米黑根毛霉脂肪酶变体及其用途

本申请要求于2021年3月30日提交的题为“Variants of Rhizomucor mieheilipase and uses thereof(米黑根毛霉(Rhizomucor miehei)脂肪酶变体及其用途)”的新加坡专利申请号10202103274U的优先权，该专利申请的全部内容通过引用纳入本文。

背景技术

三酰基甘油脂肪酶属于水解酶类，可裂解羧酸酯的酯键(E.C.3.1.1.)。脂肪酶的特殊性质是它们能够有效裂解脂质(不同类型的天然脂肪和油)的甘油和脂肪酸之间的酯键。目前，脂肪酶是在各种行业(包括清洁剂、制药、食品和生物燃料)中有应用的最广泛使用的酶类之一。

米黑根毛霉脂肪酶(RML)(成熟肽如SEQ ID No.2所示)是工业过程中更常用的脂肪酶之一。其应用之一是生产母乳脂肪替代品，其中它可以进行酯交换或酸解。不同的过程对脂肪酶性质(例如热稳定性、耐化学性、最佳活性的pH值以及底物特异性)的要求不同。为了适应RML的应用，人们在工程改造方面进行了努力以提高其热稳定性(Zhang等，2012)和对映选择性(Holmquist等，1993)。然而，无论应用如何，比活性仍然是脂肪酶最重要的特性之一，因为酶的比活性较高可以节省酶和时间。因此，我们工程改造得到了具有改良活性的RML变体。

目前已知有几种提高脂肪酶比活性的方式。最流行的方式包括操纵底物结合口袋(Lafaquière等，2009)、盖结构域(Khan等，20)和表面环(Yedavalli等，2013)。这些操作可以通过随机诱变或定点诱变来实现。此前，位点饱和诱变已成功用于鉴定在清洁剂中应用的具有更高比活性和更高稳定性的RML变体(Balumuri等，2015)。

发明内容

在本发明的第一方面，提供了一种脂肪酶变体，其包含SEQ ID No.2的脂肪酶序列的第一取代基(substituent)，其中SEQ ID No.2中的第一取代基的位置选自以下组：位置251、位置204、位置254、位置237和位置243。优选地，所述第一取代基的疏水性低于苯丙氨酸。

优选地，所述第一取代基的位置在位置251并且所述第一取代基选自以下组：天冬酰胺、丙氨酸、半胱氨酸、天冬氨酸、谷氨酸、甘氨酸、组氨酸，异亮氨酸、赖氨酸、亮氨酸、甲硫氨酸、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸和酪氨酸。有利地，在野生型位置251处的苯丙氨酸可以被任何其他天然氨基酸取代。

优选地，所述第一取代基的位置在位置204并且所述第一取代基选自以下组：疏水性较高、疏水性较低、极性且不带电、带负电和带正电，其中荷电在pH值为7时相对于第一取代基确定。优选地，所述第一取代基的位置在位置204并且所述第一取代基选自以下组：苯丙氨酸、亮氨酸、甲硫氨酸、酪氨酸、色氨酸、丙氨酸、缬氨酸、甘氨酸、甲硫氨酸、脯氨酸、丝氨酸、苏氨酸、天冬酰胺、谷氨酰胺、天冬氨酸、谷氨酸、精氨酸、组氨酸和赖氨酸。更优选地，所述第一取代基选自以下组：丙氨酸、天冬氨酸、苯丙氨酸、天冬酰胺和精氨酸。有利地，在野生型位置254的异亮氨酸可以被任何天然氨基酸取代。

优选地，所述第一取代基的位置在位置254并且所述第一取代基选自以下组：疏水性较低、极性且带负电，其中荷电在pH值为7时相对于第一取代基确定。优选地，所述第一取代基的位置在位置254并且所述第一取代基选自以下组：甲硫氨酸、酪氨酸、半胱氨酸、丙氨酸、甘氨酸、丝氨酸、苏氨酸、天冬酰胺、谷氨酰胺、天冬氨酸和谷氨酸。更优选地，所述第一取代基选自以下组：丙氨酸、天冬氨酸和天冬酰胺。

优选地，脂肪酶变体包含SEQ ID No.2的脂肪酶序列的第二取代基，其中所述第二取代基在SEQ ID No.2中的位置为位置253，其中所述第一取代基优选地在位置251。优选地，所述第二取代基是具有极性不带电侧链的氨基酸，例如苏氨酸、天冬酰胺和谷氨酰胺，优选苏氨酸。更优选地，所述第一取代基在位置251且选自以下组：天冬酰胺、丙氨酸、天冬氨酸和谷氨酰胺。

优选地，脂肪酶变体包含SEQ ID No.2的脂肪酶序列的第三取代基，其中所述第三取代基在SEQ ID No.2中的位置为位置156，其中所述第一取代基优选地在位置251。优选地，所述第三取代基可以是甘氨酸。D156X(优选D156G)取代可单独与F251X突变(其中X是任何其他氨基酸)配对或与F251X和S253X(优选S253T)突变组合。

优选地，第一取代基在位置251并且脂肪酶变体包含第四取代基和第五取代基，其中所述第四取代基和第五取代基分别位于位置237和239或者分别位于位置243和245。所述第四和第五取代基可以各自独立地是具有极性不带电侧链的氨基酸。例如，适当的为天冬酰胺、苏氨酸、丝氨酸和谷氨酰胺。在一个实例中，第四取代基是天冬酰胺，第五取代基是苏氨酸。有利地，天冬酰胺和苏氨酸的双重取代(具有居间的氨基酸，换言之，相隔2个位置)提供了与RML野生型相比具有改进活性的脂肪酶变体。

优选地，所述脂肪酶变体包含选自以下组的序列：SEQ ID No.3、SEQ ID No.4、SEQID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8、SEQ ID No.9、SEQ ID No.10、SEQ IDNo.11、SEQ ID No.12、SEQ ID No.13、SEQ ID No.14、SEQ ID No.15、SEQ ID No.16、SEQ IDNo.17、SEQ ID No.18、SEQ ID No.19.SEQ ID No.20、SEQ ID No.21、SEQ ID No.22、SEQ IDNo.23、SEQ ID No.24、SEQ ID No.25、SEQ ID No.26、SEQ ID No.27、SEQ ID No.28、SEQ IDNo.29、SEQ ID No.30、SEQ ID No.31、SEQ ID No.32、SEQ ID No.33、SEQ ID No.34、SEQ IDNo.35、SEQ ID No.36、SEQ ID No.37、SEQ ID No.38、SEQ ID No.39、SEQ ID No.40、SEQ IDNo.41、SEQ ID No.42、SEQ ID No.43、SEQ ID No.44、SEQ ID No.45、SEQ ID No.46、SEQ IDNo.47、SEQ ID No.48、SEQ ID No.49、SEQ ID No.50、SEQ ID No.51、SEQ ID No.52、SEQ IDNo.53、SEQ ID No.54、SEQ ID No.55、SEQ ID No.56、SEQ ID No.57、SEQ ID No.58和SEQID No.59。

优选地，所述脂肪酶变体包含SEQ ID No.2的脂肪酶序列的第六取代基，其中所述第一取代基和所述第六取代基位于或邻近所述脂肪酶序列的表面环区域，其中所述第一取代基是天冬酰胺并且第六取代基为苏氨酸，所述第六取代基距离第一取代基两个氨基酸位置。更优选地，第一取代基和第六取代基在位置257的上游。

优选地，所述第一取代基和第六取代基选自以下组：S237N和L239T、D243N和S245T以及F251N和S253T。

例如，所述脂肪酶变体可包含SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ IDNo.6和SEQ ID No.7中的任一个。

在各种实施方式中，脂肪酶变体与SEQ ID NO:2的序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性。

在本发明的所有方面，所述脂肪酶变体包含如说明书中所述的取代基或SEQ IDNo，或基本上由如说明书中所述的取代基或SEQ ID No组成，或由如说明书中所述的取代基或SEQ ID No组成。例如，脂肪酶变体可包含SEQ ID No.2的第一取代基和任选的其它取代基，或基本上由SEQ ID No.2的第一取代基和任选的其它取代基组成，或由SEQ ID No.2的第一取代基和任选的其它取代基组成。例如，脂肪酶变体可包含上述SEQ ID No.中的任一个，或基本上由上述SEQ ID No.中的任一个组成，或由上述SEQ ID No.中的任一个组成。

本文所述的氨基酸侧链的荷电在25℃，pH 7的水中测定。本文所述氨基酸侧链的相对疏水性在pH 7的水中测定，天然氨基酸的疏水性列表的实例描述于Monera等J.Pept.Sci.，1995，1，319-329。

在本发明的另一个方面，提供了一种方法，其包括：提供如本文或第一方面中公开的脂肪酶变体、第一酯和反应物，其中所述反应物选自以下组：水、酸和第二酯；以及在合适的反应条件下借助脂肪酶变体在第一酯和反应物之间形成反应产物。脂肪酶变体可以用于不同的反应，包括第一酯的水解，以及与酸或不同的(即第二)酯的酯交换反应。可以使用多于一种的其它酸或酯作为反应物。酯交换反应可以在不存在水或存在极少水的情况下进行以避免竞争性水解反应。脂肪酶可以催化反应物之间的反应或通过未分离的中间体的反应。

本文所用的术语“酯”包括甘油的脂肪酸酯(如甘油单酯、甘油二酯、甘油三酯)，以及由短链、中等链和长链脂肪酸与短链醇(如甲醇、乙醇、丙醇和丁醇，包括所有结构异构体)形成的单酯。

优选地，所述第一酯是脂肪酸酯，优选三酰基甘油酯。更优选地，所述脂肪酸酯或三酰基甘油酯选自以下组：橄榄油、蓖麻油、葵花籽油、高油酸葵花籽油、菜籽油、高油酸菜籽油、棕榈油、富含三棕榈酸甘油酯的棕榈级分、乳木果油、乳木果油精、芥花油、三辛酸甘油酯、三丁酸甘油酯、辛酸甲酯、癸酸甲酯、十二烷酸甲酯和月桂酸乙烯酯。

在一个实施方式中，所述反应物是中等链脂肪酸或酯。例如，所述反应物选自以下组：辛酸酯(C8)酯、癸酸酯(C10)酯、十二烷酸酯(C12)酯及其混合物。也可以使用相应的中等链脂肪酸。有利地，这提供了中等链和长链三酰基甘油(MLCT)。例如，所述反应产物可以是三酰基甘油，在三酰基甘油的1,3位处含有中等链脂肪酸，在三酰基甘油的2位处含有长链脂肪酸。换言之，所述反应产物具有式R

在一个实施方式中，所述反应物是长链脂肪酸或酯。实例包括棕榈酸、硬脂酸、油酸、亚油酸及其相应的酯。

在一个实施方式中，所述第一酯是棕榈油并且所述反应物是油酸、油酸酯、亚油酸或亚油酸酯。优选地，第一酯是富含三棕榈酸甘油酯(甘油三棕榈酸酯)的棕榈级分。棕榈级分是指棕榈油级分的分离物。

在一个实施方式中，所述脂肪酸酯或三酰甘油酯选自以下组：橄榄油、高油酸葵花籽油和高油酸菜籽油，并且所述反应物为硬脂酸或硬脂酸酯。形成的反应产物是1,3-二硬脂酰基-2-油酰基甘油(SOS)。

优选地，所述变体是SEQ ID No.5。优选地，提供水作为水性缓冲剂。优选地，变体固定在树脂上。

优选地，第一酯和反应物处于合适的反应条件下的液相或溶液相。换言之，第一酯和反应物是可混溶的液体或溶解在溶液中，例如使用溶剂来溶解第一酯和反应物，或者第一酯或反应物作为溶剂来溶解另一者。

优选地，所述反应物为第二酯，并且该反应产物为第一酯和第二酯的酯交换产物。例如，一种酯可以是棕榈油或橄榄油，另一种酯可以是C8、C10或C12甲酯中的至少一种，或其任意组合。具体地，它可以是C8、C10和C12甲酯的混合物。

应当理解，MLCT产物和SOS产物(以及通过本文的方法形成的其他产品)可以是形成的主要产物，但不太可能是形成的唯一产物。

在本发明的另一个方面，提供了由上述方法获得的产品。

发明的具体实施方式

在图中：

图1显示了RML的带状图(PDB ID-3TGL；Brzozowski等，1993；PDB ID-6QPP；Moroz等，2019；Sehnal等，2018)。箭头指示氨基酸237和239、243和245以及251和253的位置，其分别进行以下突变：(a)RML 237、(b)RML 243(b)和(c)RML 251。图1的分图(d)显示氨基酸251相对于非共价结合的前肽的位置。

图2显示了具有RML异源表达的巴斯德毕赤酵母(P.Pichia)中分泌蛋白的SDS-PAGE分析。将凝胶用考马斯蓝染色以便观察。M，标志物；WT，野生型。

图3显示了RML变体在不同pH值下的比活性。橄榄油用作脂肪酶活性试验的底物，pH值为6至9。值表示为平均值±SD(n＝3)。

图4显示了使用RML通过酸解生产1,3-二油酰-2-棕榈酰甘油(OPO)。在与固定化RML WT和RML 251孵育的前四个小时内测量反应混合物中的OPO含量(重量/重量％)。值表示为平均值±SD(n＝3)。

图5显示了使用RML和RML251(SEQ ID No.5)通过酯交换生产中等链和长链三酰基甘油(MLCT)。在与固定化RML WT和RML 251孵育的前六个小时内测量反应混合物中的C32至C46含量(重量/重量％)。值表示为平均值±SD(n＝3)。

在某一实施方式的上下文中描述的特征可相应地适用于其它实施方式中的相同或相似的特征。即使在这些其它实施方式中没有明确描述，在某一实施方式的上下文中描述的特征也可相应地应用于其它实施方式。此外，在某一实施方式的上下文中针对某一特征所描述的添加和/或组合和/或替代形式可相应地适用于其它实施方式中的相同或相似的特征。

以下的详细描述本质上仅是示例性的，并且不旨在限制本发明或本发明的应用和用途。此外，无意受本发明前述背景技术或以下详细描述中提出的任何理论的限制。

在查看以下附图和描述之后，其他技术优点对于本领域普通技术人员而言将变得显而易见。

各种氨基酸在本文中通过其全名以及本领域已知的常规1字母和3字母缩写来描述。肽中氨基酸残基的取代用常规符号描述，例如F251N表示苯丙氨酸(F)的第251个氨基酸残基(或位置)被天冬酰胺(N)取代。

如本文所用，关于某一特征或元件使用的冠词“一”，“一个/种”和“该”包括对一个/种或多个/种特征或元件的引用。如本文所用，术语“和/或”包括一个或多个相关联的所列项目的任何一项及所有组合。如本文所用，术语“第一”、“第二”和“第三”等仅用作标记，而不旨在对它们的对象提出数目要求。例如，脂肪酶变体可包含第一取代基和第三取代基，但不具有第二取代基。在另一个实例中，脂肪酶变体可包含第一取代基和第六取代基，而不具有第二取代基、第三取代基、第四取代基和第五取代基。不同取代基的其他实例也是可能的。因此，术语“第一”、“第二”和“第三”等不强加任何数字要求。

术语“多肽”和“蛋白质”在本文中可互换使用，是指氨基酸的聚合物，而不考虑聚合物的长度；因此，肽、寡肽和蛋白质都包括在多肽的定义内。该术语也不指定或排除本发明的多肽的化学或表达后修饰，尽管作为具体实施方式可以包括或排除这些多肽的化学或表达后修饰。因此，例如，包括糖基基团、乙酰基基团、磷酸基团、脂质基团等的共价连接的多肽修饰明确地涵盖在术语多肽内。此外，具有这些修饰的多肽可以被指定为要包括在本发明中或从本发明中排除的个体物种。天然或其他化学修饰(诸如上面实例中列出的那些)可以发生在多肽中的任何位置，包括肽主链、氨基酸侧链和氨基或羧基末端。应当理解相同类型的修饰可以相同或不同程度存在于给定多肽的几个位点处。此外，给定多肽可能包含许多类型的修饰。多肽可以是支化的，例如，由于泛素化，它们可以是支化或无支化的环状。修饰包括乙酰化、酰化、ADP-核糖基化、酰胺化、黄素的共价连接、血红素部分的共价连接、核苷酸或核苷酸衍生物的共价连接、脂质或脂质衍生物的共价连接、磷脂酰肌醇的共价连接、交联、环化、二硫键形成、去甲基化、共价交联的形成、半胱氨酸的形成、焦谷氨酸盐或酯的形成、甲酰化、γ-羧化、糖基化、GPI锚形成、羟基化、碘化、甲基化、肉豆蔻酰化、氧化、聚乙二醇化、蛋白水解加工、磷酸化、异戊二烯化、外消旋化、硒酰化、硫酸盐化(sulfation)、转运RNA介导的氨基酸向蛋白质的添加，例如精氨酸化和泛素化。该定义还包括含有一种或多种氨基酸类似物的多肽(包括例如非天然产生的氨基酸、仅在不相关的生物系统中天然产生的氨基酸、来自哺乳动物系统的修饰氨基酸等)、具有取代的键以及本领域已知的其他修饰的多肽，包括天然产生的和非天然产生的。

术语“序列相似性”，“序列同一性百分数”和“同源性百分数”在本文中互换使用，以指多核苷酸和多肽之间的比较，并通过在比较窗口上比较两个最佳比对的序列来确定，其中与参考序列(其不包含添加或缺失)相比，比较窗口中的多核苷酸或多肽序列部分可包含添加或缺失(即缺口)，以实现两条序列的最佳比对。该百分比可如下计算：通过测定两种序列中出现相同核酸碱基或氨基酸残基的位置数产生匹配位置数，将该匹配位置数除以比较窗口中位置的总数，得到的结果乘以100产生序列同一性百分比。使用本领域已知的任何不同序列比较算法和程序来评估同一性。此类算法和程序包括但不限于TBLASTN、BLASTP、FASTA、TFASTA、CLUSTAL W、FASTDB。

通过N-糖基化位点(天冬酰胺)的引入来修饰底物结合口袋附近的表面环(SEQ IDNo.2的位置236-243和250至253)以生成水解活性增加34％至115％的变体。此外，制备的变体在可能与前肽相互作用的区域含有取代的氨基酸。RML的结构研究表明，尽管蛋白质成熟后前肽被裂解，但它仍可以与盖区域的成熟蛋白质保持接触，从而抑制其活性(Moroz等，2019)。接触区域的氨基酸修饰可能会去除非共价连接的前肽。RML中取代的氨基酸的侧链可能会破坏与前肽的相互作用，从而导致修饰的RML的活性提高。通过这种方法，橄榄油的水解活性提高至153％。这些变体对于多种底物(包括蓖麻油、三辛酸甘油酯、三丁酸甘油酯、C12甲酯、C8/C10甲酯和月桂酸乙烯酯)的水解也表现出不同程度的改进。

实施例

进行定点诱变以在底物结合袋附近(在表面环区域处或附近)的氨基酸处引入突变，尤其是包含天冬酰胺和苏氨酸(其间有未修饰氨基酸)的一对突变。天冬酰胺修饰提供了N-糖基化位点并且苏氨酸可以促进糖基化。分别引入以下取代S237N+L239T、D243N+S245T和F251N+S253T，并确认为变体RML237、RML243和RML251(图1)。与WT RML相比，RML237、RML243和RML251对橄榄油的水解活性分别提高34％、63％和168％(结果如表1所示)。当底物改为蓖麻油时，RML237也提高了58％，RML251提高了253％(表1)。与野生型RML相比，RML 237的C8-C10甲酯水解略提高了20％，RML251进一步提高了96％，但RML243显示出水解活性降低。

表1：RML变体对橄榄油和蓖麻油的相对水解活性。这些值表示为三重复的平均值。

就最佳温度而言，在各变体之间观察到了变化。RML237和RML 251不像WT那样具有55℃的最佳水解温度，而是在50℃时达到最高活性，而RML243具有的最佳温度最低，为40℃。这些需要较低温度才能实现最佳活性的变体可以帮助最大限度地减少加热需求，从而节省能源成本。变体活性的最佳pH值仍为8，不同pH值下的活性如图4所示。

与野生型RML(WT)相比，具有组合突变RML 237+251的变体显示比活性增加了124％。RML 243+251的活性比WT高18％(表1)。

将RML 251固定化并用于酸解生产1,3-二油酰-2-棕榈酰甘油(OPO)，反应前30分钟后OPO积累量比RML WT多51.8％，并且在1小时和4小时结束时比RML分别高75.9％和41.6％(图4)。固定化的RML 251还用于酯交换反应，生产中等链和长链三酰基甘油(MLCT)。1小时反应结束时，RML 251累积的C32-C46比例比WT高20.7％，并且在4小时和6小时后依旧略高于WT，约为6％(图5)。

通过将位置251的氨基酸替换为其他疏水性较低的氨基酸或亲水性氨基酸，这些变体仍然具有与RML 251相当的活性。对于具有F251A/D/Q+S253T双突变的变体，其橄榄油水解比活性比WT高98％至128％(表2)。RML F251A+S253T的最佳温度与RML 251相同，均为50℃，而其他两个变体RML F251D+S253T和F251Q+S253T的最佳温度稍低，为45℃(表3)。

当RML 251的S253T突变被去除时，我们注意到所得RML F251N变体具有与RML 251相当的活性，并且橄榄油水解的最佳温度也为50℃。因此，为了进一步分析，仅在氨基酸位置251处进行位点饱和诱变。

与使用橄榄油作为底物的WT相比，除RML F251W和RML F251Y外，通过251位饱和诱变生成的所有变体比活性均显著提高109％至281％(表2)。然而，我们发现F251W和F251Y在60℃时的活性略高于WT，因此在某些过程中可能仍然有用。通过将水解底物改为蓖麻油、三辛酸甘油酯、三丁酸甘油酯、C12甲酯、C8/C10甲酯和月桂酸乙烯酯，这些F251变体的比活性比WT高109％至922％。RML F251Y在橄榄油水解方面表现出22％的小幅改善，对于其他七种底物也有31-77％的更适度的改善。RML F251W在水解橄榄油方面具有与WT类似的活性，在水解所研究的其他底物方面也具有类似的活性。(表2)。在这些变体中，有8个变体与WT相同维持着55℃的最佳温度，而其余11个变体则与RML 251相同具有50℃的最佳温度。

表2：氨基酸位置251处具有突变的RML变体的相对水解活性。使用橄榄油、蓖麻油、三辛酸甘油酯、三丁酸甘油酯、C12甲酯、C10甲酯、C8甲酯和月桂酸乙烯酯作为底物。这些值表示为三重复的平均值。

RML WT及其变体的橄榄水解最佳温度总结于表3中。

表3：RML WT及其变体的橄榄油水解最佳温度。

RML237、RML243和RML251上的所有突变位点均引入催化三联体残基之一(位置257处的His残基)的上游。对于RML237，突变位于由二硫键固定就位的弯曲附近(图1a)，对于RML243，突变跨越二硫键本身涉及的半胱氨酸残基(图1b)。因此，在这两种变体中，可能存在对于二硫键的形成的潜在的破坏，这可能会增加参与底物结合袋形成的链的灵活性。在RML251(F251N+S253T)中，两个突变位点位于H257附近的环区域，因此也可能有助于底物结合袋的修饰(图1c)。此外，抑制RML活性的前肽可以与位置251处的氨基酸相互作用，因此用亲水性残基或具有较小侧链的疏水性残基替换该残基可能有助于减少前肽和成熟蛋白质之间的相互作用，从而促进更高的活性。结合观察到的底物特异性和最佳活性温度的差异来看，这些变体似乎通过导致底物结合口袋的柔性增加的结构修饰以及与抑制性前肽相互作用的减少而达到更高的水解活性。可能与前肽接触的其他位置处的一个或多个氨基酸的取代也可能减少前肽与脂肪酶的相互作用。例如，将I204修饰为疏水性更高、疏水性更低、极性、带负电和带正电的其它氨基酸(分别以Phe、Ala、Asn、Asp、Arg为代表)，水解活性均显示出27-89％的提高(表4)。将V254修饰为疏水性较低、极性且带负电的残基(分别以Ala、Asn和Asp为代表)也能将水解活性提高11-90％(表4)。

表4：使用橄榄油作为底物的RML I204和RML V254变体的相对水解活性。

当在具有位置251突变的变体的氨基酸位置156处引入天冬氨酸至甘氨酸的附加突变时，观察到活性的进一步提高。RML F251W和RML F251Y突变体仅表现出小幅提高，但具有这种附加D156G突变使活性比WT提高了149-717％(表5)。对于具有D156G突变的所有其他变体，对于C8-C12甲酯和蓖麻油的水解观察到最显著的改进(表5)。D156G突变可能有助于因底物结合袋的修饰所致的更高的活性，因为Asp 156位于螺旋结构上，其中还存在催化三联体的Ser残基。

表5：在氨基酸位置251处具有D156G突变的RML变体的相对水解活性。使用橄榄油、蓖麻油、三辛酸甘油酯、三丁酸甘油酯、C12甲酯、C10甲酯、C8甲酯和月桂酸乙烯酯作为底物。这些值表示为三重复的平均值。

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RML突变和表达

为了生成RML变体，使用定点诱变将取代引入野生型(WT)RML。基于其氨基酸序列(UniProtKB-P19515)合成WT RML的密码子优化序列(SEQ ID No.1)，以在巴斯德毕赤酵母中进行最佳表达。前肽区域已被修饰以改善在巴斯德毕赤酵母中的表达。使用AOX启动子和终止子引物与设计用于替换靶位点氨基酸序列的引物配对，从pAO815W-WT RML构建体扩增突变的RML，并克隆到pAO815W载体的HindIII和EcoRI位点。pAO815W载体是对pAO815进行修饰，去除了5’-AOX启动子区域内的HindIII位点，并将其重新引入5’-AOX启动子下游。如前所述，通过电穿孔将所得pAO815W-RML变体构建体转化至巴斯德毕赤酵母GS115中(Wu等，20040)。为了表达RML变体，将来自单菌落的细胞在含有缓冲甘油复合培养基(BMGY；1％酵母提取物，2％蛋白胨，100mM磷酸钾，pH 6.0，1.34％ YNB，4x10

脂肪酶活性试验

使用橄榄油、蓖麻油、三辛酸甘油酯、三丁酸甘油酯、C12甲酯、C8/C10甲酯和月桂酸乙烯酯乳液作为底物，测定不同RML变体的比活性。使用刀式均质仪将油与4％(w/v)聚乙烯醇30 000溶液以1:3的比例均质化，制备乳液并立即使用。为了进行试验，首先将2ml油乳液、1.5ml dH

v：中和反应混合物所需的NaOH体积(ml)

v

C：NaOH浓度(μmol/ml)

t：反应时间(分钟)

A：RML添加量(mg)

1U对应于1分钟内橄榄油水解释放出1μmol游离脂肪酸。

OPO和MLCT试验

为了确定RML251用于OPO和MLCT生产的效率，首先将脂肪酶(RML251)固定在疏水性树脂上。可以使用的疏水性树脂的非限制性实例包括：Lifetech

另外，本文描述的脂肪酶变体可以用于进行两种或更多种酯的酯交换反应以产生新的酯产物(酯交换产物)。

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SEQ ID No.1:WT RML

GAAGCCGAAGCTTCCATCGACGGAGGTATTAGAGCCGCTACTTCTCAGGAAATCAACGAACTTACTTACTATACAACTTTGTCAGCTAATTCTTACTGTAGAACTGTTATTCCTGGTGCTACTTGGGATTGCATACATTGTGACGCCACTGAAGATTTAAAGATAATTAAAACCTGGTCTACTTTGATTTACGACACTAACGCTATGGTTGCTAGAGGAGATTCCGAGAAGACTATTTATATCGTGTTTAGAGGTTCTTCATCTATTCGTAATTGGATCGCTGATTTGACATTCGTTCCAGTCTCTTACCCTCCAGTTTCTGGTACTAAGGTTCACAAAGGATTTCTTGATTCTTATGGTGAAGTTCAAAACGAGTTGGTTGCTACTGTCTTGGATCAGTTTAAACAATACCCATCTTATAAGGTTGCTGTCACTGGTCACTCTTTGGGAGGTGCTACTGCCTTGCTGTGTGCTTTAGATTTATACCAGAGAGAGGAAGGATTGTCTTCAAGTAACCTATTCTTGTACACTCAAGGTCAGCCTAGAGTTGGAGATCCAGCATTTGCTAATTATGTGGTTTCTACTGGTATTCCATATAGACGTACTGTTAACGAAAGAGACATAGTACCACACTTGCCTCCAGCTGCCTTCGGATTTCTGCATGCCGGTGAAGAGTACTGGATCACAGATAATTCTCCTGAAACCGTTCAAGTGTGTACATCTGATTTAGAGACTTCCGACTGCTCTAACAGTATTGTTCCATTTACTTCAGTTCTTGATCATTTGTCTTATTTTGGAATTAACACCGGTTTGTGTACTTAA

SEQ ID NO.2：WT RML(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSVLDHLSYFGINTGLCT

SEQ ID NO.3：RML237(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTNDTETSDCSNSIVPFTSVLDHLSYFGINTGLCT

SEQ ID No.4:RML243(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSNCTNSIVPFTSVLDHLSYFGINTGLCT

SEQ ID NO.5：RML251(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPNTTVLDHLSYFGINTGLCT

SEQ ID No.6:RML 237+251(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTNDTETSDCSNSIVPNTTVLDHLSYFGINTGLCT

SED ID No.7:RML 243+251(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSNCTNSIVPNTTVLDHLSYFGINTGLCT

SEQ ID No.8:RML F251A+S253T(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPATTVLDHLSYFGINTGLCT

SEQ ID No.9:RML F251D+S253T(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPDTTVLDHLSYFGINTGLCT

SEQ ID No.10:RML F251Q+S253T(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLECSDASNSIVPQTTVLDHLSYFGINTGLCT

SEQ ID No.11:RML F251N(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPNTSVLDHLSYFGINTGLCT

SEQ ID No.12:RML F251A(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLECSDASNSIVPATSVLDHLSYFGINTGLCT

SEQ ID No.13:RML F251D(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPDTSVLDHLSYFGINTGLCT

SEQ ID No.14:RML F251Q(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPQTSVLDHLSYFGINTGLCT

SEQ ID No.15:RML F251C(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPCTSVLDHLSYFGINTGLCT

SEQ ID No.16:RML F251E(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPETSVLDHLSYFGINTGLCT

SEQ ID No.17:RML F251G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPGTSVLDHLSYFGINTGLCT

SEQ ID No.18:RML F251H(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPHTSVLDHLSYFGINTGLCT

SEQ ID No.19:RML F251I(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPITSVLDHLSYFGINTGLCT

SEQ ID No.20:RML F251K(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPKTSVLDHLSYFGINTGLCT

SEQ ID No.21:RML F251L(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPLTSVLDHLSYFGINTGLCT

SEQ ID No.22:RML F251M(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPMTSVLDHLSYFGINTGLCT

SEQ ID No.23:RML F251P(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPPTSVLDHLSYFGINTGLCT

SEQ ID No.24:RML F251R(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPRTSVLDHLSYFGINTGLCT

SEQ ID No.25:RML F251S(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPSTSVLDHLSYFGINTGLCT

SEQ ID No.26:RML F251T(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPTTSVLDHLSYFGINTGLCT

SEQ ID No.27:RML F251V(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPVTSVLDHLSYFGINTGLCT

SEQ ID No.28:RML F251W(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPWTSVLDHLSYFGINTGLCT

SEQ ID No.29:RML F251Y(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPYTSVLDHLSYFGINTGLCT

SEQ ID No.30:RML I204A(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDAVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSVLDHLSYFGINTGLCT

SEQ ID No.31:RML I204D(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDDVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSVLDHLSYFGINTGLCT

SEQ ID No.32:RML I204F(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDFVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSVLDHLSYFGINTGLCT

SEQ ID No.33:RML I204N(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDNVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSVLDHLSYFGINTGLCT

SEQ ID No.34:RML I204R(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDRVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSVLDHLSYFGINTGLCT

SEQ ID No.35:RML V254A(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSALDHLSYFGINTGLCT

SEQ ID No.36:RML V254D(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSDLDHLSYFGINTGLCT

SEQ ID No.37:RML V254F(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSFLDHLSYFGINTGLCT

SEQ ID No.38:RML V254N(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSNLDHLSYFGINTGLCT

SEQ ID No.39:RML V254R(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSRLDHLSYFGINTGLCT

SEQ ID No.40:RML 251+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPNTTVLDHLSYFGINTGLCT

SEQ ID No.41:RML F251N+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPNTSVLDHLSYFGINTGLCT

SEQ ID No.42:RML F251A+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLECSDASNSIVPATSVLDHLSYFGINTGLCT

SEQ ID No.43:RML F251D+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPDTSVLDHLSYFGINTGLCT

SEQ ID No.44:RML F251Q+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPQTSVLDHLSYFGINTGLCT

SEQ ID No.45:RML F251C+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPCTSVLDHLSYFGINTGLCT

SEQ ID No.46:RML F251E+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPETSVLDHLSYFGINTGLCT

SEQ ID No.47:RML F251G+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPGTSVLDHLSYFGINTGLCT

SEQ ID No.48:RML F251H+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPHTSVLDHLSYFGINTGLCT

SEQ ID No.49:RML F251I+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPITSVLDHLSYFGINTGLCT

SEQ ID No.50:RML F251K+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPKTSVLDHLSYFGINTGLCT

SEQ ID No.51:RML F251L+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPLTSVLDHLSYFGINTGLCT

SEQ ID No.52:RML F251M+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPMTSVLDHLSYFGINTGLCT

SEQ ID No.53:RML F251P+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPPTSVLDHLSYFGINTGLCT

SEQ ID No.54:RML F251R+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPRTSVLDHLSYFGINTGLCT

SEQ ID No.55:RML F251S+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPSTSVLDHLSYFGINTGLCT

SEQ ID No.56:RML F251T+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPTTSVLDHLSYFGINTGLCT

SEQ ID No.57:RML F251V+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPVTSVLDHLSYFGINTGLCT

SEQ ID No.58:RML F251W+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPWTSVLDHLSYFGINTGLCT

SEQ ID No.59:RML F251Y+D156G(成熟蛋白)

SIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALGLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPYTSVLDHLSYFGINTGLCT

表6：所用引物列表

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如上所示，相同的反向引物(SEQ ID No.77、93和99)可用于取代位置251的苯丙氨酸，位置204的异亮氨酸和位置254的缬氨酸。

序列表

<110>丰益国际有限公司(Wilmar International Limited)

<120>米黑根毛霉脂肪酶变体及其用途

<130>6106PCT

<150>SG10202103274U

<151>2021-03-30

<160>103

<170>PatentIn版本3.5

<210>1

<211>822

<212>DNA

<213>米黑根毛霉(Rhizomucor miehei)

<400>1

gaagccgaag cttccatcga cggaggtatt agagccgcta cttctcagga aatcaacgaa60

cttacttact atacaacttt gtcagctaat tcttactgta gaactgttat tcctggtgct 120

acttgggatt gcatacattg tgacgccact gaagatttaa agataattaa aacctggtct 180

actttgattt acgacactaa cgctatggtt gctagaggag attccgagaa gactatttat 240

atcgtgttta gaggttcttc atctattcgt aattggatcg ctgatttgac attcgttcca 300

gtctcttacc ctccagtttc tggtactaag gttcacaaag gatttcttga ttcttatggt 360

gaagttcaaa acgagttggt tgctactgtc ttggatcagt ttaaacaata cccatcttat 420

aaggttgctg tcactggtca ctctttggga ggtgctactg ccttgctgtg tgctttagat 480

ttataccaga gagaggaagg attgtcttca agtaacctat tcttgtacac tcaaggtcag 540

cctagagttg gagatccagc atttgctaat tatgtggttt ctactggtat tccatataga 600

cgtactgtta acgaaagaga catagtacca cacttgcctc cagctgcctt cggatttctg 660

catgccggtg aagagtactg gatcacagat aattctcctg aaaccgttca agtgtgtaca 720

tctgatttag agacttccga ctgctctaac agtattgttc catttacttc agttcttgat 780

catttgtctt attttggaat taacaccggt ttgtgtactt aa822

<210>2

<211>269

<212>PRT

<213>米黑根毛霉(Rhizomucor miehei)

<400>2

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>3

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>3

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

Thr Asp Asn Ser Pro Glu Thr Val Gln Val Cys Thr Asn Asp Thr Glu

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>4

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>4

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asn Cys Thr Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>5

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>5

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Asn Thr Thr Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>6

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>6

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

Thr Asp Asn Ser Pro Glu Thr Val Gln Val Cys Thr Asn Asp Thr Glu

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Asn Thr Thr Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>7

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>7

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asn Cys Thr Asn Ser Ile Val Pro Asn Thr Thr Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>8

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>8

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Ala Thr Thr Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>9

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>9

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Asp Thr Thr Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>10

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>10

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Cys Ser Asp Ala Ser Asn Ser Ile Val Pro Gln Thr Thr Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>11

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>11

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Asn Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>12

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>12

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Cys Ser Asp Ala Ser Asn Ser Ile Val Pro Ala Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>13

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>13

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Asp Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>14

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>14

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Gln Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>15

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>15

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Cys Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>16

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>16

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Glu Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>17

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>17

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Gly Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>18

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>18

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro His Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>19

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>19

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Ile Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>20

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>20

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Lys Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>21

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>21

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Leu Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>22

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>22

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Met Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>23

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>23

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Pro Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>24

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>24

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Arg Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>25

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>25

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Ser Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>26

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>26

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Thr Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>27

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>27

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Val Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>28

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>28

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Trp Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>29

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>29

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Tyr Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>30

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>30

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

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

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>31

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>31

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Asp Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>32

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>32

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

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

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>33

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>33

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Asn Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>34

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>34

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Arg Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>35

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>35

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Ala Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>36

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>36

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Asp Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>37

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>37

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Phe Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>38

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>38

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Asn Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>39

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>39

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Arg Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>40

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>40

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Asn Thr Thr Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>41

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>41

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Asn Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>42

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>42

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Cys Ser Asp Ala Ser Asn Ser Ile Val Pro Ala Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>43

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>43

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Asp Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>44

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>44

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Gln Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>45

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>45

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Cys Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>46

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>46

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Glu Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>47

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>47

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Gly Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>48

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>48

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro His Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>49

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>49

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Ile Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>50

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>50

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Lys Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>51

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>51

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Leu Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>52

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>52

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Met Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>53

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>53

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Pro Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>54

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>54

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Arg Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>55

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>55

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Ser Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>56

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>56

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Thr Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>57

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>57

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Val Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>58

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>58

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Trp Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>59

<211>269

<212>PRT

<213>人工序列

<220>

<223>取代的RML

<400>59

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

1 5 1015

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

202530

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

354045

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

505560

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65707580

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

859095

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

100 105 110

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

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

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

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Tyr Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210>60

<211>21

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>60

gactggttcc aattgacaac g21

<210>61

<211>21

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>61

gcaaatggca ttctgacatc c21

<210>62

<211>36

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>62

gtgtacaaac gatactgaga cttccgactg ctctaa36

<210>63

<211>37

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>63

aagtctcagt atcgtttgta cacacttgaa cggtttc 37

<210>64

<211>39

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>64

ttagagactt ccaactgcac taacagtatt gttccattt 39

<210>65

<211>32

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>65

agtgcagttg gaagtctcta aatcagatgt ac32

<210>66

<211>38

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>66

gtattgttcc aaacactact gttcttgatc atttgtct38

<210>67

<211>35

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>67

gaacagtagt gtttggaaca atactgttag agcag 35

<210>68

<211>38

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>68

gtattgttcc aaacacttca gttcttgatc atttgtct38

<210>69

<211>36

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>69

gaactgaagt gtttggaaca atactgttag agcagt36

<210>70

<211>38

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>70

gtattgttcc agctacttca gttcttgatc atttgtct38

<210>71

<211>35

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>71

gaactgaagt agctggaaca atactgttag agcag 35

<210>72

<211>38

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>72

gtattgttcc agatacttca gttcttgatc atttgtct38

<210>73

<211>35

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>73

gaactgaagt atctggaaca atactgttag agcag 35

<210>74

<211>38

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>74

gtattgttcc acaaacttca gttcttgatc atttgtct38

<210>75

<211>35

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>75

gaactgaagt ttgtggaaca atactgttag agcag 35

<210>76

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>76

cgactgctct aacagtattg ttccatgtac ttcagttctt gatcatttgt ct52

<210>77

<211>25

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>77

tggaacaata ctgttagagc agtcg25

<210>78

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>78

cgactgctct aacagtattg ttccagaaac ttcagttctt gatcatttgt ct52

<210>79

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>79

cgactgctct aacagtattg ttccaggtac ttcagttctt gatcatttgt ct52

<210>80

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>80

cgactgctct aacagtattg ttccacatac ttcagttctt gatcatttgt ct52

<210>81

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>81

cgactgctct aacagtattg ttccaattac ttcagttctt gatcatttgt ct52

<210>82

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>82

cgactgctct aacagtattg ttccaaagac ttcagttctt gatcatttgt ct52

<210>83

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>83

cgactgctct aacagtattg ttccattgac ttcagttctt gatcatttgt ct52

<210>84

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>84

cgactgctct aacagtattg ttccaatgac ttcagttctt gatcatttgt ct52

<210>85

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>85

cgactgctct aacagtattg ttccaccaac ttcagttctt gatcatttgt ct52

<210>86

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>86

cgactgctct aacagtattg ttccaagaac ttcagttctt gatcatttgt ct52

<210>87

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>87

cgactgctct aacagtattg ttccatctac ttcagttctt gatcatttgt ct52

<210>88

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>88

cgactgctct aacagtattg ttccaactac ttcagttctt gatcatttgt ct52

<210>89

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>89

cgactgctct aacagtattg ttccagttac ttcagttctt gatcatttgt ct52

<210>90

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>90

cgactgctct aacagtattg ttccatggac ttcagttctt gatcatttgt ct52

<210>91

<211>52

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>91

cgactgctct aacagtattg ttccatacac ttcagttctt gatcatttgt ct52

<210>92

<211>44

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>92

cgtactgtta acgaaagaga cgctgtacca cacttgcctc cagc 44

<210>93

<211>29

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>93

gtctctttcg ttaacagtac gtctatatg29

<210>94

<211>44

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>94

cgtactgtta acgaaagaga cgatgtacca cacttgcctc cagc 44

<210>95

<211>44

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>95

cgtactgtta acgaaagaga ctttgtacca cacttgcctc cagc 44

<210>96

<211>44

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>96

cgtactgtta acgaaagaga caacgtacca cacttgcctc cagc 44

<210>97

<211>44

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>97

cgtactgtta acgaaagaga cagagtacca cacttgcctc cagc 44

<210>98

<211>59

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>98

ctaacagtat tgttccattt acttcagctc ttgatcattt gtcttatttt ggaattaac 59

<210>99

<211>29

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>99

tgaagtaaat ggaacaatac tgttagagc29

<210>100

<211>59

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>100

ctaacagtat tgttccattt acttcagatc ttgatcattt gtcttatttt ggaattaac 59

<210>101

<211>59

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>101

ctaacagtat tgttccattt acttcatttc ttgatcattt gtcttatttt ggaattaac 59

<210>102

<211>59

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>102

ctaacagtat tgttccattt acttcaaacc ttgatcattt gtcttatttt ggaattaac 59

<210>103

<211>59

<212>DNA

<213>人工序列

<220>

<223>合成的引物

<400>103

ctaacagtat tgttccattt acttcaagac ttgatcattt gtcttatttt ggaattaac 59