CN116790569B - 丙酮酸脱羧酶突变体及其在制备α-羟基酮类化合物中的应用 - Google Patents
丙酮酸脱羧酶突变体及其在制备α-羟基酮类化合物中的应用 Download PDFInfo
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- CN116790569B CN116790569B CN202210361627.4A CN202210361627A CN116790569B CN 116790569 B CN116790569 B CN 116790569B CN 202210361627 A CN202210361627 A CN 202210361627A CN 116790569 B CN116790569 B CN 116790569B
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Abstract
本申请公开了一种丙酮酸脱羧酶突变体及其在制备α‑羟基酮类化合物中的应用，所述丙酮酸脱羧酶突变体是在如SEQ ID NO：1所示氨基酸序列的野生型PDC的基础上发生部分位点突变而形成的，所述丙酮酸脱羧酶突变体能够应用于制备α‑羟基酮类化合物，底物转化率可达99.7％，目标产物的e.e值可达99.8％，相较于具有如SEQ ID NO：1所示氨基酸序列的野生型PDC，所述丙酮酸脱羧酶突变体具有催化活性高、光学选择性更佳、底物耐受性更强以及热稳定性理想的优点。
Description
技术领域
本申请涉及酶工程及生物制药领域，具体涉及一种丙酮酸脱羧酶突变体及其在制备α-羟基酮类化合物中的应用。
背景技术
丙酮酸脱羧酶(pyruvate decarboxylase,PDC)属于焦磷酸硫胺素(thiamindiphosphate,TPP)依依赖性的非氧化酶，PDC是一种胞内酶，广泛存在于动植物和微生物体内。PDC能够应用于α-羟基酮类化合物的合成。
α-羟基酮类化合物是一类具有重要应用价值的化合物，α-羟基酮类化合物能够作为具有生物活性的天然产物和医药中间体，也可作为紫外光固化涂料中的光引发剂，还可以衍生转化为杂环化合物。采用PDC催化合成α-羟基酮类化合物具有反应条件温和、原料低廉、产物光学纯度高的优点，具有巨大的经济价值和环保意义。但是，采用野生型PDC催化合成α-羟基酮类化合物具有催化活性低、酶稳定性差和光学选择性不足的缺点，从而限制了PDC在α-羟基酮类化合物合成技术领域的工业应用。
因此，如何改造野生型PDC以提高其催化活性和稳定性，对PDC酶法制备α-羟基酮类化合物的发展具有重要意义。
发明内容
本申请提供了一种丙酮酸脱羧酶突变体及其在制备α-羟基酮类化合物中的应用，以改善现有技术中野生型丙酮酸脱羧酶催化合成α-羟基酮类化合物存在的催化活性低、酶稳定性差和光学选择性不足的问题。
第一方面，本申请提供了一种丙酮酸脱羧酶突变体，所述丙酮酸脱羧酶突变体的氨基酸序列为与SEQ ID NO：1所示的氨基酸序列至少具有80％、至少具有85％、至少具有90％、至少具有95％、至少具有96％、至少具有97％、至少具有98％或至少具有99％相似性的氨基酸序列，且所述丙酮酸脱羧酶突变体的氨基酸序列是由SEQ ID NO：1所示的氨基酸序列发生一个或多个点突变而获得的氨基酸序列；所述丙酮酸脱羧酶突变体具有丙酮酸脱羧酶活性。
进一步地，发生所述点突变的氨基酸包括SEQ ID NO：1中第7位氨基酸T、第8位氨基酸T、第38位氨基酸L、第169位氨基酸N、第246位氨基酸A、第294位氨基酸G、第392位氨基酸W、第450位氨基酸V、第452位氨基酸Q、第472位氨基酸I、第475位氨基酸M、第476位氨基酸I、第549位氨基酸V、第551位氨基酸W、第553位氨基酸K或第555位氨基酸V中的至少一种。
进一步地，发生所述点突变的方式包括T7R、T8W、L38M、N169Y、A246M、G294K、W392A、V450E、Q452G、I472M、M475K、I476L、V549Y、W551D、K553Y或V555P中的至少一种。
进一步地，发生所述点突变的方式为以下任意一种：
(1)W392I；
(2)I472M；
(3)W551D；
(4)G294K；
(5)I476L；
(6)V555P；
(7)M475K；
(8)W551D和M475K；
(9)V549Y和W392A；
(10)T8W和A246M；
(11)N169Y和V450E；
(12)M475K、L38M和W551D；
(13)T7R、L38M和Q452G；
(14)T7R、K553Y和M475K；或
(15)T7R、L38M、W551D和M475K。
进一步地，所述丙酮酸脱羧酶突变体的氨基酸序列选自如SEQ ID NO：3、SEQ IDNO：5、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13、SEQ ID NO：15、SEQ IDNO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：27、SEQ IDNO：29或SEQ ID NO：31任一所示的氨基酸序列。
第二方面，本申请提供了一种核酸分子，所述核酸分子包括用于编码如第一方面中任意一种所述的丙酮酸脱羧酶突变体的核苷酸序列。
进一步地，所述核苷酸序列选自如SEQ ID NO：4、SEQ ID NO：6、SEQ ID NO：8、SEQID NO：10、SEQ ID NO：12、SEQ ID NO：14、SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：20、SEQID NO：22、SEQ ID NO：24、SEQ ID NO：26、SEQ ID NO：28、SEQ ID NO：30或SEQ ID NO：32任一所示的核苷酸序列。
第三方面，本申请提供了一种重组表达载体，所述重组表达载体包括载体，以及如第二方面中任意一种所述的核酸分子；所述载体选自质粒、粘粒、噬菌体或病毒载体。
第四方面，本申请提供了一种重组表达转化体，所述重组表达转化体包括宿主，以及引入至所述宿主体内的如第二方面中任意一种所述的核酸分子、或如第三方面中所述的重组表达载体；所述宿主选自真核生物或原核生物。
第五方面，本申请提供了一种丙酮酸脱羧酶突变体的制备方法，通过培养第四方面中所述的重组表达转化体，以及从培养物中获得所述的丙酮酸脱羧酶突变体。
第六方面，本申请提供了一种手性α-羟基酮类化合物的制备方法，以如第一方面中任意一种所述的丙酮酸脱羧酶突变体或如第五方面中所述的制备方法制得的丙酮酸脱羧酶突变体作为催化剂，第一化合物和第二化合物接触反应生成α-羟基酮类化合物；其中，第一化合物具有下面通式(Ⅰ)所示的结构：
在通式(Ⅰ)，R1选自氢原子或羟基，X选自氢原子或一价金属离子。
第二化合物具有下面通式(Ⅱ)所示的结构：
在通式(Ⅱ)中，R2选自芳基或杂芳基。
进一步地，所述第一化合物选自丙酮酸、羟基丙酮酸或丙酮酸钠中的至少一种，所述第二化合物选自苯甲醛。
本申请提供了一种丙酮酸脱羧酶突变体及其在制备α-羟基酮类化合物中的应用，具有如下技术效果：
本申请中丙酮酸脱羧酶突变体是在如SEQ ID NO：1所示氨基酸序列的野生型PDC的基础上发生部分位点突变而形成的，所述丙酮酸脱羧酶突变体具有丙酮酸脱羧酶活性，所述丙酮酸脱羧酶突变体能够应用于制备α-羟基酮类化合物，以催化合成式(Ⅳ)所示结构的化合物为例，底物苯甲醛的转化率可达99.7％，目标产物的e.e值可达99.8％，相较于具有如SEQ ID NO：1所示氨基酸序列的野生型PDC，所述丙酮酸脱羧酶突变体具有催化活性高和光学选择性更佳的优点。此外，通过底物耐受性实验和热稳定性实验可知，所述丙酮酸脱羧酶突变体对底物的耐受性能和热稳定性能明显优于所述野生型PDC。
附图说明
下面结合附图，通过对本申请的具体实施方式详细描述，将使本申请的技术方案及其有益效果显而易见。
图1为实验例1中包含丙酮酸脱羧酶突变体M3的反应体系反应2h获得的反应液的HPLC图谱；
图2为实验例1中包含丙酮酸脱羧酶突变体M15的反应体系反应2h获得的反应液的HPLC图谱；
图3为实验例2中包含丙酮酸脱羧酶突变体M3的反应体系反应30min获得的反应液的HPLC图谱；
图4为实验例2中包含丙酮酸脱羧酶突变体M3的反应体系反应24h获得的反应液的HPLC图谱；
图5为实验例2中包含丙酮酸脱羧酶突变体M15的反应体系反应30min获得的反应液的HPLC图谱；
图6为实验例2中包含丙酮酸脱羧酶突变体M15的反应体系反应24h获得的反应液的HPLC图谱；
图7为实验例3中向包含丙酮酸脱羧酶突变体M3的预反应体系流加苯甲醛1h获得的反应液的HPLC图谱；
图8为实验例3中向包含丙酮酸脱羧酶突变体M3的预反应体系流加苯甲醛24h获得的反应液的HPLC图谱；
图9为实验例3中向包含丙酮酸脱羧酶突变体M15的预反应体系流加苯甲醛1h获得的反应液的HPLC图谱；
图10为实验例3中向包含丙酮酸脱羧酶突变体M15的预反应体系流加苯甲醛24h获得的反应液的HPLC图谱。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
本申请实施例提供了一种丙酮酸脱羧酶突变体及其在制备α-羟基酮类化合物中的应用，丙酮酸脱羧酶突变体的氨基酸序列与SEQ ID NO：1所示的氨基酸序列至少具有80％、至少具有85％、至少具有90％、至少具有95％、至少具有96％、至少具有97％、至少具有98％或至少具有99％相似性的氨基酸序列，且丙酮酸脱羧酶突变体的氨基酸序列是由SEQ ID NO：1所示的氨基酸序列发生一个或多个点突变而获得的氨基酸序列，丙酮酸脱羧酶突变体具有丙酮酸脱羧酶活性。
如本申请所用，“相似性”是指两个氨基酸序列或两个核苷酸序列之间的相关性，例如：丙酮酸脱羧酶突变体的氨基酸序列与SEQ ID NO：1所示的氨基酸序列之间的相关性。在本申请实施例中，至少具有80％以上相似性可以理解为80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列相似性，对应相似性的数值为整数；也可以进一步理解为80.1％、81.2％、82.3％、83.4％、84.5％、85.6％、86.7％、87.8％、88.9％、89.8％、90.3％、91.7％、92.2％、93.5％、94.8％、95.9％、96.6％、97.5％、98.4％或99.9％，但小于100％的序列相似性，对应相似性的数值为小数。
如本申请所用，“点突变”是指在SEQ ID NO：1所示的氨基酸序列中特定位点氨基酸的取代、缺失或插入。
进一步地，发生点突变的氨基酸包括SEQ ID NO：1中第7位氨基酸T、第8位氨基酸T、第38位氨基酸L、第169位氨基酸N、第246位氨基酸A、第294位氨基酸G、第392位氨基酸W、第450位氨基酸V、第452位氨基酸Q、第472位氨基酸I、第475位氨基酸M、第476位氨基酸I、第549位氨基酸V、第551位氨基酸W、第553位氨基酸K或第555位氨基酸V中的至少一种。
在本申请的一些实施例中，发生点突变的方式包括T7R、T8W、L38M、N169Y、A246M、G294K、W392A、V450E、Q452G、I472M、M475K、I476L、V549Y、W551D、K553Y或V555P中的至少一种。
如本申请所用，“氨基酸”由单字母或三字母代码表示，具有如下含义：A：Ala(丙氨酸)；R：Arg(精氨酸)；N：Asn(天冬酰胺)；D：Asp(天冬氨酸)；C：Cys(半胱氨酸)；Q：Gln(谷氨酰胺)；E：Glu(谷氨酸)；G：Gly(甘氨酸)；H：His(组氨酸)；I：Ile(异亮氨酸)；L：Leu(亮氨酸)；K：Lys(赖氨酸)；M：Met(甲硫氨酸)；F：Phe(苯丙氨酸)；P：Pro(脯氨酸)；S：Ser(丝氨酸)；T：Thr(苏氨酸)；W：Trp(色氨酸)；Y：Tyr(酪氨酸)；V：Val(缬氨酸)。
对于氨基酸取代的点突变方式，命名方法为：原始氨基酸，原始氨基酸的位点，取代氨基酸，例如：W551D表示在SEQ ID NO：1所示氨基酸序列中第551位点处采用天冬氨酸取代原始的色氨酸；M475K表示在SEQ ID NO：1所示氨基酸序列中第475位点处采用赖氨酸取代原始的赖氨酸。
在本申请的一些实施例中，所述丙酮酸脱羧酶突变体的氨基酸序列选自如SEQ IDNO：3、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13、SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23、SEQ ID NO：25、SEQ IDNO：27、SEQ ID NO：29或SEQ ID NO：31任一所示的氨基酸序列。
本申请实施例还提供了一种核酸分子，所述核酸分子包括用于编码本申请实施例中任意一种所述的丙酮酸脱羧酶突变体的核苷酸序列。
如本申请所用，“核酸分子”是指由多个核苷酸聚合而成的生物大分子化合物，可以是通过聚合酶链式反应(PCR)或通过体外翻译产生的脱氧核糖核酸(DNA)片段、核糖核酸(RNA)片段或寡核苷酸片段中的任意一种，以及通过连接、切割、内切核酸酶作用或外切核酸酶作用中的任意一种或多种产生的片段，可以是单链的或双链的。在本申请实施例中，核酸分子包括但不限于用于编码丙酮酸脱羧酶突变体的多核苷酸。
在本申请的一些实施例中，用于编码丙酮酸脱羧酶突变体的核苷酸序列选自如SEQ ID NO：4、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14、SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：20、SEQ ID NO：22、SEQ ID NO：24、SEQ ID NO：26、SEQ ID NO：28、SEQ ID NO：30或SEQ ID NO：32任一所示的核苷酸序列。
为了便于理解，下表1示出了本申请实施例中涉及的丙酮酸脱羧酶突变体的具体信息，
所述丙酮酸脱羧酶突变体相对于氨基酸序列为SEQ ID NO：1的野生型丙酮酸脱羧酶存在一个或多个突变位点，表1中提供的丙酮酸脱羧酶突变体仅作为示例：
表1本申请实施例中涉及的丙酮酸脱羧酶突变体的具体信息一览表
本申请实施例还提供了一种重组表达载体，所述重组表达载体包括载体，以及如本申请实施例中任意一种所述的核酸分子。
如本申请所用，“载体”是指能够运输另一种核酸的核酸分子，例如可以是质粒、病毒、粘粒、噬菌体等。在本申请的一个实施例中，未插入外源基因的pET24a可以作为载体。
如本申请所用，“重组表达载体”是指一种DNA构建体，其含有与合适的控制序列可操作地连接的核酸分子，所述控制序列能够实现核酸分子在合适的宿主中表达。在本申请实施例中，重组表达载体是指采用分子生物学技术将用于编码丙酮酸脱羧酶突变体的核酸分子插入至载体上，由此形成的DNA构建体。
本申请实施例还提供了一种重组表达转化体，所述重组表达转化体包括宿主，以及引入至宿主体内的如本申请实施例中任意一种所述的核酸分子或重组表达载体。
如本申请所用，“重组表达转化体”是指接受了外源遗传物质(如：质粒DNA)而使遗传特性发生变化的宿主。在本申请实施例中，接受了外源遗传物质(用于编码丙酮酸脱羧酶突变体的核酸分子或重组表达载体)的工程菌株属于重组表达转化体。在本申请的一个实施例中，T1重组工程菌至T15重组工程菌均属于重组表达转化体。
如本申请所用，“宿主”是指用于表达外源基因而产生蛋白的一类生物体，宿主例如可以是真核生物、原核生物、病毒等，其中，作为宿主的真核生物包括但不限于哺乳动物细胞、酵母、真菌、昆虫细胞和植物细胞，作为宿主的原核生物包括但不限于芽孢杆菌属、梭菌属、乳酸菌属、链霉菌属、葡萄球菌属、大肠杆菌、假单胞菌属和类芽孢杆菌属。在本申请的一个实施例中，用于表达丙酮酸脱羧酶突变体的宿主为大肠杆菌BL21(DE3)。
本申请实施例还提供了一种丙酮酸脱羧酶突变体的制备方法，具体是：通过培养本申请实施例中任意一种所述的重组表达转化体，以及从培养物中获得丙酮酸脱羧酶突变体。
本申请实施例还提供了一种α-羟基酮类化合物的制备方法，具体是：以本申请实施例中任意一种所述的丙酮酸脱羧酶突变体或任意一种所述的制备方法制得的丙酮酸脱羧酶突变体作为催化剂，第一化合物和第二化合物接触反应生成α-羟基酮类化合物，其中，第一化合物具有下面通式(Ⅰ)所示的结构：
在通式(Ⅰ)，R1选自氢原子或羟基，X选自氢原子或一价金属离子。
第二化合物具有下面通式(Ⅱ)所示的结构：
在通式(Ⅱ)中，R2选自芳基或杂芳基。
如本申请所用，“芳基”既包括未发生取代的芳基，又包括一个或多个氢原子任选地被其他基团取代的芳基，其他基团例如可以是卤素原子或烷基，允许存在多重取代度；“未发生取代的芳基”是指芳香环上仅包含碳原子的芳香基团，包括但不限于是苯基、1-萘基、2-萘基或联苯基。
如本申请所用，“杂芳基”是指芳基中一个或多个碳原子独立地被一个或多个杂原子(例如N、O、P和/或S)替代，例如杂芳基具有3至20个碳原子，又如杂芳基具有5至15个碳原子，又如杂芳基具有5至9个碳原子，杂芳基可以是未取代的，也可以是其上的一个氢原子或多个氢原子任选地被其他基团取代，其他基团例如可以是烷基、卤素等，允许存在多重取代度。
可以理解的是，用于制备α-羟基酮类化合物的丙酮酸脱羧酶突变体可以是携带有丙酮酸脱羧酶突变体的编码基因的重组工程菌的培养物(包括培养基)，也可以是通过将所述培养物分离纯化后获得的菌体细胞、菌体细胞提取物、菌体细胞破碎物或提纯的丙酮酸脱羧酶突变体。
在本申请的一些实施例中，在所述丙酮酸脱羧酶突变体催化第一化合物和第二化合物接触反应生成α-羟基酮类化合物的反应体系中，丙酮酸脱羧酶突变体：第一化合物：第二化合物的质量比值为1：(0.7～10)：(4.5～12)。若丙酮酸脱羧酶突变体的添加量过少，则对第一化合物和第二化合物的催化反应效果有限，从而出现底物过剩的现象；若丙酮酸脱羧酶突变体的添加量过多，则会造成丙酮酸脱羧酶突变体的浪费，提高了生产成本，并且为后续产物的分离纯化增加了难度。
进一步地，所述反应体系还包括焦磷酸硫胺素和Mg2+，其中，焦磷酸硫胺素用作第一化合物脱羧基反应的辅酶，Mg2+用于提升酶的反应活性，其中，催化反应如下式(Ⅲ)所示：
在本申请的一些实施例中，所述反应体系的pH为5.6至7.0，反应温度为26℃至35℃。需要说明的是，催化反应可以在振荡或搅拌的条件下进行，反应时间例如以底物残留量低于5％为准；催化反应结束后，可以依照本领域常见的分离纯化方法提取α-羟基酮类化合物，常见的分离纯化方法包括但不限于是过滤、离心、沉淀或干燥中的至少一种。
下面将对本申请实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。
除非另行定义，文中所使用的所有专业与科学用语与本领域技术人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本申请中。文中所述的较佳实施方法与材料仅作示范之用，但不能限制本申请的内容。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过本领域已知方法制备。
一、本申请实施例中涉及培养基的说明
(1)LB培养基
每100mL的LB液体培养基中，包括：1.0g的蛋白胨，0.5g的酵母粉，以及1.0g的NaCl；
对于LB固体培养基，是在LB液体培养基配方的基础上添加20g/L的琼脂；
对于含有卡那霉素抗性的LB培养基，卡那霉素的总浓度为50μg/mL。
(2)自诱导培养基
分别称取120g的酵母粉、32.25g的蛋白胨、0.75g的硫酸镁(MgSO4)、16.5g的硫酸铵((NH4)2SO4)、32.5g的磷酸二氢钾(KH2PO4)、35.5g的磷酸氢二钠(Na2HPO4)、2.5g的葡萄糖以及10g的α-乳糖，然后将各个称取好的组分全部加入至磨粉机内，充分研磨至粉状，获得粉末状的自诱导培养基。将50g粉末状的自诱导培养基溶于1L去离子水，充分混匀后调节pH至7.0，然后121℃灭菌30min。
二、本申请实施例中涉及质粒和感受态细胞的说明详见下表2:
表2本申请实施例中涉及质粒和感受态细胞的说明
三、本申请实施例中涉及的基因片段及试剂说明：
本申请实施例中涉及的基因片段，包括引物、如SEQ ID NO：2所示的核苷酸序列等由生工生物工程(上海)股份有限公司合成。
本申请实施例中涉及的限制性内切酶(如：BamH I、Nde I和Dpn I)、T4连接酶、KOD高保真酶试剂盒、胶回收试剂盒、10×T4连接酶缓冲液(Buffer)、双蒸水(ddH2O)等分子试剂均购自宝生物工程(大连)有限公司。
下面结合实施例进一步说明本申请的技术方案和有益效果。
实施例1：构建重组表达载体pET24a-ZM
运用基因挖掘技术，从NCBI数据库挖掘出源自运动发酵单胞菌(Zymomonasmobilis)的丙酮酸脱羧酶，所述丙酮酸脱羧酶的氨基酸序列如SEQ ID NO：1所示，NCBI登录号为WP_014849477.1。依据E.coli密码子偏好性进行密码子优化，以全基因合成的方法合成编码SEQ ID NO：1所示的氨基酸序列的核苷酸序列，该核苷酸序列如SEQ ID NO：2所示。
选择未插入外源基因的pET24a质粒作为载体，pET24a质粒具有酶切位点BamH I和Nde I，重组表达载体pET24a-ZM的构建方法包括如下步骤：
S1.1、在SEQ ID NO：2所示的核苷酸序列的两端分别加入酶切位点BamH I和NdeI，人工合成基因片段后，采用BamH I和Nde I限制性内切酶对合成的基因片段进行双酶切，1％琼脂糖凝胶电泳检测酶切完全后，胶回收目的基因片段，其中，双酶切后回收目的基因片段的操作根据胶回收试剂盒操作说明实施；
S1.2、采用BamH I和Nde I限制性内切酶对pET24a质粒进行双酶切，1％琼脂糖凝胶电泳检测酶切完全后，胶回收载体骨架，其中，双酶切后回收载体骨架的操作根据胶回收试剂盒操作说明实施；
S1.3、将步骤S1.1获得的目的基因片段与步骤S1.2获得的载体骨架相混合，在T4连接酶的作用下16℃连接过夜，然后将连接产物转化至DH5a感受态细胞内，挑取单克隆子测序验证，提取测序正确的重组质粒，获得包含转氨酶编码基因的重组表达载体，命名pET24a-AT，其中，重组体系为20μL，具体是：2μL的10×T4连接酶缓冲液(Buffer)、5μL的目的基因片段、5μL的载体骨架、2μL的T4连接酶以及6μL的双蒸水(ddH2O)。
实施例2构建重组工程菌的定向突变库
采用定点突变策略，以实施例1构建的pET24a-AT作为DNA模板，根据待突变的氨基酸位点利用Oligo7软件来设计点突变引物，通过在上下游突变引物的5’端以插入、替换或缺失碱基的方式引入突变，突变位点如表1所示。需要说明的是，本领域技术人员根据突变位点引入的方式，结合引物设计的基本原则可以获得突变引物的核苷酸序列。以构建W392I，上游突变引物的核苷酸序列如SEQ ID NO：33所示，下游突变引物的核苷酸序列如SEQ ID NO：34所示，共设计获得十五组突变引物对，以分别引入表1所示的十五种突变形式。
选择实施例1中构建的重组表达载体pET24a-AT为模板，分别以十五组突变引物对作为PCR引物，采用KOD高保真酶试剂盒进行反向PCR，从而获得十五种突变序列。其中，反向PCR的反应程序为：95℃预变性3min；98℃变性30s，55℃退火30s，68℃延伸3min，28个循环；72℃延伸5min。
分别使用Dpn I限制性内切酶处理十五种突变序列，酶切产物经T4连接酶连接后转化大肠杆菌BL21(DE3)感受态，随后涂布含卡那霉素的LB抗性平板，置于37℃倒置培养18h，挑选单菌落转接含卡那霉素的LB液体培养基中，挑选培养液送样测序，将测序正确的克隆子保存备用，从而获得以大肠杆菌为宿主的重组工程菌，即获得分别用于表达丙酮酸脱羧酶突变体M1至M15的重组工程菌T1至T15。
实施例3重组工程菌的诱导表达及后处理
将实施例2获得的重组工程菌接种至含50μg/mL卡那霉素的LB液体培养基中，在37℃、180r/min的条件下培养至OD600为0.6～0.8，获得种子菌液。将种子菌液以1％的体积浓度接种至新鲜的含终浓度为50μg/mL卡那霉素的自诱导培养基，置于30℃培养18h，获得培养液。将培养液在25℃、8000r/min的条件下离心10min，弃上清液以收集沉淀物，将沉淀物用pH为7.0的PB缓冲液清洗数遍，收集湿菌体备用。
通过超纯水重悬制得的湿菌体，获得菌体浓度(mg/L)为20％的菌液。采用超声波破碎法或高压均质破碎法处理菌液，破碎条件可依据实际需要自行选择。示例超声波破碎法的工作参数为：破碎1s；暂停2s；在180W的功率下，破碎10min。示例高压均质破碎法的工作参数为：在50HZ和800bar的条件下，破碎两次。
菌液破碎处理后，在4℃、12000r/min的条件下离心10min至15min，以除去细胞碎片和大分子杂质，收集上清液保存于-20℃和4℃以备用，上清液即为含有丙酮酸脱羧酶突变体的酶液。
对比例1
本对比例提供了一种丙酮酸脱羧酶，所述丙酮酸脱羧酶的氨基酸序列如SEQ IDNO：1所示，编码如SEQ ID NO：1所示的氨基酸序列的核苷酸序列如SEQ ID NO：2所示。
将重组表达载体pET24a-ZM转化入大肠杆菌BL21(DE3)感受态，随后涂布含卡那霉素的LB抗性平板，置于37℃倒置培养18h，挑选单菌落转接含卡那霉素的LB液体培养基中，挑选培养液送样测序，将测序正确的克隆子保存备用，从而获得重组工程菌T0。
将重组工程菌T0按照实施例3的方法进行诱导表达及后处理，将获得的酶液保存于-20℃和4℃以备用。
实验例1初步比较丙酮酸脱羧酶突变体M1至M15的催化反应活性和光学选择性
取100mL的烧瓶，向其中依次加入40mL的浓度为0.1mol/L的PB缓冲液(pH为6.5)、10mL的二甲基亚砜(DMSO)、50μL的浓度为50mg/mL焦磷酸硫胺素(TPP)和50μL的浓度为2mol/L的Mg2+缓冲液，温和搅拌均匀，然后加入底物：500μL的苯甲醛和0.6g的丙酮酸钠，待底物充分溶解后使用10％NaOH调整体系pH为6.5，获得母液。
将1mL的母液与5μL的含有单种丙酮酸脱羧酶突变体的酶液(实施例3制得)混合制得测活体系，一共制得十六组测活体系(分别对应为包含丙酮酸脱羧酶突变体M1的测活体系至包含丙酮酸脱羧酶突变体M15的测活体系，以及包含对比例1的丙酮酸脱羧酶的测活体系)。将各组测活体系分别置于30℃、180r/min的摇床反应2h，待反应结束后，利用高效液相色谱(High Performance Liquid Chromatography,HPLC)法对反应液进行检测分析，反应液中目标产物的结构式如下式(Ⅳ)所示：
其中，HPLC的仪器型号为岛津LC-16检测器，HPLC的工作条件如下：
(1)进样液的制备：取500μL的反应液与500μL的对二甲苯混合，充分混匀离心以收集上清液；取10μL的上清液，向其中加入990μL的流动相，震荡混匀后进样，每次进样量10μL；
(2)色谱柱：大赛璐OD-H柱，250*4.6mm，5μm。
(3)流动相的制备：将无水正己烷和无水异丙醇按照正己烷：异丙醇的体积比为9：1混合配制而成。
(4)流速：1mL/min。
(5)分析时间：20min。
(6)柱温：30℃。
根据底物化合物的减少量计算底物转化率(％)，底物转化率(％)的计算公式如下式(Ⅲ):
在式(Ⅲ)中，A0为底物峰面积，A1为产物峰面积。
作为示例，图1示出了包含丙酮酸脱羧酶突变体M3的反应体系反应2h获得的反应液的HPLC图谱，以及图2示出了包含丙酮酸脱羧酶突变体M15的反应体系反应2h获得的反应产物的HPLC图谱。
各个丙酮酸脱羧酶突变体(M1至M15)以及对比例1的丙酮酸脱羧酶对底物的转化率，以及对应生成的目标产物的光学纯度详见下表3：
表3丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15以及对比例1的丙酮酸脱羧酶的初步测活数据
由表3可知，丙酮酸脱羧酶突变体M1至M15是在对比例1的丙酮酸脱羧酶的基础上作出一个或多个位点突变而获得的突变体，相较于对比例1的丙酮酸脱羧酶，M1至M15对底物的转化率显著提升，并且对应的反应体系生成的目标产物的光学纯度也明显提高。以丙酮酸脱羧酶突变体M15为例，M15对底物的转化率可达24.32％，其是对比例1的丙酮酸脱羧酶对底物的转化率的12倍；此外，包含M15的反应体系生成的目标产物的光学纯度可达99.8％，其是包含对比例1中丙酮酸脱羧酶的反应体系生成的目标产物的光学纯度的1.1倍。
此外，对于采用单一位点突变方式获得的丙酮酸脱羧酶突变体来说，采用W551D点突变方式获得的丙酮酸脱羧酶突变体的底物转化率最高，其次是M475K；对于多位点组合突变方式获得的丙酮酸脱羧酶突变体来说，采用T7R、L38M、W551D和M475K组合突变方式获得的丙酮酸脱羧酶突变体的综合催化性能最佳。
实验例2复筛比较丙酮酸脱羧酶突变体M1至M15对底物的转化率
提供装有磁力搅拌转子的洁净三口烧瓶，向三口烧瓶中依次加入55g的纯水和6.4g的丙酮酸钠，将盛装有纯水和丙酮酸钠的三口烧瓶放置于30℃的恒温磁力搅拌水浴锅中，开启搅拌，然后向三口烧瓶内依次加入6g的苯甲醛、100μL的浓度为2mol/L的Mg2+缓冲液和100μL的浓度为50mg/mL焦磷酸硫胺素(TPP)以获得混合体系，采用10％(质量/体积,w/v)NaOH调节混合体系的pH至5.95±0.2，接着继续向三口烧瓶内加入7.0mL的含有单种丙酮酸脱羧酶突变体的酶液(实施例3制得)或对比例1的丙酮酸脱羧酶酶液以获得反应体系，采用50％(质量/体积,w/v)乙酸调节反应体系的pH至6.2±0.2。将反应体系置于30℃下恒温反应24h。
待反应结束后，利用HPLC法对反应液中的目标产物(同实验例1)进行检测分析，HPLC的检测方法参照实验例1进行。作为示例，图3和图4分别示出了包含丙酮酸脱羧酶突变体M3的反应体系反应30min和24h获得的反应液的HPLC图谱，图5和图6分别示出了包含丙酮酸脱羧酶突变体M15的反应体系反应30min和24h获得的反应液的HPLC图谱。
各个丙酮酸脱羧酶突变体(M1至M15)以及对比例1的丙酮酸脱羧酶对底物(苯甲醛)的转化率数据详见下表4：
表4丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15以及对比例1的丙酮酸脱羧酶的复筛实验数据
由表4可知，在复筛实验中，丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15对底物的转化率明显优于对比例1的丙酮酸脱羧酶。以丙酮酸脱羧酶突变体M15为例，丙酮酸脱羧酶突变体M15对底物的转化率可达95.2％，是对比例1的丙酮酸脱羧酶对底物转化率的38倍。此外，对于采用单一位点突变方式获得的丙酮酸脱羧酶突变体来说，采用W551D点突变方式获得的丙酮酸脱羧酶突变体的底物转化率最高，其次是M475K；对于多位点组合突变方式获得的丙酮酸脱羧酶突变体来说，采用T7R、L38M、W551D和M475K组合突变方式获得的丙酮酸脱羧酶突变体的底物转化率最高。
实验例3优化工艺条件下比较重组工程菌T1至T15对底物的转化率
提供装有磁力搅拌转子的洁净三口烧瓶，向三口烧瓶中加入55g的纯水和6.4g的丙酮酸钠，将盛装有纯水和丙酮酸钠的三口烧瓶放置于30℃的恒温磁力搅拌水浴锅中，开启搅拌，然后向三口烧瓶内依次加入100μL的浓度为2mol/L的Mg2+缓冲液和100μL的浓度为50mg/mL焦磷酸硫胺素(TPP)以获得混合体系，采用10％(质量/体积,w/v)NaOH调节混合体系的pH至5.95±0.2，接着继续向三口烧瓶内加入7.5mL的含有单种丙酮酸脱羧酶突变体的酶液(实施例3制得)或对比例1的丙酮酸脱羧酶酶液以获得预反应体系，然后使用蠕动泵向预反应体系中缓慢滴加6g的苯甲醛(滴加速度为0.4mL/h)以进行催化反应，在催化反应过程中，采用50％(质量/体积,w/v)乙酸控制整个反应体系的pH为6.2±0.2，30℃恒温反应24h。
待反应结束后，利用HPLC法对反应液中的目标产物(同实验例1)进行检测分析，HPLC的检测方法参照实验例1进行。作为示例，图7和图8分别示出了向包含丙酮酸脱羧酶突变体M3的预反应体系流加苯甲醛1h和流加苯甲醛24h获得的反应液的HPLC图谱，图9和图10分别示出了包含丙酮酸脱羧酶突变体M15的预反应体系流加苯甲醛1h和流加苯甲醛24h获得的反应液的HPLC图谱。
各个丙酮酸脱羧酶突变体(M1至M15)以及对比例1的丙酮酸脱羧酶对底物(苯甲醛)的转化率数据详见下表5：
表5丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15以及对比例1的丙酮酸脱羧酶的优化工艺条件实验数据
由表5可知，在优化工艺条件实验中，丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15对底物的转化率明显优于对比例1的丙酮酸脱羧酶。以丙酮酸脱羧酶突变体M15为例，丙酮酸脱羧酶突变体M15对底物的转化率可达99.7％，是对比例1的丙酮酸脱羧酶对底物转化率的11倍。
实验例4比较丙酮酸脱羧酶突变体M1至M15对底物浓度的耐受性
提供装有磁力搅拌转子的洁净三口烧瓶，向三口烧瓶中依次加入60g的纯水、100μL的浓度为2mol/L的Mg2+缓冲液和100μL的浓度为50mg/mL焦磷酸硫胺素(TPP)以获得混合体系，采用10％(质量/体积,w/v)NaOH调节混合体系的pH至5.95±0.2，接着继续向三口烧瓶内加入7.0mL的含有单种丙酮酸脱羧酶突变体的酶液(实施例3制得)或对比例1的丙酮酸脱羧酶酶液，再加入预设量的苯甲醛以获得预反应体系，每种酶液分别设置四组预反应体系，四组预反应体系的区别之处仅在于：苯甲醛的添加量不同，第一组预反应体系中苯甲醛的添加量为1g(对应反应体系中苯甲醛浓度为1％)，第二组预反应体系中苯甲醛的添加量为2g(对应反应体系中苯甲醛浓度为2％)，第三组预反应体系中苯甲醛的添加量为4g(对应反应体系中苯甲醛浓度为3％)，第四组预反应体系中苯甲醛的添加量为6g(对应反应体系中苯甲醛浓度为6％)。将盛装有预反应体系的三口烧瓶放置于30℃的恒温磁力搅拌水浴锅中，开启搅拌1h，然后向各个预反应体系中分别加入6.4g的丙酮酸钠以对应获得反应体系，采用50％(质量/体积,w/v)乙酸调节反应体系的pH至6.2±0.2。将反应体系置于30℃下恒温反应2h。
待反应结束后，利用HPLC法对反应液中的目标产物(同实验例1)进行检测分析，HPLC的检测方法参照实验例1进行。各个丙酮酸脱羧酶突变体(M1至M15)以及对比例1的丙酮酸脱羧酶在不同的底物(苯甲醛)浓度下对底物(苯甲醛)的转化率数据详见下表6：
表6丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15以及对比例1的丙酮酸脱羧酶的耐受性实验数据
由表6可知，随着反应体系中底物浓度的逐渐升高，丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15以及对比例1的丙酮酸脱羧酶对底物(苯甲醛)的转化率均呈下降趋势，但在相同的底物浓度下，丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15对底物的转化率明显高于对比例1的丙酮酸脱羧酶对底物的转化率，说明：丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15对底物的耐受性能均优于对比例1的丙酮酸脱羧酶对底物的耐受性能。
当反应体系中底物浓度由1％升高至6％时，对应丙酮酸脱羧酶突变体M15对底物的转化率下降69％，而对比例1的丙酮酸脱羧酶对底物的转化率下降80％，说明丙酮酸脱羧酶突变体M15对底物的耐受性显著优于对比例1的丙酮酸脱羧酶，并且当反应体系中底物浓度为6％时，丙酮酸脱羧酶突变体M15对底物的转化率是对比例1的丙酮酸脱羧酶对底物的转化率的60倍。
实验例5比较丙酮酸脱羧酶突变体M1至M15的热稳定性
提供装有磁力搅拌转子的洁净三口烧瓶，向三口烧瓶中依次加入55g的纯水和6.4g的丙酮酸钠，将盛装有纯水和丙酮酸钠的三口烧瓶放置于30℃的恒温磁力搅拌水浴锅中，开启搅拌，然后向三口烧瓶内依次加入6g的苯甲醛、100μL的浓度为2mol/L的Mg2+缓冲液和100μL的浓度为50mg/mL焦磷酸硫胺素(TPP)以获得混合体系，采用10％(质量/体积,w/v)NaOH调节混合体系的pH至5.95±0.2，接着继续向三口烧瓶内加入7.0mL的含有单种丙酮酸脱羧酶突变体的酶液(实施例3制得)或对比例1的丙酮酸脱羧酶酶液以获得反应体系，每种酶液设置分别设置两组反应体系，两组反应体系的区别之处仅在于：一组加入的酶液未经热处理，另一组加入的酶液是经过50℃恒温热处理60min之后的酶液。采用50％(质量/体积,w/v)乙酸调节各个反应体系的pH至6.2±0.2，将反应体系置于30℃下恒温反应2h。
待反应结束后，利用HPLC法对反应液中的目标产物(同实验例1)进行检测分析，HPLC的检测方法参照实验例1进行。各个丙酮酸脱羧酶突变体(M1至M15)以及对比例1的丙酮酸脱羧酶未经热处理对底物(苯甲醛)的转化率和热处理后对底物(苯甲醛)的转化率详见下表7：
表7丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15以及对比例1的丙酮酸脱羧酶的热稳定性实验数据
由表7可知，丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15的热稳定性能明显优于对比例1的丙酮酸脱羧酶的热稳定性能。对于丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15，虽然相较于未经热处理的底物转化率，热处理后对底物的转化率有所下降，但是下降幅度较小，以丙酮酸脱羧酶突变体M15为例，热处理后对底物的转化率仅下降6.3％，而对比例1的丙酮酸脱羧酶经热处理后基本丧失对底物的催化活性。
由实验例1至实施例5可知，丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15的综合性能明显优于对比例1的丙酮酸脱羧酶，相较于对比例1的丙酮酸脱羧酶，丙酮酸脱羧酶突变体M1至丙酮酸脱羧酶突变体M15具有催化活性更高、光学选择性更好、稳定性更佳的优点。
以上对本申请所提供的一种丙酮酸脱羧酶突变体及其在制备α-羟基酮类化合物中的应用，进行了详细介绍。本文中应用了具体个例对本申请的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本申请的技术方案及其核心思想；本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请实施例的技术方案的范围。
序列表
<110> 杭州酶易生物技术有限公司
赤峰艾克制药科技股份有限公司
<120> 丙酮酸脱羧酶突变体及其在制备α-羟基酮类化合物中的应用
<130> SUP220088CN
<141> 2022-04-07
<160> 34
<170> SIPOSequenceListing 1.0
<210> 1
<211> 568
<212> PRT
<213> Zymomonas mobilis
<400> 1
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 2
<211> 1707
<212> DNA
<213> Zymomonas mobilis
<400> 2
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 3
<211> 568
<212> PRT
<213> 人工序列
<400> 3
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Ile Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 4
<211> 1707
<212> DNA
<213> 人工序列
<400> 4
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tctatcttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 5
<211> 568
<212> PRT
<213> 人工序列
<400> 5
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Met Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 6
<211> 1707
<212> DNA
<213> 人工序列
<400> 6
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatggaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 7
<211> 568
<212> PRT
<213> 人工序列
<400> 7
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Asp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 8
<211> 1707
<212> DNA
<213> 人工序列
<400> 8
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa gatggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 9
<211> 568
<212> PRT
<213> 人工序列
<400> 9
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Lys Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 10
<211> 1707
<212> DNA
<213> 人工序列
<400> 10
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccacta agtggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 11
<211> 568
<212> PRT
<213> 人工序列
<400> 11
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Leu His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 12
<211> 1707
<212> DNA
<213> 人工序列
<400> 12
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgctgca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 13
<211> 568
<212> PRT
<213> 人工序列
<400> 13
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Pro Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 14
<211> 1707
<212> DNA
<213> 人工序列
<400> 14
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gccctgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 15
<211> 568
<212> PRT
<213> 人工序列
<400> 15
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Lys Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 16
<211> 1707
<212> DNA
<213> 人工序列
<400> 16
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttaagatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 17
<211> 568
<212> PRT
<213> 人工序列
<400> 17
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Lys Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Asp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 18
<211> 1707
<212> DNA
<213> 人工序列
<400> 18
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttaagatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa gatggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 19
<211> 568
<212> PRT
<213> 人工序列
<400> 19
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Ala Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Tyr Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 20
<211> 1707
<212> DNA
<213> 人工序列
<400> 20
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tctgcattca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attgtataaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 21
<211> 568
<212> PRT
<213> 人工序列
<400> 21
Met Ser Tyr Thr Val Gly Thr Trp Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Met Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 22
<211> 1707
<212> DNA
<213> 人工序列
<400> 22
atgagttata ctgtcggtac ctggttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctatgaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 23
<211> 568
<212> PRT
<213> 人工序列
<400> 23
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Tyr Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Glu Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 24
<211> 1707
<212> DNA
<213> 人工序列
<400> 24
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgctatact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagaa gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 25
<211> 568
<212> PRT
<213> 人工序列
<400> 25
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Met Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Lys Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Asp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 26
<211> 1707
<212> DNA
<213> 人工序列
<400> 26
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tatgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttaagatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa gatggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 27
<211> 568
<212> PRT
<213> 人工序列
<400> 27
Met Ser Tyr Thr Val Gly Arg Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Met Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gly Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 28
<211> 1707
<212> DNA
<213> 人工序列
<400> 28
atgagttata ctgtcggtcg atatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tatgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctggtatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 29
<211> 568
<212> PRT
<213> 人工序列
<400> 29
Met Ser Tyr Thr Val Gly Arg Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Lys Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Tyr Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 30
<211> 1707
<212> DNA
<213> 人工序列
<400> 30
atgagttata ctgtcggtcg atatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttaagatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggttatc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 31
<211> 568
<212> PRT
<213> 人工序列
<400> 31
Met Ser Tyr Thr Val Gly Arg Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Met Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Lys Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Asp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 32
<211> 1707
<212> DNA
<213> 人工序列
<400> 32
atgagttata ctgtcggtcg atatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tatgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttaagatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa gatggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 33
<211> 30
<212> DNA
<213> 人工序列
<400> 33
attttcaatg ctcagcgcat gaagctcccg 30
<210> 34
<211> 30
<212> DNA
<213> 人工序列
<400> 34
agagtcaccg gtttcagcaa taaccgtcgt 30
Claims (10)
1.一种丙酮酸脱羧酶突变体，其特征在于，所述丙酮酸脱羧酶突变体的氨基酸序列是由SEQ ID NO：1所示的氨基酸序列发生一个或多个点突变而获得的氨基酸序列；所述丙酮酸脱羧酶突变体具有丙酮酸脱羧酶活性；
其中，发生所述点突变的方式为以下任意一种：
(1) W392I；
(2) I472M；
(3) W551D；
(4) G294K；
(5) I476L；
(6)V555P；
(7)M475K；
(8)W551D和M475K；
(9)V549Y和W392A；
(10) T8W和A246M；
(11) N169Y和V450E；
(12) M475K、L38M和W551D；
(13)T7R、L38M和Q452G；
(14) T7R、K553Y和M475K；或
(15) T7R、L38M、W551D和M475K。
2.根据权利要求1所述的丙酮酸脱羧酶突变体，其特征在于，所述丙酮酸脱羧酶突变体的氨基酸序列选自如SEQ ID NO：3、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13、SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ IDNO：23、SEQ ID NO：25、SEQ ID NO：27、SEQ ID NO：29或SEQ ID NO：31任一所示的氨基酸序列。
3.一种核酸分子，其特征在于，所述核酸分子编码如权利要求1或2中所述的丙酮酸脱羧酶突变体。
4.根据权利要求3所述的核酸分子，其特征在于，所述核苷酸序列选自如SEQ ID NO：4、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14、SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：20、SEQ ID NO：22、SEQ ID NO：24、SEQ ID NO：26、SEQ ID NO：28、SEQ ID NO：30或SEQ ID NO：32任一所示的核苷酸序列。
5.一种重组表达载体，其特征在于，所述重组表达载体包括载体，以及如权利要求3或4所述的核酸分子；所述载体选自质粒、粘粒或病毒载体。
6.根据权利要求5所述的重组表达载体，其特征在于，所述载体选自噬菌体。
7.一种重组表达转化体，其特征在于，所述重组表达转化体包括宿主，以及引入至所述宿主体内的如权利要求3或4所述的核酸分子、或如权利要求5或6所述的重组表达载体；所述宿主选自真核生物或原核生物。
8.一种丙酮酸脱羧酶突变体的制备方法，其特征在于，通过培养如权利要求7所述的重组表达转化体，以及从培养物中获得所述的丙酮酸脱羧酶突变体。
9.一种手性α-羟基酮类化合物的制备方法，其特征在于，以如权利要求1或2中所述的丙酮酸脱羧酶突变体或如权利要求8所述的制备方法制得的丙酮酸脱羧酶突变体作为催化剂，第一化合物和第二化合物接触反应生成α-羟基酮类化合物；
其中，第一化合物具有下面通式(Ⅰ)所示的结构：
(Ⅰ)
在通式(Ⅰ)，R1选自氢原子或羟基，X选自氢原子或一价金属离子；
第二化合物具有下面通式(Ⅱ)所示的结构：
(Ⅱ)
在通式(Ⅱ)中，R2选自芳基或杂芳基。
10.根据权利要求9所述的制备方法，其特征在于，所述第一化合物选自丙酮酸钠，所述第二化合物选自苯甲醛。
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