys Phe Leu Ile Leu Lys Leu Asp His Pro Arg Val His Ser
275 280 285
Val Lys Ser Gly Gln Gln Glu Gly Lys Gly Trp Lys Ala Ile Arg Val
290 295 300
Asp Leu Val Met Cys Pro Tyr Asp Arg Arg Ala Phe Ala Leu Leu Gly
305 310 315 320
Trp Thr Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr
325 330 335
His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr
340 345 350
Lys Arg Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His
355 360 365
Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375 380
<210> 26
<211> 382
<212> PRT
<213> 金丝雀(截短型)
<400> 26
Ser Pro Pro Leu Asn Thr Pro Glu Leu Glu Met Pro Ser Phe Ile Ala
1 5 10 15
Thr Lys Val Ser Gln Tyr Ser Cys Gln Arg Lys Thr Thr Leu Asn Asn
20 25 30
Tyr Asn Lys Lys Phe Thr Asp Ala Phe Glu Val Met Ala Glu Asn Tyr
35 40 45
Glu Phe Lys Glu Asn Glu Ile Phe Cys Leu Glu Phe Leu Arg Ala Ala
50 55 60
Ser Leu Leu Lys Ser Leu Pro Phe Ser Val Thr Arg Met Lys Asp Ile
65 70 75 80
Gln Gly Leu Pro Cys Met Gly Asp Gln Val Arg Asp Val Ile Glu Ile
85 90 95
Ile Glu Glu Gly Glu Ser Ser Arg Val Lys Glu Val Leu Asn Asp Glu
100 105 110
Arg Tyr Lys Ala Phe Lys Gln Phe Thr Ser Val Phe Gly Val Gly Val
115 120 125
Lys Thr Ser Glu Lys Trp Tyr Arg Met Gly Leu Arg Thr Val Gly Glu
130 135 140
Val Lys Ala Asp Lys Thr Leu Lys Leu Ser Lys Met Gln Lys Ala Gly
145 150 155 160
Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Ser Lys Ala Glu Ala
165 170 175
Asp Ala Val Ser Leu Ile Val Lys Asn Thr Val Cys Thr Phe Leu Pro
180 185 190
Asp Ala Leu Val Thr Ile Thr Gly Gly Phe Arg Arg Gly Lys Asn Ile
195 200 205
Gly His Asp Ile Asp Phe Leu Ile Thr Asn Pro Gly Pro Arg Glu Asp
210 215 220
Asp Glu Leu Leu His Lys Val Ile Asp Leu Trp Lys Lys Gln Gly Leu
225 230 235 240
Leu Leu Tyr Cys Asp Ile Ile Glu Ser Thr Phe Val Lys Glu Gln Leu
245 250 255
Pro Ser Arg Lys Ile Asp Ala Met Asp His Phe Gln Lys Cys Phe Ala
260 265 270
Ile Leu Lys Leu Tyr Gln Pro Arg Val Asp Asn Ser Thr Cys Asn Thr
275 280 285
Ser Lys Lys Leu Glu Met Ala Glu Val Lys Asp Trp Lys Ala Ile Arg
290 295 300
Val Asp Leu Val Ile Thr Pro Phe Glu Gln Tyr Ser Tyr Ala Leu Leu
305 310 315 320
Gly Trp Thr Gly Ser Arg Gln Phe Gly Arg Asp Leu Arg Arg Tyr Ala
325 330 335
Ala His Glu Arg Arg Met Ile Leu Asp Asn His Gly Leu Tyr Asp Arg
340 345 350
Thr Lys Arg Ile Phe Leu Lys Ala Gly Ser Glu Glu Glu Ile Phe Ala
355 360 365
His Leu Gly Leu Asp Tyr Val Glu Pro Trp Glu Arg Asn Ala
370 375 380
<210> 27
<211> 370
<212> PRT
<213> 黄冠霸娇鹟(截短型)
<400> 27
Phe Ile Ala Arg Lys Val Ser Gln Tyr Ser Cys Gln Arg Lys Thr Thr
1 5 10 15
Leu Asn Asn Tyr Asn Lys Lys Phe Thr Asp Ala Phe Glu Ile Met Ala
20 25 30
Glu Asn Tyr Glu Phe Lys Glu Asn Glu Ile Phe Cys Leu Glu Phe Leu
35 40 45
Arg Ala Ala Ser Leu Leu Lys Tyr Leu Pro Phe Pro Val Thr Arg Met
50 55 60
Lys Asp Ile Gln Gly Leu Pro Cys Ile Gly Asp Gln Val Arg Asp Val
65 70 75 80
Ile Glu Gly Ile Ile Glu Glu Gly Glu Ser Ser Arg Val Lys Glu Val
85 90 95
Leu Asn Asp Glu Arg Tyr Lys Ala Phe Lys Gln Phe Thr Ser Val Phe
100 105 110
Gly Val Gly Val Lys Thr Ser Glu Lys Trp Tyr Arg Met Gly Leu Arg
115 120 125
Thr Val Glu Glu Leu Lys Ala Asp Lys Thr Leu Lys Leu Ser Lys Met
130 135 140
Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Ser
145 150 155 160
Lys Ala Glu Ala Asp Ala Val Thr Leu Ile Val Lys Asn Thr Val Ser
165 170 175
Thr Phe Leu Pro Asp Ala Leu Val Thr Ile Thr Gly Gly Phe Arg Arg
180 185 190
Gly Lys Lys Met Gly His Asp Ile Asp Phe Leu Ile Thr Asn Pro Gly
195 200 205
Pro Arg Glu Asp Asp Glu Leu Leu His Lys Val Val Asp Leu Trp Lys
210 215 220
Lys Gln Gly Leu Leu Leu Tyr Cys Asp Ile Ile Glu Ser Thr Phe Val
225 230 235 240
Glu Glu Gln Leu Pro Ser Arg Lys Val Asp Ala Met Asp Asn Phe Gln
245 250 255
Lys Cys Phe Thr Ile Leu Lys Leu Tyr Gln Pro Gly Val Asp Asn Ser
260 265 270
Ser Tyr Asn Met Ser Lys Lys Ser Asp Met Ala Glu Val Lys Asp Trp
275 280 285
Lys Ala Ile Arg Val Asp Leu Val Ile Thr Pro Phe Glu Gln Tyr Ala
290 295 300
Tyr Ala Leu Leu Gly Trp Thr Gly Ser Arg Glu Phe Gly Arg Asp Leu
305 310 315 320
Arg Arg Tyr Ala Ser His Glu Arg Lys Met Ile Leu Asp Asn His Gly
325 330 335
Leu Tyr Asp Arg Arg Lys Arg Ile Phe Leu Lys Ala Gly Ser Glu Glu
340 345 350
Glu Ile Phe Ala His Leu Gly Leu Asp Tyr Val Glu Pro Trp Glu Arg
355 360 365
Asn Ala
370
<210> 28
<211> 383
<212> PRT
<213> 扬子鳄(截短型)
<400> 28
Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Val Pro Ala Pro Ser Val
1 5 10 15
Asp Lys Val Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn
20 25 30
Tyr Asn Lys Lys Phe Thr Asp Ala Phe Glu Ile Leu Ala Glu Asn Cys
35 40 45
Glu Phe Arg Glu Asn Arg Leu Gly Cys Leu Glu Phe Leu Arg Ala Ala
50 55 60
Ser Val Leu Lys Phe Leu Pro Phe Pro Ile Val Lys Met Lys Asn Ile
65 70 75 80
Glu Gly Leu Pro Cys Met Gly Asp Lys Val Lys Cys Val Ile Glu Glu
85 90 95
Ile Leu Glu Glu Gly Glu Ser Cys Gln Ala Lys Glu Ile Leu Asn Asp
100 105 110
Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Leu Lys Thr Thr Glu Lys Trp Tyr Arg Met Gly Phe Arg Thr Leu Glu
130 135 140
Glu Val Lys Ala Glu Lys Thr Leu Lys Leu Ser Arg Met Gln Ile Ala
145 150 155 160
Gly Phe Leu His Tyr Glu Asp Ile Ile Ser Tyr Val Ser Lys Ala Glu
165 170 175
Ala Asp Ala Val Ser Leu Leu Ile Lys Asp Thr Val Cys Met Phe Leu
180 185 190
Pro Asp Ala Leu Val Thr Ile Thr Gly Gly Phe Arg Arg Gly Lys Lys
195 200 205
Thr Gly His Asp Val Asp Phe Leu Ile Thr Asn Pro Gly Pro Glu Glu
210 215 220
Glu Lys Glu Leu Leu His Lys Val Val Asp Leu Trp Glu Lys Gln Gly
225 230 235 240
Leu Leu Leu Tyr Tyr Asp Val Ile Glu Ser Thr Phe Glu Lys Glu Lys
245 250 255
Arg Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe Gln Lys Cys Phe
260 265 270
Ala Ile Leu Lys Leu His Gln Gln Arg Arg Gly Asn Ser Asn Ser Asn
275 280 285
Ile Ser Lys Glu Ser Asp Lys Ala Glu Val Lys Asp Trp Lys Ala Ile
290 295 300
Arg Val Asp Leu Val Ile Ser Pro Phe Glu Gln Tyr Ala Tyr Ala Leu
305 310 315 320
Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr
325 330 335
Ala Ser Arg Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp
340 345 350
Lys Thr Lys Arg Thr Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe
355 360 365
Ala His Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375 380
<210> 29
<211> 380
<212> PRT
<213> 爪蟾 (截短型)
<400> 29
Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Val Pro Ala Pro Ser Glu
1 5 10 15
Val Lys Val Ser Gln Tyr Ala Cys Gln Arg Cys Thr Thr Leu Gln Asp
20 25 30
Thr Asn Arg Ile Phe Thr Asp Ala Phe Asp Ile Leu Ala Glu His Phe
35 40 45
Glu Phe Cys Glu Asn Lys Gly Arg Thr Val Ala Phe Leu Arg Ala Ser
50 55 60
Ser Leu Ile Lys Ser Leu Pro Phe Pro Ile Thr Ala Met Lys Glu Leu
65 70 75 80
Glu Gly Leu Pro Trp Leu Gly Asp Gln Met Lys Gly Ile Ile Glu Glu
85 90 95
Ile Leu Glu Glu Gly Lys Ser Tyr Lys Val Leu Glu Val Met Asn Glu
100 105 110
Glu Arg Tyr Lys Ser Phe Lys Gln Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Leu Lys Thr Ser Asp Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Glu
130 135 140
Glu Ile Lys Asn Glu Lys Glu Leu Lys Leu Thr Lys Met Gln Lys Cys
145 150 155 160
Gly Leu Leu Tyr Tyr Glu Asp Ile Thr Ser Tyr Val Ser Arg Ala Glu
165 170 175
Ala Glu Thr Thr Glu Gln Leu Ile Lys Ser Ile Val Trp Lys Phe Val
180 185 190
Pro Asp Ala Ile Val Thr Leu Thr Gly Gly Phe Arg Arg Gly Lys Lys
195 200 205
Lys Gly His Asp Val Asp Ile Leu Ile Thr Cys Ala Arg Lys Gly Lys
210 215 220
Glu Lys Asn Ile Leu His Asn Thr Met Ser Val Leu Lys Asn Arg Gly
225 230 235 240
Leu Leu Leu Phe Tyr Asn Ile Ile Glu Ser Thr Phe Asp Glu Thr Lys
245 250 255
Leu Pro Ser Arg His Val Asp Ala Leu Asp His Phe Gln Lys Cys Phe
260 265 270
Thr Ile Leu Lys Leu Pro Lys Arg Gln Met Asp Ile Gly Asn Ile Ile
275 280 285
Asp Pro His Glu Cys Glu Arg Lys Asn Trp Lys Ala Val Arg Leu Asp
290 295 300
Leu Val Ile Thr Pro Tyr Glu Gln Tyr Pro Tyr Ala Leu Leu Gly Trp
305 310 315 320
Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr His
325 330 335
Glu Lys Arg Met Met Leu Asp Asn His Gly Leu Tyr Asp Lys Thr Lys
340 345 350
Asn Asn Phe Leu Lys Ala Asn Asn Glu Glu Asp Ile Phe Lys Gln Leu
355 360 365
Gly Leu Asp Tyr Leu Glu Pro Trp Glu Arg Asn Ala
370 375 380
<210> 30
<211> 383
<212> PRT
<213> 虎蛇 (截短型)
<400> 30
Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Val Pro Ala Pro Ser Val
1 5 10 15
Asp Ser Ile Ser Pro Tyr Ala Cys Gln Arg Arg Thr Thr Leu Lys Asn
20 25 30
Tyr Asn Gln Lys Phe Thr Asp Ala Phe Glu Ile Leu Val Glu Asn Tyr
35 40 45
Glu Phe Asn Glu Asn Lys Glu Phe Cys Leu Ala Phe Gly Arg Ala Ala
50 55 60
Ser Leu Leu Lys Cys Leu Pro Phe Thr Val Val Arg Val Asn Asp Ile
65 70 75 80
Glu Gly Leu Pro Trp Met Gly Lys Gln Val Arg Glu Ile Ile Glu Asp
85 90 95
Ile Leu Glu Glu Gly Glu Ser Ser Lys Val Lys Ala Val Leu Asn Asp
100 105 110
Glu Asn Tyr Arg Ser Ile Lys Leu Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Leu Arg Thr Ser Glu Lys Trp Tyr Arg Met Gly Leu Arg Thr Leu Glu
130 135 140
Glu Val Lys Cys Asn Lys Asn Leu Thr Leu Thr Arg Met Gln Lys Ala
145 150 155 160
Gly Phe Phe Tyr Tyr Asp Asp Leu Ile Ser Ser Val Ser Lys Ala Glu
165 170 175
Ala Asp Ala Ala Thr Gln Ile Val Gln Asp Thr Val Trp Lys Ile Leu
180 185 190
Pro Asn Ala Val Val Thr Leu Thr Gly Gly Phe Arg Arg Gly Lys Gln
195 200 205
Thr Gly His Asp Val Asp Phe Leu Ile Thr Val Pro Gly Ser Arg Gln
210 215 220
Glu Glu Glu Leu Leu His Pro Val Ile Asp Ile Trp Lys Lys Gln Glu
225 230 235 240
Leu Leu Leu Tyr Tyr Asp Leu Ile Glu Ser Thr Phe Glu Asn Thr Lys
245 250 255
Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe Gln Lys Cys Phe
260 265 270
Ala Ile Leu Lys Val His Lys Glu Arg Val Asn Lys Gly Ser Ala Val
275 280 285
Gln Ser Asn Val Phe Ala Glu Glu Gly Thr Lys Asp Trp Lys Ala Ile
290 295 300
Arg Val Asp Leu Val Val Thr Pro Phe Gln His Tyr Ala Phe Ala Leu
305 310 315 320
Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr
325 330 335
Ala Thr Gln Glu Lys Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp
340 345 350
Lys Thr Lys Lys Ile Phe Leu Ser Ala Ala Asn Glu Glu Glu Ile Phe
355 360 365
Ser His Leu Gly Leu Asp Tyr Leu Glu Pro Trp Glu Arg Asn Ala
370 375 380
<210> 31
<211> 367
<212> PRT
<213> 棕鳟 (截短型)
<400> 31
Thr Ala Ala Ala Asn Val Ser Gln Tyr Ala Cys Leu Arg Arg Thr Thr
1 5 10 15
Thr Glu Asn His Asn Lys Ile Phe Thr Asp Val Leu Glu Glu Leu Ala
20 25 30
Glu Asn Ser Glu Phe Asn Glu Ser Lys Gly Pro Cys Leu Ala Phe Arg
35 40 45
Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Ser Ala Val His Cys Leu
50 55 60
Gly Ala Ile Gln Gly Leu Pro Cys Leu Gly Glu His Thr Lys Ala Val
65 70 75 80
Met Glu Glu Ile Leu Ile Phe Gly Arg Ser Phe Lys Val Glu Glu Val
85 90 95
Gln Ser Asp Glu Arg Tyr Gln Ala Leu Lys Leu Phe Thr Ser Val Phe
100 105 110
Gly Val Gly Pro Lys Thr Ala Glu Lys Trp Tyr Arg Arg Gly Leu Arg
115 120 125
Ser Leu Lys Glu Ile Leu Ala Glu Pro Asn Ile Gln Leu Asn Arg Met
130 135 140
Gln Arg Ala Gly Phe Leu Tyr Tyr Arg Asp Ile Ser Lys Ala Val Ser
145 150 155 160
Lys Ala Glu Ala Lys Ala Leu Ser Ser Ile Ile Glu Glu Thr Ala His
165 170 175
Trp Ile Ala Pro Asp Ser Ile Leu Ala Leu Thr Gly Gly Phe Arg Arg
180 185 190
Gly Lys Glu Tyr Gly His Asp Val Asp Phe Leu Leu Thr Met Pro Glu
195 200 205
Met Gly Lys Glu Glu Gly Leu Leu Leu Arg Val Ile Asp Arg Leu Arg
210 215 220
Asp Gln Gly Ile Leu Leu Tyr Cys Glu His Gln Asp Ser Thr Phe Asp
225 230 235 240
Met Ser Lys Leu Pro Ser His Arg Phe Glu Ala Met Asp His Phe Glu
245 250 255
Lys Cys Phe Leu Ile Leu Arg Leu Glu Glu Gly Gln Val Glu Gly Asp
260 265 270
Gly Gly Leu Gln Lys Asp Pro Gly Glu Ser Arg Gly Trp Arg Ala Val
275 280 285
Arg Val Asp Leu Val Ala Pro Pro Val Asp Arg Tyr Ala Phe Val Leu
290 295 300
Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Phe
305 310 315 320
Ala Ser Lys Glu Arg Gly Met Cys Leu Asp Asn His Ala Leu Tyr Asp
325 330 335
Lys Thr Lys Lys Leu Phe Leu Pro Ala Thr Ser Glu Glu Asp Ile Phe
340 345 350
Ala His Leu Gly Leu Glu Tyr Val Glu Pro Trp Gln Arg Asn Ala
355 360 365
<210> 32
<211> 377
<212> PRT
<213> 电鳗 (截短型)
<400> 32
Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Val Pro Ala Pro Ser Leu
1 5 10 15
Pro Thr Val Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asp Asn
20 25 30
His Asn Lys Val Phe Thr Asp Ala Leu Glu Val Leu Ile Glu Asn Tyr
35 40 45
Glu Phe Ser Asp Asn Lys Gly Ala Cys Val Gly Phe Arg Arg Ala Ala
50 55 60
Ser Val Leu Lys Ser Leu Pro Lys Pro Leu Arg Cys Leu Lys Asp Met
65 70 75 80
Glu Gly Leu Pro Cys Leu Gly Asp Asp Thr Lys Ala Ile Ile Glu Glu
85 90 95
Ile Tyr Glu Cys Gly Ser Ser Ser Arg Val Glu Asn Ile Leu Ser Asp
100 105 110
Glu Lys Tyr Gln Thr Leu Lys Leu Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Pro Lys Thr Gly Glu Lys Trp Tyr Arg Arg Gly Leu Arg Ala Leu Glu
130 135 140
Gln Val His Ser Glu Pro Ser Ile Gln Leu Asn Lys Met Gln Ala Ala
145 150 155 160
Gly Phe Leu Tyr Tyr Glu Asp Ile Ser Lys Pro Val Ser Arg Ala Glu
165 170 175
Ala Lys Ala Val Gly Cys Ile Ile Glu Glu Val Ala Ser Cys Phe Ser
180 185 190
Ser Ser Val Thr Ile Thr Leu Thr Gly Gly Phe Arg Arg Gly Lys Glu
195 200 205
Phe Gly His Asp Val Asp Phe Leu Leu Ser Ile Pro Glu Pro Gly Lys
210 215 220
Glu Asp Gly Leu Leu Pro Ala Val Ile Asp Arg Leu Arg Lys Gln Gly
225 230 235 240
Ile Leu Leu Tyr Ser Asp Leu Gln Glu Ser Thr Leu Gln Gln Trp Lys
245 250 255
Arg Pro Ser Arg Cys Phe Asp Ser Met Asp His Phe Gln Lys Cys Phe
260 265 270
Leu Ile Val Lys Leu Trp Thr Arg Leu Val Glu Gly His Arg Glu Asp
275 280 285
Pro Ser Ser Gln Arg Asp Trp Lys Ala Val Arg Val Asp Leu Val Val
290 295 300
Pro Pro Val Asp Cys Tyr Ala Phe Ala Leu Leu Gly Trp Ser Gly Ser
305 310 315 320
Thr Gln Phe Glu Arg Asp Leu Arg Arg Phe Ala Arg Leu Glu Arg Arg
325 330 335
Met Leu Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Thr Asn Thr Phe
340 345 350
Leu Gln Ala Lys Thr Glu Glu Asp Ile Phe Ala His Leu Gly Leu Asp
355 360 365
Tyr Ile Glu Pro Trp Gln Arg Asn Ala
370 375
<210> 33
<211> 378
<212> PRT
<213> 龟壳攀鲈 (截短型)
<400> 33
Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Val Pro Ala Pro Ser Val
1 5 10 15
Ala Thr Val Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Thr Glu Asn
20 25 30
Asn Asn Lys Ile Leu Thr Asp Ala Phe Glu Val Leu Ala Glu Ser Tyr
35 40 45
Glu Leu Asn Gln Leu Glu Gly Pro Cys Leu Ala Phe Arg Arg Ala Ala
50 55 60
Ser Val Leu Lys Ser Leu Pro Trp Ala Val Gln Cys Leu Gly Ala Thr
65 70 75 80
Gln Gly Leu Pro Cys Leu Gly Glu His Thr Lys Ala Leu Ile Glu Glu
85 90 95
Ile Leu Gln Tyr Gly His Ser Phe Glu Val Glu Lys Ile Leu Ser Asp
100 105 110
Glu Arg Tyr Gln Thr Leu Lys Leu Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Pro Lys Thr Ala Glu Lys Trp Tyr Arg Arg Gly Leu Arg Ser Phe Ser
130 135 140
Asp Ile Leu Ala Glu Pro Ser Ile Gln Leu Asn Arg Met Gln Gln Ser
145 150 155 160
Gly Phe Leu His Tyr Gly Asp Ile Ser Arg Ala Val Ser Lys Ala Glu
165 170 175
Ala Arg Ala Leu Gly Asn Ile Ile Asp Glu Ala Val His Ala Ile Thr
180 185 190
Pro Asp Gly Ile Leu Ala Leu Thr Gly Gly Phe Arg Arg Gly Lys Glu
195 200 205
Phe Gly His Asp Val Asp Phe Ile Val Thr Thr Pro Glu Gln Gly Lys
210 215 220
Glu Glu Thr Leu Leu Pro Asn Ile Ile Asp Arg Leu Lys Glu Gln Gly
225 230 235 240
Ile Leu Leu Tyr Ser Asp Tyr Gln Thr Ser Thr Phe Asp Ile Ser Lys
245 250 255
Leu Pro Ser His Lys Phe Glu Ala Met Asp His Phe Ala Lys Cys Phe
260 265 270
Leu Ile Leu Arg Leu Glu Gly Ser Leu Val Asp Arg Gly Leu Asn Ser
275 280 285
Thr Glu Gly Asp Ser Arg Gly Trp Arg Ala Val Arg Val Asp Leu Val
290 295 300
Ser Pro Pro Met Glu Arg Tyr Ala Tyr Ala Leu Leu Gly Trp Thr Gly
305 310 315 320
Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Phe Ala Arg Leu Glu Gln
325 330 335
His Met Leu Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Lys Glu
340 345 350
Phe Leu Ala Ala Thr Thr Glu Arg Asp Ile Phe Ala His Leu Gly Leu
355 360 365
Glu Tyr Ile Glu Pro Trp Gln Arg Asn Ala
370 375
<210> 34
<211> 378
<212> PRT
<213> 孔雀鱼(截短型)
<400> 34
Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Val Pro Ala Pro Ser Val
1 5 10 15
Asp Lys Val Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Val Glu Asn
20 25 30
Asn Asn Arg Ile Phe Thr Asp Ala Phe Glu Val Leu Ala Glu Asn Tyr
35 40 45
Glu Phe Asn Glu Ile Glu Gly Arg Cys Leu Ala Phe Arg Arg Ala Ala
50 55 60
Ser Val Leu Lys Ser Leu Pro Trp Ala Val Arg Ser Val Gly Ala Thr
65 70 75 80
Leu Asp Leu Pro Cys Leu Gly Glu His Thr Thr Ala Val Met Lys Glu
85 90 95
Ile Leu Gln Tyr Gly Arg Ser Phe Glu Val Glu Lys Ile Leu Ser Asp
100 105 110
Glu Arg Cys Gln Thr Leu Lys Leu Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Pro Lys Thr Ala Glu Lys Trp Tyr Arg Arg Gly Leu Arg Ser Phe Ser
130 135 140
Asp Val Leu Ala Gln Pro Gly Ile His Leu Asn Arg Met Gln Gln Ser
145 150 155 160
Gly Phe Leu His Tyr Gly Asp Ile Ser Arg Ala Val Ser Lys Ala Glu
165 170 175
Ala Arg Ala Val Gly Asn Ile Ile Asp Glu Ala Val His Val Ile Thr
180 185 190
Pro Asn Ala Ile Leu Ala Leu Thr Gly Gly Phe Arg Arg Gly Lys Asp
195 200 205
Phe Gly His Asp Val Asp Phe Ile Val Thr Thr Thr Glu Leu Gly Lys
210 215 220
Glu Lys Asn Leu Leu Ile Ser Val Ile Glu Ser Leu Lys Lys Gln Gly
225 230 235 240
Leu Leu Leu Phe Ser Asp Tyr Gln Ala Ser Thr Phe Asp Ile Ser Lys
245 250 255
Leu Pro Ser His Arg Phe Glu Ala Met Asp His Phe Ala Lys Cys Phe
260 265 270
Leu Ile Leu Arg Leu Glu Gly Ser Arg Val Glu Gly Gly Leu Gln Arg
275 280 285
Ala Gln Ala Asp Gly Arg Gly Trp Arg Ala Val Arg Val Asp Leu Val
290 295 300
Ser Pro Pro Ala Asp Arg Phe Ala Phe Thr Met Leu Gly Trp Thr Gly
305 310 315 320
Ser Arg Met Phe Glu Arg Asp Leu Arg Arg Phe Ala Arg Leu Glu Arg
325 330 335
Gln Met Leu Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Lys Glu
340 345 350
Phe Leu Thr Ala Ala Thr Glu Lys Asp Ile Phe Asp His Leu Gly Leu
355 360 365
Glu Tyr Ile Glu Pro Trp Gln Arg Asn Ala
370 375
<210> 35
<211> 366
<212> PRT
<213> 短褐家鼠 (截短型)
<400> 35
Pro Leu Met Gln Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr
1 5 10 15
Leu Asn Asn His Asn Gln Leu Phe Thr Asp Ala Phe Asp Ile Leu Ala
20 25 30
Glu Asn Tyr Glu Phe Arg Glu Asn Glu Val Ser Cys Leu Pro Phe Met
35 40 45
Arg Ala Ala Ser Val Leu Lys Ser Leu Ser Phe Pro Ile Val Ser Met
50 55 60
Lys Asp Ile Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Cys Val
65 70 75 80
Ile Glu Gly Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val
85 90 95
Leu Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe
100 105 110
Gly Val Gly Leu Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg
115 120 125
Thr Leu Ser Lys Ile Lys Ser Asp Lys Ser Leu Arg Phe Thr His Met
130 135 140
Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Asn
145 150 155 160
Arg Ala Glu Ala Glu Ala Val Ser Met Leu Val Lys Glu Ala Val Val
165 170 175
Ala Phe Leu Pro Asp Ala Leu Val Thr Met Thr Gly Gly Phe Arg Arg
180 185 190
Gly Lys Met Thr Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu
195 200 205
Ala Thr Glu Glu Glu Glu Gln Gln Leu Leu His Lys Val Thr Asn Phe
210 215 220
Trp Arg Gln Gln Gly Leu Leu Leu Tyr Cys Asp Ile Ile Glu Ser Thr
225 230 235 240
Phe Glu Lys Phe Lys Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His
245 250 255
Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu His Arg Gly Leu Val Arg
260 265 270
Ser Glu Glu Ser Gly Gln Gln Glu Gly Lys Asp Trp Lys Ala Ile Arg
275 280 285
Val Asp Leu Val Met Cys Pro Tyr Glu Arg Arg Ala Phe Ala Leu Leu
290 295 300
Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala
305 310 315 320
Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys
325 330 335
Thr Lys Arg Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala
340 345 350
His Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
355 360 365
<210> 36
<211> 379
<212> PRT
<213> 长褐家鼠(截短型)
<400> 36
Ser Leu Ser Pro Val Pro Gly Ser Gln Thr Val Pro Pro Pro Leu Met
1 5 10 15
Gln Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn
20 25 30
His Asn Gln Leu Phe Thr Asp Ala Phe Asp Ile Leu Ala Glu Asn Tyr
35 40 45
Glu Phe Arg Glu Asn Glu Val Ser Cys Leu Pro Phe Met Arg Ala Ala
50 55 60
Ser Val Leu Lys Ser Leu Ser Phe Pro Ile Val Ser Met Lys Asp Ile
65 70 75 80
Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Cys Val Ile Glu Gly
85 90 95
Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp
100 105 110
Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Leu Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Ser
130 135 140
Lys Ile Lys Ser Asp Lys Ser Leu Arg Phe Thr His Met Gln Lys Ala
145 150 155 160
Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Asn Arg Ala Glu
165 170 175
Ala Glu Ala Val Ser Met Leu Val Lys Glu Ala Val Val Ala Phe Leu
180 185 190
Pro Asp Ala Leu Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Met
195 200 205
Thr Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu Ala Thr Glu
210 215 220
Glu Glu Glu Gln Gln Leu Leu His Lys Val Thr Asn Phe Trp Arg Gln
225 230 235 240
Gln Gly Leu Leu Leu Tyr Cys Asp Ile Ile Glu Ser Thr Phe Glu Lys
245 250 255
Phe Lys Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe Gln Lys
260 265 270
Cys Phe Leu Ile Leu Lys Leu His Arg Gly Leu Val Arg Ser Glu Glu
275 280 285
Ser Gly Gln Gln Glu Gly Lys Asp Trp Lys Ala Ile Arg Val Asp Leu
290 295 300
Val Met Cys Pro Tyr Glu Arg Arg Ala Phe Ala Leu Leu Gly Trp Thr
305 310 315 320
Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr His Glu
325 330 335
Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg
340 345 350
Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly
355 360 365
Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375
<210> 37
<211> 379
<212> PRT
<213> 红疣猴(截短型)
<400> 37
Ser Asp Ser Thr Asn Pro Gly Leu Pro Lys Thr Pro Pro Thr Ala Ile
1 5 10 15
Gln Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn
20 25 30
Cys Asn Gln Ile Phe Thr Asp Ala Phe Asp Ile Leu Ala Glu Asn Cys
35 40 45
Glu Phe Arg Glu Asn Glu Asp Ser Cys Val Thr Phe Met Arg Ala Ala
50 55 60
Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys Asp Thr
65 70 75 80
Glu Gly Ile Pro Cys Leu Gly Ser Lys Val Lys Cys Ile Ile Glu Glu
85 90 95
Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp
100 105 110
Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Ser
130 135 140
Lys Val Arg Ser Asp Glu Ser Leu Lys Phe Thr Arg Met Gln Arg Ala
145 150 155 160
Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu
165 170 175
Ala Glu Ala Val Ser Ala Leu Val Lys Glu Ala Val Trp Ala Phe Leu
180 185 190
Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys
195 200 205
Met Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Thr Glu
210 215 220
Asp Glu Glu Gln Gln Leu Leu Gln Lys Val Met Asn Leu Trp Glu Lys
225 230 235 240
Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys
245 250 255
Leu Arg Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe Gln Lys
260 265 270
Cys Phe Leu Ile Phe Lys Leu Pro Leu Gln Arg Val Asp Ser Asp Gln
275 280 285
Ser Ser Trp Gln Gly Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu
290 295 300
Val Met Cys Pro Tyr Glu Arg Arg Ala Phe Ala Leu Leu Gly Trp Thr
305 310 315 320
Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr His Glu
325 330 335
Arg Lys Met Ile Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg
340 345 350
Ile Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly
355 360 365
Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375
<210> 38
<211> 379
<212> PRT
<213> 野猪 (截短型)
<400> 38
Ser Ala Ser Pro Ser Pro Gly Ser Gln Asn Thr Leu Pro Pro Ala Val
1 5 10 15
Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn
20 25 30
Cys Asn His Ile Phe Thr Asp Ala Phe Glu Val Leu Ala Glu Asn Tyr
35 40 45
Glu Phe Arg Glu Asn Glu Thr Phe Cys Leu Ala Phe Met Arg Ala Ala
50 55 60
Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys Asp Thr
65 70 75 80
Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Cys Val Ile Glu Glu
85 90 95
Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp
100 105 110
Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Leu Lys Thr Ser Glu Arg Trp Phe Arg Met Gly Phe Arg Ser Leu Ser
130 135 140
Lys Ile Arg Ser Asp Lys Thr Leu Lys Phe Thr Arg Met Gln Lys Ala
145 150 155 160
Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu
165 170 175
Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Gln Ala Phe Leu
180 185 190
Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys
195 200 205
Met Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Thr Asp
210 215 220
Asp Glu Glu Gln Gln Leu Leu Pro Lys Val Val Asn Leu Trp Glu Arg
225 230 235 240
Glu Gly Leu Leu Leu Tyr Cys Asp Leu Val Glu Ser Thr Leu Glu Lys
245 250 255
Ser Lys Leu Pro Ser Arg Asn Val Asp Ala Leu Asp His Phe Gln Lys
260 265 270
Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Gly Met
275 280 285
Ser Ser Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu
290 295 300
Val Met Cys Pro Tyr Glu Leu Arg Ala Phe Ala Leu Leu Gly Trp Thr
305 310 315 320
Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr His Glu
325 330 335
Arg Lys Met Ile Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg
340 345 350
Ile Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly
355 360 365
Leu Asp Tyr Leu Glu Pro Trp Glu Arg Asn Ala
370 375
<210> 39
<211> 375
<212> PRT
<213> 西伯利亚虎 (截短型)
<400> 39
Tyr Ser Ala Ser Pro Asn Pro Glu Leu Gln Lys Thr Pro Pro Leu Val
1 5 10 15
Lys Lys Ile Pro Leu Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn
20 25 30
Phe Asn His Val Phe Thr Asp Ala Phe Glu Val Leu Ala Glu Asn Tyr
35 40 45
Glu Phe Lys Glu Asn Glu Val Ser Ser Ala Thr Phe Met Arg Ala Ala
50 55 60
Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys Asp Thr
65 70 75 80
Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Cys Val Ile Glu Glu
85 90 95
Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp
100 105 110
Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Ser
130 135 140
Lys Ile Lys Ser Asp Lys Thr Leu Lys Phe Thr Gln Met Gln Lys Ala
145 150 155 160
Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu
165 170 175
Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu
180 185 190
Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys
195 200 205
Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Thr Asp
210 215 220
Glu Glu Glu Glu Glu Leu Leu Pro Lys Val Ile Asn Leu Trp Gln Arg
225 230 235 240
Lys Glu Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys
245 250 255
Leu Lys Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe Gln Lys
260 265 270
Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Gly Lys
275 280 285
Cys Ser Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu
290 295 300
Val Met Cys Pro Tyr Glu Arg Arg Ala Phe Ala Leu Leu Gly Trp Thr
305 310 315 320
Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr His Glu
325 330 335
Arg Lys Met Ile Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Lys
340 345 350
Ile Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly
355 360 365
Leu Asp Tyr Ile Glu Pro Trp
370 375
<210> 40
<211> 365
<212> PRT
<213> 中国水牛 (截短型)
<400> 40
Leu Ala Val Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Lys Thr Thr
1 5 10 15
Leu Asn Asn Tyr Asn His Ile Phe Thr Asp Ala Phe Glu Ile Leu Ala
20 25 30
Glu Asn Ser Glu Phe Lys Glu Asn Glu Val Ser Tyr Val Thr Phe Met
35 40 45
Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met
50 55 60
Lys Asp Thr Gln Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Cys Val
65 70 75 80
Ile Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val
85 90 95
Leu Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe
100 105 110
Gly Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg
115 120 125
Ser Leu Ser Lys Ile Thr Ser Asp Lys Thr Leu Lys Phe Thr Lys Met
130 135 140
Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr
145 150 155 160
Arg Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp
165 170 175
Ala Phe Leu Pro Asp Ala Phe Ile Thr Met Thr Gly Gly Phe Arg Arg
180 185 190
Gly Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly
195 200 205
Ser Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp
210 215 220
Glu Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe
225 230 235 240
Glu Lys Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe
245 250 255
Gln Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser
260 265 270
Gly Arg Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val
275 280 285
Asp Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly
290 295 300
Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Ile Arg Arg Tyr Ala Thr
305 310 315 320
His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr
325 330 335
Lys Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His
340 345 350
Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
355 360 365
<210> 41
<211> 379
<212> PRT
<213> 黄腹旱獭 (截短型)
<400> 41
Pro Asp His Ser Asn Ser Asp Pro Gln Arg Ile Pro Pro Pro Ala Val
1 5 10 15
Gln Thr Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn
20 25 30
Cys Asn Arg Val Phe Thr Asp Ala Phe Asp Val Leu Ala Glu Asn Tyr
35 40 45
Glu Phe Arg Glu Asn Glu Ser Cys Ser Val Val Phe Met Arg Ala Ala
50 55 60
Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Arg Asp Leu
65 70 75 80
Glu Gly Ile Pro Cys Leu Glu Gly Lys Ala Lys Ser Ile Ile Glu Glu
85 90 95
Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp
100 105 110
Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly
115 120 125
Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Ser
130 135 140
Lys Ile Arg Ser Asp Lys Ser Leu Lys Phe Thr His Met Gln Lys Ala
145 150 155 160
Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu
165 170 175
Ala Glu Ala Val Ser Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu
180 185 190
Pro Asp Ala Phe Ile Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Asn
195 200 205
Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Ala Glu Ala Thr Glu
210 215 220
Glu Glu Glu Gln Gln Leu Leu His Lys Val Thr Asn Leu Trp Glu Lys
225 230 235 240
Lys Gly Leu Leu Leu Tyr Cys Asp Leu Val Glu Ser Thr Phe Glu Lys
245 250 255
Leu Lys Thr Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe Gln Lys
260 265 270
Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Asp Lys
275 280 285
Ala Ser Gln Gln Gly Gly Lys Asn Trp Lys Ala Ile Arg Val Asp Leu
290 295 300
Val Met Cys Pro Tyr Glu Arg Arg Ala Phe Ala Leu Leu Gly Trp Thr
305 310 315 320
Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr His Glu
325 330 335
Arg Lys Met Ile Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg
340 345 350
Ile Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly
355 360 365
Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375
<210> 42
<211> 378
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 没有His标签的小鼠-牛-猪嵌合体M57-N27-8-13C7
<400> 42
Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Ala Pro Ala Pro Val Val
1 5 10 15
Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn
20 25 30
Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn Asp
35 40 45
Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala Ser
50 55 60
Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Thr Ser Met Lys Asp Thr
65 70 75 80
Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu Glu
85 90 95
Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp
100 105 110
Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Gly Phe Gly Val Gly
115 120 125
Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Glu
130 135 140
Lys Ile Arg Ser Asp Lys Thr Leu Lys Phe Thr Gln Met Gln Lys Ala
145 150 155 160
Gly Phe Leu Tyr Tyr Glu Asp Leu Ala Ser Arg Val Asn Arg Ala Glu
165 170 175
Ala Glu Ala Ile Arg Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu
180 185 190
Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys Lys
195 200 205
Thr Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Thr Glu
210 215 220
Asp Glu Glu Gln Leu Leu His Lys Val Ile Asn Leu Trp Glu Lys Lys
225 230 235 240
Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe
245 250 255
Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys
260 265 270
Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser
275 280 285
Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val
290 295 300
Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly
305 310 315 320
Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg
325 330 335
Lys Gln Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val
340 345 350
Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Arg His Leu Gly Leu
355 360 365
Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
370 375
<210> 43
<211> 364
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 没有His标签的小鼠-牛嵌合体46-557
<400> 43
Ser Glu Lys Lys Ile Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu
1 5 10 15
Asn Asn Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu
20 25 30
Asn Asp Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg
35 40 45
Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys
50 55 60
Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile
65 70 75 80
Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu
85 90 95
Asn Asp Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly
100 105 110
Val Gly Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr
115 120 125
Leu Glu Glu Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln
130 135 140
Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg
145 150 155 160
Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala
165 170 175
Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly
180 185 190
Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser
195 200 205
Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu
210 215 220
Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Arg Glu Ser Thr Phe Glu
225 230 235 240
Lys Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln
245 250 255
Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser
260 265 270
Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp
275 280 285
Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp
290 295 300
Thr Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His
305 310 315 320
Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys
325 330 335
Arg Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu
340 345 350
Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
355 360
<210> 44
<211> 364
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 没有His标签的小鼠-牛嵌合体46-342
<400> 44
Ser Glu Lys Lys Ile Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu
1 5 10 15
Asn Asn Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu
20 25 30
Asn Asp Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg
35 40 45
Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys
50 55 60
Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile
65 70 75 80
Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu
85 90 95
Asn Asp Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly
100 105 110
Val Gly Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr
115 120 125
Leu Glu Glu Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln
130 135 140
Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg
145 150 155 160
Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala
165 170 175
Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly
180 185 190
Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser
195 200 205
Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu
210 215 220
Lys Lys Gly Leu Leu Leu Tyr Tyr Lys Leu Val Glu Ser Thr Phe Glu
225 230 235 240
Lys Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln
245 250 255
Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser
260 265 270
Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp
275 280 285
Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp
290 295 300
Thr Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His
305 310 315 320
Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys
325 330 335
Arg Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu
340 345 350
Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
355 360
<210> 45
<211> 364
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 没有His标签的小鼠-牛嵌合体46-571
<400> 45
Ser Glu Lys Lys Ile Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu
1 5 10 15
Asn Asn Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu
20 25 30
Asn Asp Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg
35 40 45
Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys
50 55 60
Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile
65 70 75 80
Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu
85 90 95
Asn Asp Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly
100 105 110
Val Gly Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr
115 120 125
Leu Glu Glu Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln
130 135 140
Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg
145 150 155 160
Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala
165 170 175
Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly
180 185 190
Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser
195 200 205
Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu
210 215 220
Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Gln Glu Ser Thr Phe Glu
225 230 235 240
Lys Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln
245 250 255
Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser
260 265 270
Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp
275 280 285
Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp
290 295 300
Thr Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His
305 310 315 320
Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys
325 330 335
Arg Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu
340 345 350
Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
355 360
<210> 46
<211> 390
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 没有His标签的小鼠-牛嵌合体M92
<400> 46
Ser Glu Asn Leu Tyr Phe Gln Thr Gly Ser Ser Gly Ser Pro Ser Pro
1 5 10 15
Val Pro Gly Ser Gln Asn Val Pro Ala Pro Val Val Lys Lys Ile Ser
20 25 30
Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn Tyr Asn Gln Leu
35 40 45
Phe Thr Asp Ala Leu Asp Ile Leu Ala Glu Asn Asp Glu Phe Arg Glu
50 55 60
Asn Glu Asp Ser Arg Asp Ala Phe Arg Arg Ala Ser Ser Val Leu Lys
65 70 75 80
Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp Thr Glu Gly Ile Pro
85 90 95
Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu Glu Ile Ile Glu Asp
100 105 110
Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu Arg Tyr Lys
115 120 125
Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Arg Lys Thr Ala
130 135 140
Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Glu Lys Ile Arg Ser
145 150 155 160
Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys Ala Gly Phe Leu Tyr
165 170 175
Tyr Glu Asp Leu Ala Glu Arg Val Asn Arg Pro Glu Ala Glu Ala Ile
180 185 190
Arg Met Leu Val Lys Glu Ala Val Val Thr Phe Leu Pro Asp Ala Leu
195 200 205
Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys Met Thr Gly His Asp
210 215 220
Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp Glu Glu Gln
225 230 235 240
Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys Lys Gly Leu Leu Leu
245 250 255
Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys Leu Pro Ser
260 265 270
Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe Leu Ile Leu
275 280 285
Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn Gln Gln Glu
290 295 300
Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met Cys Pro Tyr
305 310 315 320
Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Pro Gln Phe
325 330 335
Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg Lys Met Met Leu
340 345 350
Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe Leu Glu Ala
355 360 365
Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr Ile Trp
370 375 380
Pro Trp Glu Arg Asn Ala
385 390
<210> 47
<211> 364
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 没有His标签的小鼠-牛嵌合体M93
<400> 47
Ser Glu Lys Lys Ile Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu
1 5 10 15
Asn Asn Tyr Asn Glu Pro Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu
20 25 30
Asn Asp Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg
35 40 45
Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys
50 55 60
Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile
65 70 75 80
Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu
85 90 95
Asn Asp Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly
100 105 110
Val Gly Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr
115 120 125
Leu Glu Lys Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln
130 135 140
Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg
145 150 155 160
Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala
165 170 175
Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly
180 185 190
Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser
195 200 205
Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu
210 215 220
Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu
225 230 235 240
Lys Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln
245 250 255
Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser
260 265 270
Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp
275 280 285
Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp
290 295 300
Thr Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His
305 310 315 320
Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys
325 330 335
Arg Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu
340 345 350
Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
355 360
<210> 48
<211> 364
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 没有His标签的小鼠-牛嵌合体M94
<400> 48
Ser Glu Lys Lys Ile Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu
1 5 10 15
Asn Asn Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu
20 25 30
Asn Asp Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg
35 40 45
Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys
50 55 60
Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile
65 70 75 80
Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu
85 90 95
Asn Asp Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly
100 105 110
Val Gly Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr
115 120 125
Leu Glu Lys Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln
130 135 140
Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg
145 150 155 160
Ala Glu Ala Glu Ala Val Gly Val Leu Val Glu Glu Ala Val Trp Ala
165 170 175
Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly
180 185 190
Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser
195 200 205
Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu
210 215 220
Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu
225 230 235 240
Lys Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln
245 250 255
Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser
260 265 270
Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp
275 280 285
Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp
290 295 300
Thr Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His
305 310 315 320
Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys
325 330 335
Arg Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu
340 345 350
Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
355 360
<210> 49
<211> 364
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 没有His标签的小鼠-牛嵌合体M88
<400> 49
Ser Glu Lys Lys Ile Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu
1 5 10 15
Asn Asn Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu
20 25 30
Asn Asp Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg
35 40 45
Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys
50 55 60
Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile
65 70 75 80
Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu
85 90 95
Asn Asp Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly
100 105 110
Val Gly Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr
115 120 125
Leu Glu Glu Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln
130 135 140
Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg
145 150 155 160
Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala
165 170 175
Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly
180 185 190
Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser
195 200 205
Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu
210 215 220
Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Leu Glu
225 230 235 240
Lys Phe Lys Leu Pro Ser Arg Gln Val Asp Arg Leu Asp His Phe Gln
245 250 255
Lys Cys Phe Leu Ile Leu Lys Leu His Arg Gln Arg Val Asp Ser Ser
260 265 270
Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp
275 280 285
Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp
290 295 300
Thr Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His
305 310 315 320
Glu Arg Asn Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys
325 330 335
Arg Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu
340 345 350
Gly Leu Asp Tyr Ile Trp Pro Trp Glu Arg Asn Ala
355 360

Claims (7)

1.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)提供具有3’-末端核苷酸的引发物，所述3’末端核苷酸具有自由3’-羟基；
b)重复以下循环：(i)在延伸条件下使所述引发物或具有自由3’-O-羟基的延伸片段与3’-O-封闭的三磷酸核苷和TdT变体接触，使得所述引发物或延伸片段通过并入3’-O-封闭的三磷酸核苷而延伸，以形成3’-O-封闭的延伸片段，和(ii)解封闭所述延伸片段，以形成具有自由3’-羟基的延伸片段，直至形成包含一定产率的所述多核苷酸的产物；
其中所述TdT变体(i)在FMR基序中具有蛋氨酸或功能等同氨基酸的氨基酸取代，并且在GGFRR基序中具有第二精氨酸或功能等同氨基酸的氨基酸取代，并且(ii)缺少歧化作用活性。
2.如权利要求1所述的方法，其中所述TdT变体还(i)缺少BRCT样片段，(ii)能够在没有模板的情况下合成核酸片段，并且(iii)能够将3’-O-封闭的三磷酸核苷并入到核酸片段。
3.如权利要求1或2所述的方法，其中所述TdT是具有与SEQ ID NO:2、3、4、5、6、7、8、9、10、11、12、13、14、15、26、27、28、29、35、36、37、38、39、40或41之一具有至少80％同一性的氨基酸序列的TdT变体；
所述TdT变体在以下残基具有取代：相对于SEQ ID NO：2、3、4、6、7、8、11和13的第63位的蛋氨酸，或相对于SEQ ID NO:27的第48位的亮氨酸，或相对于SEQ ID NO：26、28或29的第61位的亮氨酸，或相对于SEQ ID NO:5的第62位的亮氨酸，或相对于SEQ ID NO:12的第63位的亮氨酸，或相对于SEQ ID NO:9的第64位的蛋氨酸，或相对于SEQ ID NO:15的第47位的蛋氨酸，或相对于SEQ ID NO:35和40的第48位的蛋氨酸，或相对于SEQ ID NO:9的第64位的蛋氨酸，或相对于SEQ ID NO：10、36、37、38、39和41的第61位的蛋氨酸，或相对于SEQ ID NO:14的第66位的蛋氨酸；并且
所述TdT变体在以下残基具有取代：相对于SEQ ID NO:2、3、4、6、7、8、9、11和12的第207位的第一精氨酸，或相对于SEQ ID NO:5的第206位的第一精氨酸，或相对于SEQ ID NO:9的第208位的第一精氨酸，或相对于SEQ ID NO:10、28、29、36、37、38、39和41的第205位的第一精氨酸，或相对于SEQ ID NO:15的第190位的第一精氨酸，或相对于SEQ ID NO:27、35和40的第192位的第一精氨酸，或相对于SEQ ID NO:26的第204位的第一精氨酸，或相对于SEQID NO:13的第216位的第一精氨酸，或相对于SEQ ID NO:14的第210位的第一精氨酸。
4.如权利要求3所述的方法，其中所述TdT变体还在以下残基包含取代：
相对于SEQ ID NO:2、3、4、6、7、8、11和12的第173位的半胱氨酸，或相对于SEQ ID NO:5的第172位的半胱氨酸，或相对于SEQ ID NO:9和10的第174位的半胱氨酸，或相对于SEQ IDNO:26和27的第170位的半胱氨酸，或相对于SEQ ID NO：10、36、37、38、39和41的第171位的半胱氨酸，或相对于SEQ ID NO:13的第182位的半胱氨酸，或相对于SEQ ID NO:35和40的第158位的半胱氨酸，或相对于SEQ ID NO:15的第156位的半胱氨酸，或相对于SEQ ID NO:14的第176位的半胱氨酸；或
相对于SEQ ID NO：2、8、9、12和13的第325位的第二精氨酸，或相对于SEQ ID NO 3和4的第324位的第二精氨酸，或相对于SEQ ID NO:5的第320位的第二精氨酸，或相对于SEQ IDNO:6的第331位的第二精氨酸，或相对于SEQ ID NO：10、36、37、38、39和41的第323位的第二精氨酸，或相对于SEQ ID NO:11和14的第328位的第二精氨酸，或相对于SEQ ID NO:13的第338位的第二精氨酸，或相对于SEQ ID NO:15的第308位的第二精氨酸，或相对于SEQ IDNO:40的第309位的第二精氨酸，或相对于SEQ ID NO:35的第310位的第二精氨酸；或
相对于SEQ ID NO:2、7、8和12的第328位的谷氨酸，或相对于SEQ ID NO:3和4的第327位的谷氨酸，或相对于SEQ ID NO:6和8的第334位的谷氨酸，或相对于SEQ ID NO:9的第329位的谷氨酸，或相对于SEQ ID NO：10、36、37、38、39和41的第326位的谷氨酸，或相对于SEQID NO:13的第341位的谷氨酸，或相对于SEQ ID NO:11和14的第331位的谷氨酸，或相对于SEQ ID NO:15的第311位的谷氨酸，或相对于SEQ ID NO:40的第312位的谷氨酸，或相对于SEQ ID NO:35的第313位的谷氨酸。
5.如权利要求1至4中任一项所述的方法，其中所述蛋氨酸的取代是R或Q；所述半胱氨酸的取代是G或R；所述第一精氨酸的取代是L或N；所述第二精氨酸的取代是P、N、A或V；所述谷氨酸的取代为N、L、T、S或K。
6.如权利要求1-5中任一项所述的方法，其中所述TdT变体选自SEQ ID NO:21、22、23、24、25、42、43、44、45、46、47、48和49。
7.如权利要求1-6中任一项所述的方法，其中所述3’-O-封闭的三磷酸核苷是3’-O-NH2-三磷酸核苷，3’-O-叠氮基甲基-三磷酸核苷，3’-O-烯丙基-三磷酸核苷或3’-O-(2-硝基苄基)-三磷酸核苷。
CN202080096423.1A 2019-12-16 2020-12-15 使用无歧化作用末端脱氧核苷酸转移酶变体的多核苷酸无模板酶学合成 Pending CN115135770A (zh)

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