CN113801868B

CN113801868B - 一种檀香烯合酶突变体及其制备方法

Info

Publication number: CN113801868B
Application number: CN202111085704.XA
Authority: CN
Inventors: 李迅; 张佳; 王询; 王飞
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2021-09-16
Filing date: 2021-09-16
Publication date: 2023-07-28
Anticipated expiration: 2041-09-16
Also published as: CN117004594A; CN113801868A

Abstract

本发明涉及檀香烯合酶突变酶及其在合成檀香烯中的应用。具体涉及的突变酶有SanSyn(Q527A)、SanSyn(S533A)、SanSyn(S533Q)、SanSyn(Q527A&S533A)，它们分别是将第527位的谷氨酰胺突变为丙氨酸，第533位的丝氨酸突变为丙氨酸、谷氨酰胺以及第527位的谷氨酰胺、533位的丝氨酸均突变为丙氨酸。以葡萄糖为碳源，在宿主大肠杆菌DH5α中合成α‑檀香烯的方法，主要包括构建重组质粒pETDuet‑SanSyn(Q527A)、pETDuet‑SanSyn(S533A)、pETDuet‑SanSyn(S533Q)、pETDuet‑SanSyn(Q527A&S533A)、pMVA；构建大肠杆菌重组菌株，进一步发酵培养，本发明涉及的技术方案显著提高了α‑檀香烯的产量，蛋白可溶性表达也有所提高。为进一步代谢工程改造大肠杆菌生产α‑檀香烯奠定了基础。对萜烯类合酶的改造工作提供参考。

Description

一种檀香烯合酶突变体及其制备方法

技术领域

本发明属于酶工程技术领域，具体涉及一种檀香烯合酶突变体及其制备方法。

背景技术

檀香烯，是一种倍半萜烯，是檀香醇的前体物质。檀香烯和檀香醇是檀香精油的主要成分。檀香精油多用于化妆品和香料中，并且其具有较好的抗菌、抗氧化和抗肿瘤等药理活性。此外，檀香精油在动物体内毒性较低，无致突变性，欧美多国认为是安全的食品添加剂。

目前檀香精油主要通过植物提取法获得，但由于檀香木生长条件苛刻，生长期长，树木中檀香油含量稀少，分离过程复杂繁琐，提取困难，无法满足市场需求还会造成檀香木的大量砍伐。此外，虽然已有研究报道了由溴代樟脑为底物，经过八步化学催化反应得到檀香烯，但化学反应的条件苛刻，成本高昂，路径繁琐，要想得到产物纯品，需要对中间反应产物进行一次次的分离提纯，并非理想的大规模生产檀香烯的有效手段。因此，通过微生物细胞工厂异源生物合成檀香烯和檀香醇，这对于资源稀缺、成本高昂的的萜类化合物提供了新的可持续绿色生产方式。

萜烯生物合成的关键步骤包括通过萜烯合成酶(或环化酶类)的作用将无环的GPP、FPP、GGPP环化为单帖、倍半萜或者二萜类化合物。黄皮来源的檀香烯合酶(SantaleneSynthase，SanSyn)属于植物萜烯合酶中一种，这类酶都包含相似的保守结构域DDxxD(“x”表示任意氨基酸)。目前，萜烯合酶的催化效率、对底物的特异性和稳定性等都限制着萜烯合酶的利用，我们通过对萜烯合酶的分子改造来打破这一限制。

发明内容

本部分的目的在于概述本发明的实施例的一些方面以及简要介绍一些较佳实施例。在本部分以及本申请的说明书摘要和发明名称中可能会做些简化或省略以避免使本部分、说明书摘要和发明名称的目的模糊，而这种简化或省略不能用于限制本发明的范围。

鉴于上述和/或现有技术中存在的问题，提出了本发明。

因此，本发明的目的是，克服现有技术中的不足，提供了一种檀香烯合酶的改造方法及其产品。

为解决上述技术问题，本发明提供了如下技术方案：一种改造的檀香烯合酶，其特征在于：包括，所述檀香烯合酶突变体，为突变体SanSyn(Q527A)、突变体SanSyn(S533A)、突变体SanSyn(S533Q)和突变体SanSyn(Q527A&S533A)中的任一种；

所述突变体SanSyn(Q527A)，为将氨基酸序列为SEQ ID NO.1所示的野生型SanSyn酶的第527位谷氨酰胺突变为丙氨酸；

所述突变体SanSyn(S533A)，为将氨基酸序列为SEQ ID NO.1所示的野生型SanSyn酶的第533位丝氨酸突变为丙氨酸；

所述突变体SanSyn(S533A)，为将氨基酸序列为SEQ ID NO.1所示的野生型SanSyn酶的第533位丝氨酸突变为谷氨酰胺；

所述突变体SanSyn(Q527A&S533A)为将氨基酸序列为SEQ ID NO.1所示的SanSyn酶的第527位谷氨酰胺、第533位丝氨酸均突变为丙氨酸。

作为本发明所述檀香烯合酶突变体的一种优选方案，其中：所述檀香烯合酶突变体，还包括，

所述突变体SanSyn(Q527A)的氨基酸序列为如SEQ ID NO.3所示；

所述突变体SanSyn(S533A)的氨基酸序列为如SEQ ID NO.5所示；

所述突变体SanSyn(S533Q)的氨基酸序列为如SEQ ID NO.7所示；

所述突变体SanSyn(Q527A&S533A)的氨基酸序列为如SEQ ID NO.9所示。

所述突变体SanSyn(Q527A)的核苷酸序列为如SEQ ID NO.4所示；

所述突变体SanSyn(S533A)的核苷酸序列为如SEQ ID NO.6所示；

所述突变体SanSyn(S533Q)的核苷酸序列为如SEQ ID NO.8所示；

所述突变体SanSyn(Q527A&S533A)的核苷酸序列为如SEQ ID NO.10所示。

作为本发明所述檀香烯合酶突变体的一种优选方案，其中：所述野生型SanSyn酶来源于植物黄皮(Clausena lansium)。

作为本发明所述檀香烯合酶突变体的制备方法的一种优选方案，其中：所述制备方法，包括，

将野生型SanSyn酶基因连接到质粒pETDuet-tac中，得到重组质粒pETDuet-tac-SanSyn；

设计突变引物，使用突变引物并以重组质粒为模板进行PCR扩增，酶切消化去除模板DNA，得到突变产物；

将突变产物转化到宿主细胞中，筛选得到檀香烯合酶SanSyn突变体表达菌株，诱导表达，得到檀香烯合酶突变体。

作为本发明所述檀香烯合酶突变体的制备方法的一种优选方案，其中：所述野生型SanSyn酶基因连接到质粒pETDuet-tac中，还包括，

所述野生型SanSyn酶基因核苷酸序列如SEQ ID NO.2所示；

所述连接的双酶切位点分别为NcoI和BamHI，交由上海捷瑞生物工程有限公司合成；

所述质粒pETDuet-tac的核苷酸序列如SEQ ID NO.30所示。

作为本发明所述檀香烯合酶突变体的制备方法的一种优选方案，其中：所述设计引物，其中，所述引物序列包括，

Q527A-F：氨基酸序列如SEQ ID NO.23所示；

Q527A-R：氨基酸序列如SEQ ID NO.24所示；

S533A-F：氨基酸序列如SEQ ID NO.25所示；

S533-R：氨基酸序列如SEQ ID NO.26所示；

S533Q-F：氨基酸序列如SEQ ID NO.27所示；

Q527A&S533A-F：氨基酸序列如SEQ ID NO.28所示；

Q527A&S533A-R：氨基酸序列如SEQ ID NO.29所示；

其中，引物Q527A-F和Q527A-R可用于获得突变体SanSyn(Q527A)；

引物S533A-F和S533-R可用于获得突变体SanSyn(S533A)；

引物S533Q-F和S533-R可用于获得突变体SanSyn(S533Q)；

引物Q527A&S533A-F和Q527A&S533A-R可用于获得突变体SanSyn(Q527A&S533A)。

作为本发明所述檀香烯合酶突变体的制备方法的一种优选方案，其中：所述PCR扩增，包括，

RPC反应体系：PrimeSTAR Max 25μL，正向引物、反向引物、模板DNA各1μL，加入灭菌水22μL酶切消化去除模板DNA；

PCR反应扩增程序：98℃预变性5min；随后98℃10s，退火20s温度见表1，72℃45s进行25个循环，最后72℃保温5min。

作为本发明所述檀香烯合酶突变体的制备方法的一种优选方案，其中：所述酶切消化使用消化酶为DMT酶；所述宿主细胞为大肠杆菌TOP 10的感受态细胞

作为本发明所述檀香烯合酶突变体的制备方法的一种优选方案，其中：所述筛选得到檀香烯合酶SanSyn突变体表达菌株，具体为挑转化子送测序验证氨基酸是否突变成功，进行筛选。

本发明有益效果：

本发明提供一种生产α-檀香烯的方法，方法具体为：以大肠杆菌为宿主，重组表达编码编码所述檀香烯合酶及其突变体的基因，得到重组菌；将重组大肠杆菌经种子培养基活化后转入发酵培养基，于37℃，转速200rpm条件下培养，OD600达到1.5时加入IPTG诱导酶表达，使重组菌以葡萄糖为底物生产α-檀香烯。

S533A突变菌株在诱导后发酵培养3天后α-檀香烯浓度达到1028mg/L，较未突变菌株产量提高1.9倍，Q527A突变菌株α-檀香烯浓度最终达到933mg/L，S533Q突变菌株α-檀香烯最终浓度达到959mg/L，Q527A&S533A双点突变菌株α-檀香烯浓度815mg/L。与未突变菌株浓度相比均有所提高。并通过Western Blot实验发现SanSyn的突变体S533Q、Q527A的可溶性蛋白表达也得到提高。

本发明提供了含有所述的檀香烯合酶编码基因的重组菌株，并通过定点突变SanSyn基因，提高了α-檀香烯的积累量，并且提高了可溶性蛋白表达。

附图说明

为了更清楚地说明本发明实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。其中：

图1为SanSyn酶的三维结构模拟图。

图2为实施例2中重组菌在24h、48h、72h时的α-檀香烯产量。

图3为实施例2中重组菌包含质粒。

图4为实施例4中檀香烯合酶突变体可溶性蛋白相对表达。

具体实施方式

为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合说明书附图对本发明的具体实施方式做详细的说明。

在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是本发明还可以采用不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似推广，因此本发明不受下面公开的具体实施例的限制。

其次，此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例，也不是单独的或选择性的与其他实施例互相排斥的实施例。

本发明所使用引物均由生工生物工程(上海)股份有限公司合成。

本发明所使用DMT酶购买于北京全式金生物技术有限公司。所使用PrimeSTAR Max购自Takara公司。凝胶回收试剂盒Axygen DNA购自爱思进生物技术有限公司。WesternBlot实验相关试剂均购自上海碧云天生物技术有限公司。

所使用其他原料，若无特殊说明，均为市售。

实施例1

SanSyn酶三级结构模拟模型的建立：

利用同源建模工具Swiss Model以来源于Santalum album的檀香烯合酶SaSS(PDB：5ZZJ)为参考模型对SanSyn进行同源建模。模型经评估后，获得可靠的三维结构模型，三维结构模型图如图1所示。

实施例2

构建定点突变文库：

利用PCR技术，以表达有野生型SanSyn的基因的质粒pETDuet-SanSyn为模板，对SanSyn酶第19、408、442、473、527和533位进行定点突变。

设计突变引物，正向和反向引物是根据不同突变位点进行相应设计的PCR上游和下游引物，具体引物信息如表1所示。

表1获得SanSyn突变体所用的引物

引物对F19Y-F和F19Y-R用于获得突变体SanSyn(F19Y)，突变体SanSyn(F19Y)的氨基酸序列为SEQ ID NO.11。

引物对I408L-F和I408L-R用于获得突变体SanSyn(I408L)，突变体SanSyn(I408L)的氨基酸序列为SEQ ID NO.12。

引物对F442S-F和F442S-R用于获得突变体SanSyn(F442S)，突变体SanSyn(F442S)的氨基酸序列为SEQ ID NO.13。

引物对V473A-F和V473A-R用于获得突变体SanSyn(V473A)，突变体SanSyn(V473A)的氨基酸序列为SEQ ID NO.14。

引物对Q527A-F和Q527A-R用于获得突变体SanSyn(Q527A)，突变体SanSyn(Q527A)的氨基酸序列为SEQ ID NO.3。

引物对S533A-F和S533-R用于获得突变体SanSyn(S533A)，突变体SanSyn(S533A)的氨基酸序列为SEQ ID NO.5。

引物对S533Q-F和S533-R用于获得突变体SanSyn(S533Q)，突变体SanSyn(S533Q)的氨基酸序列为SEQ ID NO.7。

引物对Q527A&S533A-F和Q527A&S533A-R用于获得突变体SanSyn(Q527A&S533A)，突变体SanSyn(Q527A&S533A)的氨基酸序列为SEQ ID NO.9。

PCR反应体系均为：PrimeSTAR Max(购自Takara公司)25μL，正向引物、反向引物、模板DNA各1μL，加入灭菌水22μL。

PCR反应扩增程序为：98℃预变性5min；随后98℃变性10s，退火20s，退火温度见表1，72℃45s进行25个循环，最后72℃保温5min。回收片段大小为7000bp左右的条带。

上述片段样品进行去模板反应，去模板反应体系为DMT酶1μL，纯化产物9μL，37℃反应1h。

磷酸化在37℃下反应3h，磷酸化反应体系：T4 Polynucleotide Kinase 1μL，10×T4 Polynucleotide Kinase Buffer 1μL，ATP 1μL，样品7μL。磷酸化后70℃灭酶活5min。

连接反应在16℃下过夜进行，连接体系：T4 DNA Ligase 1μL，10×T4 DNA LigaseBuffer 1μL，样品8μL。

将10μL连接产物全部加入大肠杆菌TOP10的感受态细胞中，充分混合，在冰上放置5min，接着将混合充分的菌液与质粒在42℃的水浴中，热激90s。热激完成后，置于冰水5min，加入1mL SOC培养基，混合均匀，在37℃下复苏45min。之后将菌液涂到含有氨苄青霉素抗性的LB平板上，37℃过夜培养。挑平板上的转化子送至生工生物工程(上海)股份有限公司进行测序验证。

其中目的基因片段使用Axygen DNA凝胶回收试剂盒回收，具体步骤为：

(1)切割含有目的DNA的琼脂糖凝胶，并计算凝胶重量，一个重量作为一个凝胶体积(100mg＝100μL)。

(2)加入三个凝胶体积的Buffer DE-A，混合均匀后置于75℃加热，间断混合，直至凝胶块完全熔化。

(3)加0.5个Buffer DE-A体积的Buffer DE-B，混合均匀，混合后溶液呈黄色。

(4)将混合后的黄色溶液转移至制备管中，12000rpm离心1min，弃滤液。

(5)将制备管置回离心管，加700μL Buffer W₁，在12000rpm离心1min，弃滤液。

(6)将制备管置回离心管，加700μL Buffer W₂，12000rpm离心1min，弃滤液；以同样的方法再用Buffer W₂洗涤一次。弃滤液。

(7)将制备管置回离心管，12000rpm离心1min，弃滤液。

(8)将制备管移入新的1.5mL离心管中，在制备管中央加20μL Eluent，室温静置1min，12000rpm离心1min。即可获得目的基因片段。

实施例3

大肠杆菌重组菌株的构建：

将具体实施例1中构建的含檀香烯合酶及其突变体质粒与质粒pMVA分别转化到大肠杆菌DH5α中(构建质粒见图3)，在菌株的平板上挑取三个单菌落，接种到5mL的带有氨苄青霉素和氯霉素抗性的液体LB中，37℃下过夜培养，然后接种到50mL的带有氨苄青霉素和氯霉素抗性的TB培养基(培养基成分包括20g/L葡萄糖，12g/L胰蛋白胨,24g/L酵母提取物,9.4g/L磷酸氢二钾，2.2g/L磷酸二氢钾)中，然后在37℃、200rpm的摇床中培养3～4h，重组大肠杆菌的OD600值达到1.5左右后，加入诱导剂IPTG进行诱导，其工作浓度为0.5mM，同时加入5mL肉豆蔻酸异丙酯萃取目的产物α-檀香烯，发酵过程在在25℃、200rpm的摇床中培养72h。每24h取样，用气相色谱测定α-檀香烯的浓度。结果见图2。

由图2看出，S533A突变菌株在诱导后发酵培养3天后，α-檀香烯浓度达到1028mg/L，较未突变菌株产量提高1.9倍，Q527A突变菌株α-檀香烯浓度最终达到933mg/L，S533Q突变菌株α-檀香烯最终浓度达到959mg/L，Q527A&S533A双点突变菌株α-檀香烯浓度815mg/L。与未突变菌株浓度相比均有所提高。

实施例4

檀香烯合酶及其突变体蛋白Western Blot分析：

按照本发明的实验步骤分别构建质粒pET28a-SanSyn-6His、pET28a-SanSyn(S533A)-6His、pET28a-SanSyn(S533Q)-6His和pET28a-SanSyn(Q527A)-6His。

构建上述质粒分为两步，其一，先在各檀香烯合酶C端添加6xHis标签，分别以pETDuet-SanSyn、pETDuet-SanSyn(S533A)、pETDuet-SanSyn(S533Q)、pET28a-SanSyn(Q527A)为载体，设计正向引物：5’-CATCACCATCATCACCACTAAGGATC-3’；反向引物：5’-ATCATCTAATTTAACCGGATCTTTCAGC-3’。PCR反应扩增程序为：98℃预变性5min；随后98℃变性10s，退火20s，退火温度59℃，72℃45s进行25个循环，最后72℃保温5min。回收大小7000bp-8000bp的条带，去模板、磷酸化反应、连接、热激转化的具体步骤见实施例2中描述。

其二，将C端含6xHis标签的檀香烯合酶(SanSyn)及其突变体基因插入质粒pET28a的NcoⅠ和BamHⅠ位点之间。

双酶切体系：基因片段36μL，10xK Buffer 5μL，BSA 5μL，NcoⅠ酶2μL，BamHⅠ酶2μL，37℃，3h，回收载体pET28a和SanSyn基因片段。回收步骤与实施例2中相同。

然后进行载体pET28a和SanSyn基因片段用Ligase进行连接反应。连接体系包括载体片段和目的片段各4μL，T4 DNA Ligase 1μL，T4 DNA Ligase Buffer1μL。于16℃过夜连接。

将连接好的片段转化到大肠杆菌BL21(DE3)中。在卡那霉素抗性的平板上挑取单菌落接种到5mL的带有卡那霉素抗性的液体LB中，37℃下过夜培养，接种到100mL的带有卡那霉素抗性的LB中(接种量1％)，在37℃、200rpm的摇床中培养2～3h，待重组大肠杆菌的OD＝600值达到0.6～0.8后，加入诱导剂IPTG进行诱导，其工作浓度为0.5mM，随后在20℃、180rpm的摇床中培养20h。

制备蛋白样品，取一定量菌液离心弃上清，用PBS缓冲冲洗菌体两次，然后每个样品添加细胞裂解液100μL，置于冰上裂解40min后，12000rpm，4℃下离心10min，吸取上清于新的EP管中，即为细胞总蛋白。

BCA法测定蛋白浓度，使用上海捷瑞生物工程有限公司的BCA法测定蛋白浓度试剂盒。将试剂A与试剂B按50：1比例混合，反应体系为200μL试剂+20μL蛋白样品，37℃反应30min，在吸光度为562nm处测定吸光值A。根据公式C＝(A-0.0131)/2.378计算蛋白浓度(μg/μL)，用PBS溶液将蛋白样品调整至浓度一致。上样量为20-50μg，上样量～20μL。

SDS-PAGE凝胶电泳，使用10％的SDS-PAGE凝胶跑胶，上样量为20-50μg，上样量～20μL。电泳结束后，取下凝胶，将其置于预冷的转膜液中。将PVDF膜裁至分离胶相同大小，并放入甲醇中活化15秒左右，再用转膜液清洗。

转膜，将PVDF膜裁至分离胶相同大小，并放入甲醇中活化15秒左右，再用转膜液清洗。将滤纸、海绵垫及凝胶制成“三明治夹心”电转装置，在转移槽内按负极、海绵、滤纸、分离胶、PVDF膜、滤纸、海绵、正极的顺序放置，确保各层之间精确对齐，保证无气泡存在，浸润在充满转移液的转膜槽中，并加冰袋降温，350mA恒流转膜1h。

封闭，待转膜结束以后，取下PVDF膜，将PVDF膜置于含5％脱脂牛奶的封闭液(TBST配制)中，正面朝下，常温震荡孵育1h。

抗体孵育及洗膜，1)将膜擦干水分，用PE手套封住后，按照marker的位置裁剪下合适的大小，做好标记；2)一抗孵育：用TBST稀释单克隆抗体(SanSyn及其突变体蛋白使用Histag小鼠单抗，内参GAPDH蛋白使用IgG抗体)，将PVDF膜放置于配制好的一抗溶液中，4℃过夜旋转结合；3)洗膜：次日取出PVDF膜用TBST洗膜3次，每次10min。4)二抗孵育：用TBST溶液分别稀释对应的辣根过氧化物酶标记IgG，将PVDF膜置于其中(操作与一抗孵育类似)，常温孵育2h；5)洗膜：TBST洗膜3次，每次10min。

显色，将ECL化学发光液(试剂盒)中的A、B液以1∶1的比例进行配制，加于然后将PVDF膜，在避光条件下反应1min或根据实际情况反应更长时间。显色液现加现用，加完后用化学发光仪拍照。实验结果见图4。

其中溶液配方10×转膜液：Tris 15.18g，甘氨酸72.08g配置成500mL溶液；1×湿转液：100mL 10×转膜液，200mL甲醇，加蒸馏水定容至1000mL；封闭液为5％脱脂奶粉：1脱脂奶粉溶于20mL TBST。Western Blot实验相关试剂：PVDF膜、6xHis标签和GAPDH的一抗及二抗、10xTBST、脱脂奶粉、ECL化学发光液均购自上海碧云天生物技术有限公司。

檀香烯合酶及其突变体的Western Blot实验中可以看到，S533Q、Q527A单突变与野生型檀香烯合酶相比，蛋白可溶性表达均有所提高(以野生型檀香烯合酶的相对蛋白表达为100％)。其中Q527A突变体蛋白可溶性表达提高1.2倍。

S533A突变菌株在诱导后发酵培养3天后α-檀香烯浓度达到1028mg/L，较未突变菌株产量提高1.9倍，Q527A突变菌株α-檀香烯浓度最终达到933mg/L，S533Q突变菌株α-檀香烯最终浓度达到959mg/L，Q527A&S533A双点突变菌株α-檀香烯浓度815mg/L。与未突变菌株浓度相比均有所提高。

本发明提供了含有所述的檀香烯合酶编码基因的重组菌株，并通过定点突变SanSyn基因，提高了α-檀香烯的积累量。通过Western Blot实验发现SanSyn的突变体S533Q、Q527A的可溶性蛋白表达也得到提高提高。

应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。

序列表

<110> 南京林业大学

<120> 一种檀香烯合酶突变体及其制备方法

<141> 2021-08-30

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Lys Asp Pro Val Lys Leu Asp Asp

545 550

<210> 6

<211> 1659

<212> DNA/RNA

<213> Artificial Sequence

<400> 6

atgggctcta cccagcaggt gtctagcgaa aatattgtgc gtaatgccgc caattttcat 60

ccgaatattt ggggtaatca ttttctgacc tgtccgtctc agaccattga ttcttggacc 120

cagcagcatc ataaagaact gaaagaagaa gtgcgcaaaa tgatggtgag cgatgccaat 180

aaaccggcac agcgcctgcg cctgattgat accgttcagc gcttaggcgt ggcctatcat 240

tttgaaaaag aaattgatga tgcactggaa aaaattggtc atgatccgtt tgatgataaa 300

gatgatctgt atattgtgag tctgtgtttt cgcctgttac gtcagcatgg cattaaaatt 360

agttgtgatg tgtttgaaaa atttaaagat gatgatggta aatttaaagc ctctttaatg 420

aatgatgttc agggtatgct gtcactgtat gaagccgcac atctggccat tcatggcgaa 480

gatattctgg atgaagccat tgtgtttacc accacccatc tgaaatcaac cgtgagtaat 540

agcccggtta atagtacctt tgcagaacag attcgtcata gcttacgcgt tccgttacgc 600

aaagcagttc cgcgcctgga atctcgctat tttctggata tttattcacg cgatgatctg 660

catgataaaa ccctgctgaa ttttgccaaa ctggatttta atattcttca agccatgcat 720

cagaaagaag cctcagaaat gacccgttgg tggcgcgatt ttgattttct gaaaaaactg 780

ccgtatattc gcgatcgtgt ggtggaactg tatttttgga ttctggtggg cgtgagctat 840

cagccgaaat ttagtaccgg tcgcattttt ctgtctaaaa ttatttgttt agaaacctta 900

gtggatgata cctttgatgc ctatggcacc tttgatgaac tggcaatttt taccgaagca 960

gtgacccgtt gggatctggg tcatcgcgat gccttaccgg aatatatgaa atttattttt 1020

aaaaccttaa ttgatgtgta tagcgaagcc gaacaggaac tggccaaaga aggtcgtagc 1080

tatagcattc attatgcaat tcgtagcttt caggaactgg ttatgaaata tttttgtgaa 1140

gccaaatggc tgaataaagg ctatgttccg agcttagatg attataaatc agtgtcactg 1200

cgctcaattg gctttctgcc gattgcagtt gcatcttttg tgtttatggg cgatattgca 1260

accaaagaag tgtttgaatg ggaaatgaat aatccgaaaa ttattattgc cgccgaaacc 1320

atttttcgct ttctggatga tattgccggt catcgctttg aacagaaacg cgaacatagt 1380

ccgagtgcaa ttgaatgtta taaaaatcag catggcgtga gcgaagaaga agcagttaaa 1440

gccctgagct tagaagttgc caatagttgg aaagatatta atgaagaact gctgctgaat 1500

ccgatggcca ttccgctgcc gctgttacag gttattctgg atctgtctcg tagcgcagat 1560

tttatgtatg gtaatgcaca ggatcgcttt acccatgcaa ccatgatgaa agatcaggtg 1620

gatctggtgc tgaaagatcc ggttaaatta gatgattaa 1659

<210> 7

<211> 552

<212> PRT

<213> Artificial Sequence

<400> 7

Met Gly Ser Thr Gln Gln Val Ser Ser Glu Asn Ile Val Arg Asn Ala

1 5 10 15

Ala Asn Phe His Pro Asn Ile Trp Gly Asn His Phe Leu Thr Cys Pro

20 25 30

Ser Gln Thr Ile Asp Ser Trp Thr Gln Gln His His Lys Glu Leu Lys

35 40 45

Glu Glu Val Arg Lys Met Met Val Ser Asp Ala Asn Lys Pro Ala Gln

50 55 60

Arg Leu Arg Leu Ile Asp Thr Val Gln Arg Leu Gly Val Ala Tyr His

65 70 75 80

Phe Glu Lys Glu Ile Asp Asp Ala Leu Glu Lys Ile Gly His Asp Pro

85 90 95

Phe Asp Asp Lys Asp Asp Leu Tyr Ile Val Ser Leu Cys Phe Arg Leu

100 105 110

Leu Arg Gln His Gly Ile Lys Ile Ser Cys Asp Val Phe Glu Lys Phe

115 120 125

Lys Asp Asp Asp Gly Lys Phe Lys Ala Ser Leu Met Asn Asp Val Gln

130 135 140

Gly Met Leu Ser Leu Tyr Glu Ala Ala His Leu Ala Ile His Gly Glu

145 150 155 160

Asp Ile Leu Asp Glu Ala Ile Val Phe Thr Thr Thr His Leu Lys Ser

165 170 175

Thr Val Ser Asn Ser Pro Val Asn Ser Thr Phe Ala Glu Gln Ile Arg

180 185 190

His Ser Leu Arg Val Pro Leu Arg Lys Ala Val Pro Arg Leu Glu Ser

195 200 205

Arg Tyr Phe Leu Asp Ile Tyr Ser Arg Asp Asp Leu His Asp Lys Thr

210 215 220

Leu Leu Asn Phe Ala Lys Leu Asp Phe Asn Ile Leu Gln Ala Met His

225 230 235 240

Gln Lys Glu Ala Ser Glu Met Thr Arg Trp Trp Arg Asp Phe Asp Phe

245 250 255

Leu Lys Lys Leu Pro Tyr Ile Arg Asp Arg Val Val Glu Leu Tyr Phe

260 265 270

Trp Ile Leu Val Gly Val Ser Tyr Gln Pro Lys Phe Ser Thr Gly Arg

275 280 285

Ile Phe Leu Ser Lys Ile Ile Cys Leu Glu Thr Leu Val Asp Asp Thr

290 295 300

Phe Asp Ala Tyr Gly Thr Phe Asp Glu Leu Ala Ile Phe Thr Glu Ala

305 310 315 320

Val Thr Arg Trp Asp Leu Gly His Arg Asp Ala Leu Pro Glu Tyr Met

325 330 335

Lys Phe Ile Phe Lys Thr Leu Ile Asp Val Tyr Ser Glu Ala Glu Gln

340 345 350

Glu Leu Ala Lys Glu Gly Arg Ser Tyr Ser Ile His Tyr Ala Ile Arg

355 360 365

Ser Phe Gln Glu Leu Val Met Lys Tyr Phe Cys Glu Ala Lys Trp Leu

370 375 380

Asn Lys Gly Tyr Val Pro Ser Leu Asp Asp Tyr Lys Ser Val Ser Leu

385 390 395 400

Arg Ser Ile Gly Phe Leu Pro Ile Ala Val Ala Ser Phe Val Phe Met

405 410 415

Gly Asp Ile Ala Thr Lys Glu Val Phe Glu Trp Glu Met Asn Asn Pro

420 425 430

Lys Ile Ile Ile Ala Ala Glu Thr Ile Phe Arg Phe Leu Asp Asp Ile

435 440 445

Ala Gly His Arg Phe Glu Gln Lys Arg Glu His Ser Pro Ser Ala Ile

450 455 460

Glu Cys Tyr Lys Asn Gln His Gly Val Ser Glu Glu Glu Ala Val Lys

465 470 475 480

Ala Leu Ser Leu Glu Val Ala Asn Ser Trp Lys Asp Ile Asn Glu Glu

485 490 495

Leu Leu Leu Asn Pro Met Ala Ile Pro Leu Pro Leu Leu Gln Val Ile

500 505 510

Leu Asp Leu Ser Arg Ser Ala Asp Phe Met Tyr Gly Asn Ala Gln Asp

515 520 525

Arg Phe Thr His Gln Thr Met Met Lys Asp Gln Val Asp Leu Val Leu

530 535 540

Lys Asp Pro Val Lys Leu Asp Asp

545 550

<210> 8

<211> 1659

<212> DNA/RNA

<213> Artificial Sequence

<400> 8

atgggctcta cccagcaggt gtctagcgaa aatattgtgc gtaatgccgc caattttcat 60

ccgaatattt ggggtaatca ttttctgacc tgtccgtctc agaccattga ttcttggacc 120

cagcagcatc ataaagaact gaaagaagaa gtgcgcaaaa tgatggtgag cgatgccaat 180

aaaccggcac agcgcctgcg cctgattgat accgttcagc gcttaggcgt ggcctatcat 240

tttgaaaaag aaattgatga tgcactggaa aaaattggtc atgatccgtt tgatgataaa 300

gatgatctgt atattgtgag tctgtgtttt cgcctgttac gtcagcatgg cattaaaatt 360

agttgtgatg tgtttgaaaa atttaaagat gatgatggta aatttaaagc ctctttaatg 420

aatgatgttc agggtatgct gtcactgtat gaagccgcac atctggccat tcatggcgaa 480

gatattctgg atgaagccat tgtgtttacc accacccatc tgaaatcaac cgtgagtaat 540

agcccggtta atagtacctt tgcagaacag attcgtcata gcttacgcgt tccgttacgc 600

aaagcagttc cgcgcctgga atctcgctat tttctggata tttattcacg cgatgatctg 660

catgataaaa ccctgctgaa ttttgccaaa ctggatttta atattcttca agccatgcat 720

cagaaagaag cctcagaaat gacccgttgg tggcgcgatt ttgattttct gaaaaaactg 780

ccgtatattc gcgatcgtgt ggtggaactg tatttttgga ttctggtggg cgtgagctat 840

cagccgaaat ttagtaccgg tcgcattttt ctgtctaaaa ttatttgttt agaaacctta 900

gtggatgata cctttgatgc ctatggcacc tttgatgaac tggcaatttt taccgaagca 960

gtgacccgtt gggatctggg tcatcgcgat gccttaccgg aatatatgaa atttattttt 1020

aaaaccttaa ttgatgtgta tagcgaagcc gaacaggaac tggccaaaga aggtcgtagc 1080

tatagcattc attatgcaat tcgtagcttt caggaactgg ttatgaaata tttttgtgaa 1140

gccaaatggc tgaataaagg ctatgttccg agcttagatg attataaatc agtgtcactg 1200

cgctcaattg gctttctgcc gattgcagtt gcatcttttg tgtttatggg cgatattgca 1260

accaaagaag tgtttgaatg ggaaatgaat aatccgaaaa ttattattgc cgccgaaacc 1320

atttttcgct ttctggatga tattgccggt catcgctttg aacagaaacg cgaacatagt 1380

ccgagtgcaa ttgaatgtta taaaaatcag catggcgtga gcgaagaaga agcagttaaa 1440

gccctgagct tagaagttgc caatagttgg aaagatatta atgaagaact gctgctgaat 1500

ccgatggcca ttccgctgcc gctgttacag gttattctgg atctgtctcg tagcgcagat 1560

tttatgtatg gtaatgcaca ggatcgcttt acccatcaga ccatgatgaa agatcaggtg 1620

gatctggtgc tgaaagatcc ggttaaatta gatgattaa 1659

<210> 9

<211> 552

<212> PRT

<213> Artificial Sequence

<400> 9

Met Gly Ser Thr Gln Gln Val Ser Ser Glu Asn Ile Val Arg Asn Ala

1 5 10 15

Ala Asn Phe His Pro Asn Ile Trp Gly Asn His Phe Leu Thr Cys Pro

20 25 30

Ser Gln Thr Ile Asp Ser Trp Thr Gln Gln His His Lys Glu Leu Lys

35 40 45

Glu Glu Val Arg Lys Met Met Val Ser Asp Ala Asn Lys Pro Ala Gln

50 55 60

Arg Leu Arg Leu Ile Asp Thr Val Gln Arg Leu Gly Val Ala Tyr His

65 70 75 80

Phe Glu Lys Glu Ile Asp Asp Ala Leu Glu Lys Ile Gly His Asp Pro

85 90 95

Phe Asp Asp Lys Asp Asp Leu Tyr Ile Val Ser Leu Cys Phe Arg Leu

100 105 110

Leu Arg Gln His Gly Ile Lys Ile Ser Cys Asp Val Phe Glu Lys Phe

115 120 125

Lys Asp Asp Asp Gly Lys Phe Lys Ala Ser Leu Met Asn Asp Val Gln

130 135 140

Gly Met Leu Ser Leu Tyr Glu Ala Ala His Leu Ala Ile His Gly Glu

145 150 155 160

Asp Ile Leu Asp Glu Ala Ile Val Phe Thr Thr Thr His Leu Lys Ser

165 170 175

Thr Val Ser Asn Ser Pro Val Asn Ser Thr Phe Ala Glu Gln Ile Arg

180 185 190

His Ser Leu Arg Val Pro Leu Arg Lys Ala Val Pro Arg Leu Glu Ser

195 200 205

Arg Tyr Phe Leu Asp Ile Tyr Ser Arg Asp Asp Leu His Asp Lys Thr

210 215 220

Leu Leu Asn Phe Ala Lys Leu Asp Phe Asn Ile Leu Gln Ala Met His

225 230 235 240

Gln Lys Glu Ala Ser Glu Met Thr Arg Trp Trp Arg Asp Phe Asp Phe

245 250 255

Leu Lys Lys Leu Pro Tyr Ile Arg Asp Arg Val Val Glu Leu Tyr Phe

260 265 270

Trp Ile Leu Val Gly Val Ser Tyr Gln Pro Lys Phe Ser Thr Gly Arg

275 280 285

Ile Phe Leu Ser Lys Ile Ile Cys Leu Glu Thr Leu Val Asp Asp Thr

290 295 300

Phe Asp Ala Tyr Gly Thr Phe Asp Glu Leu Ala Ile Phe Thr Glu Ala

305 310 315 320

Val Thr Arg Trp Asp Leu Gly His Arg Asp Ala Leu Pro Glu Tyr Met

325 330 335

Lys Phe Ile Phe Lys Thr Leu Ile Asp Val Tyr Ser Glu Ala Glu Gln

340 345 350

Glu Leu Ala Lys Glu Gly Arg Ser Tyr Ser Ile His Tyr Ala Ile Arg

355 360 365

Ser Phe Gln Glu Leu Val Met Lys Tyr Phe Cys Glu Ala Lys Trp Leu

370 375 380

Asn Lys Gly Tyr Val Pro Ser Leu Asp Asp Tyr Lys Ser Val Ser Leu

385 390 395 400

Arg Ser Ile Gly Phe Leu Pro Ile Ala Val Ala Ser Phe Val Phe Met

405 410 415

Gly Asp Ile Ala Thr Lys Glu Val Phe Glu Trp Glu Met Asn Asn Pro

420 425 430

Lys Ile Ile Ile Ala Ala Glu Thr Ile Phe Arg Phe Leu Asp Asp Ile

435 440 445

Ala Gly His Arg Phe Glu Gln Lys Arg Glu His Ser Pro Ser Ala Ile

450 455 460

Glu Cys Tyr Lys Asn Gln His Gly Val Ser Glu Glu Glu Ala Val Lys

465 470 475 480

Ala Leu Ser Leu Glu Val Ala Asn Ser Trp Lys Asp Ile Asn Glu Glu

485 490 495

Leu Leu Leu Asn Pro Met Ala Ile Pro Leu Pro Leu Leu Gln Val Ile

500 505 510

Leu Asp Leu Ser Arg Ser Ala Asp Phe Met Tyr Gly Asn Ala Ala Asp

515 520 525

Arg Phe Thr His Ala Thr Met Met Lys Asp Gln Val Asp Leu Val Leu

530 535 540

Lys Asp Pro Val Lys Leu Asp Asp

545 550

<210> 10

<211> 1659

<212> DNA/RNA

<213> Artificial Sequence

<400> 10

atgggctcta cccagcaggt gtctagcgaa aatattgtgc gtaatgccgc caattttcat 60

ccgaatattt ggggtaatca ttttctgacc tgtccgtctc agaccattga ttcttggacc 120

cagcagcatc ataaagaact gaaagaagaa gtgcgcaaaa tgatggtgag cgatgccaat 180

aaaccggcac agcgcctgcg cctgattgat accgttcagc gcttaggcgt ggcctatcat 240

tttgaaaaag aaattgatga tgcactggaa aaaattggtc atgatccgtt tgatgataaa 300

gatgatctgt atattgtgag tctgtgtttt cgcctgttac gtcagcatgg cattaaaatt 360

agttgtgatg tgtttgaaaa atttaaagat gatgatggta aatttaaagc ctctttaatg 420

aatgatgttc agggtatgct gtcactgtat gaagccgcac atctggccat tcatggcgaa 480

gatattctgg atgaagccat tgtgtttacc accacccatc tgaaatcaac cgtgagtaat 540

agcccggtta atagtacctt tgcagaacag attcgtcata gcttacgcgt tccgttacgc 600

aaagcagttc cgcgcctgga atctcgctat tttctggata tttattcacg cgatgatctg 660

catgataaaa ccctgctgaa ttttgccaaa ctggatttta atattcttca agccatgcat 720

cagaaagaag cctcagaaat gacccgttgg tggcgcgatt ttgattttct gaaaaaactg 780

ccgtatattc gcgatcgtgt ggtggaactg tatttttgga ttctggtggg cgtgagctat 840

cagccgaaat ttagtaccgg tcgcattttt ctgtctaaaa ttatttgttt agaaacctta 900

gtggatgata cctttgatgc ctatggcacc tttgatgaac tggcaatttt taccgaagca 960

gtgacccgtt gggatctggg tcatcgcgat gccttaccgg aatatatgaa atttattttt 1020

aaaaccttaa ttgatgtgta tagcgaagcc gaacaggaac tggccaaaga aggtcgtagc 1080

tatagcattc attatgcaat tcgtagcttt caggaactgg ttatgaaata tttttgtgaa 1140

gccaaatggc tgaataaagg ctatgttccg agcttagatg attataaatc agtgtcactg 1200

cgctcaattg gctttctgcc gattgcagtt gcatcttttg tgtttatggg cgatattgca 1260

accaaagaag tgtttgaatg ggaaatgaat aatccgaaaa ttattattgc cgccgaaacc 1320

atttttcgct ttctggatga tattgccggt catcgctttg aacagaaacg cgaacatagt 1380

ccgagtgcaa ttgaatgtta taaaaatcag catggcgtga gcgaagaaga agcagttaaa 1440

gccctgagct tagaagttgc caatagttgg aaagatatta atgaagaact gctgctgaat 1500

ccgatggcca ttccgctgcc gctgttacag gttattctgg atctgtctcg tagcgcagat 1560

tttatgtatg gtaatgcagc cgatcgcttt acccatgcaa ccatgatgaa agatcaggtg 1620

gatctggtgc tgaaagatcc ggttaaatta gatgattaa 1659

<210> 11

<211> 552

<212> PRT

<213> Artificial Sequence

<400> 11

Met Gly Ser Thr Gln Gln Val Ser Ser Glu Asn Ile Val Arg Asn Ala

1 5 10 15

Ala Asn Tyr His Pro Asn Ile Trp Gly Asn His Phe Leu Thr Cys Pro

20 25 30

Ser Gln Thr Ile Asp Ser Trp Thr Gln Gln His His Lys Glu Leu Lys

35 40 45

Glu Glu Val Arg Lys Met Met Val Ser Asp Ala Asn Lys Pro Ala Gln

50 55 60

Arg Leu Arg Leu Ile Asp Thr Val Gln Arg Leu Gly Val Ala Tyr His

65 70 75 80

Phe Glu Lys Glu Ile Asp Asp Ala Leu Glu Lys Ile Gly His Asp Pro

85 90 95

Phe Asp Asp Lys Asp Asp Leu Tyr Ile Val Ser Leu Cys Phe Arg Leu

100 105 110

Leu Arg Gln His Gly Ile Lys Ile Ser Cys Asp Val Phe Glu Lys Phe

115 120 125

Lys Asp Asp Asp Gly Lys Phe Lys Ala Ser Leu Met Asn Asp Val Gln

130 135 140

Gly Met Leu Ser Leu Tyr Glu Ala Ala His Leu Ala Ile His Gly Glu

145 150 155 160

Asp Ile Leu Asp Glu Ala Ile Val Phe Thr Thr Thr His Leu Lys Ser

165 170 175

Thr Val Ser Asn Ser Pro Val Asn Ser Thr Phe Ala Glu Gln Ile Arg

180 185 190

His Ser Leu Arg Val Pro Leu Arg Lys Ala Val Pro Arg Leu Glu Ser

195 200 205

Arg Tyr Phe Leu Asp Ile Tyr Ser Arg Asp Asp Leu His Asp Lys Thr

210 215 220

Leu Leu Asn Phe Ala Lys Leu Asp Phe Asn Ile Leu Gln Ala Met His

225 230 235 240

Gln Lys Glu Ala Ser Glu Met Thr Arg Trp Trp Arg Asp Phe Asp Phe

245 250 255

Leu Lys Lys Leu Pro Tyr Ile Arg Asp Arg Val Val Glu Leu Tyr Phe

260 265 270

Trp Ile Leu Val Gly Val Ser Tyr Gln Pro Lys Phe Ser Thr Gly Arg

275 280 285

Ile Phe Leu Ser Lys Ile Ile Cys Leu Glu Thr Leu Val Asp Asp Thr

290 295 300

Phe Asp Ala Tyr Gly Thr Phe Asp Glu Leu Ala Ile Phe Thr Glu Ala

305 310 315 320

Val Thr Arg Trp Asp Leu Gly His Arg Asp Ala Leu Pro Glu Tyr Met

325 330 335

Lys Phe Ile Phe Lys Thr Leu Ile Asp Val Tyr Ser Glu Ala Glu Gln

340 345 350

Glu Leu Ala Lys Glu Gly Arg Ser Tyr Ser Ile His Tyr Ala Ile Arg

355 360 365

Ser Phe Gln Glu Leu Val Met Lys Tyr Phe Cys Glu Ala Lys Trp Leu

370 375 380

Asn Lys Gly Tyr Val Pro Ser Leu Asp Asp Tyr Lys Ser Val Ser Leu

385 390 395 400

Arg Ser Ile Gly Phe Leu Pro Ile Ala Val Ala Ser Phe Val Phe Met

405 410 415

Gly Asp Ile Ala Thr Lys Glu Val Phe Glu Trp Glu Met Asn Asn Pro

420 425 430

Lys Ile Ile Ile Ala Ala Glu Thr Ile Phe Arg Phe Leu Asp Asp Ile

435 440 445

Ala Gly His Arg Phe Glu Gln Lys Arg Glu His Ser Pro Ser Ala Ile

450 455 460

Glu Cys Tyr Lys Asn Gln His Gly Val Ser Glu Glu Glu Ala Val Lys

465 470 475 480

Ala Leu Ser Leu Glu Val Ala Asn Ser Trp Lys Asp Ile Asn Glu Glu

485 490 495

Leu Leu Leu Asn Pro Met Ala Ile Pro Leu Pro Leu Leu Gln Val Ile

500 505 510

Leu Asp Leu Ser Arg Ser Ala Asp Phe Met Tyr Gly Asn Ala Gln Asp

515 520 525

Arg Phe Thr His Ser Thr Met Met Lys Asp Gln Val Asp Leu Val Leu

530 535 540

Lys Asp Pro Val Lys Leu Asp Asp

545 550

<210> 12

<211> 552

<212> PRT

<213> Artificial Sequence

<400> 12

Met Gly Ser Thr Gln Gln Val Ser Ser Glu Asn Ile Val Arg Asn Ala

1 5 10 15

Ala Asn Phe His Pro Asn Ile Trp Gly Asn His Phe Leu Thr Cys Pro

20 25 30

Ser Gln Thr Ile Asp Ser Trp Thr Gln Gln His His Lys Glu Leu Lys

35 40 45

Glu Glu Val Arg Lys Met Met Val Ser Asp Ala Asn Lys Pro Ala Gln

50 55 60

Arg Leu Arg Leu Ile Asp Thr Val Gln Arg Leu Gly Val Ala Tyr His

65 70 75 80

Phe Glu Lys Glu Ile Asp Asp Ala Leu Glu Lys Ile Gly His Asp Pro

85 90 95

Phe Asp Asp Lys Asp Asp Leu Tyr Ile Val Ser Leu Cys Phe Arg Leu

100 105 110

Leu Arg Gln His Gly Ile Lys Ile Ser Cys Asp Val Phe Glu Lys Phe

115 120 125

Lys Asp Asp Asp Gly Lys Phe Lys Ala Ser Leu Met Asn Asp Val Gln

130 135 140

Gly Met Leu Ser Leu Tyr Glu Ala Ala His Leu Ala Ile His Gly Glu

145 150 155 160

Asp Ile Leu Asp Glu Ala Ile Val Phe Thr Thr Thr His Leu Lys Ser

165 170 175

Thr Val Ser Asn Ser Pro Val Asn Ser Thr Phe Ala Glu Gln Ile Arg

180 185 190

His Ser Leu Arg Val Pro Leu Arg Lys Ala Val Pro Arg Leu Glu Ser

195 200 205

Arg Tyr Phe Leu Asp Ile Tyr Ser Arg Asp Asp Leu His Asp Lys Thr

210 215 220

Leu Leu Asn Phe Ala Lys Leu Asp Phe Asn Ile Leu Gln Ala Met His

225 230 235 240

Gln Lys Glu Ala Ser Glu Met Thr Arg Trp Trp Arg Asp Phe Asp Phe

245 250 255

Leu Lys Lys Leu Pro Tyr Ile Arg Asp Arg Val Val Glu Leu Tyr Phe

260 265 270

Trp Ile Leu Val Gly Val Ser Tyr Gln Pro Lys Phe Ser Thr Gly Arg

275 280 285

Ile Phe Leu Ser Lys Ile Ile Cys Leu Glu Thr Leu Val Asp Asp Thr

290 295 300

Phe Asp Ala Tyr Gly Thr Phe Asp Glu Leu Ala Ile Phe Thr Glu Ala

305 310 315 320

Val Thr Arg Trp Asp Leu Gly His Arg Asp Ala Leu Pro Glu Tyr Met

325 330 335

Lys Phe Ile Phe Lys Thr Leu Ile Asp Val Tyr Ser Glu Ala Glu Gln

340 345 350

Glu Leu Ala Lys Glu Gly Arg Ser Tyr Ser Ile His Tyr Ala Ile Arg

355 360 365

Ser Phe Gln Glu Leu Val Met Lys Tyr Phe Cys Glu Ala Lys Trp Leu

370 375 380

Asn Lys Gly Tyr Val Pro Ser Leu Asp Asp Tyr Lys Ser Val Ser Leu

385 390 395 400

Arg Ser Ile Gly Phe Leu Pro Leu Ala Val Ala Ser Phe Val Phe Met

405 410 415

Gly Asp Ile Ala Thr Lys Glu Val Phe Glu Trp Glu Met Asn Asn Pro

420 425 430

Lys Ile Ile Ile Ala Ala Glu Thr Ile Phe Arg Phe Leu Asp Asp Ile

435 440 445

Ala Gly His Arg Phe Glu Gln Lys Arg Glu His Ser Pro Ser Ala Ile

450 455 460

Glu Cys Tyr Lys Asn Gln His Gly Val Ser Glu Glu Glu Ala Val Lys

465 470 475 480

Ala Leu Ser Leu Glu Val Ala Asn Ser Trp Lys Asp Ile Asn Glu Glu

485 490 495

Leu Leu Leu Asn Pro Met Ala Ile Pro Leu Pro Leu Leu Gln Val Ile

500 505 510

Leu Asp Leu Ser Arg Ser Ala Asp Phe Met Tyr Gly Asn Ala Gln Asp

515 520 525

Arg Phe Thr His Ser Thr Met Met Lys Asp Gln Val Asp Leu Val Leu

530 535 540

Lys Asp Pro Val Lys Leu Asp Asp

545 550

<210> 13

<211> 552

<212> PRT

<213> Artificial Sequence

<400> 13

Met Gly Ser Thr Gln Gln Val Ser Ser Glu Asn Ile Val Arg Asn Ala

1 5 10 15

Ala Asn Phe His Pro Asn Ile Trp Gly Asn His Phe Leu Thr Cys Pro

20 25 30

Ser Gln Thr Ile Asp Ser Trp Thr Gln Gln His His Lys Glu Leu Lys

35 40 45

Glu Glu Val Arg Lys Met Met Val Ser Asp Ala Asn Lys Pro Ala Gln

50 55 60

Arg Leu Arg Leu Ile Asp Thr Val Gln Arg Leu Gly Val Ala Tyr His

65 70 75 80

Phe Glu Lys Glu Ile Asp Asp Ala Leu Glu Lys Ile Gly His Asp Pro

85 90 95

Phe Asp Asp Lys Asp Asp Leu Tyr Ile Val Ser Leu Cys Phe Arg Leu

100 105 110

Leu Arg Gln His Gly Ile Lys Ile Ser Cys Asp Val Phe Glu Lys Phe

115 120 125

Lys Asp Asp Asp Gly Lys Phe Lys Ala Ser Leu Met Asn Asp Val Gln

130 135 140

Gly Met Leu Ser Leu Tyr Glu Ala Ala His Leu Ala Ile His Gly Glu

145 150 155 160

Asp Ile Leu Asp Glu Ala Ile Val Phe Thr Thr Thr His Leu Lys Ser

165 170 175

Thr Val Ser Asn Ser Pro Val Asn Ser Thr Phe Ala Glu Gln Ile Arg

180 185 190

His Ser Leu Arg Val Pro Leu Arg Lys Ala Val Pro Arg Leu Glu Ser

195 200 205

Arg Tyr Phe Leu Asp Ile Tyr Ser Arg Asp Asp Leu His Asp Lys Thr

210 215 220

Leu Leu Asn Phe Ala Lys Leu Asp Phe Asn Ile Leu Gln Ala Met His

225 230 235 240

Gln Lys Glu Ala Ser Glu Met Thr Arg Trp Trp Arg Asp Phe Asp Phe

245 250 255

Leu Lys Lys Leu Pro Tyr Ile Arg Asp Arg Val Val Glu Leu Tyr Phe

260 265 270

Trp Ile Leu Val Gly Val Ser Tyr Gln Pro Lys Phe Ser Thr Gly Arg

275 280 285

Ile Phe Leu Ser Lys Ile Ile Cys Leu Glu Thr Leu Val Asp Asp Thr

290 295 300

Phe Asp Ala Tyr Gly Thr Phe Asp Glu Leu Ala Ile Phe Thr Glu Ala

305 310 315 320

Val Thr Arg Trp Asp Leu Gly His Arg Asp Ala Leu Pro Glu Tyr Met

325 330 335

Lys Phe Ile Phe Lys Thr Leu Ile Asp Val Tyr Ser Glu Ala Glu Gln

340 345 350

Glu Leu Ala Lys Glu Gly Arg Ser Tyr Ser Ile His Tyr Ala Ile Arg

355 360 365

Ser Phe Gln Glu Leu Val Met Lys Tyr Phe Cys Glu Ala Lys Trp Leu

370 375 380

Asn Lys Gly Tyr Val Pro Ser Leu Asp Asp Tyr Lys Ser Val Ser Leu

385 390 395 400

Arg Ser Ile Gly Phe Leu Pro Ile Ala Val Ala Ser Phe Val Phe Met

405 410 415

Gly Asp Ile Ala Thr Lys Glu Val Phe Glu Trp Glu Met Asn Asn Pro

420 425 430

Lys Ile Ile Ile Ala Ala Glu Thr Ile Ser Arg Phe Leu Asp Asp Ile

435 440 445

Ala Gly His Arg Phe Glu Gln Lys Arg Glu His Ser Pro Ser Ala Ile

450 455 460

Glu Cys Tyr Lys Asn Gln His Gly Val Ser Glu Glu Glu Ala Val Lys

465 470 475 480

Ala Leu Ser Leu Glu Val Ala Asn Ser Trp Lys Asp Ile Asn Glu Glu

485 490 495

Leu Leu Leu Asn Pro Met Ala Ile Pro Leu Pro Leu Leu Gln Val Ile

500 505 510

Leu Asp Leu Ser Arg Ser Ala Asp Phe Met Tyr Gly Asn Ala Gln Asp

515 520 525

Arg Phe Thr His Ser Thr Met Met Lys Asp Gln Val Asp Leu Val Leu

530 535 540

Lys Asp Pro Val Lys Leu Asp Asp

545 550

<210> 14

<211> 552

<212> PRT

<213> Artificial Sequence

<400> 14

Met Gly Ser Thr Gln Gln Val Ser Ser Glu Asn Ile Val Arg Asn Ala

1 5 10 15

Ala Asn Phe His Pro Asn Ile Trp Gly Asn His Phe Leu Thr Cys Pro

20 25 30

Ser Gln Thr Ile Asp Ser Trp Thr Gln Gln His His Lys Glu Leu Lys

35 40 45

Glu Glu Val Arg Lys Met Met Val Ser Asp Ala Asn Lys Pro Ala Gln

50 55 60

Arg Leu Arg Leu Ile Asp Thr Val Gln Arg Leu Gly Val Ala Tyr His

65 70 75 80

Phe Glu Lys Glu Ile Asp Asp Ala Leu Glu Lys Ile Gly His Asp Pro

85 90 95

Phe Asp Asp Lys Asp Asp Leu Tyr Ile Val Ser Leu Cys Phe Arg Leu

100 105 110

Leu Arg Gln His Gly Ile Lys Ile Ser Cys Asp Val Phe Glu Lys Phe

115 120 125

Lys Asp Asp Asp Gly Lys Phe Lys Ala Ser Leu Met Asn Asp Val Gln

130 135 140

Gly Met Leu Ser Leu Tyr Glu Ala Ala His Leu Ala Ile His Gly Glu

145 150 155 160

Asp Ile Leu Asp Glu Ala Ile Val Phe Thr Thr Thr His Leu Lys Ser

165 170 175

Thr Val Ser Asn Ser Pro Val Asn Ser Thr Phe Ala Glu Gln Ile Arg

180 185 190

His Ser Leu Arg Val Pro Leu Arg Lys Ala Val Pro Arg Leu Glu Ser

195 200 205

Arg Tyr Phe Leu Asp Ile Tyr Ser Arg Asp Asp Leu His Asp Lys Thr

210 215 220

Leu Leu Asn Phe Ala Lys Leu Asp Phe Asn Ile Leu Gln Ala Met His

225 230 235 240

Gln Lys Glu Ala Ser Glu Met Thr Arg Trp Trp Arg Asp Phe Asp Phe

245 250 255

Leu Lys Lys Leu Pro Tyr Ile Arg Asp Arg Val Val Glu Leu Tyr Phe

260 265 270

Trp Ile Leu Val Gly Val Ser Tyr Gln Pro Lys Phe Ser Thr Gly Arg

275 280 285

Ile Phe Leu Ser Lys Ile Ile Cys Leu Glu Thr Leu Val Asp Asp Thr

290 295 300

Phe Asp Ala Tyr Gly Thr Phe Asp Glu Leu Ala Ile Phe Thr Glu Ala

305 310 315 320

Val Thr Arg Trp Asp Leu Gly His Arg Asp Ala Leu Pro Glu Tyr Met

325 330 335

Lys Phe Ile Phe Lys Thr Leu Ile Asp Val Tyr Ser Glu Ala Glu Gln

340 345 350

Glu Leu Ala Lys Glu Gly Arg Ser Tyr Ser Ile His Tyr Ala Ile Arg

355 360 365

Ser Phe Gln Glu Leu Val Met Lys Tyr Phe Cys Glu Ala Lys Trp Leu

370 375 380

Asn Lys Gly Tyr Val Pro Ser Leu Asp Asp Tyr Lys Ser Val Ser Leu

385 390 395 400

Arg Ser Ile Gly Phe Leu Pro Ile Ala Val Ala Ser Phe Val Phe Met

405 410 415

Gly Asp Ile Ala Thr Lys Glu Val Phe Glu Trp Glu Met Asn Asn Pro

420 425 430

Lys Ile Ile Ile Ala Ala Glu Thr Ile Phe Arg Phe Leu Asp Asp Ile

435 440 445

Ala Gly His Arg Phe Glu Gln Lys Arg Glu His Ser Pro Ser Ala Ile

450 455 460

Glu Cys Tyr Lys Asn Gln His Gly Ala Ser Glu Glu Glu Ala Val Lys

465 470 475 480

Ala Leu Ser Leu Glu Val Ala Asn Ser Trp Lys Asp Ile Asn Glu Glu

485 490 495

Leu Leu Leu Asn Pro Met Ala Ile Pro Leu Pro Leu Leu Gln Val Ile

500 505 510

Leu Asp Leu Ser Arg Ser Ala Asp Phe Met Tyr Gly Asn Ala Gln Asp

515 520 525

Arg Phe Thr His Ser Thr Met Met Lys Asp Gln Val Asp Leu Val Leu

530 535 540

Lys Asp Pro Val Lys Leu Asp Asp

545 550

<210> 15

<211> 26

<212> PRT

<213> Artificial Sequence

<400> 15

Thr Ala Thr Cys Ala Thr Cys Cys Gly Ala Ala Thr Ala Thr Thr Thr

1 5 10 15

Gly Gly Gly Gly Thr Ala Ala Thr Cys Ala

20 25

<210> 16

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 16

Ala Thr Thr Gly Gly Cys Gly Gly Cys Ala Thr Thr Ala Cys Gly Cys

1 5 10 15

Ala Cys Ala

<210> 17

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 17

Cys Thr Gly Gly Cys Ala Gly Thr Thr Gly Cys Ala Thr Cys Thr Thr

1 5 10 15

Thr Thr Gly Thr Gly Thr Thr Thr

20

<210> 18

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 18

Cys Gly Gly Cys Ala Gly Ala Ala Ala Gly Cys Cys Ala Ala Thr Thr

1 5 10 15

Gly Ala Gly Cys

20

<210> 19

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 19

Ala Gly Cys Cys Gly Cys Thr Thr Thr Cys Thr Gly Gly Ala Thr Gly

1 5 10 15

Ala Thr Ala Thr Thr Gly Cys

20

<210> 20

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 20

Ala Ala Thr Gly Gly Thr Thr Thr Cys Gly Gly Cys Gly Gly Cys Ala

1 5 10 15

Ala Thr Ala Ala Thr Ala Ala

20

<210> 21

<211> 26

<212> PRT

<213> Artificial Sequence

<400> 21

Gly Cys Ala Ala Gly Cys Gly Ala Ala Gly Ala Ala Gly Ala Ala Gly

1 5 10 15

Cys Ala Gly Thr Thr Ala Ala Ala Gly Cys

20 25

<210> 22

<211> 28

<212> PRT

<213> Artificial Sequence

<400> 22

Gly Cys Cys Ala Thr Gly Cys Thr Gly Ala Thr Thr Thr Thr Thr Ala

1 5 10 15

Thr Ala Ala Cys Ala Thr Thr Cys Ala Ala Thr Thr

20 25

<210> 23

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 23

Gly Cys Cys Gly Ala Thr Cys Gly Cys Thr Thr Thr Ala Cys Cys Cys

1 5 10 15

Ala Thr Ala Gly Thr Ala Cys

20

<210> 24

<211> 25

<212> PRT

<213> Artificial Sequence

<400> 24

Thr Gly Cys Ala Thr Thr Ala Cys Cys Ala Thr Ala Cys Ala Thr Ala

1 5 10 15

Ala Ala Ala Thr Cys Thr Gly Cys Gly

20 25

<210> 25

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 25

Gly Cys Ala Ala Cys Cys Ala Thr Gly Ala Thr Gly Ala Ala Ala Gly

1 5 10 15

Ala Thr Cys Ala Gly Gly Thr Gly

20

<210> 26

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 26

Ala Thr Gly Gly Gly Thr Ala Ala Ala Gly Cys Gly Ala Thr Cys Cys

1 5 10 15

Thr Gly Thr Gly Cys Ala

20

<210> 27

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 27

Cys Ala Gly Ala Cys Cys Ala Thr Gly Ala Thr Gly Ala Ala Ala Gly

1 5 10 15

Ala Thr Cys Ala Gly Gly Thr Gly

20

<210> 28

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 28

Gly Cys Ala Gly Ala Thr Cys Gly Cys Thr Thr Thr Ala Cys Cys Cys

1 5 10 15

Ala Thr Gly Cys Ala Ala

20

<210> 29

<211> 26

<212> PRT

<213> Artificial Sequence

<400> 29

Thr Gly Cys Ala Thr Thr Ala Cys Cys Ala Thr Ala Cys Ala Thr Ala

1 5 10 15

Ala Ala Ala Thr Cys Thr Gly Cys Gly Cys

20 25

<210> 30

<211> 5691

<212> DNA/RNA

<213> Artificial Sequence

<400> 30

atcgattgtt gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca 60

atttcacaca ggaaacagta tccatgggca gcagccatca ccatcatcac cacagccagg 120

atccgaattc gagctcggcg cgcctgcagg tcgacaagct tgcggccgca taatgcttaa 180

gtcgaacaga aagtaatcgt attgtacacg gcctgcctgg cggcagtagc gcggtggtcc 240

cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt 300

ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa 360

gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctgttg acaattaatc 420

atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaacagtac 480

atatggcaga tctcaattgg atatcggccg gccacgcgat cgctgacgtc ggtaccctcg 540

agtgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa 600

acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc 660

atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt 720

cggtgaacgc tctcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa 780

ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc 840

ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 900

cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 960

cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 1020

tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 1080

gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 1140

cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 1200

attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 1260

tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tctggcggca 1320

cgatggcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 1380

ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 1440

cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 1500

cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 1560

accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 1620

ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 1680

ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 1740

tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 1800

acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 1860

tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 1920

actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 1980

ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 2040

aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 2100

ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 2160

cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 2220

aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 2280

actcatactc ttcctttttc aatcatgatt gaagcattta tcagggttat tgtctcatga 2340

gcggatacat atttgaatgt atttagaaaa ataaacaaat aggtcatgac caaaatccct 2400

taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 2460

tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 2520

gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 2580

agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 2640

aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 2700

gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 2760

gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 2820

tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 2880

agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 2940

cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 3000

gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 3060

gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 3120

ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 3180

cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 3240

cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg tgcactctca 3300

gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga 3360

ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 3420

tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 3480

gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg 3540

gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc 3600

cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg 3660

tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat 3720

gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga 3780

acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca 3840

gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca 3900

tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact 3960

ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca 4020

gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca 4080

accccgccag cctagccggg tcctcaacga caggagcacg atcatgctag tcatgccccg 4140

cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg 4200

tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc 4260

gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 4320

gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc 4380

ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca 4440

ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat 4500

cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc 4560

gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct 4620

cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt 4680

ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac 4740

gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga 4800

ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg 4860

gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag 4920

caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga 4980

gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca 5040

ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg 5100

cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt 5160

gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc 5220

gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga 5280

caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt 5340

gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt 5400

ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg 5460

ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac 5520

agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg 5580

aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca 5640

cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga g 5691

Claims

1.一种檀香烯合酶SanSyn突变体，其特征在于：所述檀香烯合酶SanSyn突变体为将氨基酸序列为SEQ ID NO.1所示的野生型SanSyn酶的第527位谷氨酰胺突变为丙氨酸，其中，所述野生型SanSyn酶来源于植物黄皮（Clausena lansium）。

2.如权利要求1所述的檀香烯合酶突变体，其特征在于：所述檀香烯合酶突变体的核苷酸序列如SEQ ID NO.4所示。

3.如权利要求1所述的檀香烯合酶突变体的制备方法，其特征在于：所述制备方法，包括，

将核苷酸序列如SEQ ID NO.2所示的野生型SanSyn酶基因连接到质粒pETDuet-tac中，得到重组质粒pETDuet-tac-SanSyn，其中，所述连接的双酶切位点分别为NcoI和BamHI，所述质粒pETDuet-tac的核苷酸序列如SEQ ID NO.30所示；

设计突变引物Q527A-F、Q527A-R，核苷酸序列分别为GCCGATCGCTTTACCCATAGTAC、TGCATTACCATACATAAAATCTGCG；

使用突变引物并以质粒pETDuet-tac-SanSyn为模板进行PCR扩增得到突变产物，转化到宿主细胞大肠杆菌TOP 10的感受态细胞中，筛选得到檀香烯合酶SanSyn突变体表达菌株，诱导表达，得到檀香烯合酶突变体。