CN114621982B - 香叶基二磷酸酯的生物合成方法及其在制备大麻类化合物中的应用 - Google Patents

香叶基二磷酸酯的生物合成方法及其在制备大麻类化合物中的应用 Download PDF

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CN114621982B
CN114621982B CN202210261323.0A CN202210261323A CN114621982B CN 114621982 B CN114621982 B CN 114621982B CN 202210261323 A CN202210261323 A CN 202210261323A CN 114621982 B CN114621982 B CN 114621982B
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CN114621982A (zh
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李珍珠
王筱
逯晓云
夏文豪
陈贤情
刘诗梦
杨月
黄利辉
江会锋
王文
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Jiaxing Synbiolab Biotechnology Co ltd
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    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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Abstract

本申请提供了一种香叶基二磷酸酯的生物合成方法及其在制备大麻类化合物中的应用，所述生物合成方法包括步骤：将香叶醇、腺嘌呤核苷三磷酸、1号酶以及2号酶进行接触反应，获得香叶基二磷酸酯，其中，1号酶选自羟乙基噻唑激酶、硫胺素生物合成双功能酶、胆碱激酶、十一烯醇激酶、异戊烯基磷酸激酶或甲羟戊酸激酶中的至少一种，2号酶选自异戊烯基磷酸激酶、甲羟戊酸激酶或磷酸甲羟戊酸激酶中的至少一种；所述香叶基二磷酸酯生物合成方法能够应用于制备大麻类化合物，以香叶醇作为起始原料，具有底物转化效率高、条件简单、产物单一以及无副产物等优点。

Description

香叶基二磷酸酯的生物合成方法及其在制备大麻类化合物中 的应用
技术领域
本申请涉及生物技术领域，具体涉及一种香叶基二磷酸酯的生物合成方法及其在制备大麻类化合物中的应用。
背景技术
大麻类化合物又称大麻素，是一大类具有生物活性的萜酚类化合物，包含数百种结构类似物，例如：大麻萜酚(Cannabigerol,CBG)、大麻萜酚酸(Cannabigerolic Acid,CBGA)、次大麻萜酚(Cannabigerovarin,CBGV)、次大麻萜酚酸(Cannabigerovarinic Acid,CBGVA)等。基于大麻类化合物与两种细胞受体(CB1和CB2)之间的相互作用，使得大麻类化合物在医疗领域应用广泛，可用作止吐药、抗惊厥药、镇痛药、抗抑郁药、安宁治疗药等。
目前，大麻类化合物的生物合成方法通常以葡萄糖作为起始原料，涉及十余个酶促反应步骤，其中，包括类异戊二烯前体(异戊烯基二磷酸(IPP)和二甲基烯丙基二磷酸(DMAPP))的合成步骤，所述类异戊二烯前体的合成步骤主要有两种生物合成途径，一种生物合成途径为2-甲基-D-赤藓糖醇-4-磷酸途径(2-methyl-D-erythritol-4-phosphatepathway,MEP)，包含七个酶促反应步骤，主要存在于真细菌、植物的叶绿体、蓝细菌、藻类和顶复门寄生虫中；另一种生物合成途径为甲羟戊酸(Mevalonate pathway,MVA)途径，包含七个酶促反应步骤，主要存在于大多数真核生物、古细菌和少数真细菌中。因此，现有技术中大麻类化合物的生物合成方法具有合成途径长、副产物多、产物得率低、产物分离纯化难度高的缺点。
因此，简化大麻类化合物的生物合成方法以提高产物得率对大麻类化合物的应用及发展具有重要意义。
发明内容
本申请提供一种香叶基二磷酸酯的生物合成方法及其在制备大麻类化合物中的应用，以简化香叶基二磷酸酯和大麻类化合物的生物合成方法。
第一方面，本申请提供了一种香叶基二磷酸酯的生物合成方法，包括步骤：将香叶醇、腺嘌呤核苷三磷酸、1号酶以及2号酶进行接触反应，获得香叶基二磷酸酯；
其中，所述1号酶选自羟乙基噻唑激酶、硫胺素生物合成双功能酶、胆碱激酶、十一烯醇激酶、异戊烯基磷酸激酶或甲羟戊酸激酶中的至少一种；
所述2号酶选自异戊烯基磷酸激酶、甲羟戊酸激酶或磷酸甲羟戊酸激酶中的至少一种。
进一步地，所述1号酶具有如SEQ ID NO：1至SEQ ID NO：16中任意一种所示的氨基酸序列。
进一步地，所述2号酶具有如SEQ ID NO：17至SEQ ID NO：28中任意一种所示的氨基酸序列。
进一步地，所述1号酶的氨基酸序列如SEQ ID NO：4所示，且所述2号酶的氨基酸序列如SEQ ID NO：18所示。
第二方面，本申请提供了一种用于生物合成香叶基二磷酸酯的生产系统，包括：
(a)香叶醇；
(b)腺嘌呤核苷三磷酸；
(c)1号酶，用于将香叶醇和腺嘌呤核苷三磷酸转化为香叶基磷酸；以及
(d)2号酶，用于将香叶基磷酸和腺嘌呤核苷三磷酸转化为香叶基二磷酸酯；
其中，所述1号酶选自羟乙基噻唑激酶、硫胺素生物合成双功能酶、胆碱激酶、十一烯醇激酶、异戊烯基磷酸激酶或甲羟戊酸激酶中的至少一种；
所述2号酶选自异戊烯基磷酸激酶、甲羟戊酸激酶或磷酸甲羟戊酸激酶中的至少一种。
进一步地，所述用于生物合成香叶基二磷酸酯的生产系统还包括Tris-HCl、KCl和MgCl2。
进一步地，所述香叶醇：所述1号酶：所述2号酶的摩尔比为152：(1～6)：(1～6)。
第三方面，本申请提供了一种如第一方面中任意一种所述的生物合成方法、或如第二方面中任意一种所述的用于生物合成香叶基二磷酸酯的生产系统在制备大麻类化合物中的应用，在胞质异戊烯基转移酶的催化作用下，将通过所述生物合成方法、或所述用于生物合成香叶基二磷酸酯的生产系统制得的香叶基二磷酸酯与前体化合物接触反应，生成大麻类化合物；
其中，所述前体化合物具有下面通式(Ⅰ)所示的结构：
在通式(Ⅰ)中，R1为碳原子数是1、2、3、4或5的烷基，R2和R3彼此独立地选自氢原子、羧基或甲基。
进一步地，所述胞质异戊烯基转移酶为NphB异戊烯基转移酶。
进一步地，所述前体化合物为橄榄醇，且所述大麻类化合物为大麻萜酚；
或者，所述前体化合物为橄榄酸，且所述大麻类化合物为大麻萜酚酸；
或者，所述前体化合物为4-丙基间苯二酚，且所述大麻类化合物为次大麻萜酚；
或者，所述前体化合物为2,4-二羟基丙基苯甲酸，且所述大麻类化合物为次大麻萜酚酸。
第四方面，本申请还提供了一种用于生物合成大麻类化合物的生产系统，包括：
(a)香叶醇；
(b)腺嘌呤核苷三磷酸；
(c)1号酶，用于将香叶醇和腺嘌呤核苷三磷酸转化为香叶基磷酸；
(d)2号酶，用于将香叶基磷酸和腺嘌呤核苷三磷酸转化为香叶基二磷酸酯；
(e)前体化合物；以及
(f)胞质异戊烯基转移酶，用于将所述前体化合物异戊二烯化生成大麻类化合物；
其中，所述1号酶选自羟乙基噻唑激酶、硫胺素生物合成双功能酶、胆碱激酶、十一烯醇激酶、异戊烯基磷酸激酶或甲羟戊酸激酶中的至少一种；
所述2号酶选自异戊烯基磷酸激酶、甲羟戊酸激酶或磷酸甲羟戊酸激酶中的至少一种；
所述前体化合物具有下面通式(Ⅰ)所示的结构：
在通式(Ⅰ)中，R1为碳原子数是1、2、3、4或5的烷基，R2和R3彼此独立地选自氢原子、羧基或甲基。
进一步地，所述胞质异戊烯基转移酶为NphB异戊烯基转移酶；和/或，所述香叶醇：所述1号酶：所述2号酶的摩尔比为152：(1～6)：(1～6)。
本申请提供了一种香叶基二磷酸酯的生物合成方法及其在制备大麻类化合物中的应用，具有如下技术效果：
在本申请的香叶基二磷酸酯生物合成方法中，以香叶醇作为起始原料，仅需两步酶促反应即可生成香叶基二磷酸酯，具有制备工序简单、制备周期短的优点；进一步地，当采用GK4作为第一步酶促反应的酶，且采用PGK2作为第二步酶促反应的酶时，底物相对转化率、香叶基二磷酸酯相对得率和香叶基二磷酸酯相对纯度更佳。
所述香叶基二磷酸酯生物合成方法能够应用于制备大麻类化合物，在胞质异戊烯基转移酶的催化作用下，将香叶基二磷酸酯与通式(Ⅰ)所示的前体化合物进行接触反应以生成大麻类化合物，并且能够以香叶醇作为起始原料，有效地缩短了大麻类化合物的生物合成途径，相较于传统的包含十余个酶促反应步骤的大麻类化合物生物合成途径，本申请中大麻类化合物的生物合成途径不必以葡萄糖作为起始原料，无需经过tHMG1等关键限速酶反应，同时避免使用乙酰辅酶a、丙二酰辅酶a等多种辅酶，省略了耗时长且产率低的中间反应，具有底物转化效率高、条件简单、产物单一、无副产物以及产物分离纯化难度低等优点。
附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍。
图1是本申请实施例中pET-28a-NphB重组质粒的结构示意图；
图2是本申请实施例中含有NphB的样品的SDS-PAGE电泳图，其中，泳道1表示菌液破碎后离心获得的沉淀物，泳道2表示赛默飞PageRuler蛋白marker，泳道3表示菌液破碎后离心获得的上清液，泳道4表示流穿样品，泳道5表示20mmol/L咪唑溶液的洗脱液，泳道6表示50mmol/L咪唑溶液的洗脱液，泳道7表示100mmol/L咪唑溶液的洗脱液，泳道8表示250mmol/L咪唑溶液的洗脱液，泳道9表示300mmol/L咪唑溶液的洗脱液；
图3是本申请实施例中含有1号酶(氨基酸序列为SEQ ID NO：4所示，编码基因的核苷酸序列如SEQ ID NO：31所示)的样品的SDS-PAGE电泳图，其中，泳道1表示赛默飞PageRuler蛋白marker，泳道2表示菌液破碎后离心获得的沉淀物，泳道3表示菌液破碎后离心获得的上清液，泳道4表示流穿样品，泳道5表示20mmol/L咪唑溶液的洗脱液，泳道6表示50mmol/L咪唑溶液的洗脱液，泳道7表示100mmol/L咪唑溶液的洗脱液，泳道8表示250mmol/L咪唑溶液的洗脱液，泳道9表示300mmol/L咪唑溶液的洗脱液；
图4为本申请实施例中含有2号酶(氨基酸序列为SEQ ID NO：18所示，编码基因的核苷酸序列如SEQ ID NO：32所示)的样品的SDS-PAGE电泳图，其中，泳道1表示赛默飞PageRuler蛋白marker，泳道2表示菌液破碎后离心获得的沉淀物，泳道3表示菌液破碎后离心获得的上清液，泳道4表示流穿样品，泳道5表示20mmol/L咪唑溶液的洗脱液，泳道6表示50mmol/L咪唑溶液的洗脱液，泳道7表示100mmol/L咪唑溶液的洗脱液，泳道8表示250mmol/L咪唑溶液的洗脱液，泳道9表示300mmol/L咪唑溶液的洗脱液
图5为实施例1中采用香叶醇、腺嘌呤核苷三磷酸以及GK4进行接触反应，生成的香叶基磷酸的液相色谱图；
图6为实施例1中采用香叶醇、腺嘌呤核苷三磷酸以及GK4进行接触反应，生成的香叶基磷酸的质谱图；
图7为实施例1中16种1号酶催化香叶醇和腺嘌呤核苷三磷酸生成香叶基磷酸的相对活性图；
图8为实施例2中采用香叶醇、腺嘌呤核苷三磷酸、GK4和PGK2进行接触反应，生成的香叶基磷酸的液相色谱图；
图9为实施例2中采用香叶醇、腺嘌呤核苷三磷酸、GK4和PGK2进行接触反应，生成的香叶基磷酸的质谱图；
图10为实施例2中12种酶组合催化香叶醇和腺嘌呤核苷三磷酸生成香叶基二磷酸酯的相对活性图；
图11为实施例3中香叶醇、腺嘌呤核苷三磷酸、GK4、PGK2、橄榄醇以及NphB异戊烯基转移酶进行接触反应生成的大麻萜酚的液相色谱图；
图12为实施例3中香叶醇、腺嘌呤核苷三磷酸、GK4、PGK2、橄榄醇以及NphB异戊烯基转移酶进行接触反应生成的大麻萜酚的质谱图；
图13为实施例4中香叶醇、腺嘌呤核苷三磷酸、GK4、PGK2、橄榄醇以及NphB异戊烯基转移酶进行接触反应生成的大麻萜酚的液相色谱图；
图14为实施例4中香叶醇、腺嘌呤核苷三磷酸、GK4、PGK2、橄榄醇以及NphB异戊烯基转移酶进行接触反应生成的大麻萜酚的质谱图。
具体实施方式
下面将结合本申请实施例，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。
本申请实施例提供一种香叶基二磷酸酯的生物合成方法及其在制备大麻类化合物中的应用。以下分别进行详细说明。需说明的是，以下实施例的描述顺序不作为对实施例优选顺序的限定。另外，在本申请的描述中，术语“包括”是指“包括但不限于”。
本申请实施例提供了一种香叶基二磷酸酯的生物合成方法，包括步骤：将香叶醇(Geraniol,CAS号为106-24-1)、腺嘌呤核苷三磷酸(Adenosine Triphosphate,ATP)、1号酶以及2号酶进行接触反应，获得香叶基二磷酸酯；其中，1号酶选自羟乙基噻唑激酶(Hydroxyethylthiazole kinase)、硫胺素生物合成双功能酶(Thiamine biosynthesisbifunctional)、胆碱激酶(Choline kinase)、十一烯醇激酶(Undecaprenol kinase)、异戊烯基磷酸激酶(Isopentenyl phosphate kinase)或甲羟戊酸激酶(Mevalonate kinase)中的至少一种，2号酶选自异戊烯基磷酸激酶(Isopentenyl phosphate kinase)、甲羟戊酸激酶(Mevalonate kinase)或磷酸甲羟戊酸激酶中的至少一种(Phosphomevalonatekinase)。
反应式如下式(Ⅱ)所示：
在反应式(Ⅱ)中，1号酶催化香叶醇和腺嘌呤核苷三磷酸生成香叶基磷酸，2号酶催化香叶基磷酸和腺嘌呤核苷三磷酸生成香叶基二磷酸酯。在本申请实施例的生物合成方法中，以香叶醇作为起始原料，仅需两步酶促反应即可生成香叶基磷酸，具有制备工序简单、制备周期短的优点。
对上述香叶基二磷酸酯的生物合成方法需要说明的是，可以将香叶醇、腺嘌呤核苷三磷酸、1号酶以及2号酶一起混合进行酶促反应；也可以先将香叶醇、腺嘌呤核苷三磷酸和1号酶混合以进行第一步酶促反应，然后再加入2号酶以进行第二步酶促反应。
在本申请的一些实施例中，1号酶具有如SEQ ID NO：1至SEQ ID NO：16中任意一种所示的氨基酸序列。
在本申请的一些实施例中，2号酶具有如SEQ ID NO：17至SEQ ID NO：28中任意一种所示的氨基酸序列。
在本申请的一个实施例中，1号酶的氨基酸序列如SEQ ID NO：4所示，且2号酶的氨基酸序列如SEQ ID NO：18所示。
本申请实施例还提供了一种用于生物合成香叶基二磷酸酯的生产系统，所述生产系统包括：
(a)香叶醇；
(b)腺嘌呤核苷三磷酸；
(c)1号酶，用于将香叶醇和腺嘌呤核苷三磷酸转化为香叶基磷酸；以及
(d)2号酶，用于将香叶基磷酸和腺嘌呤核苷三磷酸转化为香叶基二磷酸酯；
其中，所述1号酶选自羟乙基噻唑激酶、硫胺素生物合成双功能酶、胆碱激酶、十一烯醇激酶、异戊烯基磷酸激酶或甲羟戊酸激酶中的至少一种；
所述2号酶选自异戊烯基磷酸激酶、甲羟戊酸激酶或磷酸甲羟戊酸激酶中的至少一种。
在本申请的一些实施例中，所述用于生物合成香叶基二磷酸酯的生产系统还包括Tris-HCl、KCl和MgCl2，其中，Tris-HCl用作整个反应体系的缓冲液；KCl用作缓冲液的组成部分之一，并且能够作为电解质；MgCl2用作缓冲液的组成部分之一，提供离子的同时，能够促进骨架聚合。
在本申请的一些实施例中，在所述用于生物合成香叶基二磷酸酯的生产系统中，香叶醇：1号酶：2号酶的摩尔比为152：(1～6)：(1～6)，若1号酶或2号酶的添加量过少，则香叶醇的转化率有限；若1号酶或2号酶的添加量过多，则会造成酶浪费，并且为产物的分离纯化增加了难度。
需要说明的是，所述用于生物合成香叶基二磷酸酯的生产系统例如可以是盛装有香叶醇、腺嘌呤核苷三磷酸、1号酶和2号酶的酶反应器，其中，1号酶和2号酶可以分别以纯种酶的形式添加于酶反应器中，1号酶和2号酶还可以分别以细胞裂解物、细胞提取物或其他制备物的形式添加于酶反应器中，其中，细胞裂解物、细胞提取物或其他制备物中所有的细胞已被破坏或以其他方式处理，使得所有或选择的细胞组分，例如细胞器、蛋白、核酸、细胞膜本身(或其片段或组分)等从细胞中释放出来或重悬于合适的培养基中和/或从细胞环境中纯化出来。此外，1号酶和2号酶可以来源于同一种细胞裂解物、细胞提取物或其他制备物，也可以分别来源于不同的细胞裂解物、细胞提取物或其他制备物中。
可以理解的是，也可以利用工程细胞生物合成香叶基二磷酸酯，用于生物合成香叶基二磷酸酯的工程细胞包括：
(ⅰ)宿主；
(ⅱ)编码1号酶的第一多核苷酸，位于宿主体内；
(ⅲ)编码2号酶的第二多核苷酸，位于宿主体内。
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，例如选自曲霉、毛霉、根霉、青霉、毕赤酵母、假丝酵母或汉逊酵母中的至少一种，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。
在宿主体内，第一多核苷酸和第二多核苷酸可以彼此独立地存在于一自主复制表达载体中，第一多核苷酸和第二多核苷酸也可以存在于同一个自主复制表达载体中，第一多核苷酸和/或第二多核苷酸也可以整合至宿主的基因组中。
如本申请所用，“表达载体”是指一种DNA构建体，其为基因组中包含目的基因的载体，并且载体的基因组中含有合适的控制序列，所述控制序列能够实现目的基因在合适的宿主中表达，载体例如可以是质粒、病毒、粘粒、噬菌体等。
作为示例，编码1号酶的第一多核苷酸具有如SEQ ID NO：31所示的核苷酸序列，编码2号酶的第二多核苷酸具有如SEQ ID NO：32所示的核苷酸序列。
采用工程细胞生物合成香叶基二磷酸酯的方法例如包括步骤：在合适的培养基中培养包含编码1号酶的第一多核苷酸和编码2号酶的第二多核苷酸的宿主，回收产生的香叶基二磷酸酯，其中，合适的培养基例如包括腺嘌呤核苷三磷酸、Tris-HCl、KCl和MgCl2。
本申请实施例还提供了本申请实施例中任意一种所述的香叶基二磷酸酯的生物合成方法、或如本申请实施例中任意一种所述的用于生物合成香叶基二磷酸酯的生产系统在制备大麻类化合物中的应用，具体为：在胞质异戊烯基转移酶的催化作用下，将通过所述香叶基二磷酸酯的生物合成方法、或所述用于生物合成香叶基二磷酸酯的生产系统制得的香叶基二磷酸酯与前体化合物接触反应，生成大麻类化合物，其中，前体化合物具有下面通式(Ⅰ)所示的结构：
在通式(Ⅰ)中，R1为碳原子数是1、2、3、4或5的烷基，R2和R3彼此独立地选自氢原子、羧基或甲基。
其中，香叶基二磷酸酯与前体化合物接触反应生成大麻类化合物的反应式如下式(Ⅲ)所示：
在本申请的一些实施例中，胞质异戊烯基转移酶为NphB异戊烯基转移酶。
在本申请的一个实施例中，前体化合物为橄榄醇，且大麻类化合物为大麻萜酚。
在本申请的另一个实施例中，前体化合物为橄榄酸，且大麻类化合物为大麻萜酚酸。
在本申请的另一个实施例中，前体化合物为4-丙基间苯二酚，且大麻类化合物为次大麻萜酚。
在本申请的另一个实施例中，前体化合物为2,4-二羟基丙基苯甲酸，且大麻类化合物为次大麻萜酚酸。
本申请实施例还提供了一种用于生物合成大麻类化合物的生产系统，所述生产系统包括：
(a)香叶醇；
(b)腺嘌呤核苷三磷酸；
(c)1号酶，用于将香叶醇和腺嘌呤核苷三磷酸转化为香叶基磷酸；
(d)2号酶，用于将香叶基磷酸和腺嘌呤核苷三磷酸转化为香叶基二磷酸酯；
(e)前体化合物；以及
(f)胞质异戊烯基转移酶，用于将所述前体化合物异戊二烯化生成大麻类化合物。
其中，1号酶、2号酶和前体化合物参照前述描述。
在本申请的一些实施例中，所述用于生物合成大麻类化合物的生产系统还包括Tris-HCl、KCl和MgCl2。
在本申请的一些实施例中，所述胞质异戊烯基转移酶为NphB异戊烯基转移酶。
在本申请的一些实施例中，在所述用于生物合成大麻类化合物的生产系统中，香叶醇：1号酶：2号酶的摩尔比为152：(1～6)：(1～6)，以尽可能提高香叶醇和前体化合物的转化率的同时，避免酶的浪费，降低产物的分离纯化难度。
需要说明的是，所述用于生物合成大麻类化合物的生产系统例如可以是盛装有香叶醇、腺嘌呤核苷三磷酸、1号酶、2号酶、前体化合物和胞质异戊烯基转移酶的酶反应器，其中，1号酶、2号酶和胞质异戊烯基转移酶可以分别以纯种酶的形式添加于酶反应器中，1号酶、2号酶和胞质异戊烯基转移酶还可以分别以细胞裂解物、细胞提取物或其他制备物的形式添加于酶反应器中。此外，1号酶、2号酶和胞质异戊烯基转移酶可以全部来源于同一种细胞裂解物、细胞提取物或其他制备物，也可以部分来源于同一种细胞裂解物、细胞提取物或其他制备物，也可以全部来源于不同的细胞裂解物、细胞提取物或其他制备物中。
可以理解的是，也可以利用工程细胞生物合成大麻类化合物，用于生物合成大麻类化合物的工程细胞包括：
(ⅰ)宿主；
(ⅱ)编码1号酶的第一多核苷酸，位于宿主体内；
(ⅲ)编码2号酶的第二多核苷酸，位于宿主体内；
(ⅳ)编码胞质异戊烯基转移酶的第三多核苷酸，位于宿主体内。
其中，第一多核苷酸、第二多核苷酸以及第三多核苷酸可以彼此独立地存在于一自主复制表达载体中，也可以部分存在于同一自主复制表达载体中，也可以全部存在于同一个自主复制表达载体中。此外，第一多核苷酸、第二多核苷酸或第三多核苷酸中的一种或多种也可以整合至宿主的基因组中。
作为示例，编码1号酶的第一多核苷酸具有如SEQ ID NO：31所示的核苷酸序列，且编码2号酶的第二多核苷酸具有如SEQ ID NO：32所示的核苷酸序列，且编码胞质异戊烯基转移酶的第三多核苷酸具有如SEQ ID NO：30所示的核苷酸序列。
采用工程细胞生物合成大麻类化合物的方法例如包括步骤：在合适的培养基中培养包含编码1号酶的第一多核苷酸、编码2号酶的第二多核苷酸以及编码第三多核苷酸的宿主，回收产生的香叶基二磷酸酯，其中，合适的培养基例如包括腺嘌呤核苷三磷酸、Tris-HCl、KCl和MgCl2。
相较于传统的包含十余个酶促反应步骤的大麻类化合物生物合成途径，本申请实施例中大麻类化合物的生物合成途径不必以葡萄糖作为起始原料，无需经过tHMG1等关键限速酶反应，同时避免使用乙酰辅酶a、丙二酰辅酶a等多种辅酶，省略了耗时长且产率低的中间反应，具有底物转化效率高、条件简单、产物单一、无副产物以及产物分离纯化难度低等优点。
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的液相及质谱检测方法说明
本申请实施例中对反应产物(例如香叶基磷酸、香叶基二磷酸酯、大麻萜酚或大麻萜酚酸)的鉴定均采用液相及质谱检测方法，检测条件如下：
1.1检测仪器：超高效液相色谱-质谱联用仪(Shimadzu LC-30A，SCIEXTripleTOF6600)；
1.2液相色谱条件如下：
1.2.1进样液的制备：将纯化后的反应产物置于12000r/min的条件下离心20min，取上清液作为进样液检测；
1.2.2流动相：流动相A为5mmol/L的碳酸氢铵水溶液；流动相B为甲酸-乙腈溶液，甲酸的体积百分数为0.005％；
1.2.3梯度：在0min至2min的范围内，99％的流动相A和1％的流动相B；在2min至2.1min的范围内，流动相A由99％降至90％，流动相B由1％升至10％；在2.1min至12min的范围内，流动相A由90％降至20％，流动相B由10％升至80％；在12min至12.1min的范围内，流动相A由20％降至0，流动相B由80％升至100％；在12.1min至20min，100％的流动相B；
1.2.3色谱柱：Agilent Eclipse plus C18(100mm×2.1mm,3.5μm)；
1.2.4柱温：30℃；
1.2.5流速：0.4mL/min。
1.3质谱条件
1.3.1离子源种类：ESI离子源；
1.3.2离子源参数：离子电压(Ion Voltage)为4500V；去簇电压(DeclusteringPotential)为80V；离子源温度(Source Temperature)为600℃；帘气(Curtain Gas)的压强为35psi；喷雾气(Nebulizer Gas)的压强为55psi；加热气(Heater Gas)的压强为55psi；一级扫描范围为m/z 200至600，二级扫描范围为m/z30至600；
1.3.3检测模式：负离子IDA。
1.4数据分析：Peakview软件(SCIEX,美国)
二、本申请实施例中涉及的酶的制备方法
本申请实施例中涉及的1号酶、2号酶以及NphB异戊烯基转移酶的制备方法的流程相同，下面以NphB异戊烯基转移酶的制备方法为例进行详细说明，1号酶和2号酶的制备方法参照NphB异戊烯基转移酶的制备方法进行。
2.1构建表达质粒
根据大肠杆菌(E.coli)的密码子偏爱性，对编码NphB的核苷酸序列(SEQ ID NO：30所示)进行密码子优化，然后按照图1连接至pET-28a质粒(购自北京索莱宝生物科技有限公司)上，获得pET-28a-NphB重组质粒，将pET-28a-NphB重组质粒转化大肠杆菌BL21(DE3)感受态(购自南京诺唯赞生物科技有限公司)，培养8h至12h后，划线铺板，挑取单菌落测序，测序结果正确的菌株即为pET-28a-NphB单克隆菌株。
2.2蛋白的表达纯化：
S2.2.1、挑取pET-28a-NphB的单克隆菌株(一环)或其-80℃保存的菌株(融化后取菌液1mL)接种于含5mL LB液体培养基(Kan+,100μg/mL)的试管中，置于37℃、220r/min过夜培养，作为种子液；
S2.2.2、将步骤S2.2.1制得的种子液按照1:50～100的接种量转入50mL的LB液体培养基(Kan+,100μg/mL)中，置于37℃、220r/min活化培养5h至7h，获得活化菌液；
S2.2.3、将步骤S2.2.2制得的活化菌液按照1％的接种量转入800mL的2×YT液体培养基(Kan+,100μg/mL)中，置于37℃、220r/min的条件下培养至OD600为0.6至0.8，获得培养液；
S2.2.4、待培养液的温度降至16℃时，向培养液中加入异丙基硫代-β-D-半乳糖苷(Isopropylthio-β-D-galactoside,IPTG)至终浓度为0.5mmol/L，在16℃下诱导表达16h至20h，获得诱导表达液；
S2.2.5、将步骤S2.2.4的诱导表达液置于4℃、5500r/min的条件下离心10min，去除上清液，获得沉淀物；
S2.2.6、提供蛋白纯化缓冲液(50mmol/L Tris,150mmol/NaCl,pH＝8)，向步骤S2.2.5的沉淀物中加入30mL的蛋白纯化缓冲液，并采用旋涡振荡器重悬菌体，获得重悬菌体液；
S2.2.7、将步骤S2.2.6的重悬菌体液置于4℃、5500r/min的条件下离心10min，去除上清液，再加入30mL的蛋白纯化缓冲液，并采用旋涡振荡器重悬菌体，重复离心和重悬操作直至无固体颗粒，获得的菌液保存于-80℃冰箱。
2.3蛋白纯化
S2.3.1、采用高压低温破碎仪处理上述步骤S2.2.7制得的菌液，工艺参数为：处理压力为800bar至1000bar，处理温度为4℃，处理时间为3min至5min；
S2.3.2、将完成破碎的菌液置于4℃、8000r/min的条件下离心60min，分别收集上清液和沉淀物；
S2.3.3、将步骤S2.3.2收集的上清液进行镍亲和层析纯化(目的蛋白携带有6个组氨酸标签)，采用不同浓度的咪唑溶液洗脱目标蛋白，所述不同浓度的咪唑溶液分别为20mmol/L的咪唑溶液(溶剂为步骤S2.2.6中所述的蛋白纯化缓冲液)、50mmol/L的咪唑溶液、100mmol/L的咪唑溶液、250mmol/L的咪唑溶液和300mmol/L的咪唑溶液，并收集上清液经过镍柱后的流穿样品、20mmol/L咪唑溶液的洗脱液、50mmol/L咪唑溶液的洗脱液、100mmol/L咪唑溶液的洗脱液、250mmol/L咪唑溶液的洗脱液和300mmol/L咪唑溶液的洗脱液；
S2.3.4、将步骤S2.3.2收集的上清液和沉淀物，以及步骤S2.3.3收集的流穿样品、20mmol/L咪唑溶液的洗脱液、50mmol/L咪唑溶液的洗脱液、100mmol/L咪唑溶液的洗脱液、250mmol/L咪唑溶液的洗脱液和300mmol/L咪唑溶液的洗脱液分别进行12％的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulfate-Polyacrylamide GelElectrophoresis,SDS-PAGE)验证分析；
S2.3.5、浓缩验证结果正确的咪唑洗脱液：将咪唑洗脱液置于Amicon超滤管(10kDa,Millipore公司)，然后在4℃、3400r/min的条件下离心浓缩，直至含有目的蛋白的洗脱液的体积浓缩至1mL，然后向其中加入10mL的蛋白缓冲液，再在4℃、3400r/min的条件下离心浓缩至体积为1mL，可重复数次直至将咪唑浓度降低至2mmol/L以下，获得纯化的目的蛋白。
其中，步骤S2.3.3的具体步骤如下：
S2.3.3.1、柱平衡：采用双蒸水(ddH2O)清洗镍亲和层析柱(购自GE Healthcare)2个柱体积，再用蛋白缓冲液平衡镍亲和层析柱1个柱体积；
S2.3.3.2、上样：取上述步骤S2.3.2收集的上清液，将其缓慢通过镍亲和层析柱，流穿(可重复一次)，并取前2-3滴流穿样品；
S2.3.3.3、洗脱目的蛋白：取30mL的浓度为50mmol/L咪唑通过镍亲和层析柱，以洗脱镍柱上结合的杂蛋白，然后依次采用50mmol/L的咪唑溶液、100mmol/L的咪唑溶液、250mmol/L的咪唑溶液和300mmol/L的咪唑溶液分别通过镍亲和层析柱，并分别收集洗脱液，每一种洗脱液分别取50μL用于SDS-PAGE验证分析。
作为示例，NphB的SDS-PAGE电泳图如图2所示，1号酶(氨基酸序列为SEQ ID NO：4所示，编码基因的核苷酸序列如SEQ ID NO：31所示)的SDS-PAGE电泳图如图3所示，2号酶(氨基酸序列为SEQ ID NO：18所示，编码基因的核苷酸序列如SEQ ID NO：32所示)的SDS-PAGE电泳图如图4所示。
三、本申请实施例中涉及的酶相对活性、底物相对转化率、产物得率和产物相对纯度的计算方法
3.1、酶相对活性的计算方法
酶相对活性的计算公式如下式(Ⅰ)所示：
在式(Ⅰ)中，Sx是各个实施例中相对活性为1的单种酶或酶组合所对应的液相检测中目标产物的峰面积，Sa是被比较的酶所对应的液相检测中目标产物的峰面积。
作为示例，在实施例1中，Sx为GK1所对应的液相检测中香叶基磷酸的峰面积，GK2的相对活性为：GK1所对应的液相检测中香叶基磷酸的峰面积除以GK2所对应的液相检测中香叶基磷酸的峰面积。
作为示例，在实施例2中，Sx为GK4+PGK1酶组合所对应的液相检测中香叶基二磷酸酯的峰面积，GK4+PGK2的相对活性为：GK4+PGK1酶组合所对应的液相检测中香叶基二磷酸酯的峰面积除以GK4+PGK2酶组合所对应的液相检测中香叶基二磷酸酯的峰面积。
3.2底物相对转化率
底物相对转化率的计算公式如下式(Ⅱ)所示：
在式(Ⅱ)中，Sx1是本申请各个实施例中某种酶或某种酶组合所对应的酶促反应初始时(反应0h)所对应的液相检测中底物的峰面积减去酶促反应结束后反应产物的液相检测中底物的峰面积，Sx2是本申请各个实施例中某种酶或某种酶组合所对应的酶促反应初始时(反应0h)所对应的液相检测中底物的峰面积。
3.3产物相对得率
底物相对转化率的计算公式如下式(Ⅲ)所示：
在式(Ⅲ)中，Sy1是本申请各个实施例中某种酶或某种酶组合所对应的酶促反应结束后反应产物的液相检测中目标产物的峰面积，Sy2是本申请各个实施例中某种酶或某种酶组合所对应的酶促反应初始时(反应0h)所对应的液相检测中底物的峰面积。
3.4产物相对纯度
底物相对转化率的计算公式如下式(Ⅳ)所示：
在式(Ⅳ)中，Sx是本申请各个实施例中某种酶或某种酶组合所对应的酶促反应结束后反应产物的液相检测中目标产物的峰面积，S1+S2+S3+...是本申请各个实施例中某种酶或某种酶组合所对应的酶促反应结束后反应产物的液相检测中所有峰面积之和。
实施例1生物合成香叶基磷酸
本实施例提供了一种香叶基磷酸的生物合成方法，包括步骤：将香叶醇、腺嘌呤核苷三磷酸以及1号酶进行接触反应，反应生成香叶基磷酸。
本实施例采用16种1号酶分别与香叶醇和腺嘌呤核苷三磷酸进行接触反应，并比较这16种1号酶催化香叶醇和腺嘌呤核苷三磷酸生成香叶基磷酸的相对活性。
其中，16种1号酶的具体信息如下表1所示：
表1 实施例1中16种1号酶的具体信息一览表
在本实施例中，将香叶醇、腺嘌呤核苷三磷酸、单种1号酶、Tris-HCl、KCl和MgCl2混合获得反应体系，并将反应体系置于30℃下反应1h，获得包含香叶基磷酸的反应产物，其中，反应体系的初始pH为8，反应体系的总体积量为40μL(除香叶醇、腺嘌呤核苷三磷酸、单种1号酶、Tris-HCl、KCl和MgCl2之外，余量为水)，反应体系中各个组分的添加量如下表2所示：
表2 实施例1中生物合成香叶基磷酸的反应体系一览表
成分名称 在反应体系中的终浓度
香叶醇 2mmol/L
腺嘌呤核苷三磷酸 5mmol/L
单种1号酶 2mg/mL
Tris-HCl(pH为8.0) 50mmol/L
KCl 30mmol/L
MgCl2 10mmol/L
将反应产物置于12000r/min的条件下离心20min，并收集上清液，然后将收集的上清液进行液相及质谱检测，检测结果如下表3和图5至图7所示：
表3 实施例1中16种1号酶的催化活性一览表
由表3和图7可知，GK4对底物香叶醇的催化活性最高，其次是GK1，GK14最低。此外，相较于反应体系中的1号酶选自GK1至GK3或GK5至GK16中的任意一种，反应体系中的1号酶选自GK4更有利于提高产物香叶基磷酸的得率和纯度。
实施例2生物合成香叶基二磷酸酯
本实施例提供了一种香叶基磷酸的生物合成方法，包括步骤：将香叶醇、腺嘌呤核苷三磷酸、1号酶以及2号酶进行接触反应，获得香叶基二磷酸酯。
具体的，1号酶选自实施例1中的GK4，并采用12种2号酶分别与香叶基磷酸和腺嘌呤核苷三磷酸进行接触反应。
其中，12种2号酶的具体信息如下表4所示：
表4 实施例2中12种2号酶的具体信息一览表
2号酶的标号 酶的名称 EC编号 氨基酸序列
PGK1 磷酸甲羟戊酸激酶 2.7.4.26 SEQ ID NO：17
PGK2 异戊烯基磷酸激酶 2.7.4.26 SEQ ID NO：18
PGK3 磷酸甲羟戊酸激酶 2.7.4.26 SEQ ID NO：19
PGK4 异戊烯基磷酸激酶 2.7.4.26 SEQ ID NO：20
PGK5 磷酸甲羟戊酸激酶 2.7.4.26 SEQ ID NO：21
PGK6 甲羟戊酸激酶 2.7.1.36 SEQ ID NO：22
PGK7 磷酸甲羟戊酸激酶 2.7.4.26 SEQ ID NO：23
PGK8 磷酸甲羟戊酸激酶 2.7.4.26 SEQ ID NO：24
PGK9 磷酸甲羟戊酸激酶 2.7.4.26 SEQ ID NO：25
PGK10 磷酸甲羟戊酸激酶 2.7.4.26 SEQ ID NO：26
PGK11 磷酸甲羟戊酸激酶 2.7.4.26 SEQ ID NO：27
PGK12 磷酸甲羟戊酸激酶 2.7.4.26 SEQ ID NO：28
在本实施例中，将香叶醇、腺嘌呤核苷三磷酸、GK4、单种2号酶、Tris-HCl、KCl和MgCl2混合获得反应体系，并将反应体系置于30℃下反应1h，获得包含香叶基二磷酸酯的反应产物，其中，反应体系的初始pH为8，反应体系的总体积量为40μL(除香叶醇、腺嘌呤核苷三磷酸、GK4、单种2号酶、Tris-HCl、KCl和MgCl2之外，余量为水)，反应体系中各个组分的添加量如下表5所示：
表5 实施例2中生物合成香叶基二磷酸酯的反应体系一览表
成分名称 在反应体系中的终浓度
香叶醇 2mmol/L
腺嘌呤核苷三磷酸 5mmol/L
GK4 2mg/mL
单种2号酶 2mg/mL
Tris-HCl(pH为8.0) 50mmol/L
KCl 30mmol/L
MgCl2 10mmol/L
将反应产物置于12000r/min的条件下离心20min，并收集上清液，然后将收集的上清液进行液相及质谱检测，检测结果如下表6和图8至图10所示：
表6 实施例2中12种酶组合的催化活性一览表
由表4和图10可知，当反应体系中的1号酶选自GK4且2号酶选自PGK2时，对底物香叶醇的催化活性最高，其次是1号酶选自GK4且2号酶选自PGK5。此外，在反应体系中的1号酶均为GK4的前提下，相较于反应体系中的2号酶选自PGK1或PGK3至PGK12中的任意一种时，反应体系中的2号酶选自PGK2更有利于提高产物香叶基二磷酸酯的得率和纯度。
实施例3 生物合成大麻萜酚
本实施例提供了一种大麻萜酚的生物合成方法，包括步骤：将香叶醇、腺嘌呤核苷三磷酸、1号酶、2号酶、橄榄醇以及NphB异戊烯基转移酶进行接触反应，获得大麻萜酚。
其中，1号酶选自GK4，2号酶选自PGK2；NphB异戊烯基转移酶的氨基酸序列如SEQID NO：29所示。
在本实施例中，将香叶醇、腺嘌呤核苷三磷酸、GK4、PGK2、橄榄醇、NphB异戊烯基转移酶Tris-HCl、KCl和MgCl2混合获得反应体系，并将反应体系置于30℃下反应1h，获得包含大麻萜酚的反应产物，其中，反应体系的初始pH为8，反应体系的总体积量为40(除香叶醇、腺嘌呤核苷三磷酸、GK4、PGK2、橄榄醇、NphB异戊烯基转移酶Tris-HCl、KCl和MgCl2之外，余量为水)，反应体系中各个组分的添加量如下表7所示：
表7 实施例3中生物合成大麻萜酚的反应体系一览表
成分名称 在反应体系中的终浓度
香叶醇 2mmol/L
腺嘌呤核苷三磷酸 5mmol/L
GK4 2mg/mL
PGK2 2mg/mL
橄榄醇 0.1g/L
NphB异戊烯基转移酶 2mg/mL
Tris-HCl(pH为8.0) 50mmol/L
KCl 30mmol/L
MgCl2 10mmol/L
将反应产物置于12000r/min的条件下离心20min，并收集上清液，然后将收集的上清液进行液相及质谱检测，获得的液相色谱图如图11所示，获得的质谱图如图12所示，反应生成的大麻萜酚的相对得率为19.3％，大麻萜酚的相对纯度为96.5％。
实施例4 生物合成大麻萜酚
本实施例提供了一种大麻萜酚的生物合成方法，包括步骤：将香叶醇、腺嘌呤核苷三磷酸、1号酶、2号酶、橄榄醇以及NphB异戊烯基转移酶进行接触反应，获得大麻萜酚。
其中，1号酶选自GK4，2号酶选自PGK2；NphB异戊烯基转移酶的氨基酸序列如SEQID NO：29所示。
在本实施例中，将香叶醇、腺嘌呤核苷三磷酸、GK4、PGK2、橄榄醇、NphB异戊烯基转移酶Tris-HCl、KCl和MgCl2混合获得反应体系，并将反应体系置于30℃下反应1h，获得包含大麻萜酚的反应产物，其中，反应体系的初始pH为8，反应体系的总体积量为40μL(除香叶醇、腺嘌呤核苷三磷酸、GK4、PGK2、橄榄醇、NphB异戊烯基转移酶Tris-HCl、KCl和MgCl2之外，余量为水)，反应体系中各个组分的添加量如下表8所示：
表8 实施例4中生物合成大麻萜酚的反应体系一览表
成分名称 在反应体系中的终浓度
香叶醇 2mmol/L
腺嘌呤核苷三磷酸 5mmol/L
GK4 2mg/mL
PGK2 2mg/mL
橄榄醇 0.2g/L
NphB异戊烯基转移酶 2mg/mL
Tris-HCl(pH为8.0) 50mmol/L
KCl 30mmol/L
MgCl2 10mmol/L
将反应产物置于12000r/min的条件下离心20min，并收集上清液，然后将收集的上清液进行液相及质谱检测，反应产物的液相色谱图如图13所示，反应产物的质谱图如图14所示，反应生成的大麻萜酚的相对得率为25.4％，大麻萜酚的相对纯度为95.2％。
以上对本申请所提供的一种香叶基二磷酸酯的生物合成方法及其在制备大麻类化合物中的应用，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。
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<110> 嘉兴欣贝莱生物科技有限公司
<120> 香叶基二磷酸酯的生物合成方法及其在制备大麻类化合物中的应用
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85 90 95
Glu Thr Arg Thr Arg Ala Ala Leu Glu Leu Leu Glu Arg Val Arg Val
100 105 110
Thr Ala Val Arg Gly Asn Ala Gly Glu Ile Leu Ala Leu Ala Gly Arg
115 120 125
Asp Gly Leu Val Arg Gly Val Asp Gly Pro Asp Gly Arg Pro Gly Pro
130 135 140
Gln Thr Glu Arg Ala Ala Arg Ala Val Ala Arg Arg Phe Gly Cys Leu
145 150 155 160
Val Ala Val Thr Gly Ala Thr Asp Leu Val Thr Asp Gly Arg Arg Thr
165 170 175
Leu Ala Val Arg Ala Gly His Pro Leu Met Ser Gln Val Pro Gly Thr
180 185 190
Gly Cys Leu Ala Thr Ala Leu Val Ala Ala Ala Leu Ala Ala Gly Thr
195 200 205
Gly Ala Gly Pro Ala Gly Arg Asp Arg Pro Met Glu Asp Val Asp Val
210 215 220
Val Ala Glu Ala Leu Leu Trp Ala Gly Trp Ala Gly Glu Gln Ala Ala
225 230 235 240
Ser Ala Ala Ser Gly Pro Gly Thr Phe Ala Ala Ala Phe Leu Asp Arg
245 250 255
Leu Ala Leu Arg Gly Pro Leu Pro Pro Gly Arg Ile Ala Pro Pro Leu
260 265 270
Ser Glu Arg Leu Ser Leu Tyr Val Leu Val Ser Gly Ala Thr Pro Pro
275 280 285
Asp Val Leu Glu Ala Val Leu Gln Ala Gly Cys Arg Met Ile Gln Phe
290 295 300
Arg Glu Lys Arg Leu Pro Leu Pro Ala Gln Leu Glu Ala Ala Ala Arg
305 310 315 320
Val Arg Glu Ala Cys Arg Arg His Gly Ala Leu Leu Val Val Asn Asp
325 330 335
Arg Val Asp Leu Ala Leu Ala Val Gly Ala Asp Gly Val His Leu Gly
340 345 350
Gln Glu Asp Leu Pro Val Ala Ala Ala Arg Arg Ile Leu Gly Pro Asp
355 360 365
Ala Val Ile Gly Ala Thr Cys Glu Thr Ala Gly Glu Ala Arg Ala Ala
370 375 380
Arg Asp Ala Gly Ala Asp Tyr Ile Gly Ala Gly Pro Val Tyr Val Thr
385 390 395 400
Pro Ser Lys Pro Asp Ala Gly Glu Pro Tyr Gly Pro Asp Val Val Arg
405 410 415
Arg Val Ser Glu Ala Ala Asp Leu Pro Val Val Gly Ile Gly Gly Ile
420 425 430
Gly Pro Gly Arg Ala Ala Pro Val Ile Ala Ala Gly Ala Ala Gly Val
435 440 445
Ala Val Ile Ser Ala Val Leu Gly Ala Pro Asp Pro Gly Ala Ala Ala
450 455 460
Arg Ala Ile Leu Asp Glu Val Arg Arg Ala Lys Gly Glu Val Ser Ala
465 470 475 480
<210> 4
<211> 296
<212> PRT
<213> 人工序列
<400> 4
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Met Gln Val Asp Leu Leu Gly Ser Ala Gln Ser Ala His Ala
35 40 45
Leu His Leu Phe His Gln His Ser Pro Leu Val His Cys Met Thr Asn
50 55 60
Asp Val Val Gln Thr Phe Thr Ala Asn Thr Leu Leu Ala Leu Gly Ala
65 70 75 80
Ser Pro Ala Met Val Ile Glu Thr Glu Glu Ala Ser Gln Phe Ala Ala
85 90 95
Ile Ala Ser Ala Leu Leu Ile Asn Val Gly Thr Leu Thr Gln Pro Arg
100 105 110
Ala Gln Ala Met Arg Ala Ala Val Glu Gln Ala Lys Ser Ser Gln Thr
115 120 125
Pro Trp Thr Leu Asp Pro Val Ala Val Gly Ala Leu Asp Tyr Arg Arg
130 135 140
His Phe Cys His Glu Leu Leu Ser Phe Lys Pro Ala Ala Ile Arg Gly
145 150 155 160
Asn Ala Ser Glu Ile Met Ala Leu Ala Gly Ile Ala Asn Gly Gly Arg
165 170 175
Gly Val Asp Thr Thr Asp Ala Ala Ala Asn Ala Ile Pro Ala Ala Gln
180 185 190
Thr Leu Ala Arg Glu Thr Gly Ala Ile Val Val Val Thr Gly Glu Met
195 200 205
Asp Tyr Val Thr Asp Gly His Arg Ile Ile Gly Ile His Gly Gly Asp
210 215 220
Pro Leu Met Thr Lys Val Val Gly Thr Gly Cys Ala Leu Ser Ala Val
225 230 235 240
Val Ala Ala Cys Cys Ala Leu Pro Gly Asp Thr Leu Glu Asn Val Ala
245 250 255
Ser Ala Cys His Trp Met Lys Gln Ala Gly Glu Arg Ala Val Ala Arg
260 265 270
Ser Glu Gly Pro Gly Ser Phe Val Pro His Phe Leu Asp Ala Leu Trp
275 280 285
Gln Leu Thr Gln Glu Val Gln Ala
290 295
<210> 5
<211> 610
<212> PRT
<213> 人工序列
<400> 5
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Val Gln Glu Ser Arg Pro Gly Ser Val Arg Ser
20 25 30
Tyr Ser Val Gly Tyr Gln Ala Arg Ser Arg Ser Ser Ser Gln Arg Arg
35 40 45
His Ser Leu Thr Arg Gln Arg Ser Ser Gln Arg Leu Ile Arg Thr Ile
50 55 60
Ser Ile Glu Ser Asp Val Ser Asn Ile Thr Asp Asp Asp Asp Leu Arg
65 70 75 80
Ala Val Asn Glu Gly Val Ala Gly Val Gln Leu Asp Val Ser Glu Thr
85 90 95
Ala Asn Lys Gly Pro Arg Arg Ala Ser Ala Thr Asp Val Thr Asp Ser
100 105 110
Leu Gly Ser Thr Ser Ser Glu Tyr Ile Glu Ile Pro Phe Val Lys Glu
115 120 125
Thr Leu Asp Ala Ser Leu Pro Ser Asp Tyr Leu Lys Gln Asp Ile Leu
130 135 140
Asn Leu Ile Gln Ser Leu Lys Ile Ser Lys Trp Tyr Asn Asn Lys Lys
145 150 155 160
Ile Gln Pro Val Ala Gln Asp Met Asn Leu Val Lys Ile Ser Gly Ala
165 170 175
Met Thr Asn Ala Ile Phe Lys Val Glu Tyr Pro Lys Leu Pro Ser Leu
180 185 190
Leu Leu Arg Ile Tyr Gly Pro Asn Ile Asp Asn Ile Ile Asp Arg Glu
195 200 205
Tyr Glu Leu Gln Ile Leu Ala Arg Leu Ser Leu Lys Asn Ile Gly Pro
210 215 220
Ser Leu Tyr Gly Cys Phe Val Asn Gly Arg Phe Glu Gln Phe Leu Glu
225 230 235 240
Asn Ser Lys Thr Leu Thr Lys Asp Asp Ile Arg Asn Trp Lys Asn Ser
245 250 255
Gln Arg Ile Ala Arg Arg Met Lys Glu Leu His Val Gly Val Pro Leu
260 265 270
Leu Ser Ser Glu Arg Lys Asn Gly Ser Ala Cys Trp Gln Lys Ile Asn
275 280 285
Gln Trp Leu Arg Thr Ile Glu Lys Val Asp Gln Trp Val Gly Asp Pro
290 295 300
Lys Asn Ile Glu Asn Ser Leu Leu Cys Glu Asn Trp Ser Lys Phe Met
305 310 315 320
Asp Ile Val Asp Arg Tyr His Lys Trp Leu Ile Ser Gln Glu Gln Gly
325 330 335
Ile Glu Gln Val Asn Lys Asn Leu Ile Phe Cys His Asn Asp Ala Gln
340 345 350
Tyr Gly Asn Leu Leu Phe Thr Ala Pro Val Met Asn Thr Pro Ser Leu
355 360 365
Tyr Thr Ala Pro Ser Ser Thr Ser Leu Thr Ser Gln Ser Ser Ser Leu
370 375 380
Phe Pro Ser Ser Ser Asn Val Ile Val Asp Asp Ile Ile Asn Pro Pro
385 390 395 400
Lys Gln Glu Gln Ser Gln Asp Ser Lys Leu Val Val Ile Asp Phe Glu
405 410 415
Tyr Ala Gly Ala Asn Pro Ala Ala Tyr Asp Leu Ala Asn His Leu Ser
420 425 430
Glu Trp Met Tyr Asp Tyr Asn Asn Ala Lys Ala Pro His Gln Cys His
435 440 445
Ala Asp Arg Tyr Pro Asp Lys Glu Gln Val Leu Asn Phe Leu Tyr Ser
450 455 460
Tyr Val Ser His Leu Arg Gly Gly Ala Lys Glu Pro Ile Asp Glu Glu
465 470 475 480
Val Gln Arg Leu Tyr Lys Ser Ile Ile Gln Trp Arg Pro Thr Val Gln
485 490 495
Leu Phe Trp Ser Leu Trp Ala Ile Leu Gln Ser Gly Lys Leu Glu Lys
500 505 510
Lys Glu Ala Ser Thr Ala Ile Thr Arg Glu Glu Ile Gly Pro Asn Gly
515 520 525
Lys Lys Tyr Ile Ile Lys Thr Glu Pro Glu Ser Pro Glu Glu Asp Phe
530 535 540
Val Glu Asn Asp Asp Glu Pro Glu Ala Gly Val Ser Ile Asp Thr Phe
545 550 555 560
Asp Tyr Met Ala Tyr Gly Arg Asp Lys Ile Ala Val Phe Trp Gly Asp
565 570 575
Leu Ile Gly Leu Gly Ile Ile Thr Glu Glu Glu Cys Lys Asn Phe Ser
580 585 590
Ser Phe Lys Phe Leu Asp Thr Ser Tyr Leu Leu Glu His His His His
595 600 605
His His
610
<210> 6
<211> 165
<212> PRT
<213> 人工序列
<400> 6
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Pro Met Asp Leu Arg Asp Asn Lys Gln Ser Gln
20 25 30
Lys Lys Trp Lys Asn Arg Thr Leu Thr Ser Ser Leu Glu Phe Ala Leu
35 40 45
Thr Gly Ile Phe Thr Ala Phe Lys Glu Glu Arg Asn Met Lys Lys His
50 55 60
Ala Val Ser Ala Leu Leu Ala Val Ile Ala Gly Leu Val Phe Lys Val
65 70 75 80
Ser Val Ile Glu Trp Leu Phe Leu Leu Leu Ser Ile Phe Leu Val Ile
85 90 95
Thr Phe Glu Ile Val Asn Ser Ala Ile Glu Asn Val Val Asp Leu Ala
100 105 110
Ser Asp Tyr His Phe Ser Met Leu Ala Lys Asn Ala Lys Asp Met Ala
115 120 125
Ala Gly Ala Val Leu Val Ile Ser Gly Phe Ala Ala Leu Thr Gly Leu
130 135 140
Ile Ile Phe Leu Leu Lys Ile Trp Phe Leu Leu Phe His Leu Glu His
145 150 155 160
His His His His His
165
<210> 7
<211> 244
<212> PRT
<213> 人工序列
<400> 7
Met Ile Leu Lys Ile Gly Gly Ser Val Ile Thr Asp Lys Ser Ala Tyr
1 5 10 15
Arg Thr Ala Arg Thr Tyr Ala Ile Arg Ser Ile Val Lys Val Leu Ser
20 25 30
Gly Ile Glu Asp Leu Val Cys Val Val His Gly Gly Gly Ser Phe Gly
35 40 45
His Ile Lys Ala Met Glu Phe Gly Leu Pro Gly Pro Lys Asn Pro Arg
50 55 60
Ser Ser Ile Gly Tyr Ser Ile Val His Arg Asp Met Glu Asn Leu Asp
65 70 75 80
Leu Met Val Ile Asp Ala Met Ile Glu Met Gly Met Arg Pro Ile Ser
85 90 95
Val Pro Ile Ser Ala Leu Arg Tyr Asp Gly Arg Phe Asp Tyr Thr Pro
100 105 110
Leu Ile Arg Tyr Ile Asp Ala Gly Phe Val Pro Val Ser Tyr Gly Asp
115 120 125
Val Tyr Ile Lys Asp Glu His Ser Tyr Gly Ile Tyr Ser Gly Asp Asp
130 135 140
Ile Met Ala Asp Met Ala Glu Leu Leu Lys Pro Asp Val Ala Val Phe
145 150 155 160
Leu Thr Asp Val Asp Gly Ile Tyr Ser Lys Asp Pro Lys Arg Asn Pro
165 170 175
Asp Ala Val Leu Leu Arg Asp Ile Asp Thr Asn Ile Thr Phe Asp Arg
180 185 190
Val Gln Asn Asp Val Thr Gly Gly Ile Gly Lys Lys Phe Glu Ser Met
195 200 205
Val Lys Met Lys Ser Ser Val Lys Asn Gly Val Tyr Leu Ile Asn Gly
210 215 220
Asn His Pro Glu Arg Ile Gly Asp Ile Gly Lys Glu Ser Phe Ile Gly
225 230 235 240
Thr Val Ile Arg
<210> 8
<211> 356
<212> PRT
<213> 人工序列
<400> 8
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Met Asn Ile Lys Lys Gln Gly Leu Gly Gln Ala Thr Gly Lys
35 40 45
Ile Ile Leu Met Gly Glu His Ala Val Val Tyr Gly Glu Pro Ala Ile
50 55 60
Ala Phe Pro Phe Gln Ala Thr Glu Ile Thr Ala Val Phe Thr Pro Ala
65 70 75 80
Lys Thr Met Gln Ile Asp Cys Ala Tyr Phe Thr Gly Leu Leu Glu Asp
85 90 95
Val Pro Gln Glu Leu Ala Asn Ile Lys Glu Val Val Gln Gln Thr Leu
100 105 110
His Phe Leu Lys Glu Asp Thr Phe Lys Gly Thr Leu Thr Leu Thr Ser
115 120 125
Thr Ile Pro Ala Glu Arg Gly Met Gly Ser Ser Ala Ala Thr Ala Val
130 135 140
Ala Ile Val Arg Ser Leu Phe Asp Tyr Phe Asp Tyr Ala Tyr Thr Tyr
145 150 155 160
Gln Glu Leu Phe Glu Leu Val Ser Leu Ser Glu Lys Ile Ala His Gly
165 170 175
Asn Pro Ser Gly Ile Asp Ala Ala Ala Thr Ser Gly Ala Asp Pro Leu
180 185 190
Phe Phe Thr Arg Gly Phe Pro Pro Thr His Phe Ser Met Asn Leu Ser
195 200 205
Asn Ala Tyr Leu Val Val Ala Asp Thr Gly Ile Lys Gly Gln Thr Arg
210 215 220
Glu Ala Val Lys Asp Ile Ala Gln Leu Ala Gln Asn Asn Pro Thr Ala
225 230 235 240
Ile Ala Glu Thr Met Lys Gln Leu Gly Ser Phe Thr Lys Glu Ala Lys
245 250 255
Gln Ala Ile Leu Gln Asp Asp Lys Gln Lys Leu Gly Gln Leu Met Thr
260 265 270
Leu Ala Gln Glu Gln Leu Gln Gln Leu Thr Val Ser Asn Asp Met Leu
275 280 285
Asp Arg Leu Val Ala Leu Ser Leu Glu His Gly Ala Leu Gly Ala Lys
290 295 300
Leu Thr Gly Gly Gly Arg Gly Gly Cys Met Ile Ala Leu Thr Asp Asn
305 310 315 320
Lys Lys Thr Ala Gln Thr Ile Ala Gln Thr Leu Glu Glu Asn Gly Ala
325 330 335
Val Ala Thr Trp Ile Gln Ser Leu Glu Val Lys Lys Leu Glu His His
340 345 350
His His His His
355
<210> 9
<211> 438
<212> PRT
<213> 人工序列
<400> 9
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Met Leu Ser Glu Val Leu Leu Val Ser Ala Pro Gly Lys Val
35 40 45
Ile Leu His Gly Glu His Ala Val Val His Gly Lys Val Ala Leu Ala
50 55 60
Val Ser Leu Asn Leu Arg Thr Phe Leu Arg Leu Gln Pro His Ser Asn
65 70 75 80
Gly Lys Val Asp Leu Ser Leu Pro Asn Ile Gly Ile Lys Arg Ala Trp
85 90 95
Asp Val Ala Arg Leu Gln Ser Leu Asp Thr Ser Phe Leu Glu Gln Gly
100 105 110
Asp Val Thr Thr Pro Thr Ser Glu Gln Val Glu Lys Leu Lys Glu Val
115 120 125
Ala Gly Leu Pro Asp Asp Cys Ala Val Thr Glu Arg Leu Ala Val Leu
130 135 140
Ala Phe Leu Tyr Leu Tyr Leu Ser Ile Cys Arg Lys Gln Arg Ala Leu
145 150 155 160
Pro Ser Leu Asp Ile Val Val Trp Ser Glu Leu Pro Pro Gly Ala Gly
165 170 175
Leu Gly Ser Ser Ala Ala Tyr Ser Val Cys Leu Ala Ala Ala Leu Leu
180 185 190
Thr Val Cys Glu Glu Ile Pro Asn Pro Leu Lys Asp Gly Asp Cys Val
195 200 205
Asn Arg Trp Thr Lys Glu Asp Leu Glu Leu Ile Asn Lys Trp Ala Phe
210 215 220
Gln Gly Glu Arg Met Ile His Gly Asn Pro Ser Gly Val Asp Asn Ala
225 230 235 240
Val Ser Thr Trp Gly Gly Ala Leu Arg Tyr His Gln Gly Lys Ile Ser
245 250 255
Ser Leu Lys Arg Ser Pro Ala Leu Gln Ile Leu Leu Thr Asn Thr Lys
260 265 270
Val Pro Arg Asn Thr Arg Ala Leu Val Ala Gly Val Arg Asn Arg Leu
275 280 285
Leu Lys Phe Pro Glu Ile Val Ala Pro Leu Leu Thr Ser Ile Asp Ala
290 295 300
Ile Ser Leu Glu Cys Glu Arg Val Leu Gly Glu Met Gly Glu Ala Pro
305 310 315 320
Ala Pro Glu Gln Tyr Leu Val Leu Glu Glu Leu Ile Asp Met Asn Gln
325 330 335
His His Leu Asn Ala Leu Gly Val Gly His Ala Ser Leu Asp Gln Leu
340 345 350
Cys Gln Val Thr Arg Ala Arg Gly Leu His Ser Lys Leu Thr Gly Ala
355 360 365
Gly Gly Gly Gly Cys Gly Ile Thr Leu Leu Lys Pro Gly Leu Glu Gln
370 375 380
Pro Glu Val Glu Ala Thr Lys Gln Ala Leu Thr Ser Cys Gly Phe Asp
385 390 395 400
Cys Leu Glu Thr Ser Ile Gly Ala Pro Gly Val Ser Ile His Ser Ala
405 410 415
Thr Ser Leu Asp Ser Arg Val Gln Gln Ala Leu Asp Gly Leu Leu Glu
420 425 430
His His His His His His
435
<210> 10
<211> 414
<212> PRT
<213> 人工序列
<400> 10
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Glu Val Arg Ala Arg Ala Pro Gly Lys Ile Ile
20 25 30
Leu Ala Gly Glu His Ala Val Val His Gly Ser Thr Ala Val Ala Ala
35 40 45
Ala Ile Asp Leu Tyr Thr Tyr Ile Ser Leu His Phe Pro Thr Pro Ala
50 55 60
Glu Asn Asp Asp Ala Leu Lys Leu His Leu Lys Asp Met Gly Leu Glu
65 70 75 80
Phe Ser Trp Pro Val Gly Arg Ile Lys Asp Val Leu Pro Glu Val Ser
85 90 95
Ser His Asp Val Ser Ser Pro Ser Ser Cys Ser Leu Glu Thr Leu Lys
100 105 110
Ala Ile Ala Ala Leu Val Glu Glu Gln Asn Ile Pro Glu Ala Asn Val
115 120 125
Gly Leu Ala Ser Gly Val Ser Thr Phe Leu Trp Met Tyr Ser Ser Ile
130 135 140
His Gly Tyr Lys Pro Ala Lys Val Val Val Thr Ser Glu Leu Pro Leu
145 150 155 160
Gly Ser Gly Leu Gly Ser Ser Ala Ala Phe Cys Val Ser Leu Ser Ala
165 170 175
Ala Leu Leu Ala Leu Ser Asp Ser Val Lys Leu Asp Phe Ser Asn Gln
180 185 190
Gly Trp Gln Met Phe Ala Glu Thr Glu Leu Glu Leu Val Asn Lys Trp
195 200 205
Ala Phe Glu Gly Glu Lys Ile Ile His Gly Lys Pro Ser Gly Ile Asp
210 215 220
Asn Thr Val Ser Thr Tyr Gly Asn Met Ile Lys Phe Lys Ser Gly Glu
225 230 235 240
Met Val Arg Ile Lys Thr Asn Met Pro Leu Lys Met Leu Ile Thr Asn
245 250 255
Thr Lys Val Gly Arg Asn Thr Lys Ala Leu Val Ala Gly Val Ser Glu
260 265 270
Arg Thr Val Arg His Ser Asn Ala Met Ser Ser Val Phe Asn Ala Val
275 280 285
Asp Cys Ile Ser Asn Glu Leu Ala Ala Ile Ile Gln Ser Pro Val Ser
290 295 300
Asp Asp Leu Ala Ile Thr Glu Lys Glu Glu Lys Leu Gly Glu Leu Met
305 310 315 320
Glu Met Asn Gln Gly Leu Leu Gln Cys Met Gly Val Ser His Ala Ser
325 330 335
Ile Glu Thr Val Ile Arg Thr Thr Leu Lys Tyr Lys Leu Ala Thr Lys
340 345 350
Leu Thr Gly Ala Gly Gly Gly Gly Cys Val Leu Ser Leu Leu Pro Thr
355 360 365
Leu Leu Ser Gly Thr Val Val Asp Ile Val Ile Ser Glu Leu Glu Ala
370 375 380
Cys Gly Phe Gln Cys Leu Ile Ala Gly Ile Gly Gly Asn Gly Val Glu
385 390 395 400
Ile Ser Phe Ser Pro Ser Leu Glu His His His His His His
405 410
<210> 11
<211> 469
<212> PRT
<213> 人工序列
<400> 11
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Leu Pro Phe Leu Thr Ser Ala Pro Gly Lys
20 25 30
Val Ile Ile Phe Gly Glu His Ser Ala Val Tyr Asn Lys Pro Ala Val
35 40 45
Ala Ala Ser Val Ser Ala Leu Arg Thr Tyr Leu Leu Ile Ser Glu Ser
50 55 60
Ser Ala Pro Asp Thr Ile Glu Leu Asp Phe Pro Asp Ile Ser Phe Asn
65 70 75 80
His Lys Trp Ser Ile Asn Asp Phe Asn Ala Ile Thr Glu Asp Gln Val
85 90 95
Asn Ser Gln Lys Leu Ala Lys Ala Gln Gln Ala Thr Asp Gly Leu Ser
100 105 110
Gln Glu Leu Val Ser Leu Leu Asp Pro Leu Leu Ala Gln Leu Ser Glu
115 120 125
Ser Phe His Tyr His Ala Ala Phe Cys Phe Leu Tyr Met Phe Val Cys
130 135 140
Leu Cys Pro His Ala Lys Asn Ile Lys Phe Ser Leu Lys Ser Thr Leu
145 150 155 160
Pro Ile Gly Ala Gly Leu Gly Ser Ser Ala Ser Ile Ser Val Ser Leu
165 170 175
Ala Leu Ala Met Ala Tyr Leu Gly Gly Leu Ile Gly Ser Asn Asp Leu
180 185 190
Glu Lys Leu Ser Glu Asn Asp Lys His Ile Val Asn Gln Trp Ala Phe
195 200 205
Ile Gly Glu Lys Cys Ile His Gly Thr Pro Ser Gly Ile Asp Asn Ala
210 215 220
Val Ala Thr Tyr Gly Asn Ala Leu Leu Phe Glu Lys Asp Ser His Asn
225 230 235 240
Gly Thr Ile Asn Thr Asn Asn Phe Lys Phe Leu Asp Asp Phe Pro Ala
245 250 255
Ile Pro Met Ile Leu Thr Tyr Thr Arg Ile Pro Arg Ser Thr Lys Asp
260 265 270
Leu Val Ala Arg Val Arg Val Leu Val Thr Glu Lys Phe Pro Glu Val
275 280 285
Met Lys Pro Ile Leu Asp Ala Met Gly Glu Cys Ala Leu Gln Gly Leu
290 295 300
Glu Ile Met Thr Lys Leu Ser Lys Cys Lys Gly Thr Asp Asp Glu Ala
305 310 315 320
Val Glu Thr Asn Asn Glu Leu Tyr Glu Gln Leu Leu Glu Leu Ile Arg
325 330 335
Ile Asn His Gly Leu Leu Val Ser Ile Gly Val Ser His Pro Gly Leu
340 345 350
Glu Leu Ile Lys Asn Leu Ser Asp Asp Leu Arg Ile Gly Ser Thr Lys
355 360 365
Leu Thr Gly Ala Gly Gly Gly Gly Cys Ser Leu Thr Leu Leu Arg Arg
370 375 380
Asp Ile Thr Gln Glu Gln Ile Asp Ser Phe Lys Lys Lys Leu Gln Asp
385 390 395 400
Asp Phe Ser Tyr Glu Thr Phe Glu Thr Asp Leu Gly Gly Thr Gly Cys
405 410 415
Cys Leu Leu Ser Ala Lys Asn Leu Asn Lys Asp Pro Lys Ile Lys Ser
420 425 430
Leu Val Phe Gln Leu Phe Glu Asn Lys Thr Thr Thr Lys Gln Gln Ile
435 440 445
Asp Asp Leu Leu Leu Pro Gly Asn Thr Asn Leu Pro Trp Thr Ser His
450 455 460
His His His His His
465
<210> 12
<211> 485
<212> PRT
<213> 人工序列
<400> 12
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Thr Arg Lys Ala Ser Ser Pro Met Ala Pro Ala
20 25 30
Phe Met Val Ser Ala Pro Gly Lys Val Ile Val Tyr Gly Glu His Ala
35 40 45
Val Val His Gly Lys Ala Ala Leu Ala Ala Ala Ile Ser Leu Arg Ser
50 55 60
Tyr Leu Leu Val Thr Thr Leu Ser Lys Ser His Arg Thr Val Thr Leu
65 70 75 80
Asn Phe Arg Asp Ile Gly Leu Asn His Thr Trp Asn Ile Asp Asp Leu
85 90 95
Pro Trp Ala Ile Phe His His Pro Ser Lys Lys Lys Phe Tyr Tyr Asp
100 105 110
Leu Ile Asn Ser Leu Asp Pro Glu Leu Val Ala Ala Ile Gln Pro His
115 120 125
Val Asp Ala Val Ser Pro Asp Ala Ser Pro Glu Gln Arg Lys Ile His
130 135 140
Arg Asn Ser Ala Ser Ser Phe Leu Tyr Leu Phe Leu Ser Leu Gly Ser
145 150 155 160
Pro Gln Ser Pro Gly Ala Ile Tyr Thr Leu Arg Ser Thr Ile Pro Thr
165 170 175
Gly Ala Gly Leu Gly Ser Ser Ala Ser Val Asn Val Cys Val Ser Ala
180 185 190
Ala Leu Leu Leu Gln Ile Arg Thr Leu Ala Gly Pro His Pro Asp Gln
195 200 205
Pro Pro Asp Glu Ala Glu Val Gln Ile Glu Arg Ile Asn Arg Trp Ala
210 215 220
Phe Val Gly Glu Leu Cys Ile His Gly Asn Pro Ser Gly Val Asp Asn
225 230 235 240
Thr Val Ala Ala Gly Gly Lys Ala Val Ile Phe Arg Arg Gly Asp Tyr
245 250 255
Ser Lys Pro Pro Ser Val Lys Ser Leu Pro Ser Phe Pro Glu Leu Pro
260 265 270
Leu Leu Leu Val Asp Thr Arg Gln Ser Arg Ser Thr Ala Thr Glu Val
275 280 285
Ala Lys Val Gly Lys Leu Arg Glu Asp His Pro Val Val Thr Glu Ser
290 295 300
Ile Leu Asn Gly Ile Asp Gln Val Thr Val Ser Ala Glu Glu Leu Val
305 310 315 320
Glu Ser Pro Asp Phe Asp Gly Leu Ser Glu Lys Thr Phe Asp His Leu
325 330 335
Gly Ala Leu Phe Arg Ile Asn His Gly Phe Leu Val Ser Leu Gly Val
340 345 350
Ser His Pro Arg Leu Glu Arg Ile Arg Glu Ile Val Asp Phe Ala Asp
355 360 365
Ile Gly Trp Thr Lys Leu Thr Gly Ala Gly Gly Gly Gly Cys Ala Ile
370 375 380
Thr Leu Leu Arg Pro Asp Ala Lys Glu Ser Val Ile Lys Asp Leu Glu
385 390 395 400
Thr His Phe Gln Glu Glu Gly Phe Gly Lys Phe Glu Thr Thr Leu Gly
405 410 415
Gly Asp Gly Val Gly Val Leu Tyr Pro Ala Val Leu Arg Asn Gly Ser
420 425 430
Asp Glu Glu Gly Gly Glu Glu Ile Asp Gln Gln Lys Phe Glu Leu Ala
435 440 445
Asp Gly Thr Glu Gly Ile Glu Gln Leu Val Gly Val Gly Val Gln Glu
450 455 460
Arg Arg Glu Gly Trp Lys Phe Trp Lys Arg Ala Thr Arg Leu Glu His
465 470 475 480
His His His His His
485
<210> 13
<211> 365
<212> PRT
<213> 人工序列
<400> 13
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Thr Val Ser Ser Ala Pro Gly Lys Val Tyr Leu
20 25 30
Phe Gly Glu His Ala Val Val Tyr Gly Glu Pro Ala Val Pro Cys Ala
35 40 45
Ile Glu Arg Arg Ala Arg Val Thr Val Glu Glu Arg Ala Asp Ser Lys
50 55 60
Leu Arg Val His Ala Glu Asp Leu Ser Leu Asp Gly Phe Thr Val Glu
65 70 75 80
Tyr Ser Gly Asp Ala Gly Gly Arg Pro Asp Val Asp Val Ser Glu Thr
85 90 95
Leu Val Glu Ala Ala Met Gly Tyr Val Asp Ala Ala Val Glu Gln Ala
100 105 110
Arg Asp Ala Ala Asp Ala Pro Asp Ala Gly Phe Glu Val Thr Ile Glu
115 120 125
Ser Asp Ile Pro Leu Gly Ala Gly Leu Gly Ser Ser Ala Ala Val Thr
130 135 140
Val Ala Gly Ile Asp Ala Ala Thr Arg Glu Leu Gly Val Glu Leu Ser
145 150 155 160
Thr Glu Asp Leu Ala Asp Arg Ala Phe Arg Ala Glu Tyr Glu Val Gln
165 170 175
Glu Gly Gln Ala Ser Arg Ala Asp Thr Phe Cys Ser Ala Met Gly Gly
180 185 190
Ala Val Arg Val Glu Gly Asp Asp Cys Arg Ser Ile Asp Ala Pro Asp
195 200 205
Leu Pro Phe Val Ile Gly Phe Asp Gly Gly Ala Gly Asp Thr Gly Lys
210 215 220
Leu Val Ala Gly Val Arg Gly Leu Arg Glu Glu Tyr Gly Phe Ala Ala
225 230 235 240
Asp Thr Val Glu Ala Ile Gly Asp Val Val Arg Arg Gly Glu Gln Ile
245 250 255
Leu Ala Asp Ala Ala Glu Gly Glu Glu Gly Asp Glu Trp Leu Ala Glu
260 265 270
Leu Gly Asp Leu Leu Asn Phe Asn His Gly Leu Leu Glu Ala Leu Gly
275 280 285
Val Ser Ser Arg Ser Leu Asp Ala Met Val Trp Ala Ala Arg Asp Ala
290 295 300
Gly Ala His Gly Ala Lys Leu Thr Gly Ala Gly Gly Gly Gly Cys Ile
305 310 315 320
Val Ala Leu Asp Glu Thr Glu Glu Thr Glu Thr Ala Leu Lys Phe Thr
325 330 335
Pro Gly Cys Glu Glu Ala Phe Arg Ala Glu Leu Ala Thr Asp Gly Val
340 345 350
Arg Val Glu Ser Arg Leu Glu His His His His His His
355 360 365
<210> 14
<211> 424
<212> PRT
<213> 人工序列
<400> 14
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Leu Ser Glu Val Leu Leu Val Ser Ala Pro Gly
20 25 30
Lys Val Ile Leu His Gly Glu His Ala Val Val His Gly Lys Val Ala
35 40 45
Leu Ala Val Ala Leu Asn Leu Arg Thr Phe Leu Arg Leu Gln Pro His
50 55 60
Ser Asn Gly Lys Val Gly Leu Asn Leu Pro Asn Ile Gly Ile Gln Arg
65 70 75 80
Val Trp Asp Val Ala Lys Leu Gln Met Leu Asp Thr Ser Phe Leu Lys
85 90 95
Gln Gly Asp Val Thr Ala Leu Thr Pro Glu Gln Met Glu Lys Leu Lys
100 105 110
Glu Val Ala Gly Phe Pro Glu Asp Cys Ala Asp His Glu His Leu Ala
115 120 125
Val Leu Ser Phe Leu Tyr Leu Tyr Leu Ser Ile Cys Gln Ser Gln Arg
130 135 140
Ala Leu Pro Ser Leu Asp Ile Ala Val Trp Ser Glu Leu Pro Thr Gly
145 150 155 160
Ala Gly Leu Gly Ser Ser Ala Ala Tyr Ser Val Cys Leu Ala Ala Ala
165 170 175
Leu Leu Thr Ala Cys Lys Glu Ile Pro Asn Pro Leu Lys Asp Gly Glu
180 185 190
Ala Ala Ser Arg Trp Thr Glu Glu Ser Leu Glu Leu Ile Asn Lys Trp
195 200 205
Ala Phe Gln Gly Glu Arg Val Ile His Gly Asn Pro Ser Gly Val Asp
210 215 220
Asn Ala Val Ser Thr Trp Gly Gly Ala Leu Arg Tyr Gln Gln Gly Lys
225 230 235 240
Ile Ser Ser Leu Asn Arg Pro Pro Ala Leu Lys Ile Leu Leu Thr Asn
245 250 255
Thr Lys Val Pro Arg Ser Thr Lys Ala Leu Val Ala Gly Val Arg Ser
260 265 270
Arg Leu Leu Lys Phe Pro Glu Ile Val Ala Pro Leu Leu Thr Ser Ile
275 280 285
Asp Ala Ile Ser Leu Glu Cys Glu Arg Leu Leu Gly Glu Met Ala Ala
290 295 300
Ala Pro Ala Pro Glu His Tyr Leu Val Leu Glu Glu Leu Ile Asp Met
305 310 315 320
Asn Gln His His Leu Asn Ala Leu Gly Val Gly His Ala Ser Leu Asp
325 330 335
Arg Leu Cys Gln Val Thr Met Ala His Gly Leu His Ser Lys Leu Thr
340 345 350
Gly Ala Gly Gly Gly Gly Cys Gly Ile Thr Leu Leu Arg Pro Asp Leu
355 360 365
Glu Arg Pro Glu Val Glu Ala Met Lys Gln Ala Leu Thr Ser Cys Gly
370 375 380
Phe Asp Cys Trp Glu Thr Ser Ile Gly Ala Pro Gly Val Ser Val His
385 390 395 400
Thr Ala Ala Ser Leu Asp Ala Pro Val Arg Gln Ala Leu Asp Gly Leu
405 410 415
Leu Glu His His His His His His
420
<210> 15
<211> 320
<212> PRT
<213> 人工序列
<400> 15
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Lys Lys Ile Gly Ile Gly Lys Ala His Ser
20 25 30
Lys Ile Ile Leu Met Gly Glu His Ser Val Val Tyr Gly Phe Pro Ala
35 40 45
Ile Ala Leu Pro Leu Lys Asp Ile Glu Val Val Cys Arg Ile Arg Arg
50 55 60
Ala Glu Lys Lys Leu Glu Phe Asp Phe Tyr Asp Thr Leu Ser Thr Ala
65 70 75 80
Ile Phe Ser Ala Leu Asp Tyr Leu Lys Ile Lys Asn Gln Pro Ile Ser
85 90 95
Tyr Glu Ile Thr Ser Gln Val Pro Gln Arg Arg Gly Met Gly Ser Ser
100 105 110
Ala Ala Val Ser Ile Ala Ala Ile Arg Ala Val Phe Ser Tyr Phe Asp
115 120 125
Gln Glu Leu Ser Asp Gln Leu Leu Glu Ile Leu Val Asn Lys Ala Glu
130 135 140
Ile Ile Ala His Thr Asn Pro Ser Gly Leu Asp Ala Lys Thr Cys Leu
145 150 155 160
Ser Asp Gln Ala Ile Lys Phe Ile Arg Asn Val Gly Phe Val Ser Leu
165 170 175
Glu Ile Asn Leu Asp Ala Tyr Leu Val Ile Ala Asp Thr Gly Ile His
180 185 190
Gly His Thr Arg Glu Ala Val Asn Lys Val Ala Lys Phe Glu Glu Ser
195 200 205
Asn Leu Pro His Leu Ala Ala Leu Gly Gln Leu Thr Glu Asp Val Glu
210 215 220
Glu Ala Ile Lys Ala Lys Asp Val Ile Ser Ile Gly Gln Ser Met Thr
225 230 235 240
Gln Ala His Glu His Leu Lys Ala Ile Gly Val Ser Val Glu Lys Ser
245 250 255
Asn Gln Leu Val Glu Glu Ala Leu Lys Gln Gly Ala Leu Gly Ala Lys
260 265 270
Met Ser Gly Gly Gly Leu Gly Gly Cys Ile Ile Ala Leu Ala Asn Ser
275 280 285
Gln Asn Asp Ala Val Val Ile Ser Gln Ala Leu Glu Glu Lys Gly Ala
290 295 300
Val Asn Thr Trp Ile Gln Lys Leu Leu Glu His His His His His His
305 310 315 320
<210> 16
<211> 336
<212> PRT
<213> 人工序列
<400> 16
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Asn Pro Leu Phe Tyr Ala Lys Ile Leu Leu
20 25 30
Phe Gly Glu Tyr Gly Ile Ile Glu Asp Ser Gln Gly Leu Thr Leu Pro
35 40 45
Tyr Ser Phe Tyr Lys Gly Thr Leu Lys Phe Ser Asp Leu Glu Ser Asp
50 55 60
Phe Glu Lys Thr Ser Asn Ile Ser Leu Gln Lys Tyr Ala Asp Tyr Leu
65 70 75 80
Gly Ser Leu Asn Leu Pro Asp Ser Phe Lys Leu Asn Ile Ser Lys Leu
85 90 95
Gln Lys Asp Leu Lys Lys Gly Leu Phe Phe Asp Ser Asn Ile Pro Gln
100 105 110
Gly Tyr Gly Val Gly Ser Ser Gly Ala Leu Val Ala Ala Ile Phe Glu
115 120 125
Lys Tyr Ser Val Lys Thr Tyr Leu Pro Glu His Ile Ser Lys Asp Gln
130 135 140
Leu Lys Glu Leu Lys Lys Val Phe Gly Glu Met Glu Ser Tyr Phe His
145 150 155 160
Gly Lys Ser Ser Gly Ile Asp Pro Leu Ile Cys Tyr Met Asn Leu Pro
165 170 175
Ile Leu Ile Glu Asn Lys Glu Asn Val Asp Lys Val Ser Ile Pro Glu
180 185 190
Ser His Glu Gly Lys Gly Ala Ile Phe Leu Ile Asp Ser Gly Met Thr
195 200 205
Gly Glu Thr Gly Pro Met Val Gln Ile Phe Phe Glu Lys Met Lys Thr
210 215 220
Glu Gly Phe Arg Lys Thr Met Lys Glu Glu Phe Ile Arg Tyr Asn Asn
225 230 235 240
Ala Cys Ile Asp Ala Phe Leu Lys Lys Glu Met Asn Pro Leu Phe Arg
245 250 255
Asn Leu Lys Ser Leu Ser Val Trp Ala Tyr Glu His Phe Lys Pro Met
260 265 270
Ile Pro Glu Ser Ile Tyr Asn Ala Trp Lys Lys Gly Leu Asp Thr Asn
275 280 285
Ala Tyr Tyr Leu Lys Leu Cys Gly Ser Gly Gly Gly Gly Tyr Ile Leu
290 295 300
Gly Phe Thr Lys Asp Tyr Lys Lys Ala Glu Lys Met Leu Glu Gly Phe
305 310 315 320
His Lys Glu Val Ile Tyr Arg Phe Leu Glu His His His His His His
325 330 335
<210> 17
<211> 234
<212> PRT
<213> 人工序列
<400> 17
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Met Ala Pro Leu Gly Gly Ala Pro Arg Leu Val Leu Leu Phe
35 40 45
Ser Gly Lys Arg Lys Ser Gly Lys Asp Phe Val Thr Glu Ala Leu Gln
50 55 60
Ser Arg Leu Gly Ala Asp Val Cys Ala Val Leu Arg Leu Ser Gly Pro
65 70 75 80
Leu Lys Glu Gln Tyr Ala Gln Glu His Gly Leu Asn Phe Gln Arg Leu
85 90 95
Leu Asp Thr Ser Thr Tyr Lys Glu Ala Phe Arg Lys Asp Met Ile Arg
100 105 110
Trp Gly Glu Glu Lys Arg Gln Ala Asp Pro Gly Phe Phe Cys Arg Lys
115 120 125
Ile Val Glu Gly Ile Ser Gln Pro Ile Trp Leu Val Ser Asp Thr Arg
130 135 140
Arg Val Ser Asp Ile Gln Trp Phe Arg Glu Ala Tyr Gly Ala Val Thr
145 150 155 160
Gln Thr Val Arg Val Val Ala Leu Glu Gln Ser Arg Gln Gln Arg Gly
165 170 175
Trp Val Phe Thr Pro Gly Val Asp Asp Ala Glu Ser Glu Cys Gly Leu
180 185 190
Asp Asn Phe Gly Asp Phe Asp Trp Val Ile Glu Asn His Gly Val Glu
195 200 205
Gln Arg Leu Glu Glu Gln Leu Glu Asn Leu Ile Glu Phe Ile Arg Ser
210 215 220
Arg Leu Leu Glu His His His His His His
225 230
<210> 18
<211> 276
<212> PRT
<213> 人工序列
<400> 18
Gly Ser Met Ile Ile Leu Lys Leu Gly Gly Ser Val Ile Thr Arg Lys
1 5 10 15
Asp Ser Glu Glu Pro Ala Ile Asp Arg Asp Asn Leu Glu Arg Ile Ala
20 25 30
Ser Glu Ile Gly Asn Ala Ser Pro Ser Ser Leu Met Ile Val His Gly
35 40 45
Ala Gly Ser Phe Gly His Pro Phe Ala Gly Glu Tyr Arg Ile Gly Ser
50 55 60
Glu Ile Glu Asn Glu Glu Asp Leu Arg Arg Arg Arg Phe Gly Phe Ala
65 70 75 80
Leu Thr Gln Asn Trp Val Lys Lys Leu Asn Ser His Val Cys Asp Ala
85 90 95
Leu Leu Ala Glu Gly Ile Pro Ala Val Ser Met Gln Pro Ser Ala Phe
100 105 110
Ile Arg Ala His Ala Gly Arg Ile Ser His Ala Asp Ile Ser Leu Ile
115 120 125
Arg Ser Tyr Leu Glu Glu Gly Met Val Pro Val Val Tyr Gly Asp Val
130 135 140
Val Leu Asp Ser Asp Arg Arg Leu Lys Phe Ser Val Ile Ser Gly Asp
145 150 155 160
Gln Leu Ile Asn His Phe Ser Leu Arg Leu Met Pro Glu Arg Val Ile
165 170 175
Leu Gly Thr Asp Val Asp Gly Val Tyr Thr Arg Asn Pro Lys Lys His
180 185 190
Pro Asp Ala Arg Leu Leu Asp Val Ile Gly Ser Leu Asp Asp Leu Glu
195 200 205
Ser Leu Asp Gly Thr Leu Asn Thr Asp Val Thr Gly Gly Met Val Gly
210 215 220
Lys Ile Arg Glu Leu Leu Leu Leu Ala Glu Lys Gly Val Glu Ser Glu
225 230 235 240
Ile Ile Asn Ala Ala Val Pro Gly Asn Ile Glu Arg Ala Leu Leu Gly
245 250 255
Glu Glu Val Arg Gly Thr Arg Ile Thr Gly Lys His Leu Glu His His
260 265 270
His His His His
275
<210> 19
<211> 391
<212> PRT
<213> 人工序列
<400> 19
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ile Thr Val Lys Ala Pro Gly Lys Leu Tyr Ile
20 25 30
Ala Gly Glu Tyr Ala Val Val Glu Ser Gly Phe Pro Ala Ile Ile Val
35 40 45
Ala Leu Asp Gln Phe Val Thr Ala Thr Ile Ser Ser Ser Asp Thr Val
50 55 60
Gly Ser Ile Val Ser Lys Gln Tyr Gln Glu Asn Ser Ile Val Trp Arg
65 70 75 80
Arg Lys Gly Asp Ala Met Val Phe Asp Asn Arg Asp Asn Pro Phe His
85 90 95
Tyr Ile Leu Ser Ala Ile Ser Leu Thr Glu Ala Tyr Ala His Glu Leu
100 105 110
Gly Arg Asp Leu Gly Val Tyr His Leu Gly Ile Asn Ser Glu Leu Asp
115 120 125
Ser Ala Asp Gly Lys Lys Tyr Gly Leu Gly Ser Ser Ala Ala Val Thr
130 135 140
Val Ala Thr Val Lys Ala Leu Cys Arg Phe Tyr His Leu Pro Met Asp
145 150 155 160
Lys Pro Lys Leu Phe Lys Leu Ala Ala Ile Ala His Leu Ser Val Gln
165 170 175
Gly Asn Gly Ser Leu Gly Asp Ile Ala Ala Ser Val Tyr Gly Gly Trp
180 185 190
Ile Ala Tyr His Ser Phe Asp Arg Asp Trp Leu His Val Gln Arg Lys
195 200 205
Gln Thr Ser Leu Ser Glu Leu Leu Asp Met Asp Trp Pro Asp Leu Lys
210 215 220
Ile Asp Leu Leu Thr Pro Pro Ala Asp Leu Arg Leu Met Ile Gly Trp
225 230 235 240
Thr Gly Ser Pro Ala Ser Thr Ser His Leu Val Asp Lys Val Ala Leu
245 250 255
Val Lys Ala Lys Gln Arg Gly Glu Tyr Gln Thr Phe Leu Gln Asp Ser
260 265 270
Lys Ala Cys Leu Lys Arg Met Ile Ala Gly Phe His Ala Gly Asp Leu
275 280 285
Gln Ala Ile Gln Thr Glu Ile Arg Cys Asn Arg His Leu Leu Gln Thr
290 295 300
Leu Gly Ala Phe Ser His Val Thr Ile Glu Thr Pro Ile Leu Lys Lys
305 310 315 320
Met Ile His Leu Ala Glu Ala Ala Gly Ala Ala Ala Lys Thr Ser Gly
325 330 335
Ala Gly Gly Gly Asp Cys Gly Ile Val Leu Ile Asp Gln Ala Ile Asn
340 345 350
Pro Ala Asn Leu Leu Lys Gln Trp Ala His Asp Gly Ile Glu Gln Leu
355 360 365
Asn Leu Thr Val His Thr Val Ser Asp Asp Ser Ile Ser Asn Ala Leu
370 375 380
Glu His His His His His His
385 390
<210> 20
<211> 270
<212> PRT
<213> 人工序列
<400> 20
Gly Ser Met Leu Thr Ile Leu Lys Leu Gly Gly Ser Ile Leu Ser Asp
1 5 10 15
Lys Asn Val Pro Tyr Ser Ile Lys Trp Asp Asn Leu Glu Arg Ile Ala
20 25 30
Met Glu Ile Lys Asn Ala Leu Asp Tyr Tyr Lys Asn Gln Asn Lys Glu
35 40 45
Ile Lys Leu Ile Leu Val His Gly Gly Gly Ala Phe Gly His Pro Val
50 55 60
Ala Lys Lys Tyr Leu Lys Ile Glu Asp Gly Lys Lys Ile Phe Ile Asn
65 70 75 80
Met Glu Lys Gly Phe Trp Glu Ile Gln Arg Ala Met Arg Arg Phe Asn
85 90 95
Asn Ile Ile Ile Asp Thr Leu Gln Ser Tyr Asp Ile Pro Ala Val Ser
100 105 110
Ile Gln Pro Ser Ser Phe Val Val Phe Gly Asp Lys Leu Ile Phe Asp
115 120 125
Thr Ser Ala Ile Lys Glu Met Leu Lys Arg Asn Leu Val Pro Val Ile
130 135 140
His Gly Asp Ile Val Ile Asp Asp Lys Asn Gly Tyr Arg Ile Ile Ser
145 150 155 160
Gly Asp Asp Ile Val Pro Tyr Leu Ala Asn Glu Leu Lys Ala Asp Leu
165 170 175
Ile Leu Tyr Ala Thr Asp Val Asp Gly Val Leu Ile Asp Asn Lys Pro
180 185 190
Ile Lys Arg Ile Asp Lys Asn Asn Ile Tyr Lys Ile Leu Asn Tyr Leu
195 200 205
Ser Gly Ser Asn Ser Ile Asp Val Thr Gly Gly Met Lys Tyr Lys Ile
210 215 220
Asp Met Ile Arg Lys Asn Lys Cys Arg Gly Phe Val Phe Asn Gly Asn
225 230 235 240
Lys Ala Asn Asn Ile Tyr Lys Ala Leu Leu Gly Glu Val Glu Gly Thr
245 250 255
Glu Ile Asp Phe Ser Glu Leu Glu His His His His His His
260 265 270
<210> 21
<211> 532
<212> PRT
<213> 人工序列
<400> 21
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Val Val Ala Ser Ala Pro Gly Lys Val Leu
20 25 30
Ile Thr Gly Gly Tyr Leu Ile Leu Glu Arg Pro Asn Ala Gly Ile Val
35 40 45
Leu Ser Thr Asn Ala Arg Phe Tyr Ala Ile Val Lys Pro Leu Tyr Glu
50 55 60
Gln Val Lys Pro Asp Ser Trp Ser Trp Gly Trp Thr Asp Val Lys Leu
65 70 75 80
Thr Ser Pro Gln Leu Leu Arg Glu Ser Met Tyr Lys Leu Ser Leu Lys
85 90 95
Asn Leu Thr Leu Gln Ala Val Ser Leu Ser Glu Ser Arg Asn Pro Phe
100 105 110
Val Glu Tyr Ala Val Gln Tyr Ala Val Ala Ala Ala Tyr Ala Ile Phe
115 120 125
Asp Lys Asn Lys Lys Asp Ala Leu His Lys Leu Leu Leu Gln Gly Leu
130 135 140
Asp Ile Thr Ile Leu Gly Cys Asn Asp Phe Tyr Ser Tyr Arg Asn Gln
145 150 155 160
Ile Glu Ala Arg Gly Leu Pro Leu Thr Pro Glu Ala Leu Ala Ala Leu
165 170 175
Pro Pro Phe Ala Ser Ile Thr Phe Asn Ala Asp Glu Ser Asn Gly Gly
180 185 190
Asn Cys Lys Pro Glu Val Ala Lys Thr Gly Leu Gly Ser Ser Ala Ala
195 200 205
Met Thr Thr Ala Val Val Ala Ala Leu Leu His Tyr Leu Gly Val Val
210 215 220
Asn Leu Ser Ser Ser Ile Asp Gln Gln His Asp Gly Asp Leu Asp Met
225 230 235 240
Val His Met Ile Ala Gln Ser Ala His Cys Ile Ala Gln Gly Lys Ile
245 250 255
Gly Ser Gly Phe Asp Val Ser Ser Ala Val Tyr Gly Ser Gln Arg Tyr
260 265 270
Val Arg Phe Ser Pro Glu Val Leu Ser Ser Ala Gln Val Ala Val Lys
275 280 285
Glu Thr Pro Leu Gln Glu Val Ile Thr Gly Ile Leu Lys Gly Lys Trp
290 295 300
Asp His Glu Arg Ala Met Phe Ser Leu Pro Pro Leu Met Thr Leu Leu
305 310 315 320
Leu Gly Glu Pro Gly Thr Gly Gly Ser Ser Thr Pro Ser Met Val Gly
325 330 335
Ala Val Lys Lys Trp Gln Lys Ser Asp Pro Gln Lys Ser Gln Glu Thr
340 345 350
Trp Lys Lys Leu Ser Glu Ser Asn Ser Ala Leu Glu Thr Gln Leu Asn
355 360 365
Met Leu Ser Lys Leu Ala Glu Glu His Trp Asn Ala Tyr Lys Gln Val
370 375 380
Ile Glu Ser Cys Ser Lys Leu Lys Ser Glu Lys Trp Met Glu Gln Ala
385 390 395 400
Thr Glu Pro Thr Gln Glu Ala Val Val Lys Ser Leu Leu Gly Ala Arg
405 410 415
Asp Ala Met Leu Gly Ile Arg Tyr His Met Arg Leu Met Gly Glu Ala
420 425 430
Ala Gly Val Pro Ile Glu Pro Glu Ser Gln Thr Gln Leu Leu Asn Ala
435 440 445
Thr Met Asp Met Glu Gly Val Leu Leu Ala Gly Val Pro Gly Ala Gly
450 455 460
Gly Phe Asp Ala Val Phe Ala Val Thr Leu Gly Asp Ser Gly Ser Asn
465 470 475 480
Val Thr Lys Ala Trp Ser Ser Val Asn Val Leu Ala Leu Leu Val Arg
485 490 495
Glu Asp Pro His Gly Val Ser Leu Glu Ser Cys Asp Pro Arg Thr Thr
500 505 510
Glu Ile Thr Ser Ala Val Ser Ala Val His Ile Glu Leu Glu His His
515 520 525
His His His His
530
<210> 22
<211> 340
<212> PRT
<213> 人工序列
<400> 22
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ile Ile Glu Thr Pro Ser Lys Val Ile Leu Phe
20 25 30
Gly Glu His Ala Val Val Tyr Gly Tyr Arg Ala Ile Ser Met Ala Ile
35 40 45
Asp Leu Thr Ser Thr Ile Glu Ile Lys Glu Thr Gln Glu Asp Glu Ile
50 55 60
Ile Leu Asn Leu Asn Asp Leu Asn Lys Ser Leu Gly Leu Asn Leu Asn
65 70 75 80
Glu Ile Lys Asn Ile Asn Pro Asn Asn Phe Gly Asp Phe Lys Tyr Cys
85 90 95
Leu Cys Ala Ile Lys Asn Thr Leu Asp Tyr Leu Asn Ile Glu Pro Lys
100 105 110
Thr Gly Phe Lys Ile Asn Ile Ser Ser Lys Ile Pro Ile Ser Cys Gly
115 120 125
Leu Gly Ser Ser Ala Ser Ile Thr Ile Gly Thr Ile Lys Ala Val Ser
130 135 140
Gly Phe Tyr Asn Lys Glu Leu Lys Asp Asp Glu Ile Ala Lys Leu Gly
145 150 155 160
Tyr Met Val Glu Lys Glu Ile Gln Gly Lys Ala Ser Ile Thr Asp Thr
165 170 175
Ser Thr Ile Thr Tyr Lys Gly Ile Leu Glu Ile Lys Asn Asn Lys Phe
180 185 190
Arg Lys Ile Lys Gly Glu Phe Glu Glu Phe Leu Lys Asn Cys Lys Phe
195 200 205
Leu Ile Val Tyr Ala Glu Lys Arg Lys Lys Lys Thr Ala Glu Leu Val
210 215 220
Asn Glu Val Ala Lys Ile Glu Asn Lys Asp Glu Ile Phe Lys Glu Ile
225 230 235 240
Asp Lys Val Ile Asp Glu Ala Leu Lys Ile Lys Asn Lys Glu Asp Phe
245 250 255
Gly Lys Leu Met Thr Lys Asn His Glu Leu Leu Lys Lys Leu Asn Ile
260 265 270
Ser Thr Pro Lys Leu Asp Arg Ile Val Asp Ile Gly Asn Arg Phe Gly
275 280 285
Phe Gly Ala Lys Leu Thr Gly Ala Gly Gly Gly Gly Cys Val Ile Ile
290 295 300
Leu Val Asn Glu Glu Lys Glu Lys Glu Leu Leu Lys Glu Leu Asn Lys
305 310 315 320
Glu Asp Val Arg Ile Phe Asn Cys Arg Met Met Asn Leu Glu His His
325 330 335
His His His His
340
<210> 23
<211> 494
<212> PRT
<213> 人工序列
<400> 23
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Ser Asn Pro Ala Leu Glu Pro Lys Ala Val
20 25 30
Ser Ala Pro Gly Lys Val Phe Val Ala Gly Gly Tyr Leu Val Leu Asp
35 40 45
Arg Lys Tyr Thr Ala Leu Val Phe Gly Leu Asp Ala Arg Ile His Val
50 55 60
Glu Ile Glu Pro Ile Arg Thr Lys Ser Gly Val Thr Leu Ser Glu Ile
65 70 75 80
Ile Val Arg Ser Pro Gln Phe Arg Glu Ala Ala Trp Glu Tyr Gly Tyr
85 90 95
Arg Leu Thr Glu Ala Gly Gly Gly Ile Ala Val Thr Gln Leu Arg Ala
100 105 110
Ser His Asp Asp Lys Leu Asn Lys Asn Pro Phe Val Glu Thr Ala Leu
115 120 125
Thr Tyr Ala Leu Thr Tyr Val Ser Thr Leu Leu Pro Gly Met Pro Ile
130 135 140
Ser Pro Ser Ser Ile Ser Ile Leu Ala Asp Gln Ala Tyr Tyr Ser Asn
145 150 155 160
Pro Gly Thr Ala Pro Ser Pro Thr Ser Arg Phe Leu Asn Phe Asn Val
165 170 175
Thr Ile Ser Glu Ala His Lys Thr Gly Leu Gly Ser Ser Ala Ala Leu
180 185 190
Val Thr Ser Phe Thr Ala Ala Leu Leu Ser Tyr Tyr Leu Pro Gln Thr
195 200 205
His Phe Asp Leu Ala Ser Glu Ala Ser Leu Arg Ile Leu His Asn Leu
210 215 220
Ser Gln Ala Ser His Cys Ala Ala Gln Gly Lys Val Gly Ser Gly Phe
225 230 235 240
Asp Ile Ala Ser Ala Val Tyr Gly Ser Cys Leu Tyr Arg Arg Phe Ser
245 250 255
Pro Ser Val Leu Ser Ser Leu Pro Glu Pro Gly Asn Pro Asp Phe Ser
260 265 270
Lys Lys Leu Arg Ala Leu Val Glu Gly Ser Asp Trp Asp Thr Glu Ile
275 280 285
Arg Lys Ala Ala Val Lys Met Pro Thr Gly Leu Arg Leu Val Met Cys
290 295 300
Asp Val Asp Cys Gly Ser Gln Thr Pro Gly Met Val Lys Lys Val Leu
305 310 315 320
Lys Trp Arg Ala Glu Asn Gly Glu Glu Ala Glu Arg Ile Trp Gly Gln
325 330 335
Leu Gln Lys Gly Asn Glu Ala Leu Ala Thr Glu Leu Thr Arg Leu Ala
340 345 350
Thr Ala Asp Ala Glu Asn Gly Glu Gly Ser Glu Lys Cys Ala Lys Leu
355 360 365
Arg Gln Ile Ile Gly Glu Asn Arg Thr Leu Ile Arg Gln Met Ser Val
370 375 380
Ala Ser Arg Val Pro Ile Glu Pro Pro Gln Gln Thr Ala Leu Leu Asp
385 390 395 400
Ala Cys Ser Ser Val Pro Gly Val Ile Gly Gly Val Val Pro Gly Ala
405 410 415
Gly Gly Tyr Asp Ala Ile Val Leu Leu Leu Glu Asp Lys Val Glu Val
420 425 430
Leu Phe Glu Leu Glu Glu Leu Leu Ala Gly Trp Lys Val Glu Gly Glu
435 440 445
Asp Glu Gly Glu Glu Gly Val Lys Ile Gly Lys Val Gly Ile Ile Gly
450 455 460
Val Arg Glu Glu Met Val Gly Val Lys Gln Glu Asp Pro Ala Ile Tyr
465 470 475 480
Lys Glu Trp Ile Lys Thr Leu Glu His His His His His His
485 490
<210> 24
<211> 523
<212> PRT
<213> 人工序列
<400> 24
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Val Arg Thr Thr Val Val Ser Ala Pro Gly Lys
20 25 30
Val Leu Ile Ala Gly Gly Tyr Leu Val Leu Asp Pro Ala Tyr Pro Gly
35 40 45
Thr Val Val Ser Thr Ser Ser Arg Phe Tyr Thr Val Ile Gln Ser Gln
50 55 60
Glu Phe Leu Ser Lys Asn Ser Ile Arg Val Arg Ser Pro Gln Phe Leu
65 70 75 80
Glu Ala Thr Trp Ser Tyr Ser Val Leu Phe Glu Pro Ala Val Ala Val
85 90 95
Glu Ala Ser Pro Glu Asn Ser Ser Lys Asn Lys Phe Val His Leu Ala
100 105 110
Leu Gln Lys Thr Ile Ala Leu Ala Val Glu Leu Arg Gly Ala Ala Gln
115 120 125
Ile Gln Glu Ala Leu Thr His Gly Phe Asp Ile Ala Ile Val Gly Asp
130 135 140
Asn Asp Phe Tyr Ser Gln Arg Ala Lys Leu Glu Ser Leu Gly Leu Pro
145 150 155 160
Arg Thr Leu Asp Ser Leu Thr Gln Ile Thr Pro Phe Cys Ala Thr Glu
165 170 175
Val His Leu Ser Asp Val His Lys Thr Gly Leu Gly Ser Ser Ala Ala
180 185 190
Leu Ile Thr Ser Leu Thr Ser Ala Ile Leu Val His Leu Ser Val Ile
195 200 205
Ser Glu Ser Ser Leu Ala Glu Asp Asp Ser Arg Asp Arg Arg Gln Ala
210 215 220
His Asn Leu Ala Gln Tyr Val His Cys Leu Ala Gln Gly Lys Val Gly
225 230 235 240
Ser Gly Phe Asp Val Ser Ala Ala Val Phe Gly Ser His Leu Tyr Ser
245 250 255
Arg Phe Asp Pro Ala Val Ile Gln Asp Leu Met Ser Asp Asp Ala Leu
260 265 270
Pro Ser Gln Leu Pro Ser Val Leu Ser Pro Ser Asn Ala Ala Trp Asn
275 280 285
Tyr Arg Ile Glu Gln Phe Lys Leu Pro Pro Leu Thr Arg Ile Met Leu
290 295 300
Ala Asp Val Asp Ala Gly Ser Asp Thr Pro Ser Leu Val Gly Lys Val
305 310 315 320
Leu Lys Trp Arg Lys Glu Asn Ser Thr Glu Ala Glu Ala Leu Trp Thr
325 330 335
Asn Leu Asp Gln Gln Asn Gln Ser Leu Ala Gln Thr Leu Leu His Leu
340 345 350
Gly Lys Leu Ala Glu Asp Asp His Glu Asn Tyr Ala Ser Ala Val Lys
355 360 365
Tyr Ile Cys Ser Leu Gln Pro Val Gln Trp Val Ala Asn Pro Leu Gln
370 375 380
Pro Thr Ser Glu Arg Pro Ile Ile Thr Ala Phe Tyr Glu Ala His Arg
385 390 395 400
Ile Cys Glu Ala Ile Arg Gly Lys Met Arg Glu Met Gly Asn Leu Ser
405 410 415
Gly Val Pro Ile Glu Pro Val Glu Gln Thr Thr Leu Leu Asp Ala Cys
420 425 430
Val Ser Gln Ala Gly Val Ile Gly Gly Gly Val Pro Gly Ala Gly Gly
435 440 445
Tyr Asp Ala Ile Trp Leu Leu Val Cys Asp Pro Pro Ser Cys Ala Pro
450 455 460
Asp Gln Ser Pro Leu Glu Arg Ile Glu His Leu Trp Ser His Tyr Glu
465 470 475 480
Lys Leu Asp Val Ser Pro Leu Ser Ala Gln Glu Ser Met Ala Lys Gly
485 490 495
Val Arg Val Glu Ala Leu Asp Asp Val Pro Gly Leu Lys Asn Ala Ile
500 505 510
Ser Val Ser Leu Glu His His His His His His
515 520
<210> 25
<211> 399
<212> PRT
<213> 人工序列
<400> 25
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Thr Ser Gly Arg Thr Val Val Arg Ser Ala Pro
20 25 30
Gly Lys Leu Phe Val Ala Gly Glu Tyr Ala Val Val Glu Pro Gly Thr
35 40 45
Pro Ala Ile Leu Val Ala Val Asp Arg Glu Ile Thr Val Thr Val Ser
50 55 60
Gly Ser Glu Gly Ala Asp Val Val Ile Ser Ser Asp Leu Gly Pro Arg
65 70 75 80
Ala Val Arg Trp Gln Trp Arg Glu Gly Val Leu His Val Gln Asp Glu
85 90 95
Glu Asp Glu Glu Gly Ala Arg Gly Ala Leu Ala His Val Val Ser Ala
100 105 110
Val Glu Thr Val Ala Gly Leu Leu Ala Glu Arg Gly Leu Pro Val Pro
115 120 125
Thr Leu Asp Ile Gly Val Ser Ser Arg Leu His Glu Asp Gly Arg Lys
130 135 140
Phe Gly Leu Gly Ser Ser Gly Ala Val Thr Val Ala Thr Val Asp Ala
145 150 155 160
Val Ser Ala Phe Cys Gly Val Glu Leu Ser Leu Asp Ala Arg Phe Arg
165 170 175
Leu Ala Leu Ile Ala Asn Ala Arg Ile Asp Pro Lys Gly Ser Gly Gly
180 185 190
Asp Leu Ala Ala Ser Thr Trp Gly Gly Trp Ile Ala Tyr Gln Gly Pro
195 200 205
Asp Arg Ala Phe Val Leu Asp Leu Val Arg Arg Glu Gly Val Glu Glu
210 215 220
Ala Leu Arg Ala Pro Trp Pro Gly Phe Ala Val Arg Arg Leu Pro Pro
225 230 235 240
Pro Asn Gly Leu Ser Leu Glu Val Gly Trp Thr Gly Asn Pro Ala Ser
245 250 255
Thr Thr Ser Leu Val Ser Gly Leu His Arg Arg Thr Trp Arg Gly Thr
260 265 270
Asp Ser His Arg Ala Phe Val Glu Thr Ser Thr Asp Leu Val Arg Ala
275 280 285
Ala Ile Ala Ala Leu Glu Ala Gly Asp Gly Gln Ala Leu Leu His Gln
290 295 300
Val Arg Arg Ala Arg Gln Glu Leu Ala Arg Leu Asp Glu Glu Val Gly
305 310 315 320
Leu Gly Ile Phe Thr Thr Glu Leu Thr Ala Leu Cys Glu Ala Ala Glu
325 330 335
Gly Val Gly Gly Ala Ala Lys Pro Ser Gly Ala Gly Gly Gly Asp Cys
340 345 350
Gly Ile Ala Leu Leu Asp Thr Gly Ala Ala His Asp Ile Ser His Val
355 360 365
Arg Gln Arg Trp Ala Ala Ala Gly Val Leu Pro Leu Leu Val Arg Pro
370 375 380
Thr Ser Glu Gly Ile Glu Glu Leu Glu His His His His His His
385 390 395
<210> 26
<211> 392
<212> PRT
<213> 人工序列
<400> 26
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ile Glu Thr Arg Ala Pro Gly Lys Leu Tyr Ile
20 25 30
Ala Gly Glu Tyr Ala Val Val Glu Pro Gly Glu Pro Ala Ala Leu Val
35 40 45
Ala Val Asp Arg Tyr Leu Thr Val Arg Leu Thr Ala Ser Glu Gly Ala
50 55 60
Gly Arg Val His Ser Arg Glu Tyr Gly Arg Leu Pro Leu Val Trp Thr
65 70 75 80
Arg Glu Pro Gly Gly Ala Arg Ile Val Leu Asp His His Pro Tyr Asp
85 90 95
Tyr Val Met Ala Ala Ile Ala Thr Val Asp Arg Leu Arg Leu Glu Leu
100 105 110
Gly Leu Ala Pro Arg Tyr Phe Asp Leu Arg Ile Glu Ser Glu Leu Asp
115 120 125
Asp Ala Asp Gly Arg Lys Phe Gly Leu Gly Ser Ser Ala Ala Val Thr
130 135 140
Val Ala Thr Ile Ala Ala Leu Asp Glu Phe Tyr Arg Leu Gly Leu Ser
145 150 155 160
Arg Ala Glu Arg Phe Lys Leu Ala Leu Leu Ala Thr Val Gln Val Ala
165 170 175
Pro Thr Ala Ser Gly Gly Asp Leu Ala Ala Ser Thr Phe Gly Gly Trp
180 185 190
Val Arg Tyr Thr Ala Pro Asp Arg Asp Ala Leu Arg Ala His Gly Asp
195 200 205
Glu His Gly Val Ala Arg Thr Leu Thr Ser Pro Glu Ala Trp Glu Gly
210 215 220
Cys Ser Val Thr Arg Leu Pro Pro Pro Asp Ala Leu Arg Leu Val Val
225 230 235 240
Gly Trp Thr Gly Ser Pro Ala Ser Thr Glu Asp Leu Val Asp Arg Val
245 250 255
Arg Pro His Gly Gln Asp Ala Gly Arg Arg Tyr Gly Thr Phe Val Glu
260 265 270
Asp Ser Arg Val Cys Val Asp Ala Leu Val Asp Ala Leu Arg Glu Asn
275 280 285
Asp Ala Pro Gly Ala Leu Arg Val Ile Arg Arg Ala Arg Arg Leu Leu
290 295 300
Gln Thr Leu Gly Glu Thr Arg Gly Ile Ser Ile Glu Thr Glu Lys Leu
305 310 315 320
Ala Thr Leu Cys Asp Ile Ala Glu Asn Ala Gly Ala Ala Ala Lys Pro
325 330 335
Ser Gly Ala Gly Gly Gly Asp Cys Gly Ile Val Leu Ala Pro Arg Asp
340 345 350
Leu Pro Glu Ser Ala Ile Leu Arg Ala Trp Glu Ala His Asp Ile Arg
355 360 365
Arg Leu Ser Leu Ala Val His Pro Ala Glu Gly Gln Val Asp Gly Ile
370 375 380
Leu Glu His His His His His His
385 390
<210> 27
<211> 393
<212> PRT
<213> 人工序列
<400> 27
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ile Glu Val Ser Thr Pro Gly Lys Leu Phe Ile
20 25 30
Ala Gly Glu Tyr Ala Val Val Glu Pro Gly His Pro Ala Ile Ile Val
35 40 45
Ala Val Asp Gln Phe Val Thr Val Thr Leu Glu Lys Ala Glu Asn Val
50 55 60
Gly Ser Ile Gln Ser Ala Gln Phe Ser Ser Leu Pro Val Arg Trp Thr
65 70 75 80
Arg Arg Asn Asn Glu Leu Val Leu Asp Ile Arg Glu Asn Pro Phe His
85 90 95
Tyr Val Leu Ala Ala Ile Arg Leu Thr Glu Lys Tyr Ala Gln Glu Gln
100 105 110
Tyr Lys Glu Leu Ser Phe Tyr His Leu Lys Val Thr Ser Glu Leu Asp
115 120 125
Asn Ser Asn Gly Arg Lys Tyr Gly Leu Gly Ser Ser Gly Ala Val Thr
130 135 140
Val Ala Thr Val Lys Ala Leu Ser Leu Phe Tyr Gly Leu Glu Leu Ser
145 150 155 160
Glu Glu Glu Ile Phe Lys Leu Ser Ala Leu Ala His Leu Glu Val Gln
165 170 175
Gly Asn Gly Ser Cys Gly Asp Ile Ala Ala Ser Cys Tyr Gly Gly Trp
180 185 190
Ile Ala Phe Ser Thr Phe Asp His Gln Trp Val Asn Glu Gln Val Gln
195 200 205
Lys Gln Thr Leu Thr Thr Leu Leu Gln Ala Thr Trp Pro Lys Leu Met
210 215 220
Ile Gln Pro Leu Thr Val Pro Lys Lys Leu Arg Leu Leu Ile Gly Trp
225 230 235 240
Thr Gly Ser Pro Ala Ser Thr Ser Asp Leu Val Asp Gln Val Asn Gln
245 250 255
Ser Lys Glu Val Gln Glu Thr Ala Tyr Gln Gln Phe Leu Asp Asp Ser
260 265 270
Lys Ala Cys Val Glu Thr Met Ile Thr Gly Phe Asn Thr Glu Asn Ile
275 280 285
Ser Leu Ile Gln Ala Gln Ile Arg Lys Asn Arg Lys Leu Leu Gln Gln
290 295 300
Leu Thr Ser Ile Thr Gly Val Thr Ile Glu Thr Pro Ala Leu Lys Lys
305 310 315 320
Leu Cys Asn Leu Ala Gln Thr Tyr Gly Gly Ala Ala Lys Ser Ser Gly
325 330 335
Ala Gly Gly Gly Asp Cys Gly Ile Val Leu Phe Lys Gln Lys Ser Gly
340 345 350
Ile Leu Pro Leu Met Ser Ser Trp Glu Lys Glu Gly Ile Thr Pro Leu
355 360 365
Pro Leu His Val Tyr Phe Tyr Gly Lys Gln Glu Lys Asp Glu Ser Lys
370 375 380
Arg Leu Glu His His His His His His
385 390
<210> 28
<211> 394
<212> PRT
<213> 人工序列
<400> 28
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Asn Val Leu Ala Ile Thr Ile Lys Val Pro Gly
20 25 30
Lys Leu Met Ile Ala Gly Glu Phe Ala Val Leu Glu Pro Tyr His Asn
35 40 45
Leu Ala Val Leu Ala Val Asp Arg Phe Val Tyr Ala Lys Ile Glu Gly
50 55 60
His His Glu Asn Arg Leu Thr Leu Gln Asp Phe Gly Leu Glu Asn Leu
65 70 75 80
Asn Phe His Phe Thr Asn Ser Lys Val Glu Ile Ala Ser Asn Asp Arg
85 90 95
Arg Thr Arg Phe Val Gly Asp Ala Met Thr Ile Val Leu Thr Tyr Leu
100 105 110
Lys Glu Lys Gly Ile Thr Pro Asp Pro Phe His Leu Ser Ile Lys Ser
115 120 125
Glu Leu Asp Asp Ala Ser Gly Val Lys Tyr Gly Leu Gly Ser Ser Ala
130 135 140
Ala Val Val Thr Ser Val Ile Thr Ala Ile Leu Thr Lys Leu Leu Pro
145 150 155 160
Ser Lys Pro Glu Lys Glu Leu Ile Phe Lys Leu Ala Ala Ile Ser His
165 170 175
Val Glu Thr Gln Gly Asn Gly Ser Gly Ala Asp Val Ala Ala Ser Ser
180 185 190
Tyr Gly Gly Leu Leu Lys Tyr Ala Ser Phe Gln Ala Glu Trp Leu His
195 200 205
Ser Glu Tyr Ile Ala Ser Asn Ser Ile Ser Glu Leu Val Ala Lys Glu
210 215 220
Trp Arg Tyr Phe Ser Val Lys Pro Met Arg Leu Pro Gln Asn Ile His
225 230 235 240
Phe Cys Val Gly Trp Thr Gly Lys Pro Ala Ser Thr Ala Lys Leu Val
245 250 255
Asp Val Ile Gly Gln Leu Lys Ser Asp Asn Leu Glu Gln Tyr Glu Lys
260 265 270
Phe Leu Gln Asp Ser Glu Ala Ala Val Arg Thr Phe Phe Lys Gly Met
275 280 285
Glu Glu Glu Ser Ile Ala Asp Leu Leu Glu Gly Val Lys Ala Asn Arg
290 295 300
Gln Ala Leu Ala Thr Val Gly Lys His Ala Asn Ala Ser Ile Glu Thr
305 310 315 320
Pro Leu Leu Thr Thr Leu Cys Asp Leu Ala Glu Gln Phe Gly Gly Ala
325 330 335
Gly Lys Pro Ser Gly Ala Gly Gly Gly Asp Cys Gly Ile Ala Phe Met
340 345 350
Pro Ser His Glu Gln Ala Lys Lys Leu Met His Ala Trp Glu Glu Ala
355 360 365
Gly Ile Lys Pro Leu Ala Ile Arg Pro Asn Ser Gln Gly Ala Ile Glu
370 375 380
Ile Gly Leu Glu His His His His His His
385 390
<210> 29
<211> 349
<212> PRT
<213> 人工序列
<400> 29
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met
35 40 45
Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile
50 55 60
Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser
65 70 75 80
Val Val Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp
85 90 95
Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val
100 105 110
Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu
115 120 125
Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp
130 135 140
Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr
145 150 155 160
Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro
165 170 175
Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp
180 185 190
Lys Val Arg Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu
195 200 205
Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu
210 215 220
Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu
225 230 235 240
Lys Phe Cys Lys Arg Ser Phe His Val Tyr Pro Thr Leu Asn Trp Glu
245 250 255
Thr Gly Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro
260 265 270
Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn
275 280 285
Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu
290 295 300
Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Gly
305 310 315 320
Ala Val Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe
325 330 335
Asp Ser Leu Glu Asp Leu Glu His His His His His His
340 345
<210> 30
<211> 948
<212> DNA
<213> 人工序列
<400> 30
atgagcgaag cagcagatgt tgaacgtgtt tatgcagcaa tggaagaagc agccggtctg 60
ctgggtgttg catgtgcacg tgataaaatc tatccgctgc tgagcacctt tcaggatacc 120
ctggttgaag gtggtagcgt tgttgttttt agcatggcaa gcggtcgtca tagcaccgaa 180
ctggatttta gcattagcgt tccgaccagc catggtgatc cgtatgcaac cgttgttgaa 240
aaaggtctgt ttccggcaac cggtcatccg gttgatgatc tgctggcaga tacccagaaa 300
catctgccgg ttagcatgtt tgcaattgat ggtgaagtta ccggtggctt caaaaaaacc 360
tatgcatttt ttccgaccga taatatgcct ggtgttgcag aactgagcgc aattccgagc 420
atgcctccgg cagttgcaga aaatgccgaa ctgtttgcac gttatggtct ggataaagtt 480
cgtatgacca gcatggatta caaaaaacgt caggtgaacc tgtattttag cgaactgagt 540
gcacagaccc tggaagcaga aagcgttctg gcactggttc gtgaactggg tctgcatgtt 600
ccgaatgaac tgggcctgaa attttgtaaa cgtagctttc atgtttatcc gacgctgaat 660
tgggaaaccg gtaaaattga tcgtctgtgc tttgccgtta ttagcaatga tccgacactg 720
gttccgagca gtgatgaagg tgatatcgaa aaatttcaca actacgcaac caaagcaccg 780
tatgcatatg ttggtgaaaa acgtaccctg gtgtatggtc tgaccctgag tccgaaagaa 840
gaatattaca aactgggtgc cgtttaccat attaccgatg ttcagcgtgg tctgctgaaa 900
gcatttgata gcctggaaga tctggaacat catcatcacc atcactaa 948
<210> 31
<211> 801
<212> DNA
<213> 人工序列
<400> 31
ggatccatgc aggttgatct gctgggtagc gcacagagcg cacatgcact gcacctgttt 60
catcagcata gtccgctggt tcattgtatg accaatgatg ttgttcagac ctttaccgca 120
aataccctgc tggcactggg tgcaagtccg gcaatggtta ttgaaaccga agaagcaagc 180
cagtttgcag caattgcaag cgcactgctg attaatgttg gtacactgac ccagcctcgt 240
gcacaggcaa tgcgtgcagc agttgaacag gcaaaaagca gtcagacccc gtggacctta 300
gatccggttg cagttggtgc actggattat cgtcgtcatt tttgtcatga actgctgagc 360
tttaaaccgg cagcaattcg tggtaatgca agcgaaatta tggcactggc aggtattgca 420
aatggtggtc gtggtgttga taccaccgat gcagcagcaa atgcaattcc ggcagcacag 480
accctggcac gtgaaaccgg tgcaattgtt gttgttaccg gtgaaatgga ttatgtgacc 540
gatggtcatc gtattattgg tattcatggt ggtgatccgc tgatgaccaa agttgttggc 600
accggttgtg cactgagcgc agttgttgca gcatgttgtg ccctgcctgg tgataccctg 660
gaaaatgttg ccagcgcatg tcattggatg aaacaagccg gtgaacgtgc agttgcacgt 720
agcgaaggtc cgggtagctt tgttccgcat tttctggatg cactgtggca gctgacccaa 780
gaagttcagg cataagaatt c 801
<210> 32
<211> 837
<212> DNA
<213> 人工序列
<400> 32
ggatccatga tcattctgaa acttggtggt agcgttatta cccgtaaaga tagcgaagaa 60
ccggcaattg atcgtgataa tctggaacgt attgcaagcg aaattggtaa tgcaagcccg 120
agcagcctga tgattgttca tggtgcaggt agctttggtc atccgtttgc cggtgaatat 180
cgtattggtt ccgaaatcga aaacgaagaa gatctgcgtc gtcgtcgttt tggttttgca 240
ctgacccaga attgggtgaa aaaactgaat agccatgttt gtgatgcact gctggcagaa 300
ggtattccgg cagttagcat gcagccgagc gcatttattc gtgcacatgc aggtcgtatt 360
agccatgcag atattagcct gattcgtagc tatctggaag aaggcatggt tccggttgtt 420
tatggtgatg ttgttctgga tagcgatcgt cgtctgaaat ttagcgtgat tagcggtgat 480
caactgatca atcattttag cctgcgtctg atgccggaac gtgttattct gggcaccgat 540
gttgatggtg tttatacccg taatccgaaa aaacatcctg atgcacgtct gctggatgtt 600
attggtagcc tggatgatct ggaaagtctg gatggcaccc tgaataccga tgttaccggt 660
ggtatggttg gcaaaattcg tgaactgctg ctgctggccg aaaaaggtgt tgaaagcgaa 720
atcattaatg cagccgttcc gggtaatatt gaacgtgccc tgctgggtga agaagttcgc 780
ggtacacgta ttacaggtaa acatctggaa catcatcatc accatcacta agaattc 837

Claims (8)

1.一种香叶基二磷酸酯的生物合成方法，其特征在于，所述生物合成方法包括步骤：将香叶醇、腺嘌呤核苷三磷酸、1号酶以及2号酶进行接触反应，获得香叶基二磷酸酯；
其中，所述1号酶选自羟乙基噻唑激酶，所述羟乙基噻唑激酶的氨基酸序列如SEQ IDNO：4所示；所述2号酶选自异戊烯基磷酸激酶，所述异戊烯基磷酸激酶的氨基酸序列如SEQID NO：18所示。
2.一种用于生物合成香叶基二磷酸酯的生产系统，其特征在于，包括：
(a)香叶醇；
(b)腺嘌呤核苷三磷酸；
(c)1号酶，用于将香叶醇和腺嘌呤核苷三磷酸转化为香叶基磷酸；以及
(d)2号酶，用于将香叶基磷酸和腺嘌呤核苷三磷酸转化为香叶基二磷酸酯；
其中，所述1号酶选自羟乙基噻唑激酶，所述羟乙基噻唑激酶的氨基酸序列如SEQ IDNO：4所示；所述2号酶选自异戊烯基磷酸激酶，所述异戊烯基磷酸激酶的氨基酸序列如SEQID NO：18所示。
3.根据权利要求2所述的用于生物合成香叶基二磷酸酯的生产系统，其特征在于，所述用于生物合成香叶基二磷酸酯的生产系统还包括Tris-HCl、KCl和MgCl2。
4.根据权利要求2或3所述的用于生物合成香叶基二磷酸酯的生产系统，其特征在于，所述香叶醇：所述1号酶：所述2号酶的摩尔比为152：(1～6)：(1～6)。
5.一种如权利要求1所述的生物合成方法、或如权利要求2至4任一项中所述的用于生物合成香叶基二磷酸酯的生产系统在制备大麻类化合物中的应用，其特征在于，在NphB异戊烯基转移酶的催化作用下，将通过所述生物合成方法、或所述用于生物合成香叶基二磷酸酯的生产系统制得的香叶基二磷酸酯与前体化合物接触反应，生成大麻类化合物；
其中，所述前体化合物的结构如下面通式(Ⅰ)所示：
在通式(Ⅰ)中，R1为碳原子数是1、2、3、4或5的烷基，R2和R3彼此独立地选自氢原子、羧基或甲基。
6.根据权利要求5所述的应用，其特征在于，所述前体化合物为橄榄醇，且所述大麻类化合物为大麻萜酚；
或者，所述前体化合物为橄榄酸，且所述大麻类化合物为大麻萜酚酸；
或者，所述前体化合物为4-丙基间苯二酚，且所述大麻类化合物为次大麻萜酚；
或者，所述前体化合物为2,4-二羟基丙基苯甲酸，且所述大麻类化合物为次大麻萜酚酸。
7.一种用于生物合成大麻类化合物的生产系统，其特征在于，包括：
(a)香叶醇；
(b)腺嘌呤核苷三磷酸；
(c)1号酶，用于将香叶醇和腺嘌呤核苷三磷酸转化为香叶基磷酸；
(d)2号酶，用于将香叶基磷酸和腺嘌呤核苷三磷酸转化为香叶基二磷酸酯；
(e)前体化合物；以及
(f)NphB异戊烯基转移酶，用于将所述前体化合物异戊二烯化生成大麻类化合物；
其中，所述1号酶选自羟乙基噻唑激酶，所述羟乙基噻唑激酶的氨基酸序列如SEQ IDNO：4所示；所述2号酶选自异戊烯基磷酸激酶，所述异戊烯基磷酸激酶的氨基酸序列如SEQID NO：18所示；
所述前体化合物的结构如下面通式(Ⅰ)所示：
在通式(Ⅰ)中，R1为碳原子数是1、2、3、4或5的烷基，R2和R3彼此独立地选自氢原子、羧基或甲基。
8.根据权利要求7所述的用于生物合成大麻类化合物的生产系统，其特征在于，所述香叶醇：所述1号酶：所述2号酶的摩尔比为152：(1～6)：(1～6)。
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