CN108368151B - 二聚逆转录酶 - Google Patents

二聚逆转录酶 Download PDF

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CN108368151B
CN108368151B CN201680071876.2A CN201680071876A CN108368151B CN 108368151 B CN108368151 B CN 108368151B CN 201680071876 A CN201680071876 A CN 201680071876A CN 108368151 B CN108368151 B CN 108368151B
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龚小松
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Abstract

提供了包含共价连接在一起的至少两种DNA聚合酶的多肽。与单体DNA聚合酶相比，这种多肽具有增加的稳定性并且可以具有减少的反应时间。具体地，提供了包含通过异源接头共价连接的第一DNA聚合酶和第二DNA聚合酶的多肽，其中第一或第二DNA聚合酶是逆转录酶或DNA依赖性聚合酶。

Description

二聚逆转录酶
相关申请的交叉引用
本专利申请要求2015年12月7日提交的美国临时专利申请号62/264,223的优先权，其通过引用纳入本文用于所有目的。
以ASCII文本文件提交的“序列表”的引用
根据37C.F.R.§§1.821-1.825，在机型IBM-PC，MS-Windows操作系统上，2016年11月28日创建的200,388字节的文件094260-110910PC-1031618_SeqList.txt的序列表通过引用全文纳入本文用于所有目的。
背景技术
核酸的检测，分析，转录和扩增是现代分子生物学中经常使用的程序。DNA聚合酶可用于检测和扩增DNA或RNA。这种RNA分析程序的应用可能涉及基因表达的研究，传染原或遗传疾病的诊断以及cDNA的产生，仅举几例应用。RNA的逆转录(“RT”)因此具有许多用途。在一些情况下，RT之后是聚合酶链式反应扩增，其可以用于RNA的快速检测和定量。这个程序通常被称为“RT-PCR”。
发明内容
提供了包含共价连接在一起的至少两种DNA聚合酶的多肽。与单体DNA聚合酶相比，这种多肽具有增加的稳定性并且可以具有减少的反应时间。在一些实施方式中，提供了包含通过异源接头共价连接的第一DNA聚合酶和第二DNA聚合酶的多肽。在一些实施方式中，第一DNA聚合酶是第一逆转录酶，第DNA聚合酶是第二逆转录酶。
在一些实施方式中，多肽是融合蛋白，并且异源接头是将第一逆转录酶的羧基末端连接至第二逆转录酶的氨基末端的氨基酸接头。在一些实施方式中，多肽还包含通过第二接头共价连接至第一或第二逆转录酶的第三逆转录酶。
在一些实施方式中，多肽的长度不超过1400、1500、1600、1700、1800、1900或2000个氨基酸。
在一些实施方式中，第一逆转录酶和第二逆转录酶是相同的。在一些实施方式中，第一逆转录酶和第二逆转录酶至少80％，85％，90％或95％相同。在一些实施方式中，第一和第二逆转录酶是鼠白血病病毒(MLV)逆转录酶。在一些实施方式中，第一和第二逆转录酶是猫白血病病毒(FLV)逆转录酶。
在一些实施方式中，第一逆转录酶和第二逆转录酶有不到70％，75％，80％，95％或90％相同。
在一些实施方式中，氨基酸接头长度为1-30或1-50(例如1-15，3-25)个氨基酸。
在一些实施方式中，第一或第二逆转录酶选自鼠白血病病毒(MLV)逆转录酶，猫白血病病毒(FLV)逆转录酶，牛白血病病毒(BLV)，禽成髓细胞瘤病毒(AMV)逆转录酶，呼吸道合胞病毒(RSV)逆转录酶，马传染性贫血病毒(EIAV)逆转录酶，Rous相关病毒-2(RAV2)逆转录酶，SUPERSCRIPTII逆转录酶，SUPERSCRIPT I逆转录酶，THERMOSCRIPT逆转录酶和MMLVRNA酶H-逆转录酶。
在一些实施方式中，第一逆转录酶是鼠白血病病毒(MLV)逆转录酶，第二逆转录酶是猫白血病病毒(FLV)逆转录酶。在一些实施方式中，第一逆转录酶是猫白血病病毒(FLV)逆转录酶，第二逆转录酶是鼠白血病病毒(MLV)逆转录酶。
在一些实施方式中，与天然存在的逆转录酶相比，至少第一或第二逆转录酶具有至少一个突变。在一些实施方式中，第一或第二逆转录酶是RNA酶H-逆转录酶。
在一些实施方式中，第一DNA聚合酶是第一DNA依赖性聚合酶和/或第二DNA聚合酶是第二DNA依赖性聚合酶。在一些实施方式中，第一DNA聚合酶是DNA依赖性聚合酶，第二DNA聚合酶是逆转录酶。在一些实施方式中，第一DNA聚合酶是逆转录酶，第二DNA聚合酶是DNA依赖性聚合酶。
还提供了编码如上文或本文其它地方所述的多肽的核酸(任选地分离或纯化的)。还提供了包含如本文所述的核酸的多核苷酸载体。还提供了包含该载体的宿主细胞(例如，原核细胞，真菌，酵母或真核细胞)。
还提供了一种反应混合物，其包含：纯化的mRNA；和上文或本文其他地方所述的多肽。在一些实施方式中，反应混合物还包含选自Tris，HEPES，ACES，PIPES，MOPSO，BES，MOPS，TES，TAPSO，POPSO，BICINE，TAPS和AMPSO的缓冲剂。在一些实施方式中，反应混合物还包含至少一种寡核苷酸引物和/或脱氧核苷酸。
还提供了包含如上文或本文其它地方所述的多肽的试剂盒。在一些实施方式中，所述多肽包含一种或两种逆转录酶，并且所述试剂盒还包含DNA依赖性DNA聚合酶。
还提供了进行聚合酶反应的方法。在一些实施方式中，所述方法包括使靶核酸与本文其他地方如上所述的多肽和寡核苷酸引物接触，该寡核苷酸引物在允许多肽以模板依赖性方式延伸寡核苷酸引物的条件下与靶核酸杂交，由此进行聚合酶反应。在一些实施方式中，靶核酸是RNA并且聚合酶包含一种(或两种)逆转录酶。在一些实施方式中，靶核酸是DNA并且聚合酶包含一种(或两种)DNA依赖性DNA聚合酶。
定义
除非另有说明，本文所用的所有科技术语具有本发明所属领域普通技术人员通常所理解的含义。通常，本文所用的命名和下述细胞培养、分子遗传学、有机化学和核酸化学以及杂交中的实验室步骤均为本领域熟知和常用的。使用标准技术进行核酸和肽合成。按照本领域和各种通用参考文献所述的常规方法进行这些技术和步骤(通常参见，Sambrook等，《分子克隆：实验室手册》(MOLECULAR CLONING：A LABORATORY MANUAL)，第2版(1989)冷泉港实验室出版社(Cold Spring Harbor Laboratory Press)，纽约冷泉港(Cold SpringHarbor，N.Y.)，其通过引用纳入本文)，将它们引入本文全文中。本文所用的命名以及下述分析化学和有机合成中的实验室步骤均为本领域熟知且常用。也可使用标准技术或其变型进行化学合成或化学分析。
用于蛋白质部分时，“异源”指该蛋白质包含在天然情况下彼此不存在于同一关系中的两个或更多结构域。这样的蛋白质，例如融合蛋白，含有两个或多个通过肽键或肽接头序列共价连接的序列，以排列成新的功能性蛋白质。
“引物”指与靶核酸上的序列杂交并且用作核酸合成的起始点的多核苷酸序列。引物可以是各种长度的并且通常长度小于50个核苷酸，例如长度为12-30个核苷酸。可基于本领域技术人员已知的原理设计用于PCR的引物的长度和序列，参见例如Innis等(同上)。
“聚合酶”是指进行模板引导的多核苷酸合成的酶。该术语同时包括全长多肽和具有聚合酶活性的结构域。
“模板”指多核苷酸序列，其包含任选侧接一个或两个引物杂交位点的待扩增的多核苷酸。
本文所用的“核酸”表示DNA、RNA、单链、双链、或更高度聚集的杂交基序及其任意化学修饰。修饰包括但不限于，提供整合入其它电荷、极化性、氢键、静电相互作用、与核酸配体碱基或核酸配体整体的连接点和作用点的化学基团的那些修饰。这类修饰包括但不限于，肽核酸(PNA)、磷酸二酯基团修饰(例如，硫代磷酸酯、甲基膦酸酯)、2′-位糖修饰、5-位嘧啶修饰、8-位嘌呤修饰、环外胺处的修饰、4-硫尿核苷的取代、5-溴或5-碘-尿嘧啶的取代、骨架修饰、甲基化、不常见的碱基配对组合如异碱基(isobases)、异胞苷和异胍(isoguanidine)等。核酸也可包含非天然碱基，如硝基吲哚。修饰还可包括3′和5′修饰，例如用荧光团(例如，量子点)或其他部分加帽。
术语“多肽”、“肽”和“蛋白质”在本文中互换使用，指氨基酸残基的聚合物。该术语适用于其中一个或多个氨基酸残基是相应天然产生氨基酸的人造化学模拟物的氨基酸聚合物，以及天然产生的氨基酸聚合物和非天然产生的氨基酸聚合物。
术语“氨基酸”指天然产生的和合成的氨基酸，以及作用方式类似于天然产生氨基酸的氨基酸类似物和氨基酸模拟物。天然产生的氨基酸是由遗传密码编码的那些氨基酸，以及随后经修饰的那些氨基酸，例如，羟基脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。氨基酸类似物指与天然产生的氨基酸具有相同基本化学结构的化合物，即碳原子与氢原子、羧基、氨基和R基结合，如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。这种类似物具有修饰的R基(如正亮氨酸)或修饰的肽主链，但保留了与天然产生的氨基酸基本相同的化学结构。氨基酸模拟物指结构不同于氨基酸的普通化学结构，但作用方式类似于天然产生氨基酸的化合物。
本文中氨基酸可按IUPAC-IUB生物化学命名委员会推荐的通常已知的三字母符号或单字母符号表述。核苷酸同样可由其普遍接受的单字母代码表述。
术语“启动子”指定位于转录起始上游和/或下游且涉及识别和结合RNA聚合酶和其他蛋白质以起始转录的区域或序列。
“载体”指多核苷酸，其独立于宿主染色体时能够在宿主生物体中复制。优选的载体包括质粒且通常具有复制起始点。载体可包括，例如，转录和翻译终止子、转录和翻译起始序列和用于调控特定核酸表达的启动子。
对于序列比较，一般将一条序列用作与测试序列比较的参比序列。使用序列比较算法时，测试和参比序列均输入计算机，必要时指定子序列坐标，指定序列算法程序参数。可使用默认的程序参数，或者可指定另外的参数。然后，该序列比较算法基于程序参数计算出测试序列相对于参比序列的序列相同性百分数。
本文所用的“比较窗口”包括参考选自20-600，通常约50-200，更常见约100-150的任一数量的毗连位置的区段，其中对两个序列进行最优比对后，可将序列与具有相同数量毗连位置的参比序列作比较。比对序列的比较方法是本领域熟知的。可进行最优序列比对以作比较，例如，通过Smith和Waterman的局部同源性算法，Adv.Appl.Math.2：482(1981)；通过Needleman和Wunsch的同源性比对算法，J.Mol.Biol.48：443(1970)；通过Pearson和Lipman的相似性搜索法，Proc.Nat’l.Acad.Sci.USA 85：2444(1988)；通过计算机执行这些算法(阿克赛勒里公司(Accelrys)的威斯康星遗传学软件包(Wisconsin GeneticsSoftware Package)中的GAP、BESTFIT、FASTA和TFASTA)，或通过手工比对和目测。
使用BLAST算法确定百分比序列相同性和序列相似性，其在Altschul等，J.Mol.Biol.215：403-410(1990)中描述。进行BLAST分析的软件可从国家生物技术信息中心(National Center for Biotechnology Information)公开获得(http：//ncbi.nlm.nih.gov/)。此算法包括：首先通过鉴定查询序列中长度为W的短字来鉴定高评分序列对(HSP)，与数据库序列中长度相同的字比对时它们能匹配或满足一些正值的阈值评分T。T称为相邻字评分阈值(Altschul等，同上)。这些初始相邻字命中用作启动搜索的种子，以便找到含有它们的较长HSP。只要可提高累积比对评分，该字命中在两个方向上沿各序列延伸。出现以下情况时中止字命中在各个方向上的延伸：累积比对评分比其最大获得值降低X；由于一个或多个负评分残基比对的累积，累积评分变为零或零以下；或者达到任一序列的末端。BLAST算法参数W、T和X决定比对的灵敏度和速度。BLAST程序使用的默认值为：字长(W)11，BLOSUM62评分矩阵(参见Henikoff和Henikoff，Proc.Natl.Acad.Sci.USA89：10915(1989))比对(B)50，期望值(E)10，M＝5，N＝-4，以及比较两条链。
发明详述
I.引言
已经令人惊讶地发现，两种DNA聚合酶的共价连接产生热稳定性更高的多肽，并且在一些情况下与单体聚合酶相比具有改善的(更短的)反应时间。例如，如实施例中所示，相同或不同逆转录酶的蛋白质融合产生与对照单体逆转录酶相比具有更大热稳定性和在一些情况下更低反应时间的多肽。
II.二聚蛋白质
本文提供了二聚(和三聚)DNA聚合酶。这样的多肽可以是分离的或纯化的，或者可以处于复杂的混合物中，或者例如在细胞中。如本文所述的多肽可以包含，例如，至少两种并且在一些实施方式中，包含三种不同的DNA聚合酶。不同的DNA聚合酶可以是相同的，或者它们可以具有不同的序列。
为方便起见，在二聚多肽中，最靠近多肽氨基末端的DNA聚合酶被称为“第一”DNA聚合酶，离羧基末端最近的DNA聚合酶被称为“第二”DNA聚合酶。在多肽是三聚体的情况下，中间的DNA聚合酶是“第二”，最靠近多肽羧基末端的DNA聚合酶是“第三”DNA聚合酶。“DNA聚合酶”是指具有DNA聚合酶活性的任何蛋白质(如天然存在的全长或其片段或变体)。类似地，“逆转录酶”是指具有逆转录酶活性的任何蛋白质(如天然存在的全长或其片段或变体)。
在一些实施方式中，多肽中的一种或两种DNA聚合酶(或在三聚体情况下，一种，两种或全部三种)可以是逆转录酶，即RNA依赖性DNA聚合酶。在一些实施方式中，本文所述的多肽包含两种逆转录酶。在其中多肽包含两种逆转录酶的实施方式中，逆转录酶可以是序列相同的，或逆转录酶可以具有不同的序列。在一些实施方式中，两种逆转录酶彼此至少80％，85％，90％，95％或99％相同。在其他实施方式中，两种逆转录酶彼此相同性小于70％，80％或90％(例如40-70，40-80，40-90％)。例如，可以选择互补但活性不同的两种不同的逆转录酶(或一种逆转录酶和一种DNA依赖性DNA聚合酶)，使得包含两种逆转录酶(或一种RT和一种DNA依赖性DNA聚合酶)的多肽具有与单独的单体酶中的任一种相比优异的活性。
可以是第一或第二(或第三)逆转录酶的示例性逆转录酶包括但不限于，鼠白血病病毒(MLV)逆转录酶(Gerard和Grandgenett，Journal of Virology 15：785-797，1975；Verma，Journal ofVirology 15：843-854，1975)或SEQ ID NO：1，猫白血病病毒(FLV)逆转录酶(Rho和Gallo，Cancer Lett.，10：207-221，1980)或SEQ ID NO：1，牛白血病病毒(BLV)(Demirhan等，Anticancer Res.，16：2501-5，1996；Drescher等，Arch Geschwulstforsch.，49：569-79，1979)，禽成髓细胞瘤病毒(AMV)逆转录酶，呼吸道合胞病毒(RSV)逆转录酶，马传染性贫血病毒(EIAV)逆转录酶，Rous相关病毒-2(RAV2)逆转录酶，SUPERSCRIPT II逆转录酶，SUPERSCRIPT III逆转录酶(US8541219，US7056716，US7078208)，THERMOSCRIPT转录酶和MMLV RNA酶H-逆转录酶。
在某些情况中，DNA聚合酶中的一种或两种(例如逆转录酶)是天然聚合酶。在其他实施方式中，DNA聚合酶中的一种或两种(例如逆转录酶)包含一个或多个(例如1，2，3，4，5，6，7，8，9，10，1-20，1-10，1-5，1-2个)突变(包括但不限于SEQ ID NO：1或SEQ ID NO：2)。示例性突变包括但不限于降低(例如降低至少50，70或95％)或消除RNA酶H活性(称为“RNA酶H-突变”)的突变。也可以引入增加RT中热稳定性的突变。这种类型的示例性突变描述于例如Arezi等，Nucleic Acids Res.2009年2月；37(2)：473-481。
在一些实施方式中，聚合酶包含至少两种(例如2或3种)DNA依赖性DNA聚合酶。示例性的DNA依赖性DNA聚合酶包括但不限于五个DNA依赖性DNA聚合酶家族的任何聚合酶，尽管大部分将归入A，B和C家族。各家族之间很少或没有结构或序列相似性。大多数A家族聚合酶是可含有多重酶促功能(包括聚合酶、3′到5′核酸外切酶活性和5′到3′核酸外切酶活性)的单链蛋白质。B家族聚合酶通常有具有聚合酶和3′到5′核酸外切酶活性的单个催化结构域，以及辅助因子。C家族聚合酶通常是具有聚合和3′到5′核酸外切酶活性的多亚基蛋白质。在大肠杆菌中，已经发现了3种类型的DNA聚合酶，DNA聚合酶I(A家族)、DNA聚合酶II(B家族)和DNA聚合酶III(C家族)。在真核细胞中，核复制中涉及3种不同的B家族聚合酶，DNA聚合酶α、δ和ε，并且A家族聚合酶，聚合酶γ用于线粒体DNA复制。示例性的DNA依赖性聚合酶可以包括但不限于，Taq聚合酶或iProofDNA聚合酶。参见例如，美国专利号8,916,352。
任选地，DNA聚合酶可以连接至非特异性DNA结合结构域。这类蛋白质的示例包括但不限于古细菌小基础DNA结合蛋白Sso7d和Sso7d样蛋白(参见例如，Choli等，Biochimicaet Biophysica Acta 950：193-203，1988；Baumann等，StructuralBiol.1：808-819，1994；以及Gao等，Nature Struc.Biol.5：782-786，1998)，古细菌HMf样蛋白(参见例如，Starich等，J.Molec.Biol.255：187-203，1996；Sandman等，Gene 150：207-208，1994)，以及PCNA同源物(参见例如，Cann等，J.Bacteriology 181：6591-6599，1999；Shamoo和Steitz，Cell；99，155-166，1999；De Felice等，J.Molec.Biol.291，47-57，1999；以及Zhang等，Biochemistry 34：10703-10712.1995)。
Sso7d和Sso7d样蛋白、Sac7d和Sac7d样蛋白(例如Sac7a、Sac7b、Sac7d和Sac7e)是分别来自极端嗜热性古细菌硫磺矿硫化叶菌(Sulfolobus solfataricus)和酸热脂环酸硫化叶菌(S.acidocaldarius)的小(约7,000kd MW)、基础染色体蛋白。这些蛋白质是富赖氨酸的且具有高度的热、酸和化学稳定性。其以不依赖序列的方式结合DNA，在一些情况下，一旦结合，使得DNA的Tm升高最多达40℃(McAfee，Biochemistry 34：10063-10077，1995；Gao等，Nat.Struct.Biol.5(9)：782-786，1998)。这些蛋白质及其同源物通常被认为参与在升高的温度下稳定基因组DNA。用于本发明的合适的Sso7d样DNA结合结构域可基于其与Sso7d的序列同源性进行修饰。通常，在约25个氨基酸的比较窗口上(任选约50-100个氨基酸或整个蛋白质的长度)与已知的DNA结合蛋白相同或基本相同的DNA结合结构域可用于本发明。该序列可针对比较窗口或指定区域上的最大对应性进行比较和比对，所述最大对应性的测量方法为使用描述的比较算法之一或通过手动比对和视觉观察。Sso7结合结构域中的多种突变已描述于例如美国专利申请号2005/0048530和2007/0141591。
除了两种(或三种)DNA聚合酶和接头之外，多肽还可以包含额外的氨基酸序列。例如，在一些实施方式中，多肽在多肽的氨基或羧基末端包含额外的序列。这样的序列的示例包括但不限于用于亲和纯化多肽的序列或荧光蛋白。另外，在一些实施方式中，多肽可以包括一个或多个荧光标记物或荧光标记物/猝灭剂对。
III.接头
所描述的两种DNA聚合酶可通过接头通过本领域技术人员熟知的方法接合。这些方法可包括化学和重组手段。
化学连接可以例如如《生物偶联技术》(Bioconjugate Techniques)，Hermanson编，学术出版社(Academic Press)(1996)中所述进行。接合可包括，例如，出于将2种蛋白质互相连接的目的的衍生化，所述衍生化通过蛋白质化学领域所熟知的方法以直接或通过连接化合物的方式进行。例如，在一个化学偶联实施方式中，连接催化结构域和核酸结合结构域的手段包括杂双官能-偶联剂，其最终导致在2个基团之间形成分子间二硫键。可用于本发明的这种能力的其他类型的偶联剂描述于，例如，美国专利号4,545,985。或者，分子间二硫键可在各基团中的半胱氨酸之间方便地形成，其是天然产生的或者通过遗传工程改造插入的。连接基团的手段也可使用双官能交联剂或特定低pH可切割交联剂之间的硫醚连接或特定蛋白酶可切割接头或其他可切割或不可切割化学连接。其他化学接头包括碳水化合物接头、脂质接头、脂肪酸接头、聚醚接头，如PEG，等。例如，聚(乙二醇)接头可获自阿拉巴马州汉茨维尔的谢氏聚合物有限公司(Shearwater Polymers，Inc.)。这些接头任选具有酰胺键、巯基键或杂双官能键。
连接两种DNA聚合酶也可包括在基团之间形成肽基键，其通过标准肽合成化学或重组手段分开合成。也可使用化学方法来产生偶联蛋白质本身以整体或部分合成氨基酸序列。例如，可通过固相技术来合成肽，例如，Merrifield固相合成方法，其中，向氨基酸的生长链连续加入氨基酸(参见，Merrifield(1963)J.Am.Chem.Soc.，85：2149-2146)。用于自动化合成多肽的设备可购自供应商如PE公司(加利福尼亚州福斯特城(Foster City，CA))并且一般可按照生产商的说明操作。然后合成的肽可从树脂上切下，并且纯化，例如，通过制备型高效液相色谱(参见Creighton，Proteins Structures and Molecular Principles(《蛋白质、结构和分子原理》)，50-60(1983))。可通过氨基酸合成或测序来确认合成多肽或多肽亚片段的组成(例如，埃得曼降解步骤；参见，Creighton，Proteins，Structures andMolecular Principles(《蛋白质、结构和分子原理》)，第34-49页(1983))。
另外，可以取代或添加向序列中导入非经典氨基酸或化学氨基酸类似物。非经典氨基酸一般包括但不限于：普通氨基酸的D-异构体、α-氨基异丁酸、4-氨基丁酸、Abu、2-氨基丁酸、γ-Abu、ε-Ahx、6-氨基己酸、Aib、2-氨基异丁酸、3-氨基丙酸、鸟氨酸、正亮氨酸、正缬氨酸、羟基脯氨酸、肌氨酸、瓜氨酸、磺基丙氨酸、叔丁基甘氨酸、叔丁基丙氨酸、苯基甘氨酸、环己基丙氨酸、β-丙氨酸、氟代-氨基酸、设计氨基酸如β-甲基氨基酸、Cα-甲基氨基酸、Nα-甲基氨基酸和氨基酸类似物。此外，氨基酸可以是D(右旋)或L(左旋)。
在一些实施方式中，两种DNA聚合酶通过连接基团连接。连接基团可以是化学交联剂，包括，例如琥珀酰亚胺基-(N-马来酰亚胺甲基)-环己烷-1-羧酸酯(SMCC)。连接基团也可以是其他氨基酸序列，包括，例如，聚丙氨酸、聚甘氨酸或类似的连接基团。
或者，在一些实施方式中，多肽中每种DNA聚合酶的编码序列直接连接并表达为融合蛋白。或者，氨基酸接头序列也可编码在多肽编码序列中并用于将第一和第二多肽组分分开足够的距离以确保每个多肽折叠成其二级和三级结构。使用本领域熟知的重组技术将这类氨基酸接头序列整合至融合蛋白中。合适的肽接头序列可基于以下因素选择：(1)其采取挠性延伸构象的能力；(2)其采取可与第一和第二多肽上功能性表位相互作用的二级结构的能力；以及(3)缺少可与多肽功能性表位反应的疏水性或带电残基。典型的肽接头序列含有Gly、Ser、Val和Thr残基。其他的近中性氨基酸(如Ala)也可用于接头序列。可用作接头的氨基酸序列包括下述中公开的那些：Maratea等，(1985)Gene 40：39-46；Murphy等，(1986)Proc.Natl.Acad.Sci.USA83：8258-8262；美国专利号4,935,233和4,751，180。该接头序列的长度通常可以是1至约50个氨基酸，例如长度为3、4、6或10个氨基酸，但长度也可以是100或200个氨基酸。当第二多肽具有可用于分隔功能性结构域且阻止位阻干扰的非必需N端氨基酸区域时，可以不需要接头序列。
IV.表达和纯化
编码DNA聚合酶的核酸可以使用重组遗传学领域的常规技术获得。公开本发明所用一般方法的基础文本包括Sambrook和Russell，Molecular Cloning，A LaboratoryManual(《分子克隆，实验室手册》)(第3版，2001)；Kriegler，Gene Transfer andExpression：A Laboratory Manual(《基因转移和表达：实验室手册》)(1990)；以及CurrentProtocols in Molecular Biology(《新编分子生物学实验指南》)(Ausubel等编，1994-1999))。这类核酸还可通过体外扩增方法获得，例如描述于以下文献中的那些：Berger，Sambrook，和Ausubel，以及Mullis等(1987)美国专利号4,683,202；PCR ProtocolsAGuideto Methods and Applications(《PCR实验方法：方法和应用指南》)(Innis等编)学术出版社公司，加利福尼亚州圣地亚哥(1990)(Innis)；Arnheim和Levinson(1990年10月1日)C&EN36-47；The Journal OfNIHResearch(1991)3：81-94；Kwoh等(1989)Proc.Natl.Acad.Sci.USA 86：1173；Guatelli等(1990)Proc.Natl.Acad.Sci.USA 87，1874；Lomell等(1989)J.Clin.Chem.，35：1826；Landegren等(1988)Science 241：1077-1080；Van Brunt(1990)Biotechnology 8：291-294；Wu和Wallace(1989)Gene 4：560；以及Barringer等(1990)Gene 89：117，其各自通过引用全文纳入本文用于所有目的且尤其是用于所有涉及扩增方法的教导。
技术人员将认识到可以对DNA聚合酶进行另外的修饰而不降低其生物活性。可进行一些修饰以促进克隆、表达或将结构域整合至融合蛋白中。这类修饰为本领域技术人员所熟知并且包括，例如，在编码结合结构域的多核苷酸的一端添加密码子(例如在氨基端添加甲硫氨酸)以提供起始位点，或将额外的氨基酸(如聚His)置于一端以建立方便地设置的限制性酶切位点或终止密码子或纯化序列。
本发明所述的融合多肽可在多种宿主细胞中表达，包括大肠杆菌、其他细菌宿主、酵母、丝状真菌和多种高级真核细胞如COS、CHO和HeLa细胞系和骨髓瘤细胞系。在微生物中进行基因表达的技术描述于例如Smith，Gene Expression in RecombinantMicroorganisms(重组微生物中的基因表达)(Bioprocess Technology(《生物加工技术》)，卷22)，Marcel Dekker，1994。可用于表达的细菌的示例包括但不限于，埃希菌属，肠杆菌，固氮菌属，欧文氏菌属，杆菌属，假单胞菌属，克雷伯氏菌属，变形杆菌属，沙门氏菌属，沙雷氏菌属，志贺氏菌属，根瘤菌属，透明颤菌属和副球菌属。可用作表达宿主的丝状真菌包括例如下列属：曲霉属，木霉属，链孢霉属，青霉属，头孢霉属，绵霉属，柄孢壳菌属，毛霉属，旋孢腔菌属和梨孢属。参见，例如，美国专利号5,679,543以及Stahl和Tudzynski编，《丝状真菌分子生物学》(Molecular Biology in Filamentous Fungi)，约翰韦利父子公司(JohnWiley&Sons)，1992。在酵母中合成异源蛋白质是众所周知的并描述于文献中。Sherman，F等，《酵母遗传方法》(Methods in Yeast Genetics)，冷泉港实验室(1982)是一项熟知的工作，描述了可用于在酵母中产生酶的各种方法。
存在许多本领域普通技术人员已知的生成多肽的表达系统。(参见例如，GeneExpression Systems(《基因表达系统》)Fernandex和Hoeffler编，学术出版社，1999；Sambrook和Russell，同上；以及Ausubel等，同上)。通常，编码多肽的多核苷酸位于在所需宿主细胞中具有功能的启动子的控制下。许多不同的启动子是可用的且是本领域技术人员已知的，且可用于本发明的表达载体中，这取决于具体应用。通常，选择的启动子依赖于其中启动子将具有活性的细胞。还可任选地包括其他表达控制序列，如核糖体结合位点、转录终止位点等。包含一种或多种这些控制序列的构建体称作“表达盒”。因此，编码接合的多肽的核酸经整合以在所需宿主细胞中高水平表达。
通常通过克隆在特定细胞中表达的基因来将获得适用于特定宿主细胞的表达控制序列。通常使用的原核控制序列(其在本发明中定义为包含用于转录起始的启动子(任选具有操纵子)以及核糖体结合位点序列)包括这类通常使用的启动子作为β-内酰胺酶(青霉素酶)和乳糖(1ac)启动子系统(Change等，Nature(1977)198：1056)、色氨酸(trp)启动子系统(Goeddel等，Nucleic Acids Res.(1980)8：4057)、tac启动子(DeBoer等，Proc.Natl.Acad.Sci.U.S.A.(1983)80：21-25)；以及λ衍生的PL启动子和N基因核糖体结合位点(Shimatake等，Nature(1981)292：128)。具体的启动子系统并不重要，可使用在原核细胞中具有功能并提供所需活性水平的任何可用的启动子。标准的细菌表达载体包括质粒，如基于pBR322的质粒(如pBLUESCRIPTTM)、pSKF、pET23D、λ噬菌体衍生的载体和融合表达系统(如GST和LacZ)。还可向重组蛋白添加表位标签以提供方便的分离方法，例如c-myc、HA-标签、6-His标签、麦芽糖结合蛋白、VSV-G标签、抗DYKDDDDK标签或任何这类标签，许多标签是本领域技术人员熟知的。
为了在大肠杆菌以外的原核细胞中表达，需要在特定的原核生物物种中起作用的启动子。这种启动子可以从已从该物种克隆的基因获得，或者可以使用异源启动子。例如，除了大肠杆菌以外，杂交trp-lac启动子在芽孢杆菌属中起作用。这些和其它合适的细菌启动子是本领域熟知的，参见例如Sambrook等和Ausubel等。用于表达本发明的蛋白质的细菌表达系统可获自，例如大肠杆菌、芽孢杆菌(Bacillus sp.)和沙门菌(Salmonella)中(Palva等，Gene 22：229-235(1983)；Mosbach等，Nature 302：543-545(1983)。这类表达系统的试剂盒市售可得。
哺乳动物细胞、酵母和昆虫细胞的真核表达系统是本领域众所周知的，并且也可从市场上购得。在酵母中，载体包括酵母整合质粒(例如YIp5)和酵母复制质粒(YRp系列质粒)和pGPD-2。一般将含有真核病毒调控元件的表达载体用于真核表达载体，如SV40载体、乳头瘤病毒载体和衍生自EB病毒的载体中。其它示范性真核载体包括pMSG、pAV009/A+、pMTO10/A+、pMAMneo-5、杆状病毒pDSVE以及能够在CMV启动子、SV40早期启动子、SV40晚期启动子、金属硫蛋白启动子、鼠乳房肿瘤病毒启动子、劳氏肉瘤病毒启动子、多角体蛋白启动子或在真核细胞中显示有效表达的其它启动子的指导下表达蛋白质的任何其它载体。
可以使用组成型或调节型启动子。调控的启动子可能是有利的，因为在诱导融合多肽的表达之前，宿主细胞可以生长至高密度。在某些情况下，异源蛋白的高水平表达会减缓细胞生长。诱导型启动子是在表达水平由环境或发育因子如温度，pH，厌氧或需氧条件，光，转录因子和化学品改变的情况下指导基因表达的启动子。
对于大肠杆菌和其他细菌宿主细胞，诱导型启动子是本领域技术人员已知的。这些包括例如lac启动子，噬菌体λPL启动子，杂合trp-lac启动子(Amann等，(1983)Gene 25：167；de Boer等，(1983)Proc.Nat′1.Acad.Sci.USA 80：21)和噬菌体T7启动子(Studier等，(1986)J.Mol.Biol.；Tabor等，(1985)Proc.Nat′l Acad.Sci.USA 82：1074-8)。这些启动子及其用途也在上文的Sambrook等中进行了讨论。
翻译偶联可用于增强表达。该策略使用来源于翻译系统天然高度表达基因的短上游开放阅读框，其位于启动子的下游，和核糖体结合位点，其在几个氨基酸密码子之后有终止密码子。恰好在终止密码子之前是第二核糖体结合位点，并且在终止密码子之后是起始翻译的起始密码子。该系统溶解RNA中的二级结构，从而高效启动翻译。参见Squires等，(1988)，J.Biol.Chem.263：16297-16302。
多核苷酸构建体的构建通常需要使用能够在细菌中复制的载体。这种载体通常用于本领域。可商购获得多种试剂盒用于从细菌纯化质粒(例如，来自法玛西亚生物技术公司(Pharmacia Biotech)的EasyPrepTM，FlexiPrepTM；来自司查塔基公司(Stratagene)的StrataCleanTM；和QIAexpress Expression System，凯捷(Qiagen))。然后可以进一步操纵分离和纯化的质粒以产生其他质粒，并用于转化细胞。
本文所述的多肽可以在细胞内表达，或可以从细胞分泌。细胞内表达常常导致高产量。如果需要，可通过进行重折叠程序来增加可溶性活性融合多肽的量(参见例如Sambrook等，同上；Marston等，Bio/Technology(1984)2：800；Schoner等，Bio/Technology(1985)3：151)。多肽可在多种宿主细胞中表达，包括大肠杆菌、其他细菌宿主、酵母、和多种高级真核细胞如COS、CHO和HeLa细胞系和骨髓瘤细胞系。宿主细胞可以是哺乳动物细胞，昆虫细胞或微生物，例如酵母细胞，细菌细胞或真菌细胞。
一旦表达，可按照本领域的标准程序纯化多肽，包括硫酸铵沉淀，亲和柱，柱层析，凝胶电泳等(一般参见R.Scopes，“蛋白质纯化”(Protein Purification)，Springer-Verlag，N.Y.(1982)，Deutscher，Methods in Enzymology卷182：《蛋白质纯化指南》(Guideto Protein Purification)，学术出版公司，纽约(1990))。优选至少约90-95％均一性的基本纯化的组合物，并且最优选98-99％或更高的均一性。一旦根据需要部分纯化或均一化，然后可以使用多肽(例如，作为用于抗体生产的免疫原)。
为了便于多肽的纯化，编码多肽的核酸还可以包括可用亲和结合试剂针对的表位或“标签”的编码序列。合适的表位的示例包括myc和V-5报道基因；可用于重组生产具有这些表位的融合多肽的表达载体是可商购的(例如，英杰(Invitrogen)(加利福尼亚州卡尔斯巴德)载体pcDNA3.1/Myc-His和pcDNA3.1/V5-His适于在哺乳动物细胞中表达)。适用于将标签附着到本发明的融合蛋白上的其他表达载体和相应的检测系统是本领域技术人员已知的，并且一些是可商购的(例如，“FLAG”(柯达公司(Kodak)，纽约州罗切斯特(RochesterN.Y.))。合适的标签的另一个示例是能够结合金属螯合亲和配体的聚组氨酸序列。通常使用六个相邻的组氨酸，尽管可以使用多于或少于六个。可用作聚组氨酸标签的结合部分的合适金属螯合亲和配体包括次氮基三乙酸(NTA)(Hochuli，E.(1990)“用金属螯合吸附剂纯化重组蛋白”，在《基因工程改造：原理和方法》(Gene Engineering：Principlesand Methods)中，J.K.Setlow编，普莱南出版社(Plenum Press)，纽约；可从凯捷(Qiagen(加利福尼亚州圣克拉丽塔(Santa Clarita，Calif.))商购获得))。
本领域技术人员将认识到，在生物表达或纯化之后，聚合酶肽可具有与组成多肽的天然构象显著不同的构象。在这种情况下，可能需要或希望变性并还原多肽，然后使多肽重新折叠成优选的构象。减少和变性蛋白质和诱导重折叠的方法是本领域技术人员熟知的(参见Debinski等，(1993)J.Bio1.Chem.268：14065-14070；Kreitman和Pastan(1993)Bioconiug.Chem.4：581-585；和Buchner等，(1992)Anal.Biochem.205：263-270)。例如，Debinski等描述了胍-DTE中包涵体蛋白的变性和还原。然后将蛋白质在含有氧化型谷胱甘肽和L-精氨酸的氧化还原缓冲液中重折叠。
V.使用方法
逆转录(RT)是将RNA复制成DNA的扩增方法。RT反应可以如本文所述用反应混合物进行。例如，本发明提供了在允许逆转录和产生第一链和任选的第二链cDNA的条件下逆转录一种或多种RNA(包括例如，细胞中的所有RNA，例如制备cDNA文库)。RT反应可以用随机引物，寡聚dT或RNA特异性引物引发。RT反应的组分和条件通常是已知的。
如果需要，反应可以进一步包括RT-PCR。用于进行PCR测定的标准技术是本领域已知的(《PCR技术：DNA扩增的原理和应用》(Principles and Applications for DNAAmplification)(Erlich编，1989)；《PCR方案：方法和应用指南》(PCR Protocols：A Guideto Methods and Applications)(Innis，Gelfland，Sninsky，和White编，1990)；Mattila等，Nucleic Acids Res.19：4967(1991)；Eckert和Kunkel，《PCR方法和应用》(PCR Methodsand Applications)1：17(1991)；Wallace等，连接酶链反应(Ligase Chain Reaction)，在《DNA损伤和突变检测技术》(Technologies for Detection of DNA Damage andMutations)中，第307-322页(Pfiefer编，1996))。RT和PCR反应通常用于相同的测定中，并被称为RT-PCR。RT-PCR将RNA逆转录成DNA与随后在单个反应中进行DNA扩增反应结合在一起。最佳的逆转录，杂交和扩增条件将根据所使用的引物和靶标的序列组成和长度，以及实施者选择的实验方法而变化。可以使用各种指南来选择合适的引物序列和杂交条件(参见例如Sambrook等，《分子克隆：实验室手册》(Molecular Cloning：A Laboratory Manual)(第2版)(1989)；Ausubel，F.M.等编，(1999)《分子生物学简明方案》(Short Protocols inMolecular Biology)，第4版，约翰威利父子有限公司(John Wiley&Sons))；Ausubel，F.M.等编，(1999-2010)《新编分子生物学方案》(Current Protocols in Molecular Biology)，约翰威利父子有限公司)。
本发明的实施可采用化学、生物化学、分子生物学、细胞生物学、遗传学、免疫学和药理学的常规方法，这些方法在本领域技术范围内。这些技术在文献中已有充分描述。参见例如Gennaro，A.R.编，(1990)《雷明顿药物科学》(Remington′s PharmaceuticalSciences)，第18版，马克出版公司(Mack Publishing Co.)；Hardman，J.G.，Limbird，L.E.和Gilman，A.G.编，(2001)《治疗的药理学基础》(The Pharmacological Basis ofTherapeutics)，第10版，麦格劳希尔公司(McGraw-Hill Co.)；Colowick，S.等编，《酶学方法》(Methods In Enzymology)，学术出版公司；Weir，D.M.和Blackwell，C.C.编，(1986)实验免疫学手册(Handbook ofExperimental Immunology)，卷I-IV，布莱克威尔科学出版公司(Blackwell Scientific Publications)；Maniatis，T.等编，(1989)《分子克隆：实验室手册》(Molecular Cloning：A Laboratory Manual)，第2版，卷I-III，冷泉港实验室出版社；Ausubel，F.M.等编，(1999-2010)《新编分子生物学方案》(Current Protocols inMolecular Biology)，约翰威利父子有限公司；Ream等编，(1998)《分子生物学技术：详细实验室课程》(Molecular Biology Techniques：An Intensive Laboratory Course)，学术出版社；Newton，C.R.和Graham，A.编，(1997)《PCR(生物技术入门系列)》(PCR(Introductionto Biotechniques Series))，第2版，施普林格出版社(Springer Verlag)；Sambrook等，《分子克隆：实验室手册》(Molecular Cloning：A Laboratory Manual)(第2版)(1989)。
本发明反应混合物的一个优点是反应混合物允许在比可能的温度更高的温度下于RT或DNA依赖性DNA聚合酶反应中使用逆转录酶。因此，在实施方式中，本文所述的二聚或三聚酶可以在37℃或42℃或高于42℃的温度下使用，例如43-55℃，45-56℃，45-65℃等。较高温度的RT反应在模板RNA在部分或完全抑制逆转录的正常RT温度(例如37℃或42℃)下形成二级结构的情况下特别有用。
在其中如本文所述的多肽包含一种或两种DNA依赖性DNA聚合酶的情况下，所述多肽可以以模板依赖性方式用于引物延伸反应中。在一些实施方式中，引物延伸反应是扩增反应。在一些实施方式中，实时监测扩增反应，并任选用于定量靶核酸的初始量。
这类扩增反应包括但不限于聚合酶链式反应(PCR)、DNA连接酶链式反应(LCR)、和基于RNA转录(如TAS和3SR)的扩增反应以及本领域技术人员已知的其他反应。可使用本发明所述组合物进行的聚合酶链式反应包括但不限于逆转录PCR(rt-PCR)和定量PCR(qPCR)。
在一些实施方式中，使用基于染料的qPCR检测方法来监测利用本发明组分的扩增反应。这种检测方法通常依赖于监测由DNA结合染料与扩增的DNA结合导致的荧光信号的增加。例如，SYBR Green I是一种常用的荧光DNA结合染料，其结合所有双链DNA，并通过测量整个周期内荧光的增加来监测检测结果。SYBR Green I分别具有494nm和521nm的激发和发射最大值。
在其它实施方式中，使用基于探针的qPCR检测方法来监测利用本发明组分的扩增反应。这样的检测方法通常依赖于期望的PCR产物的序列特异性检测。与检测所有双链DNA的基于染料的qPCR方法不同，基于探针的qPCR使用荧光标记的靶标特异性探针，其检测扩增的DNA中的特定序列。
VI.反应混合物
提供了包含本文所述多肽的反应混合物。反应混合物可以包含例如靶核酸，例如发生逆转录的RNA靶标或发生DNA依赖性聚合酶反应的DNA。反应混合物可以包含合适的核苷酸(例如，脱氧核苷酸(dNTP)或双脱氧核苷酸)，并且在一些实施方式中，包含至少一种缓冲剂。示例性的缓冲剂可包括例如但不限于，Tris，HEPES，ACES，PIPES，MOPSO，BES，MOPS，TES，TAPSO，POPSO，BICINE，TAPS或AMPSO。反应混合物可以任选地包含一种或多种用作模板依赖性核酸延伸的引物的寡核苷酸，一种或多种用作探针的寡核苷酸(例如，与标记物例如猝灭剂，荧光染料等连接)，和/或双链DNA结合染料(例如SYBRGREEN)。在一些实施方式中，反应混合物将进一步包含单独的DNA依赖性DNA聚合酶。在一些实施方式中，反应混合物将进一步包含镁(Mg++)。
VII.试剂盒
在一个方面，提供了用于进行核酸延伸(和任选的环状扩增，例如PCR)反应的试剂盒。在一些实施方式中，这类试剂盒包含聚合酶，和任选的dNTP，以及至少一种缓冲剂。这类试剂盒也可包含稳定剂和其他添加剂(例如，肝素和/或肌氨酸)以提高扩增反应的效率。这类试剂盒还可包含一种或多种引物以及使用试剂盒的组分进行核酸扩增反应的指南。在一些实施方式中，试剂盒将进一步包含单独的DNA依赖性DNA聚合酶。
实施例
方法和材料
克隆和表达：
通过PCR将MLV逆转录酶基因克隆到pET28载体中以扩增该基因，然后进行限制性内切酶消化和连接。表达构建体含有编码组氨酸标签的基因，该基因与逆转录酶的N末端融合。两个MLV基因通过含有限制性位点HindIII，NheI和SacI的接头序列连接在一起。
将表达质粒转化到大肠杆菌BL21/DE3细胞中。将含有表达质粒的细胞接种在LB培养基中过夜培养，第二天早上用1/100的培养物接种新鲜的LB培养基。细胞在16℃下生长。细胞密度达到OD600＝0.6至0.8后，加入IPTG至0.1mM以诱导蛋白质表达。诱导后15小时收集细胞。
MLV RT氨基酸序列-SEQ ID NO：1：
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL
FLV RT氨基酸序列-SEQ ID NO：2：
TLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTIL
MM二聚RT序列(接头加下划线)-SEQ ID NO：3：
EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSAS
MNM二聚RT序列(接头加下划线)-SEQ ID NO：4：
EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSAS
MNMN二聚RT序列(接头加下划线)-SEQ ID NO：5：
EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSAS
FF二聚RT序列(接头加下划线)-SEQ ID NO：6：
EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSAS
FNF二聚RT序列(接头加下划线)-SEQ ID NO：7：
EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSAS
FNFN二聚RT序列(接头加下划线)-SEQ ID NO：8：
EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSAS
FM二聚RT序列(接头加下划线)-SEQ ID NO：9：
EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSAS
FNM二聚RT序列(接头加下划线)-SEQ ID NO：10：
EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSAS
FNMN二聚RT序列(接头加下划线)-SEQ ID NO：11：
EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSAS
MF二聚RT序列(接头加下划线)-SEQ ID NO：12：
EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSAS
MNF二聚RT序列(接头加下划线)-SEQ ID NO：13：
EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSAS
MNFN二聚RT序列(接头加下划线)-SEQ ID NO：14：
EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSAS
FFN二聚RT序列(接头加下划线)-SEQ ID NO：15：
EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSAS
FMN二聚RT序列(接头加下划线)-SEQ ID NO：16：
EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSAS
MFN二聚RT序列(接头加下划线)-SEQ ID NO：17：
EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSAS
MMN二聚RT序列(接头加下划线)-SEQ ID NO：18：
EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSAS
酶纯化：
表达该酶的大肠杆菌细胞重新悬浮在裂解缓冲液中。用超声波脉冲(2x 30秒，占空比80％，输出10)在冰上破碎细胞，并以19000rpm离心10分钟。通过0.22um过滤单元过滤上清液，用10x缓冲液A稀释以进行iMAC层析(50mM KPO4，pH 6.5，300mM KCl，0.1％吐温20)，并上样到Nuvia iMAC柱上。用线性梯度将酶洗脱至缓冲液B(缓冲液A加500mM咪唑)。合并含有酶的级分并用Amicon Ultra-15离心过滤单元浓缩。将样品施加于尺寸排阻柱(SEC650)，收集含有纯化酶的级分。
RT-qPCR测定：
iScript高级逆转录混合物用于cDNA的逆转录。将纯化的酶加入到反应中。iScript高级逆转录酶用作对照。为了研究温度对cDNA合成的影响，逆转录反应分别在42，55和60℃下进行30分钟。纯化酶的cDNA合成速度也与iScript高级逆转录酶相比较，其中逆转录分别在iScript高级反应混合物中进行30、2、1分钟。cDNA合成后，对不同的靶标进行qPCR。为了评估cDNA合成的性能，使用42℃的cDNA反应30分钟作为对照计算ΔCq(dCq)。
表1.二聚MLV RT(MNM和MM)显示比野生型对照更好的热稳定性。两步RT-qPCR在6种不同的靶标上进行。
Figure BDA0001688558490000381
表2：另外的二聚RT显示比野生型对照更好的热稳定性。模板靶标是人GAPDH。
Figure BDA0001688558490000382
MF：MLVRT-FLVRT二聚体；
MNF：MLVRT RNA酶H(-)-FLVRT二聚体；
MM：MLVRT-MLVRT二聚体；
MNM：MLVRT RNA酶H(-)-MLVRT二聚体；
FNM：FLVRT RNA酶H(-)-MLVRT二聚体；
RT对照：纯化的单体RT。
全部五种二聚RT在两个温度下表现出比单体RT对照更好的稳定性，例如由较低的dCq值所显示。与RT对照相比，全部五种二聚RT还表现出缩短的反应时间。
本文所述的实施例和实施方式仅用于说明目的，本领域技术人员应了解据此作出的各种修饰或改变，且它们包括在本申请的主旨和权益以及所附权利要求书的范围内。本文引用的所有出版物、专利和专利申请通过引用全文纳入本文以用于所有目的。
序列表
<110> 生物辐射实验室股份有限公司（Bio-Rad Laboratories, Inc.）
<120> 二聚逆转录酶
<130> 094260-110910PC-1031618
<150> US 62/264,223
<151> 2015-12-07
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 671
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 1
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
<210> 2
<211> 667
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 2
Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu Phe Glu Pro Glu Ser Thr
1 5 10 15
Gln Lys Gln Glu Met Asp Ile Trp Leu Lys Asn Phe Pro Gln Ala Trp
20 25 30
Ala Glu Thr Gly Gly Met Gly Met Ala His Cys Gln Ala Pro Val Leu
35 40 45
Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile Ser Ile Arg Gln Tyr Pro
50 55 60
Met Pro His Glu Ala Tyr Gln Gly Ile Lys Pro His Ile Arg Arg Met
65 70 75 80
Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln Ser Pro Trp Asn Thr Pro
85 90 95
Leu Leu Pro Val Lys Lys Pro Gly Thr Lys Asp Tyr Arg Pro Val Gln
100 105 110
Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr Val
115 120 125
Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu Pro Pro Ser His Pro Trp
130 135 140
Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu His
145 150 155 160
Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu Ile
165 170 175
Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys
180 185 190
Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Ser Asp Leu Ala Asp
195 200 205
Phe Arg Val Arg Tyr Pro Ala Leu Val Leu Leu Gln Tyr Val Asp Asp
210 215 220
Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu Cys Leu Glu Gly Thr Lys
225 230 235 240
Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly Tyr Arg Ala Ser Ala Lys
245 250 255
Lys Ala Gln Ile Cys Leu Gln Glu Val Thr Tyr Leu Gly Tyr Ser Leu
260 265 270
Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala Arg Lys Glu Ala Ile Leu
275 280 285
Ser Ile Pro Val Pro Lys Asn Pro Arg Gln Val Arg Glu Phe Leu Gly
290 295 300
Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Leu Ala
305 310 315 320
Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly Thr Leu Phe Gln Trp Gly
325 330 335
Thr Glu Gln Gln Leu Ala Phe Glu Asn Ile Arg Lys Ala Leu Leu Ser
340 345 350
Ser Pro Ala Leu Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu Leu Phe
355 360 365
Ile Asp Glu Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln Lys Leu
370 375 380
Gly Pro Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Thr
385 390 395 400
Val Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala
405 410 415
Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro Leu Thr
420 425 430
Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro Pro Asn
435 440 445
Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met Leu Leu
450 455 460
Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn Pro Ala
465 470 475 480
Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro Arg Leu Ser Pro Asp
485 490 495
Leu Ala Glu Thr Met Ala Gln Thr Asp Leu Thr Asp Gln Pro Leu Pro
500 505 510
Asp Ala Asp Leu Thr Trp Tyr Thr Asp Gly Ser Ser Phe Ile Arg Asn
515 520 525
Gly Glu Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Ser Glu Val Ile
530 535 540
Trp Ala Ala Ser Leu Pro Pro Gly Thr Ser Ala Gln Arg Ala Glu Leu
545 550 555 560
Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Lys Gly Lys Lys Leu Thr
565 570 575
Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Val His Gly
580 585 590
Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile
595 600 605
Lys Asn Lys Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe Leu Pro
610 615 620
Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly Asp Ser
625 630 635 640
Pro Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys Lys Ala
645 650 655
Ala Thr Glu Thr Gln Ser Ser Leu Thr Ile Leu
660 665
<210> 3
<211> 1358
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 3
Glu Phe Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser
1 5 10 15
Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro
20 25 30
Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala
35 40 45
Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys
50 55 60
Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile
65 70 75 80
Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
85 90 95
Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg
100 105 110
Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His
115 120 125
Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser
130 135 140
His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu
145 150 155 160
Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp
165 170 175
Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln
180 185 190
Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp
195 200 205
Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
210 215 220
Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln
225 230 235 240
Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala
245 250 255
Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly
260 265 270
Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu
275 280 285
Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu
290 295 300
Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala
305 310 315 320
Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe
325 330 335
Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala
340 345 350
Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe
355 360 365
Glu Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr
370 375 380
Gln Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys
385 390 395 400
Leu Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala
405 410 415
Ala Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln
420 425 430
Pro Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln
435 440 445
Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala
450 455 460
Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu
465 470 475 480
Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn
485 490 495
Cys Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr
500 505 510
Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser
515 520 525
Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr
530 535 540
Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala
545 550 555 560
Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu
565 570 575
Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr
580 585 590
Ala His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser
595 600 605
Glu Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys
610 615 620
Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His
625 630 635 640
Gln Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln
645 650 655
Ala Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu
660 665 670
Leu Ser Ser Ser Ala Ser Lys Leu Glu Phe Thr Leu Asn Ile Glu Asp
675 680 685
Glu His Arg Leu His Glu Thr Ser Lys Glu Pro Asp Val Ser Leu Gly
690 695 700
Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly
705 710 715 720
Met Gly Leu Ala Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala
725 730 735
Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr Pro Met Ser Gln Glu Ala
740 745 750
Arg Leu Gly Ile Lys Pro His Ile Gln Arg Leu Leu Asp Gln Gly Ile
755 760 765
Leu Val Pro Cys Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys
770 775 780
Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val
785 790 795 800
Asn Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn
805 810 815
Leu Leu Ser Gly Leu Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp
820 825 830
Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu His Pro Thr Ser Gln Pro
835 840 845
Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu Met Gly Ile Ser Gly Gln
850 855 860
Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu
865 870 875 880
Phe Asp Glu Ala Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln His
885 890 895
Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala
900 905 910
Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr Arg Ala Leu Leu Gln Thr
915 920 925
Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys
930 935 940
Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu Leu Lys Glu Gly Gln Arg
945 950 955 960
Trp Leu Thr Glu Ala Arg Lys Glu Thr Val Met Gly Gln Pro Thr Pro
965 970 975
Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys
980 985 990
Arg Leu Trp Ile Pro Gly Phe Ala Glu Met Ala Ala Pro Leu Tyr Pro
995 1000 1005
Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp Gly Pro Asp Gln Gln
1010 1015 1020
Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu Thr Ala Pro Ala
1025 1030 1035
Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu Phe Val Asp
1040 1045 1050
Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys Leu Gly
1055 1060 1065
Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Pro
1070 1075 1080
Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
1085 1090 1095
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
1100 1105 1110
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln
1115 1120 1125
Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln
1130 1135 1140
Ala Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val
1145 1150 1155
Ala Leu Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu
1160 1165 1170
Gln His Asn Cys Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg
1175 1180 1185
Pro Asp Leu Thr Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp
1190 1195 1200
Tyr Thr Asp Gly Ser Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala
1205 1210 1215
Gly Ala Ala Val Thr Thr Glu Thr Glu Val Ile Trp Ala Lys Ala
1220 1225 1230
Leu Pro Ala Gly Thr Ser Ala Gln Arg Ala Glu Leu Ile Ala Leu
1235 1240 1245
Thr Gln Ala Leu Lys Met Ala Glu Gly Lys Lys Leu Asn Val Tyr
1250 1255 1260
Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Ile His Gly Glu
1265 1270 1275
Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile
1280 1285 1290
Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu Phe Leu
1295 1300 1305
Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly
1310 1315 1320
His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
1325 1330 1335
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
1340 1345 1350
Ser Ser Ser Ala Ser
1355
<210> 4
<211> 1358
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 4
Glu Phe Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser
1 5 10 15
Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro
20 25 30
Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala
35 40 45
Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys
50 55 60
Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile
65 70 75 80
Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
85 90 95
Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg
100 105 110
Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His
115 120 125
Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser
130 135 140
His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu
145 150 155 160
Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp
165 170 175
Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln
180 185 190
Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp
195 200 205
Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
210 215 220
Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln
225 230 235 240
Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala
245 250 255
Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly
260 265 270
Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu
275 280 285
Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu
290 295 300
Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala
305 310 315 320
Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe
325 330 335
Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala
340 345 350
Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe
355 360 365
Glu Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr
370 375 380
Gln Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys
385 390 395 400
Leu Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala
405 410 415
Ala Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln
420 425 430
Pro Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln
435 440 445
Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala
450 455 460
Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu
465 470 475 480
Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn
485 490 495
Cys Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr
500 505 510
Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asn Gly Ser
515 520 525
Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr
530 535 540
Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala
545 550 555 560
Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu
565 570 575
Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr
580 585 590
Ala His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser
595 600 605
Glu Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys
610 615 620
Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His
625 630 635 640
Gln Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln
645 650 655
Ala Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu
660 665 670
Leu Ser Ser Ser Ala Ser Lys Leu Glu Phe Thr Leu Asn Ile Glu Asp
675 680 685
Glu His Arg Leu His Glu Thr Ser Lys Glu Pro Asp Val Ser Leu Gly
690 695 700
Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly
705 710 715 720
Met Gly Leu Ala Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala
725 730 735
Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr Pro Met Ser Gln Glu Ala
740 745 750
Arg Leu Gly Ile Lys Pro His Ile Gln Arg Leu Leu Asp Gln Gly Ile
755 760 765
Leu Val Pro Cys Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys
770 775 780
Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val
785 790 795 800
Asn Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn
805 810 815
Leu Leu Ser Gly Leu Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp
820 825 830
Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu His Pro Thr Ser Gln Pro
835 840 845
Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu Met Gly Ile Ser Gly Gln
850 855 860
Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu
865 870 875 880
Phe Asp Glu Ala Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln His
885 890 895
Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala
900 905 910
Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr Arg Ala Leu Leu Gln Thr
915 920 925
Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys
930 935 940
Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu Leu Lys Glu Gly Gln Arg
945 950 955 960
Trp Leu Thr Glu Ala Arg Lys Glu Thr Val Met Gly Gln Pro Thr Pro
965 970 975
Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys
980 985 990
Arg Leu Trp Ile Pro Gly Phe Ala Glu Met Ala Ala Pro Leu Tyr Pro
995 1000 1005
Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp Gly Pro Asp Gln Gln
1010 1015 1020
Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu Thr Ala Pro Ala
1025 1030 1035
Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu Phe Val Asp
1040 1045 1050
Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys Leu Gly
1055 1060 1065
Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Pro
1070 1075 1080
Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
1085 1090 1095
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
1100 1105 1110
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln
1115 1120 1125
Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln
1130 1135 1140
Ala Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val
1145 1150 1155
Ala Leu Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu
1160 1165 1170
Gln His Asn Cys Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg
1175 1180 1185
Pro Asp Leu Thr Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp
1190 1195 1200
Tyr Thr Asp Gly Ser Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala
1205 1210 1215
Gly Ala Ala Val Thr Thr Glu Thr Glu Val Ile Trp Ala Lys Ala
1220 1225 1230
Leu Pro Ala Gly Thr Ser Ala Gln Arg Ala Glu Leu Ile Ala Leu
1235 1240 1245
Thr Gln Ala Leu Lys Met Ala Glu Gly Lys Lys Leu Asn Val Tyr
1250 1255 1260
Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Ile His Gly Glu
1265 1270 1275
Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile
1280 1285 1290
Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu Phe Leu
1295 1300 1305
Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly
1310 1315 1320
His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
1325 1330 1335
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
1340 1345 1350
Ser Ser Ser Ala Ser
1355
<210> 5
<211> 1358
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 5
Glu Phe Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser
1 5 10 15
Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro
20 25 30
Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala
35 40 45
Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys
50 55 60
Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile
65 70 75 80
Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
85 90 95
Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg
100 105 110
Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His
115 120 125
Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser
130 135 140
His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu
145 150 155 160
Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp
165 170 175
Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln
180 185 190
Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp
195 200 205
Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
210 215 220
Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln
225 230 235 240
Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala
245 250 255
Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly
260 265 270
Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu
275 280 285
Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu
290 295 300
Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala
305 310 315 320
Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe
325 330 335
Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala
340 345 350
Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe
355 360 365
Glu Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr
370 375 380
Gln Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys
385 390 395 400
Leu Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala
405 410 415
Ala Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln
420 425 430
Pro Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln
435 440 445
Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala
450 455 460
Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu
465 470 475 480
Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn
485 490 495
Cys Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr
500 505 510
Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asn Gly Ser
515 520 525
Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr
530 535 540
Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala
545 550 555 560
Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu
565 570 575
Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr
580 585 590
Ala His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser
595 600 605
Glu Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys
610 615 620
Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His
625 630 635 640
Gln Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln
645 650 655
Ala Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu
660 665 670
Leu Ser Ser Ser Ala Ser Lys Leu Glu Phe Thr Leu Asn Ile Glu Asp
675 680 685
Glu His Arg Leu His Glu Thr Ser Lys Glu Pro Asp Val Ser Leu Gly
690 695 700
Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly
705 710 715 720
Met Gly Leu Ala Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala
725 730 735
Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr Pro Met Ser Gln Glu Ala
740 745 750
Arg Leu Gly Ile Lys Pro His Ile Gln Arg Leu Leu Asp Gln Gly Ile
755 760 765
Leu Val Pro Cys Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys
770 775 780
Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val
785 790 795 800
Asn Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn
805 810 815
Leu Leu Ser Gly Leu Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp
820 825 830
Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu His Pro Thr Ser Gln Pro
835 840 845
Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu Met Gly Ile Ser Gly Gln
850 855 860
Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu
865 870 875 880
Phe Asp Glu Ala Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln His
885 890 895
Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala
900 905 910
Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr Arg Ala Leu Leu Gln Thr
915 920 925
Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys
930 935 940
Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu Leu Lys Glu Gly Gln Arg
945 950 955 960
Trp Leu Thr Glu Ala Arg Lys Glu Thr Val Met Gly Gln Pro Thr Pro
965 970 975
Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys
980 985 990
Arg Leu Trp Ile Pro Gly Phe Ala Glu Met Ala Ala Pro Leu Tyr Pro
995 1000 1005
Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp Gly Pro Asp Gln Gln
1010 1015 1020
Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu Thr Ala Pro Ala
1025 1030 1035
Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu Phe Val Asp
1040 1045 1050
Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys Leu Gly
1055 1060 1065
Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Pro
1070 1075 1080
Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
1085 1090 1095
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
1100 1105 1110
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln
1115 1120 1125
Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln
1130 1135 1140
Ala Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val
1145 1150 1155
Ala Leu Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu
1160 1165 1170
Gln His Asn Cys Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg
1175 1180 1185
Pro Asp Leu Thr Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp
1190 1195 1200
Tyr Thr Asn Gly Ser Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala
1205 1210 1215
Gly Ala Ala Val Thr Thr Glu Thr Glu Val Ile Trp Ala Lys Ala
1220 1225 1230
Leu Pro Ala Gly Thr Ser Ala Gln Arg Ala Glu Leu Ile Ala Leu
1235 1240 1245
Thr Gln Ala Leu Lys Met Ala Glu Gly Lys Lys Leu Asn Val Tyr
1250 1255 1260
Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Ile His Gly Glu
1265 1270 1275
Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile
1280 1285 1290
Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu Phe Leu
1295 1300 1305
Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly
1310 1315 1320
His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
1325 1330 1335
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
1340 1345 1350
Ser Ser Ser Ala Ser
1355
<210> 6
<211> 1350
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 6
Glu Phe Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu Phe Glu Pro Glu
1 5 10 15
Ser Thr Gln Lys Gln Glu Met Asp Ile Trp Leu Lys Asn Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Met Ala His Cys Gln Ala Pro
35 40 45
Val Leu Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile Ser Ile Arg Gln
50 55 60
Tyr Pro Met Pro His Glu Ala Tyr Gln Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Met Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Lys Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu Pro Pro Ser His
130 135 140
Pro Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Ile Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Ser Asp Leu
195 200 205
Ala Asp Phe Arg Val Arg Tyr Pro Ala Leu Val Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu Cys Leu Glu Gly
225 230 235 240
Thr Lys Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Leu Gln Glu Val Thr Tyr Leu Gly Tyr
260 265 270
Ser Leu Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala Arg Lys Glu Ala
275 280 285
Ile Leu Ser Ile Pro Val Pro Lys Asn Pro Arg Gln Val Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Leu Ala Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly Thr Leu Phe Gln
325 330 335
Trp Gly Thr Glu Gln Gln Leu Ala Phe Glu Asn Ile Arg Lys Ala Leu
340 345 350
Leu Ser Ser Pro Ala Leu Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu
355 360 365
Leu Phe Ile Asp Glu Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln
370 375 380
Lys Leu Gly Pro Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Thr Val Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro
420 425 430
Leu Thr Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro
435 440 445
Pro Asn Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met
450 455 460
Leu Leu Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro Arg Leu Ser
485 490 495
Pro Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu Thr Asp Gln Pro
500 505 510
Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asp Gly Ser Ser Phe Ile
515 520 525
Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Ser Glu
530 535 540
Val Ile Trp Ala Ala Ser Leu Pro Pro Gly Thr Ser Ala Gln Arg Ala
545 550 555 560
Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Lys Gly Lys Lys
565 570 575
Leu Thr Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Val
580 585 590
His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys
595 600 605
Glu Ile Lys Asn Lys Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe
610 615 620
Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly
625 630 635 640
Asp Ser Pro Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys
645 650 655
Lys Ala Ala Thr Glu Thr Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser
660 665 670
Ala Ser Lys Leu Glu Phe Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu
675 680 685
Phe Glu Pro Glu Ser Thr Gln Lys Gln Glu Met Asp Ile Trp Leu Lys
690 695 700
Asn Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Met Ala His
705 710 715 720
Cys Gln Ala Pro Val Leu Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile
725 730 735
Ser Ile Arg Gln Tyr Pro Met Pro His Glu Ala Tyr Gln Gly Ile Lys
740 745 750
Pro His Ile Arg Arg Met Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln
755 760 765
Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Lys
770 775 780
Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu
785 790 795 800
Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu
805 810 815
Pro Pro Ser His Pro Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe
820 825 830
Phe Cys Leu Arg Leu His Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu
835 840 845
Trp Arg Asp Pro Glu Ile Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg
850 855 860
Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu
865 870 875 880
His Ser Asp Leu Ala Asp Phe Arg Val Arg Tyr Pro Ala Leu Val Leu
885 890 895
Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu
900 905 910
Cys Leu Glu Gly Thr Lys Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly
915 920 925
Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys Leu Gln Glu Val Thr
930 935 940
Tyr Leu Gly Tyr Ser Leu Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala
945 950 955 960
Arg Lys Glu Ala Ile Leu Ser Ile Pro Val Pro Lys Asn Pro Arg Gln
965 970 975
Val Arg Glu Phe Leu Gly Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro
980 985 990
Gly Phe Ala Glu Leu Ala Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly
995 1000 1005
Thr Leu Phe Gln Trp Gly Thr Glu Gln Gln Leu Ala Phe Glu Asn
1010 1015 1020
Ile Arg Lys Ala Leu Leu Ser Ser Pro Ala Leu Gly Leu Pro Asp
1025 1030 1035
Ile Thr Lys Pro Phe Glu Leu Phe Ile Asp Glu Asn Ser Gly Phe
1040 1045 1050
Ala Lys Gly Val Leu Val Gln Lys Leu Gly Pro Trp Lys Arg Pro
1055 1060 1065
Val Ala Tyr Leu Ser Lys Lys Leu Asp Thr Val Ala Ser Gly Trp
1070 1075 1080
Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala Ile Leu Val Lys
1085 1090 1095
Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro Leu Thr Ile Leu Thr
1100 1105 1110
Ser His Pro Val Glu Ala Leu Val Arg Gln Pro Pro Asn Lys Trp
1115 1120 1125
Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met Leu Leu Asp
1130 1135 1140
Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn Pro Ala
1145 1150 1155
Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro Arg Leu Ser Pro
1160 1165 1170
Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu Thr Asp Gln Pro
1175 1180 1185
Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asp Gly Ser Ser Phe
1190 1195 1200
Ile Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
1205 1210 1215
Ser Glu Val Ile Trp Ala Ala Ser Leu Pro Pro Gly Thr Ser Ala
1220 1225 1230
Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala
1235 1240 1245
Lys Gly Lys Lys Leu Thr Val Tyr Thr Asp Ser Arg Tyr Ala Phe
1250 1255 1260
Ala Thr Ala His Val His Gly Glu Ile Tyr Arg Arg Arg Gly Leu
1265 1270 1275
Leu Thr Ser Glu Gly Lys Glu Ile Lys Asn Lys Asn Glu Ile Leu
1280 1285 1290
Ala Leu Leu Glu Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile
1295 1300 1305
His Cys Pro Gly His Gln Lys Gly Asp Ser Pro Gln Ala Lys Gly
1310 1315 1320
Asn Arg Leu Ala Asp Asp Thr Ala Lys Lys Ala Ala Thr Glu Thr
1325 1330 1335
Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser Ala Ser
1340 1345 1350
<210> 7
<211> 1350
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 7
Glu Phe Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu Phe Glu Pro Glu
1 5 10 15
Ser Thr Gln Lys Gln Glu Met Asp Ile Trp Leu Lys Asn Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Met Ala His Cys Gln Ala Pro
35 40 45
Val Leu Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile Ser Ile Arg Gln
50 55 60
Tyr Pro Met Pro His Glu Ala Tyr Gln Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Met Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Lys Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu Pro Pro Ser His
130 135 140
Pro Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Ile Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Ser Asp Leu
195 200 205
Ala Asp Phe Arg Val Arg Tyr Pro Ala Leu Val Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu Cys Leu Glu Gly
225 230 235 240
Thr Lys Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Leu Gln Glu Val Thr Tyr Leu Gly Tyr
260 265 270
Ser Leu Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala Arg Lys Glu Ala
275 280 285
Ile Leu Ser Ile Pro Val Pro Lys Asn Pro Arg Gln Val Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Leu Ala Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly Thr Leu Phe Gln
325 330 335
Trp Gly Thr Glu Gln Gln Leu Ala Phe Glu Asn Ile Arg Lys Ala Leu
340 345 350
Leu Ser Ser Pro Ala Leu Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu
355 360 365
Leu Phe Ile Asp Glu Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln
370 375 380
Lys Leu Gly Pro Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Thr Val Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro
420 425 430
Leu Thr Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro
435 440 445
Pro Asn Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met
450 455 460
Leu Leu Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro Arg Leu Ser
485 490 495
Pro Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu Thr Asp Gln Pro
500 505 510
Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asn Gly Ser Ser Phe Ile
515 520 525
Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Ser Glu
530 535 540
Val Ile Trp Ala Ala Ser Leu Pro Pro Gly Thr Ser Ala Gln Arg Ala
545 550 555 560
Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Lys Gly Lys Lys
565 570 575
Leu Thr Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Val
580 585 590
His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys
595 600 605
Glu Ile Lys Asn Lys Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe
610 615 620
Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly
625 630 635 640
Asp Ser Pro Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys
645 650 655
Lys Ala Ala Thr Glu Thr Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser
660 665 670
Ala Ser Lys Leu Glu Phe Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu
675 680 685
Phe Glu Pro Glu Ser Thr Gln Lys Gln Glu Met Asp Ile Trp Leu Lys
690 695 700
Asn Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Met Ala His
705 710 715 720
Cys Gln Ala Pro Val Leu Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile
725 730 735
Ser Ile Arg Gln Tyr Pro Met Pro His Glu Ala Tyr Gln Gly Ile Lys
740 745 750
Pro His Ile Arg Arg Met Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln
755 760 765
Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Lys
770 775 780
Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu
785 790 795 800
Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu
805 810 815
Pro Pro Ser His Pro Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe
820 825 830
Phe Cys Leu Arg Leu His Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu
835 840 845
Trp Arg Asp Pro Glu Ile Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg
850 855 860
Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu
865 870 875 880
His Ser Asp Leu Ala Asp Phe Arg Val Arg Tyr Pro Ala Leu Val Leu
885 890 895
Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu
900 905 910
Cys Leu Glu Gly Thr Lys Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly
915 920 925
Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys Leu Gln Glu Val Thr
930 935 940
Tyr Leu Gly Tyr Ser Leu Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala
945 950 955 960
Arg Lys Glu Ala Ile Leu Ser Ile Pro Val Pro Lys Asn Pro Arg Gln
965 970 975
Val Arg Glu Phe Leu Gly Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro
980 985 990
Gly Phe Ala Glu Leu Ala Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly
995 1000 1005
Thr Leu Phe Gln Trp Gly Thr Glu Gln Gln Leu Ala Phe Glu Asn
1010 1015 1020
Ile Arg Lys Ala Leu Leu Ser Ser Pro Ala Leu Gly Leu Pro Asp
1025 1030 1035
Ile Thr Lys Pro Phe Glu Leu Phe Ile Asp Glu Asn Ser Gly Phe
1040 1045 1050
Ala Lys Gly Val Leu Val Gln Lys Leu Gly Pro Trp Lys Arg Pro
1055 1060 1065
Val Ala Tyr Leu Ser Lys Lys Leu Asp Thr Val Ala Ser Gly Trp
1070 1075 1080
Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala Ile Leu Val Lys
1085 1090 1095
Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro Leu Thr Ile Leu Thr
1100 1105 1110
Ser His Pro Val Glu Ala Leu Val Arg Gln Pro Pro Asn Lys Trp
1115 1120 1125
Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met Leu Leu Asp
1130 1135 1140
Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn Pro Ala
1145 1150 1155
Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro Arg Leu Ser Pro
1160 1165 1170
Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu Thr Asp Gln Pro
1175 1180 1185
Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asp Gly Ser Ser Phe
1190 1195 1200
Ile Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
1205 1210 1215
Ser Glu Val Ile Trp Ala Ala Ser Leu Pro Pro Gly Thr Ser Ala
1220 1225 1230
Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala
1235 1240 1245
Lys Gly Lys Lys Leu Thr Val Tyr Thr Asp Ser Arg Tyr Ala Phe
1250 1255 1260
Ala Thr Ala His Val His Gly Glu Ile Tyr Arg Arg Arg Gly Leu
1265 1270 1275
Leu Thr Ser Glu Gly Lys Glu Ile Lys Asn Lys Asn Glu Ile Leu
1280 1285 1290
Ala Leu Leu Glu Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile
1295 1300 1305
His Cys Pro Gly His Gln Lys Gly Asp Ser Pro Gln Ala Lys Gly
1310 1315 1320
Asn Arg Leu Ala Asp Asp Thr Ala Lys Lys Ala Ala Thr Glu Thr
1325 1330 1335
Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser Ala Ser
1340 1345 1350
<210> 8
<211> 1350
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 8
Glu Phe Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu Phe Glu Pro Glu
1 5 10 15
Ser Thr Gln Lys Gln Glu Met Asp Ile Trp Leu Lys Asn Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Met Ala His Cys Gln Ala Pro
35 40 45
Val Leu Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile Ser Ile Arg Gln
50 55 60
Tyr Pro Met Pro His Glu Ala Tyr Gln Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Met Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Lys Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu Pro Pro Ser His
130 135 140
Pro Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Ile Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Ser Asp Leu
195 200 205
Ala Asp Phe Arg Val Arg Tyr Pro Ala Leu Val Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu Cys Leu Glu Gly
225 230 235 240
Thr Lys Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Leu Gln Glu Val Thr Tyr Leu Gly Tyr
260 265 270
Ser Leu Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala Arg Lys Glu Ala
275 280 285
Ile Leu Ser Ile Pro Val Pro Lys Asn Pro Arg Gln Val Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Leu Ala Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly Thr Leu Phe Gln
325 330 335
Trp Gly Thr Glu Gln Gln Leu Ala Phe Glu Asn Ile Arg Lys Ala Leu
340 345 350
Leu Ser Ser Pro Ala Leu Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu
355 360 365
Leu Phe Ile Asp Glu Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln
370 375 380
Lys Leu Gly Pro Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Thr Val Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro
420 425 430
Leu Thr Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro
435 440 445
Pro Asn Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met
450 455 460
Leu Leu Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro Arg Leu Ser
485 490 495
Pro Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu Thr Asp Gln Pro
500 505 510
Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asn Gly Ser Ser Phe Ile
515 520 525
Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Ser Glu
530 535 540
Val Ile Trp Ala Ala Ser Leu Pro Pro Gly Thr Ser Ala Gln Arg Ala
545 550 555 560
Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Lys Gly Lys Lys
565 570 575
Leu Thr Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Val
580 585 590
His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys
595 600 605
Glu Ile Lys Asn Lys Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe
610 615 620
Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly
625 630 635 640
Asp Ser Pro Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys
645 650 655
Lys Ala Ala Thr Glu Thr Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser
660 665 670
Ala Ser Lys Leu Glu Phe Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu
675 680 685
Phe Glu Pro Glu Ser Thr Gln Lys Gln Glu Met Asp Ile Trp Leu Lys
690 695 700
Asn Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Met Ala His
705 710 715 720
Cys Gln Ala Pro Val Leu Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile
725 730 735
Ser Ile Arg Gln Tyr Pro Met Pro His Glu Ala Tyr Gln Gly Ile Lys
740 745 750
Pro His Ile Arg Arg Met Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln
755 760 765
Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Lys
770 775 780
Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu
785 790 795 800
Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu
805 810 815
Pro Pro Ser His Pro Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe
820 825 830
Phe Cys Leu Arg Leu His Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu
835 840 845
Trp Arg Asp Pro Glu Ile Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg
850 855 860
Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu
865 870 875 880
His Ser Asp Leu Ala Asp Phe Arg Val Arg Tyr Pro Ala Leu Val Leu
885 890 895
Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu
900 905 910
Cys Leu Glu Gly Thr Lys Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly
915 920 925
Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys Leu Gln Glu Val Thr
930 935 940
Tyr Leu Gly Tyr Ser Leu Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala
945 950 955 960
Arg Lys Glu Ala Ile Leu Ser Ile Pro Val Pro Lys Asn Pro Arg Gln
965 970 975
Val Arg Glu Phe Leu Gly Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro
980 985 990
Gly Phe Ala Glu Leu Ala Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly
995 1000 1005
Thr Leu Phe Gln Trp Gly Thr Glu Gln Gln Leu Ala Phe Glu Asn
1010 1015 1020
Ile Arg Lys Ala Leu Leu Ser Ser Pro Ala Leu Gly Leu Pro Asp
1025 1030 1035
Ile Thr Lys Pro Phe Glu Leu Phe Ile Asp Glu Asn Ser Gly Phe
1040 1045 1050
Ala Lys Gly Val Leu Val Gln Lys Leu Gly Pro Trp Lys Arg Pro
1055 1060 1065
Val Ala Tyr Leu Ser Lys Lys Leu Asp Thr Val Ala Ser Gly Trp
1070 1075 1080
Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala Ile Leu Val Lys
1085 1090 1095
Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro Leu Thr Ile Leu Thr
1100 1105 1110
Ser His Pro Val Glu Ala Leu Val Arg Gln Pro Pro Asn Lys Trp
1115 1120 1125
Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met Leu Leu Asp
1130 1135 1140
Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn Pro Ala
1145 1150 1155
Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro Arg Leu Ser Pro
1160 1165 1170
Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu Thr Asp Gln Pro
1175 1180 1185
Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asn Gly Ser Ser Phe
1190 1195 1200
Ile Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
1205 1210 1215
Ser Glu Val Ile Trp Ala Ala Ser Leu Pro Pro Gly Thr Ser Ala
1220 1225 1230
Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala
1235 1240 1245
Lys Gly Lys Lys Leu Thr Val Tyr Thr Asp Ser Arg Tyr Ala Phe
1250 1255 1260
Ala Thr Ala His Val His Gly Glu Ile Tyr Arg Arg Arg Gly Leu
1265 1270 1275
Leu Thr Ser Glu Gly Lys Glu Ile Lys Asn Lys Asn Glu Ile Leu
1280 1285 1290
Ala Leu Leu Glu Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile
1295 1300 1305
His Cys Pro Gly His Gln Lys Gly Asp Ser Pro Gln Ala Lys Gly
1310 1315 1320
Asn Arg Leu Ala Asp Asp Thr Ala Lys Lys Ala Ala Thr Glu Thr
1325 1330 1335
Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser Ala Ser
1340 1345 1350
<210> 9
<211> 1354
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 9
Glu Phe Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu Phe Glu Pro Glu
1 5 10 15
Ser Thr Gln Lys Gln Glu Met Asp Ile Trp Leu Lys Asn Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Met Ala His Cys Gln Ala Pro
35 40 45
Val Leu Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile Ser Ile Arg Gln
50 55 60
Tyr Pro Met Pro His Glu Ala Tyr Gln Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Met Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Lys Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu Pro Pro Ser His
130 135 140
Pro Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Ile Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Ser Asp Leu
195 200 205
Ala Asp Phe Arg Val Arg Tyr Pro Ala Leu Val Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu Cys Leu Glu Gly
225 230 235 240
Thr Lys Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Leu Gln Glu Val Thr Tyr Leu Gly Tyr
260 265 270
Ser Leu Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala Arg Lys Glu Ala
275 280 285
Ile Leu Ser Ile Pro Val Pro Lys Asn Pro Arg Gln Val Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Leu Ala Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly Thr Leu Phe Gln
325 330 335
Trp Gly Thr Glu Gln Gln Leu Ala Phe Glu Asn Ile Arg Lys Ala Leu
340 345 350
Leu Ser Ser Pro Ala Leu Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu
355 360 365
Leu Phe Ile Asp Glu Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln
370 375 380
Lys Leu Gly Pro Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Thr Val Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro
420 425 430
Leu Thr Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro
435 440 445
Pro Asn Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met
450 455 460
Leu Leu Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro Arg Leu Ser
485 490 495
Pro Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu Thr Asp Gln Pro
500 505 510
Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asp Gly Ser Ser Phe Ile
515 520 525
Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Ser Glu
530 535 540
Val Ile Trp Ala Ala Ser Leu Pro Pro Gly Thr Ser Ala Gln Arg Ala
545 550 555 560
Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Lys Gly Lys Lys
565 570 575
Leu Thr Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Val
580 585 590
His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys
595 600 605
Glu Ile Lys Asn Lys Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe
610 615 620
Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly
625 630 635 640
Asp Ser Pro Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys
645 650 655
Lys Ala Ala Thr Glu Thr Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser
660 665 670
Ala Ser Lys Leu Glu Phe Thr Leu Asn Ile Glu Asp Glu His Arg Leu
675 680 685
His Glu Thr Ser Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu
690 695 700
Ser Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala
705 710 715 720
Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro
725 730 735
Val Ser Ile Lys Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile
740 745 750
Lys Pro His Ile Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys
755 760 765
Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr
770 775 780
Asn Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val
785 790 795 800
Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly
805 810 815
Leu Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala
820 825 830
Phe Phe Cys Leu Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe
835 840 845
Glu Trp Arg Asp Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr
850 855 860
Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala
865 870 875 880
Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile
885 890 895
Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu
900 905 910
Asp Cys Gln Gln Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu
915 920 925
Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val
930 935 940
Lys Tyr Leu Gly Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu
945 950 955 960
Ala Arg Lys Glu Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg
965 970 975
Gln Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile
980 985 990
Pro Gly Phe Ala Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr
995 1000 1005
Gly Thr Leu Phe Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln
1010 1015 1020
Glu Ile Lys Gln Ala Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro
1025 1030 1035
Asp Leu Thr Lys Pro Phe Glu Leu Phe Val Asp Glu Lys Gln Gly
1040 1045 1050
Tyr Ala Lys Gly Val Leu Thr Gln Lys Leu Gly Pro Trp Arg Arg
1055 1060 1065
Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Pro Val Ala Ala Gly
1070 1075 1080
Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala Val Leu Thr
1085 1090 1095
Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu Val Ile Leu
1100 1105 1110
Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro Asp Arg
1115 1120 1125
Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu Leu
1130 1135 1140
Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
1145 1150 1155
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
1160 1165 1170
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr
1175 1180 1185
Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly
1190 1195 1200
Ser Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val
1205 1210 1215
Thr Thr Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly
1220 1225 1230
Thr Ser Ala Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu
1235 1240 1245
Lys Met Ala Glu Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg
1250 1255 1260
Tyr Ala Phe Ala Thr Ala His Ile His Gly Glu Ile Tyr Arg Arg
1265 1270 1275
Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile Lys Asn Lys Asp
1280 1285 1290
Glu Ile Leu Ala Leu Leu Lys Ala Leu Phe Leu Pro Lys Arg Leu
1295 1300 1305
Ser Ile Ile His Cys Pro Gly His Gln Lys Gly His Ser Ala Glu
1310 1315 1320
Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala Arg Lys Ala Ala
1325 1330 1335
Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ser Ser Ser Ala
1340 1345 1350
Ser
<210> 10
<211> 1354
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 10
Glu Phe Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu Phe Glu Pro Glu
1 5 10 15
Ser Thr Gln Lys Gln Glu Met Asp Ile Trp Leu Lys Asn Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Met Ala His Cys Gln Ala Pro
35 40 45
Val Leu Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile Ser Ile Arg Gln
50 55 60
Tyr Pro Met Pro His Glu Ala Tyr Gln Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Met Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Lys Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu Pro Pro Ser His
130 135 140
Pro Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Ile Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Ser Asp Leu
195 200 205
Ala Asp Phe Arg Val Arg Tyr Pro Ala Leu Val Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu Cys Leu Glu Gly
225 230 235 240
Thr Lys Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Leu Gln Glu Val Thr Tyr Leu Gly Tyr
260 265 270
Ser Leu Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala Arg Lys Glu Ala
275 280 285
Ile Leu Ser Ile Pro Val Pro Lys Asn Pro Arg Gln Val Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Leu Ala Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly Thr Leu Phe Gln
325 330 335
Trp Gly Thr Glu Gln Gln Leu Ala Phe Glu Asn Ile Arg Lys Ala Leu
340 345 350
Leu Ser Ser Pro Ala Leu Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu
355 360 365
Leu Phe Ile Asp Glu Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln
370 375 380
Lys Leu Gly Pro Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Thr Val Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro
420 425 430
Leu Thr Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro
435 440 445
Pro Asn Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met
450 455 460
Leu Leu Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro Arg Leu Ser
485 490 495
Pro Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu Thr Asp Gln Pro
500 505 510
Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asn Gly Ser Ser Phe Ile
515 520 525
Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Ser Glu
530 535 540
Val Ile Trp Ala Ala Ser Leu Pro Pro Gly Thr Ser Ala Gln Arg Ala
545 550 555 560
Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Lys Gly Lys Lys
565 570 575
Leu Thr Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Val
580 585 590
His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys
595 600 605
Glu Ile Lys Asn Lys Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe
610 615 620
Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly
625 630 635 640
Asp Ser Pro Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys
645 650 655
Lys Ala Ala Thr Glu Thr Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser
660 665 670
Ala Ser Lys Leu Glu Phe Thr Leu Asn Ile Glu Asp Glu His Arg Leu
675 680 685
His Glu Thr Ser Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu
690 695 700
Ser Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala
705 710 715 720
Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro
725 730 735
Val Ser Ile Lys Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile
740 745 750
Lys Pro His Ile Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys
755 760 765
Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr
770 775 780
Asn Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val
785 790 795 800
Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly
805 810 815
Leu Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala
820 825 830
Phe Phe Cys Leu Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe
835 840 845
Glu Trp Arg Asp Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr
850 855 860
Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala
865 870 875 880
Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile
885 890 895
Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu
900 905 910
Asp Cys Gln Gln Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu
915 920 925
Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val
930 935 940
Lys Tyr Leu Gly Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu
945 950 955 960
Ala Arg Lys Glu Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg
965 970 975
Gln Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile
980 985 990
Pro Gly Phe Ala Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr
995 1000 1005
Gly Thr Leu Phe Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln
1010 1015 1020
Glu Ile Lys Gln Ala Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro
1025 1030 1035
Asp Leu Thr Lys Pro Phe Glu Leu Phe Val Asp Glu Lys Gln Gly
1040 1045 1050
Tyr Ala Lys Gly Val Leu Thr Gln Lys Leu Gly Pro Trp Arg Arg
1055 1060 1065
Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Pro Val Ala Ala Gly
1070 1075 1080
Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala Val Leu Thr
1085 1090 1095
Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu Val Ile Leu
1100 1105 1110
Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro Asp Arg
1115 1120 1125
Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu Leu
1130 1135 1140
Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
1145 1150 1155
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
1160 1165 1170
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr
1175 1180 1185
Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly
1190 1195 1200
Ser Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val
1205 1210 1215
Thr Thr Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly
1220 1225 1230
Thr Ser Ala Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu
1235 1240 1245
Lys Met Ala Glu Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg
1250 1255 1260
Tyr Ala Phe Ala Thr Ala His Ile His Gly Glu Ile Tyr Arg Arg
1265 1270 1275
Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile Lys Asn Lys Asp
1280 1285 1290
Glu Ile Leu Ala Leu Leu Lys Ala Leu Phe Leu Pro Lys Arg Leu
1295 1300 1305
Ser Ile Ile His Cys Pro Gly His Gln Lys Gly His Ser Ala Glu
1310 1315 1320
Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala Arg Lys Ala Ala
1325 1330 1335
Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ser Ser Ser Ala
1340 1345 1350
Ser
<210> 11
<211> 1354
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 11
Glu Phe Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu Phe Glu Pro Glu
1 5 10 15
Ser Thr Gln Lys Gln Glu Met Asp Ile Trp Leu Lys Asn Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Met Ala His Cys Gln Ala Pro
35 40 45
Val Leu Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile Ser Ile Arg Gln
50 55 60
Tyr Pro Met Pro His Glu Ala Tyr Gln Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Met Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Lys Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu Pro Pro Ser His
130 135 140
Pro Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Ile Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Ser Asp Leu
195 200 205
Ala Asp Phe Arg Val Arg Tyr Pro Ala Leu Val Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu Cys Leu Glu Gly
225 230 235 240
Thr Lys Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Leu Gln Glu Val Thr Tyr Leu Gly Tyr
260 265 270
Ser Leu Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala Arg Lys Glu Ala
275 280 285
Ile Leu Ser Ile Pro Val Pro Lys Asn Pro Arg Gln Val Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Leu Ala Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly Thr Leu Phe Gln
325 330 335
Trp Gly Thr Glu Gln Gln Leu Ala Phe Glu Asn Ile Arg Lys Ala Leu
340 345 350
Leu Ser Ser Pro Ala Leu Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu
355 360 365
Leu Phe Ile Asp Glu Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln
370 375 380
Lys Leu Gly Pro Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Thr Val Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro
420 425 430
Leu Thr Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro
435 440 445
Pro Asn Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met
450 455 460
Leu Leu Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro Arg Leu Ser
485 490 495
Pro Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu Thr Asp Gln Pro
500 505 510
Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asn Gly Ser Ser Phe Ile
515 520 525
Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Ser Glu
530 535 540
Val Ile Trp Ala Ala Ser Leu Pro Pro Gly Thr Ser Ala Gln Arg Ala
545 550 555 560
Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Lys Gly Lys Lys
565 570 575
Leu Thr Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Val
580 585 590
His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys
595 600 605
Glu Ile Lys Asn Lys Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe
610 615 620
Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly
625 630 635 640
Asp Ser Pro Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys
645 650 655
Lys Ala Ala Thr Glu Thr Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser
660 665 670
Ala Ser Lys Leu Glu Phe Thr Leu Asn Ile Glu Asp Glu His Arg Leu
675 680 685
His Glu Thr Ser Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu
690 695 700
Ser Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala
705 710 715 720
Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro
725 730 735
Val Ser Ile Lys Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile
740 745 750
Lys Pro His Ile Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys
755 760 765
Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr
770 775 780
Asn Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val
785 790 795 800
Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly
805 810 815
Leu Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala
820 825 830
Phe Phe Cys Leu Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe
835 840 845
Glu Trp Arg Asp Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr
850 855 860
Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala
865 870 875 880
Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile
885 890 895
Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu
900 905 910
Asp Cys Gln Gln Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu
915 920 925
Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val
930 935 940
Lys Tyr Leu Gly Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu
945 950 955 960
Ala Arg Lys Glu Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg
965 970 975
Gln Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile
980 985 990
Pro Gly Phe Ala Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr
995 1000 1005
Gly Thr Leu Phe Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln
1010 1015 1020
Glu Ile Lys Gln Ala Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro
1025 1030 1035
Asp Leu Thr Lys Pro Phe Glu Leu Phe Val Asp Glu Lys Gln Gly
1040 1045 1050
Tyr Ala Lys Gly Val Leu Thr Gln Lys Leu Gly Pro Trp Arg Arg
1055 1060 1065
Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Pro Val Ala Ala Gly
1070 1075 1080
Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala Val Leu Thr
1085 1090 1095
Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu Val Ile Leu
1100 1105 1110
Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro Asp Arg
1115 1120 1125
Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu Leu
1130 1135 1140
Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
1145 1150 1155
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
1160 1165 1170
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr
1175 1180 1185
Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asn Gly
1190 1195 1200
Ser Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val
1205 1210 1215
Thr Thr Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly
1220 1225 1230
Thr Ser Ala Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu
1235 1240 1245
Lys Met Ala Glu Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg
1250 1255 1260
Tyr Ala Phe Ala Thr Ala His Ile His Gly Glu Ile Tyr Arg Arg
1265 1270 1275
Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile Lys Asn Lys Asp
1280 1285 1290
Glu Ile Leu Ala Leu Leu Lys Ala Leu Phe Leu Pro Lys Arg Leu
1295 1300 1305
Ser Ile Ile His Cys Pro Gly His Gln Lys Gly His Ser Ala Glu
1310 1315 1320
Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala Arg Lys Ala Ala
1325 1330 1335
Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ser Ser Ser Ala
1340 1345 1350
Ser
<210> 12
<211> 1354
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 12
Glu Phe Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser
1 5 10 15
Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro
20 25 30
Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala
35 40 45
Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys
50 55 60
Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile
65 70 75 80
Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
85 90 95
Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg
100 105 110
Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His
115 120 125
Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser
130 135 140
His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu
145 150 155 160
Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp
165 170 175
Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln
180 185 190
Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp
195 200 205
Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
210 215 220
Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln
225 230 235 240
Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala
245 250 255
Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly
260 265 270
Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu
275 280 285
Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu
290 295 300
Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala
305 310 315 320
Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe
325 330 335
Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala
340 345 350
Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe
355 360 365
Glu Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr
370 375 380
Gln Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys
385 390 395 400
Leu Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala
405 410 415
Ala Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln
420 425 430
Pro Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln
435 440 445
Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala
450 455 460
Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu
465 470 475 480
Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn
485 490 495
Cys Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr
500 505 510
Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser
515 520 525
Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr
530 535 540
Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala
545 550 555 560
Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu
565 570 575
Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr
580 585 590
Ala His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser
595 600 605
Glu Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys
610 615 620
Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His
625 630 635 640
Gln Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln
645 650 655
Ala Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu
660 665 670
Leu Ser Ser Ser Ala Ser Lys Leu Glu Phe Thr Leu Gln Leu Glu Glu
675 680 685
Glu Tyr Arg Leu Phe Glu Pro Glu Ser Thr Gln Lys Gln Glu Met Asp
690 695 700
Ile Trp Leu Lys Asn Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly Met
705 710 715 720
Gly Met Ala His Cys Gln Ala Pro Val Leu Ile Gln Leu Lys Ala Thr
725 730 735
Ala Thr Pro Ile Ser Ile Arg Gln Tyr Pro Met Pro His Glu Ala Tyr
740 745 750
Gln Gly Ile Lys Pro His Ile Arg Arg Met Leu Asp Gln Gly Ile Leu
755 760 765
Lys Pro Cys Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys Lys
770 775 780
Pro Gly Thr Lys Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn
785 790 795 800
Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu
805 810 815
Leu Ser Thr Leu Pro Pro Ser His Pro Trp Tyr Thr Val Leu Asp Leu
820 825 830
Lys Asp Ala Phe Phe Cys Leu Arg Leu His Ser Glu Ser Gln Leu Leu
835 840 845
Phe Ala Phe Glu Trp Arg Asp Pro Glu Ile Gly Leu Ser Gly Gln Leu
850 855 860
Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe
865 870 875 880
Asp Glu Ala Leu His Ser Asp Leu Ala Asp Phe Arg Val Arg Tyr Pro
885 890 895
Ala Leu Val Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Ala
900 905 910
Thr Arg Thr Glu Cys Leu Glu Gly Thr Lys Ala Leu Leu Glu Thr Leu
915 920 925
Gly Asn Lys Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys Leu
930 935 940
Gln Glu Val Thr Tyr Leu Gly Tyr Ser Leu Lys Asp Gly Gln Arg Trp
945 950 955 960
Leu Thr Lys Ala Arg Lys Glu Ala Ile Leu Ser Ile Pro Val Pro Lys
965 970 975
Asn Pro Arg Gln Val Arg Glu Phe Leu Gly Thr Ala Gly Tyr Cys Arg
980 985 990
Leu Trp Ile Pro Gly Phe Ala Glu Leu Ala Ala Pro Leu Tyr Pro Leu
995 1000 1005
Thr Arg Pro Gly Thr Leu Phe Gln Trp Gly Thr Glu Gln Gln Leu
1010 1015 1020
Ala Phe Glu Asn Ile Arg Lys Ala Leu Leu Ser Ser Pro Ala Leu
1025 1030 1035
Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu Leu Phe Ile Asp Glu
1040 1045 1050
Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln Lys Leu Gly Pro
1055 1060 1065
Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Thr Val
1070 1075 1080
Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala
1085 1090 1095
Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro Leu
1100 1105 1110
Thr Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro
1115 1120 1125
Pro Asn Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala
1130 1135 1140
Met Leu Leu Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser
1145 1150 1155
Leu Asn Pro Ala Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro
1160 1165 1170
Arg Leu Ser Pro Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu
1175 1180 1185
Thr Asp Gln Pro Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asp
1190 1195 1200
Gly Ser Ser Phe Ile Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala
1205 1210 1215
Val Thr Thr Glu Ser Glu Val Ile Trp Ala Ala Ser Leu Pro Pro
1220 1225 1230
Gly Thr Ser Ala Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala
1235 1240 1245
Leu Lys Met Ala Lys Gly Lys Lys Leu Thr Val Tyr Thr Asp Ser
1250 1255 1260
Arg Tyr Ala Phe Ala Thr Ala His Val His Gly Glu Ile Tyr Arg
1265 1270 1275
Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile Lys Asn Lys
1280 1285 1290
Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe Leu Pro Lys Arg
1295 1300 1305
Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly Asp Ser Pro
1310 1315 1320
Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys Lys Ala
1325 1330 1335
Ala Thr Glu Thr Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser Ala
1340 1345 1350
Ser
<210> 13
<211> 1354
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 13
Glu Phe Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser
1 5 10 15
Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro
20 25 30
Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala
35 40 45
Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys
50 55 60
Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile
65 70 75 80
Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
85 90 95
Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg
100 105 110
Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His
115 120 125
Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser
130 135 140
His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu
145 150 155 160
Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp
165 170 175
Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln
180 185 190
Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp
195 200 205
Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
210 215 220
Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln
225 230 235 240
Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala
245 250 255
Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly
260 265 270
Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu
275 280 285
Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu
290 295 300
Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala
305 310 315 320
Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe
325 330 335
Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala
340 345 350
Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe
355 360 365
Glu Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr
370 375 380
Gln Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys
385 390 395 400
Leu Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala
405 410 415
Ala Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln
420 425 430
Pro Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln
435 440 445
Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala
450 455 460
Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu
465 470 475 480
Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn
485 490 495
Cys Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr
500 505 510
Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asn Gly Ser
515 520 525
Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr
530 535 540
Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala
545 550 555 560
Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu
565 570 575
Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr
580 585 590
Ala His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser
595 600 605
Glu Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys
610 615 620
Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His
625 630 635 640
Gln Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln
645 650 655
Ala Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu
660 665 670
Leu Ser Ser Ser Ala Ser Lys Leu Glu Phe Thr Leu Gln Leu Glu Glu
675 680 685
Glu Tyr Arg Leu Phe Glu Pro Glu Ser Thr Gln Lys Gln Glu Met Asp
690 695 700
Ile Trp Leu Lys Asn Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly Met
705 710 715 720
Gly Met Ala His Cys Gln Ala Pro Val Leu Ile Gln Leu Lys Ala Thr
725 730 735
Ala Thr Pro Ile Ser Ile Arg Gln Tyr Pro Met Pro His Glu Ala Tyr
740 745 750
Gln Gly Ile Lys Pro His Ile Arg Arg Met Leu Asp Gln Gly Ile Leu
755 760 765
Lys Pro Cys Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys Lys
770 775 780
Pro Gly Thr Lys Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn
785 790 795 800
Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu
805 810 815
Leu Ser Thr Leu Pro Pro Ser His Pro Trp Tyr Thr Val Leu Asp Leu
820 825 830
Lys Asp Ala Phe Phe Cys Leu Arg Leu His Ser Glu Ser Gln Leu Leu
835 840 845
Phe Ala Phe Glu Trp Arg Asp Pro Glu Ile Gly Leu Ser Gly Gln Leu
850 855 860
Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe
865 870 875 880
Asp Glu Ala Leu His Ser Asp Leu Ala Asp Phe Arg Val Arg Tyr Pro
885 890 895
Ala Leu Val Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Ala
900 905 910
Thr Arg Thr Glu Cys Leu Glu Gly Thr Lys Ala Leu Leu Glu Thr Leu
915 920 925
Gly Asn Lys Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys Leu
930 935 940
Gln Glu Val Thr Tyr Leu Gly Tyr Ser Leu Lys Asp Gly Gln Arg Trp
945 950 955 960
Leu Thr Lys Ala Arg Lys Glu Ala Ile Leu Ser Ile Pro Val Pro Lys
965 970 975
Asn Pro Arg Gln Val Arg Glu Phe Leu Gly Thr Ala Gly Tyr Cys Arg
980 985 990
Leu Trp Ile Pro Gly Phe Ala Glu Leu Ala Ala Pro Leu Tyr Pro Leu
995 1000 1005
Thr Arg Pro Gly Thr Leu Phe Gln Trp Gly Thr Glu Gln Gln Leu
1010 1015 1020
Ala Phe Glu Asn Ile Arg Lys Ala Leu Leu Ser Ser Pro Ala Leu
1025 1030 1035
Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu Leu Phe Ile Asp Glu
1040 1045 1050
Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln Lys Leu Gly Pro
1055 1060 1065
Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Thr Val
1070 1075 1080
Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala
1085 1090 1095
Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro Leu
1100 1105 1110
Thr Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro
1115 1120 1125
Pro Asn Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala
1130 1135 1140
Met Leu Leu Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser
1145 1150 1155
Leu Asn Pro Ala Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro
1160 1165 1170
Arg Leu Ser Pro Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu
1175 1180 1185
Thr Asp Gln Pro Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asp
1190 1195 1200
Gly Ser Ser Phe Ile Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala
1205 1210 1215
Val Thr Thr Glu Ser Glu Val Ile Trp Ala Ala Ser Leu Pro Pro
1220 1225 1230
Gly Thr Ser Ala Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala
1235 1240 1245
Leu Lys Met Ala Lys Gly Lys Lys Leu Thr Val Tyr Thr Asp Ser
1250 1255 1260
Arg Tyr Ala Phe Ala Thr Ala His Val His Gly Glu Ile Tyr Arg
1265 1270 1275
Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile Lys Asn Lys
1280 1285 1290
Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe Leu Pro Lys Arg
1295 1300 1305
Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly Asp Ser Pro
1310 1315 1320
Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys Lys Ala
1325 1330 1335
Ala Thr Glu Thr Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser Ala
1340 1345 1350
Ser
<210> 14
<211> 1354
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 14
Glu Phe Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser
1 5 10 15
Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro
20 25 30
Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala
35 40 45
Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys
50 55 60
Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile
65 70 75 80
Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
85 90 95
Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg
100 105 110
Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His
115 120 125
Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser
130 135 140
His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu
145 150 155 160
Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp
165 170 175
Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln
180 185 190
Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp
195 200 205
Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
210 215 220
Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln
225 230 235 240
Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala
245 250 255
Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly
260 265 270
Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu
275 280 285
Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu
290 295 300
Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala
305 310 315 320
Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe
325 330 335
Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala
340 345 350
Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe
355 360 365
Glu Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr
370 375 380
Gln Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys
385 390 395 400
Leu Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala
405 410 415
Ala Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln
420 425 430
Pro Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln
435 440 445
Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala
450 455 460
Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu
465 470 475 480
Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn
485 490 495
Cys Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr
500 505 510
Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asn Gly Ser
515 520 525
Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr
530 535 540
Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala
545 550 555 560
Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu
565 570 575
Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr
580 585 590
Ala His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser
595 600 605
Glu Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys
610 615 620
Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His
625 630 635 640
Gln Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln
645 650 655
Ala Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu
660 665 670
Leu Ser Ser Ser Ala Ser Lys Leu Glu Phe Thr Leu Gln Leu Glu Glu
675 680 685
Glu Tyr Arg Leu Phe Glu Pro Glu Ser Thr Gln Lys Gln Glu Met Asp
690 695 700
Ile Trp Leu Lys Asn Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly Met
705 710 715 720
Gly Met Ala His Cys Gln Ala Pro Val Leu Ile Gln Leu Lys Ala Thr
725 730 735
Ala Thr Pro Ile Ser Ile Arg Gln Tyr Pro Met Pro His Glu Ala Tyr
740 745 750
Gln Gly Ile Lys Pro His Ile Arg Arg Met Leu Asp Gln Gly Ile Leu
755 760 765
Lys Pro Cys Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys Lys
770 775 780
Pro Gly Thr Lys Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn
785 790 795 800
Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu
805 810 815
Leu Ser Thr Leu Pro Pro Ser His Pro Trp Tyr Thr Val Leu Asp Leu
820 825 830
Lys Asp Ala Phe Phe Cys Leu Arg Leu His Ser Glu Ser Gln Leu Leu
835 840 845
Phe Ala Phe Glu Trp Arg Asp Pro Glu Ile Gly Leu Ser Gly Gln Leu
850 855 860
Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe
865 870 875 880
Asp Glu Ala Leu His Ser Asp Leu Ala Asp Phe Arg Val Arg Tyr Pro
885 890 895
Ala Leu Val Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Ala
900 905 910
Thr Arg Thr Glu Cys Leu Glu Gly Thr Lys Ala Leu Leu Glu Thr Leu
915 920 925
Gly Asn Lys Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys Leu
930 935 940
Gln Glu Val Thr Tyr Leu Gly Tyr Ser Leu Lys Asp Gly Gln Arg Trp
945 950 955 960
Leu Thr Lys Ala Arg Lys Glu Ala Ile Leu Ser Ile Pro Val Pro Lys
965 970 975
Asn Pro Arg Gln Val Arg Glu Phe Leu Gly Thr Ala Gly Tyr Cys Arg
980 985 990
Leu Trp Ile Pro Gly Phe Ala Glu Leu Ala Ala Pro Leu Tyr Pro Leu
995 1000 1005
Thr Arg Pro Gly Thr Leu Phe Gln Trp Gly Thr Glu Gln Gln Leu
1010 1015 1020
Ala Phe Glu Asn Ile Arg Lys Ala Leu Leu Ser Ser Pro Ala Leu
1025 1030 1035
Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu Leu Phe Ile Asp Glu
1040 1045 1050
Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln Lys Leu Gly Pro
1055 1060 1065
Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Thr Val
1070 1075 1080
Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala
1085 1090 1095
Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro Leu
1100 1105 1110
Thr Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro
1115 1120 1125
Pro Asn Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala
1130 1135 1140
Met Leu Leu Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser
1145 1150 1155
Leu Asn Pro Ala Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro
1160 1165 1170
Arg Leu Ser Pro Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu
1175 1180 1185
Thr Asp Gln Pro Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asn
1190 1195 1200
Gly Ser Ser Phe Ile Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala
1205 1210 1215
Val Thr Thr Glu Ser Glu Val Ile Trp Ala Ala Ser Leu Pro Pro
1220 1225 1230
Gly Thr Ser Ala Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala
1235 1240 1245
Leu Lys Met Ala Lys Gly Lys Lys Leu Thr Val Tyr Thr Asp Ser
1250 1255 1260
Arg Tyr Ala Phe Ala Thr Ala His Val His Gly Glu Ile Tyr Arg
1265 1270 1275
Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile Lys Asn Lys
1280 1285 1290
Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe Leu Pro Lys Arg
1295 1300 1305
Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly Asp Ser Pro
1310 1315 1320
Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys Lys Ala
1325 1330 1335
Ala Thr Glu Thr Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser Ala
1340 1345 1350
Ser
<210> 15
<211> 1350
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 15
Glu Phe Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu Phe Glu Pro Glu
1 5 10 15
Ser Thr Gln Lys Gln Glu Met Asp Ile Trp Leu Lys Asn Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Met Ala His Cys Gln Ala Pro
35 40 45
Val Leu Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile Ser Ile Arg Gln
50 55 60
Tyr Pro Met Pro His Glu Ala Tyr Gln Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Met Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Lys Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu Pro Pro Ser His
130 135 140
Pro Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Ile Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Ser Asp Leu
195 200 205
Ala Asp Phe Arg Val Arg Tyr Pro Ala Leu Val Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu Cys Leu Glu Gly
225 230 235 240
Thr Lys Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Leu Gln Glu Val Thr Tyr Leu Gly Tyr
260 265 270
Ser Leu Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala Arg Lys Glu Ala
275 280 285
Ile Leu Ser Ile Pro Val Pro Lys Asn Pro Arg Gln Val Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Leu Ala Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly Thr Leu Phe Gln
325 330 335
Trp Gly Thr Glu Gln Gln Leu Ala Phe Glu Asn Ile Arg Lys Ala Leu
340 345 350
Leu Ser Ser Pro Ala Leu Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu
355 360 365
Leu Phe Ile Asp Glu Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln
370 375 380
Lys Leu Gly Pro Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Thr Val Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro
420 425 430
Leu Thr Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro
435 440 445
Pro Asn Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met
450 455 460
Leu Leu Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro Arg Leu Ser
485 490 495
Pro Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu Thr Asp Gln Pro
500 505 510
Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asp Gly Ser Ser Phe Ile
515 520 525
Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Ser Glu
530 535 540
Val Ile Trp Ala Ala Ser Leu Pro Pro Gly Thr Ser Ala Gln Arg Ala
545 550 555 560
Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Lys Gly Lys Lys
565 570 575
Leu Thr Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Val
580 585 590
His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys
595 600 605
Glu Ile Lys Asn Lys Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe
610 615 620
Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly
625 630 635 640
Asp Ser Pro Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys
645 650 655
Lys Ala Ala Thr Glu Thr Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser
660 665 670
Ala Ser Lys Leu Glu Phe Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu
675 680 685
Phe Glu Pro Glu Ser Thr Gln Lys Gln Glu Met Asp Ile Trp Leu Lys
690 695 700
Asn Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Met Ala His
705 710 715 720
Cys Gln Ala Pro Val Leu Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile
725 730 735
Ser Ile Arg Gln Tyr Pro Met Pro His Glu Ala Tyr Gln Gly Ile Lys
740 745 750
Pro His Ile Arg Arg Met Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln
755 760 765
Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Lys
770 775 780
Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu
785 790 795 800
Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu
805 810 815
Pro Pro Ser His Pro Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe
820 825 830
Phe Cys Leu Arg Leu His Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu
835 840 845
Trp Arg Asp Pro Glu Ile Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg
850 855 860
Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu
865 870 875 880
His Ser Asp Leu Ala Asp Phe Arg Val Arg Tyr Pro Ala Leu Val Leu
885 890 895
Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu
900 905 910
Cys Leu Glu Gly Thr Lys Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly
915 920 925
Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys Leu Gln Glu Val Thr
930 935 940
Tyr Leu Gly Tyr Ser Leu Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala
945 950 955 960
Arg Lys Glu Ala Ile Leu Ser Ile Pro Val Pro Lys Asn Pro Arg Gln
965 970 975
Val Arg Glu Phe Leu Gly Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro
980 985 990
Gly Phe Ala Glu Leu Ala Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly
995 1000 1005
Thr Leu Phe Gln Trp Gly Thr Glu Gln Gln Leu Ala Phe Glu Asn
1010 1015 1020
Ile Arg Lys Ala Leu Leu Ser Ser Pro Ala Leu Gly Leu Pro Asp
1025 1030 1035
Ile Thr Lys Pro Phe Glu Leu Phe Ile Asp Glu Asn Ser Gly Phe
1040 1045 1050
Ala Lys Gly Val Leu Val Gln Lys Leu Gly Pro Trp Lys Arg Pro
1055 1060 1065
Val Ala Tyr Leu Ser Lys Lys Leu Asp Thr Val Ala Ser Gly Trp
1070 1075 1080
Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala Ile Leu Val Lys
1085 1090 1095
Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro Leu Thr Ile Leu Thr
1100 1105 1110
Ser His Pro Val Glu Ala Leu Val Arg Gln Pro Pro Asn Lys Trp
1115 1120 1125
Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met Leu Leu Asp
1130 1135 1140
Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn Pro Ala
1145 1150 1155
Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro Arg Leu Ser Pro
1160 1165 1170
Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu Thr Asp Gln Pro
1175 1180 1185
Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asn Gly Ser Ser Phe
1190 1195 1200
Ile Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
1205 1210 1215
Ser Glu Val Ile Trp Ala Ala Ser Leu Pro Pro Gly Thr Ser Ala
1220 1225 1230
Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala
1235 1240 1245
Lys Gly Lys Lys Leu Thr Val Tyr Thr Asp Ser Arg Tyr Ala Phe
1250 1255 1260
Ala Thr Ala His Val His Gly Glu Ile Tyr Arg Arg Arg Gly Leu
1265 1270 1275
Leu Thr Ser Glu Gly Lys Glu Ile Lys Asn Lys Asn Glu Ile Leu
1280 1285 1290
Ala Leu Leu Glu Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile
1295 1300 1305
His Cys Pro Gly His Gln Lys Gly Asp Ser Pro Gln Ala Lys Gly
1310 1315 1320
Asn Arg Leu Ala Asp Asp Thr Ala Lys Lys Ala Ala Thr Glu Thr
1325 1330 1335
Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser Ala Ser
1340 1345 1350
<210> 16
<211> 1354
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 16
Glu Phe Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu Phe Glu Pro Glu
1 5 10 15
Ser Thr Gln Lys Gln Glu Met Asp Ile Trp Leu Lys Asn Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Met Ala His Cys Gln Ala Pro
35 40 45
Val Leu Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile Ser Ile Arg Gln
50 55 60
Tyr Pro Met Pro His Glu Ala Tyr Gln Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Met Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Lys Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu Pro Pro Ser His
130 135 140
Pro Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Ile Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Ser Asp Leu
195 200 205
Ala Asp Phe Arg Val Arg Tyr Pro Ala Leu Val Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu Cys Leu Glu Gly
225 230 235 240
Thr Lys Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Leu Gln Glu Val Thr Tyr Leu Gly Tyr
260 265 270
Ser Leu Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala Arg Lys Glu Ala
275 280 285
Ile Leu Ser Ile Pro Val Pro Lys Asn Pro Arg Gln Val Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Leu Ala Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly Thr Leu Phe Gln
325 330 335
Trp Gly Thr Glu Gln Gln Leu Ala Phe Glu Asn Ile Arg Lys Ala Leu
340 345 350
Leu Ser Ser Pro Ala Leu Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu
355 360 365
Leu Phe Ile Asp Glu Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln
370 375 380
Lys Leu Gly Pro Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Thr Val Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro
420 425 430
Leu Thr Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro
435 440 445
Pro Asn Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met
450 455 460
Leu Leu Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro Arg Leu Ser
485 490 495
Pro Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu Thr Asp Gln Pro
500 505 510
Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asp Gly Ser Ser Phe Ile
515 520 525
Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Ser Glu
530 535 540
Val Ile Trp Ala Ala Ser Leu Pro Pro Gly Thr Ser Ala Gln Arg Ala
545 550 555 560
Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Lys Gly Lys Lys
565 570 575
Leu Thr Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Val
580 585 590
His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys
595 600 605
Glu Ile Lys Asn Lys Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe
610 615 620
Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly
625 630 635 640
Asp Ser Pro Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys
645 650 655
Lys Ala Ala Thr Glu Thr Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser
660 665 670
Ala Ser Lys Leu Glu Phe Thr Leu Asn Ile Glu Asp Glu His Arg Leu
675 680 685
His Glu Thr Ser Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu
690 695 700
Ser Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala
705 710 715 720
Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro
725 730 735
Val Ser Ile Lys Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile
740 745 750
Lys Pro His Ile Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys
755 760 765
Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr
770 775 780
Asn Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val
785 790 795 800
Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly
805 810 815
Leu Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala
820 825 830
Phe Phe Cys Leu Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe
835 840 845
Glu Trp Arg Asp Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr
850 855 860
Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala
865 870 875 880
Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile
885 890 895
Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu
900 905 910
Asp Cys Gln Gln Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu
915 920 925
Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val
930 935 940
Lys Tyr Leu Gly Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu
945 950 955 960
Ala Arg Lys Glu Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg
965 970 975
Gln Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile
980 985 990
Pro Gly Phe Ala Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr
995 1000 1005
Gly Thr Leu Phe Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln
1010 1015 1020
Glu Ile Lys Gln Ala Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro
1025 1030 1035
Asp Leu Thr Lys Pro Phe Glu Leu Phe Val Asp Glu Lys Gln Gly
1040 1045 1050
Tyr Ala Lys Gly Val Leu Thr Gln Lys Leu Gly Pro Trp Arg Arg
1055 1060 1065
Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Pro Val Ala Ala Gly
1070 1075 1080
Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala Val Leu Thr
1085 1090 1095
Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu Val Ile Leu
1100 1105 1110
Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro Asp Arg
1115 1120 1125
Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu Leu
1130 1135 1140
Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
1145 1150 1155
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
1160 1165 1170
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr
1175 1180 1185
Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asn Gly
1190 1195 1200
Ser Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val
1205 1210 1215
Thr Thr Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly
1220 1225 1230
Thr Ser Ala Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu
1235 1240 1245
Lys Met Ala Glu Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg
1250 1255 1260
Tyr Ala Phe Ala Thr Ala His Ile His Gly Glu Ile Tyr Arg Arg
1265 1270 1275
Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile Lys Asn Lys Asp
1280 1285 1290
Glu Ile Leu Ala Leu Leu Lys Ala Leu Phe Leu Pro Lys Arg Leu
1295 1300 1305
Ser Ile Ile His Cys Pro Gly His Gln Lys Gly His Ser Ala Glu
1310 1315 1320
Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala Arg Lys Ala Ala
1325 1330 1335
Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ser Ser Ser Ala
1340 1345 1350
Ser
<210> 17
<211> 1354
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 17
Glu Phe Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser
1 5 10 15
Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro
20 25 30
Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala
35 40 45
Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys
50 55 60
Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile
65 70 75 80
Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
85 90 95
Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg
100 105 110
Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His
115 120 125
Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser
130 135 140
His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu
145 150 155 160
Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp
165 170 175
Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln
180 185 190
Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp
195 200 205
Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
210 215 220
Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln
225 230 235 240
Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala
245 250 255
Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly
260 265 270
Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu
275 280 285
Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu
290 295 300
Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala
305 310 315 320
Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe
325 330 335
Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala
340 345 350
Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe
355 360 365
Glu Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr
370 375 380
Gln Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys
385 390 395 400
Leu Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala
405 410 415
Ala Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln
420 425 430
Pro Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln
435 440 445
Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala
450 455 460
Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu
465 470 475 480
Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn
485 490 495
Cys Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr
500 505 510
Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser
515 520 525
Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr
530 535 540
Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala
545 550 555 560
Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu
565 570 575
Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr
580 585 590
Ala His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser
595 600 605
Glu Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys
610 615 620
Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His
625 630 635 640
Gln Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln
645 650 655
Ala Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu
660 665 670
Leu Ser Ser Ser Ala Ser Lys Leu Glu Phe Thr Leu Gln Leu Glu Glu
675 680 685
Glu Tyr Arg Leu Phe Glu Pro Glu Ser Thr Gln Lys Gln Glu Met Asp
690 695 700
Ile Trp Leu Lys Asn Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly Met
705 710 715 720
Gly Met Ala His Cys Gln Ala Pro Val Leu Ile Gln Leu Lys Ala Thr
725 730 735
Ala Thr Pro Ile Ser Ile Arg Gln Tyr Pro Met Pro His Glu Ala Tyr
740 745 750
Gln Gly Ile Lys Pro His Ile Arg Arg Met Leu Asp Gln Gly Ile Leu
755 760 765
Lys Pro Cys Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys Lys
770 775 780
Pro Gly Thr Lys Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn
785 790 795 800
Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu
805 810 815
Leu Ser Thr Leu Pro Pro Ser His Pro Trp Tyr Thr Val Leu Asp Leu
820 825 830
Lys Asp Ala Phe Phe Cys Leu Arg Leu His Ser Glu Ser Gln Leu Leu
835 840 845
Phe Ala Phe Glu Trp Arg Asp Pro Glu Ile Gly Leu Ser Gly Gln Leu
850 855 860
Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe
865 870 875 880
Asp Glu Ala Leu His Ser Asp Leu Ala Asp Phe Arg Val Arg Tyr Pro
885 890 895
Ala Leu Val Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Ala
900 905 910
Thr Arg Thr Glu Cys Leu Glu Gly Thr Lys Ala Leu Leu Glu Thr Leu
915 920 925
Gly Asn Lys Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys Leu
930 935 940
Gln Glu Val Thr Tyr Leu Gly Tyr Ser Leu Lys Asp Gly Gln Arg Trp
945 950 955 960
Leu Thr Lys Ala Arg Lys Glu Ala Ile Leu Ser Ile Pro Val Pro Lys
965 970 975
Asn Pro Arg Gln Val Arg Glu Phe Leu Gly Thr Ala Gly Tyr Cys Arg
980 985 990
Leu Trp Ile Pro Gly Phe Ala Glu Leu Ala Ala Pro Leu Tyr Pro Leu
995 1000 1005
Thr Arg Pro Gly Thr Leu Phe Gln Trp Gly Thr Glu Gln Gln Leu
1010 1015 1020
Ala Phe Glu Asn Ile Arg Lys Ala Leu Leu Ser Ser Pro Ala Leu
1025 1030 1035
Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu Leu Phe Ile Asp Glu
1040 1045 1050
Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln Lys Leu Gly Pro
1055 1060 1065
Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Thr Val
1070 1075 1080
Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala
1085 1090 1095
Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro Leu
1100 1105 1110
Thr Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro
1115 1120 1125
Pro Asn Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala
1130 1135 1140
Met Leu Leu Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser
1145 1150 1155
Leu Asn Pro Ala Thr Leu Leu Pro Leu Pro Ser Gly Lys Pro Pro
1160 1165 1170
Arg Leu Ser Pro Asp Leu Ala Glu Thr Met Ala Gln Thr Asp Leu
1175 1180 1185
Thr Asp Gln Pro Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asn
1190 1195 1200
Gly Ser Ser Phe Ile Arg Asn Gly Glu Arg Lys Ala Gly Ala Ala
1205 1210 1215
Val Thr Thr Glu Ser Glu Val Ile Trp Ala Ala Ser Leu Pro Pro
1220 1225 1230
Gly Thr Ser Ala Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala
1235 1240 1245
Leu Lys Met Ala Lys Gly Lys Lys Leu Thr Val Tyr Thr Asp Ser
1250 1255 1260
Arg Tyr Ala Phe Ala Thr Ala His Val His Gly Glu Ile Tyr Arg
1265 1270 1275
Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile Lys Asn Lys
1280 1285 1290
Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe Leu Pro Lys Arg
1295 1300 1305
Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly Asp Ser Pro
1310 1315 1320
Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys Lys Ala
1325 1330 1335
Ala Thr Glu Thr Gln Ser Ser Leu Thr Ile Leu Ser Ser Ser Ala
1340 1345 1350
Ser
<210> 18
<211> 1358
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 18
Glu Phe Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser
1 5 10 15
Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro
20 25 30
Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala
35 40 45
Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys
50 55 60
Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile
65 70 75 80
Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
85 90 95
Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg
100 105 110
Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His
115 120 125
Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser
130 135 140
His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu
145 150 155 160
Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp
165 170 175
Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln
180 185 190
Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp
195 200 205
Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
210 215 220
Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln
225 230 235 240
Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala
245 250 255
Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly
260 265 270
Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu
275 280 285
Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu
290 295 300
Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala
305 310 315 320
Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe
325 330 335
Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala
340 345 350
Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe
355 360 365
Glu Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr
370 375 380
Gln Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys
385 390 395 400
Leu Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala
405 410 415
Ala Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln
420 425 430
Pro Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln
435 440 445
Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala
450 455 460
Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu
465 470 475 480
Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn
485 490 495
Cys Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr
500 505 510
Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser
515 520 525
Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr
530 535 540
Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala
545 550 555 560
Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu
565 570 575
Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr
580 585 590
Ala His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser
595 600 605
Glu Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys
610 615 620
Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His
625 630 635 640
Gln Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln
645 650 655
Ala Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu
660 665 670
Leu Ser Ser Ser Ala Ser Lys Leu Glu Phe Thr Leu Asn Ile Glu Asp
675 680 685
Glu His Arg Leu His Glu Thr Ser Lys Glu Pro Asp Val Ser Leu Gly
690 695 700
Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly
705 710 715 720
Met Gly Leu Ala Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala
725 730 735
Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr Pro Met Ser Gln Glu Ala
740 745 750
Arg Leu Gly Ile Lys Pro His Ile Gln Arg Leu Leu Asp Gln Gly Ile
755 760 765
Leu Val Pro Cys Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys
770 775 780
Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val
785 790 795 800
Asn Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn
805 810 815
Leu Leu Ser Gly Leu Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp
820 825 830
Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu His Pro Thr Ser Gln Pro
835 840 845
Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu Met Gly Ile Ser Gly Gln
850 855 860
Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu
865 870 875 880
Phe Asp Glu Ala Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln His
885 890 895
Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala
900 905 910
Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr Arg Ala Leu Leu Gln Thr
915 920 925
Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys
930 935 940
Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu Leu Lys Glu Gly Gln Arg
945 950 955 960
Trp Leu Thr Glu Ala Arg Lys Glu Thr Val Met Gly Gln Pro Thr Pro
965 970 975
Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys
980 985 990
Arg Leu Trp Ile Pro Gly Phe Ala Glu Met Ala Ala Pro Leu Tyr Pro
995 1000 1005
Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp Gly Pro Asp Gln Gln
1010 1015 1020
Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu Thr Ala Pro Ala
1025 1030 1035
Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu Phe Val Asp
1040 1045 1050
Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys Leu Gly
1055 1060 1065
Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Pro
1070 1075 1080
Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
1085 1090 1095
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
1100 1105 1110
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln
1115 1120 1125
Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln
1130 1135 1140
Ala Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val
1145 1150 1155
Ala Leu Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu
1160 1165 1170
Gln His Asn Cys Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg
1175 1180 1185
Pro Asp Leu Thr Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp
1190 1195 1200
Tyr Thr Asn Gly Ser Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala
1205 1210 1215
Gly Ala Ala Val Thr Thr Glu Thr Glu Val Ile Trp Ala Lys Ala
1220 1225 1230
Leu Pro Ala Gly Thr Ser Ala Gln Arg Ala Glu Leu Ile Ala Leu
1235 1240 1245
Thr Gln Ala Leu Lys Met Ala Glu Gly Lys Lys Leu Asn Val Tyr
1250 1255 1260
Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Ile His Gly Glu
1265 1270 1275
Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile
1280 1285 1290
Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu Phe Leu
1295 1300 1305
Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly
1310 1315 1320
His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
1325 1330 1335
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
1340 1345 1350
Ser Ser Ser Ala Ser
1355

Claims (13)

1.一种多肽，由氨基酸序列SEQ ID NO:3、4、10、12或13构成，所述多肽具有相比单体逆转录酶改进的热稳定性并具有逆转录酶活性。
2.一种编码权利要求1所述的多肽的核酸。
3.一种包含权利要求2所述核酸的多核苷酸载体。
4.一种包含权利要求3所述载体的宿主细胞。
5.如权利要求4所述的宿主细胞，其中，所述宿主细胞是原核细胞或真核细胞。
6.如权利要求4所述的宿主细胞，其中，所述宿主细胞是真菌细胞。
7.如权利要求4所述的宿主细胞，其中，所述宿主细胞是酵母细胞。
8.一种反应混合物，其包含：
RNA或DNA模板；和
权利要求1所述的多肽。
9.如权利要求8所述的反应混合物，所述反应混合物还包含选自Tris，HEPES，ACES，PIPES，MOPSO，BES，MOPS，TES，TAPSO，POPSO，BICINE，TAPS和AMPSO的缓冲剂。
10.如权利要求8或9所述的反应混合物，还包含至少一种寡核苷酸引物和/或脱氧核苷酸。
11.一种包含权利要求1所述的多肽的试剂盒。
12.如权利要求11所述的试剂盒，其中所述试剂盒还包含DNA依赖性DNA聚合酶。
13.一种进行聚合酶反应的方法，所述方法包括，
使靶核酸与权利要求1所述的多肽和寡核苷酸引物接触，所述寡核苷酸引物在允许所述多肽以模板依赖性方式延伸所述寡核苷酸引物的条件下与所述靶核酸杂交，由此进行聚合酶反应。
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