本发明涉及工程菌构建技术领域，具体涉及一种以甲醇为原料的丁二烯生产菌及其生产丁二烯的方法。
背景技术：
1,3-丁二烯(下文称为丁二烯)是分子式为C4H6的有机化合物，由两个乙烯基构成，它是最简单的共轭二烯烃(图1)。它是一种容易液化的无色气体，有特殊气味。稍溶于水，溶于乙醇、甲醇，易溶于丙酮、乙醚和氯仿等。(https://en.wikipedia.org/wiki/1,3-Butadiene)丁二烯是一种重要的平台化合物，是用于生产合成橡胶(包括苯乙烯-丁二烯橡胶(SBR)、聚丁二烯(PB)、苯乙烯-丁二烯胶乳(SBL)、丙烯腈-丁二烯-苯乙烯树脂(ABS)、丁腈橡胶和己二腈)的重要单体，其也用在制造Nylon-66(WHITE W C.Butadiene production process overview[J].Chemico-biological interactions,2007,166(1-3):10-4)。
随着我国高性能轮胎、绿色轮胎日益增多，对于橡胶，尤其是高性能稀土聚丁二烯橡胶的需求量将日益增加。此外，随着国内电器电子行业的迅速发展，ABS树脂和HIPS对聚丁二烯橡胶的需求量也将进一步增加。近年来，我国丁二烯橡胶的表观消费量一直呈现递增的发展态势。预计到2020年对丁二烯的需求量将达到约320万t，依然有着较大的需求量，而其最直接的影响因素是供给少需求多(崔小明.国内外聚丁二烯橡胶市场分析[J].中国石油和化工经济分析,2014(07):48-51)。由于自然条件的限制，我国的天然橡胶资源一直处于供不应求的状态，进口依赖度较高，在2016年我国丁二烯橡胶的进口量为27.33万t，进口金额约3亿美元，进口金额消费较高(谭捷,刘博超等.国内聚丁二烯橡胶生产技术进展及市场分析[J].弹性体,2018,28(01):80-6)。
丁二烯的生产方法包括从通过石脑油裂解的C4馏分的提取方法、正丁烷和正丁烯的脱氢(Houdry方法)和正丁烯的氧化脱氢(O-X-D方法)。石脑油裂解的C4馏分的提取方法，通常作为蒸汽裂化过程的副产品产生，蒸馏成粗制丁二烯流，并经萃取蒸馏纯化。目前，工业上丁二烯的主要生产方法是石脑油裂解的C4馏分的提取方法，该方法受石油资源的限制，超过95％的丁二烯采用石化路线制备，这种生产方式不可持续，环境危害大。随着原油价格的剧烈波动，丁二烯的市场价格波动巨大，其供应存在不稳定性(WHITEW C.Butadiene production process overview[J].Chemico-biological interactions,2007,166(1-3):10-4)。此外，通过正丁烷和正丁烯的脱氢和正丁烯的氧化脱氢的生产方法不但在热力学上不利，而且，作为吸热反应，需要高温和低压的条件来生产高产率丁二烯，并且因此不适合生产丁二烯的工业化过程。
在追求更加可持续的化学品生产工艺过程中，利用微生物从可再生原料来生产丁二烯将会是一个重大的进步。目前为止，微生物合成高附加值产品主要依赖六碳糖等作为碳源，然而由于大规模化生产存在与人畜争粮争地的原料瓶颈问题，以及纤维素转化成糖类技术瓶颈，利用微生物合成丁二烯仍成本过高，目前并没有一种成本低的高效合成丁二烯的生产方法。
技术实现要素：
本发明的目的在于提供一种丁二烯生产菌及其生产丁二烯的方法。本发明提供的丁二烯生产菌能够以甲醇作为原料生产丁二烯，能够充分利用具有竞争力价格和供应充足的原料甲醇，通过微生物代谢，直接合成丁二烯产品，成本大大降低，有利于实现由甲醇合成丁二烯的工业化生产。
本发明提供了一种丁二烯生产菌，所述生产菌属于甲基杆菌属，所述生产菌由甲基杆菌经表达以下几种酶的外源基因导入修饰表达获得：丁二烯合成酶MTS或ISPS；巴豆醇单磷酸酯激酶IPK；巴豆醇激酶THK或MK或TK；以及辅酶A还原酶FAR或BLD-BDH或ADHE2；所述BLD-BDH为BLD和BDH两种酶同时表达；
所述甲基杆菌含有丝氨酸循环途径和乙酰丙二酰辅酶A途径。
优选的是，所述生产菌由甲基杆菌经表达以下几种酶的外源基因导入修饰表达获得：丁二烯合成酶MTS；巴豆醇单磷酸酯激酶IPK；巴豆醇激酶THK和辅酶A还原酶FAR。
优选的是，所述甲基杆菌包括扭脱甲基杆菌AM1、扭脱甲基杆菌PA1或耐辐射甲基杆菌。
优选的是，所述辅酶A还原酶FAR来自Marinobacter manganoxydans或Hahella chejuensis。
优选的是，所述丁二烯合成酶MTS的氨基酸序列如SEQ ID NO.1所示；所述丁二烯合成酶ISPS的氨基酸序列如SEQ ID NO.2所示。
优选的是，所述巴豆醇单磷酸酯激酶IPK的氨基酸序列如SEQ ID NO.3所示。
优选的是，所述巴豆醇激酶THK的氨基酸序列如SEQ ID NO.4所示；所述巴豆醇激酶MK的氨基酸序列如SEQ ID NO.5所示；所述巴豆醇激酶TK的氨基酸序列如SEQ ID NO.6所示。
优选的是，所述巴豆醇激酶THK的氨基酸序列如SEQ ID NO.12所示。
优选的是，来自Marinobacter manganoxydans的辅酶A还原酶FAR的氨基酸序列如SEQ ID NO.7所示；来自Hahella chejuensis的辅酶A还原酶FAR的氨基酸序列如SEQ ID NO.8所示的序列；BLD的氨基酸序列如SEQ ID NO.9所示；BDH的氨基酸序列如SEQ ID NO.10所示；ADHE2的氨基酸序列如SEQ ID NO.11所示的序列。
本发明还提供了一种基于上述技术方案所述丁二烯生产菌的丁二烯生产方法，包括以下步骤：以甲醇为原料对所述丁二烯生产菌进行培养。
本发明提供了一种丁二烯生产菌，所述生产菌使用甲基杆菌，能够代谢甲醇并产生巴豆酰辅酶A这一中间产物，辅酶A还原酶FAR或辅酶A还原酶丁醛脱氢酶-丁醇脱氢酶BLD-BDH或辅酶A还原酶醛醇脱氢酶ADHE2将巴豆酰辅酶A还原成巴豆醇，巴豆醇被巴豆醇激酶磷酸化成巴豆醇单磷酸酯、巴豆醇单磷酸酯激酶催化巴豆醇单磷酸酯磷酸化生成巴豆醇焦磷酸酯，丁二烯合成酶催化巴豆醇焦磷酸酯生成丁二烯。本发明提供的丁二烯生产菌能够以甲醇作为原料生产丁二烯，能够充分利用具有竞争力价格和供应充足的原料甲醇，通过微生物代谢，直接合成丁二烯产品，成本大大降低，有利于实现由甲醇合成丁二烯的工业化生产。
附图说明
图1为丁二烯的结构式及分子模型图；
图2为本发明提供的丁二烯合成途径示意图；
图3为本发明提供的高通量筛选方法图；
图4为本发明提供的同源建模分析突变酶催化效力提高机制图；
图5为本发明提供的IPK酶活检测级联反应图；
图6为本发明提供的测定丁二烯含量的标准曲线。
具体实施方式
本发明提供了一种丁二烯生产菌，所述生产菌属于甲基杆菌属，所述生产菌由甲基杆菌经表达以下几种酶的外源基因导入修饰表达获得：丁二烯合成酶MTS或ISPS；巴豆醇单磷酸酯激酶IPK；巴豆醇激酶THK或MK或TK；以及辅酶A还原酶FAR或BLD-BDH或ADHE2；所述BLD-BDH为BLD和BDH两种酶同时表达；
所述甲基杆菌含有丝氨酸循环途径和乙酰丙二酰辅酶A途径。
在本发明中，所述生产菌有优选由甲基杆菌经外源基因导入修饰表达：丁二烯合成酶MTS；巴豆醇单磷酸酯激酶IPK；巴豆醇激酶THK和辅酶A还原酶FAR。
本发明所述甲基杆菌能够代谢甲醇并产生巴豆酰辅酶A这一中间产物，甲基杆菌代谢甲醇经过一碳化合物的氧化模块将甲醇催化氧化为甲醛，然后甲醛从周质空间进入细胞质中，经过一碳中间代谢物(甲醛)的异化模块产生甲酸，甲酸一部分在甲酸脱氢酶的作用下被氧化成CO2，产生用于细胞生长的能量和NADH，另一部分甲酸转化成次亚甲基-四氢叶酸，经三个催化反应(FtfL，Fch和MtdA)生成亚甲基-四氢叶酸，然后进入一碳中间物(甲醛或甲酸)的同化模块，经丝氨酸循环生成乙酰辅酶A，乙酰辅酶A经乙酰丙二酰辅酶A途径(EMCP)在三个酶连续催化作用下生成巴豆酰辅酶A，将巴豆酰辅酶A作为前体物异源构建丁二烯生产途径。
本发明所述丁二烯合成途径包括4步反应(如图2所示)：以巴豆酰辅酶A为前体物，本发明辅酶A还原酶(辅酶A还原酶FAR或丁醛脱氢酶-丁醇脱氢酶BLD-BDH或醛醇脱氢酶ADHE2)将巴豆酰辅酶A还原成巴豆醇；巴豆醇被巴豆醇激酶(THK或MK或TK)磷酸化成巴豆醇单磷酸酯；巴豆醇单磷酸酯激酶IPK催化巴豆醇单磷酸酯磷酸化生成巴豆醇焦磷酸酯；丁二烯合成酶MTS或ISPS催化巴豆醇焦磷酸酯生成丁二烯。
本发明通过将辅酶A还原酶FAR或丁醛脱氢酶-丁醇脱氢酶BLD-BDH或醛醇脱氢酶ADHE2，以及丁二烯合成酶、巴豆醇单磷酸酯激酶和巴豆醇激酶相应的基因转入甲基杆菌，实现丁二烯生产菌的构建，所述丁二烯生产菌能够以甲醇作为生产原料，实现丁二烯的工业化生产。本发明对上述酶对应的外源基因(如far、thim、ipk或mts)的转化导入方法没有特殊的限定，采用本领域技术人员熟知的基因转化方法进行转化即可。在本发明中，上述技术方案所述酶对应的外源基因包括far(辅酶A还原酶FAR)、bld(丁醛脱氢酶BLD)、bdh(丁醇脱氢酶BDH)、adhe2(醛醇脱氢酶ADHE2)、ipk(巴豆醇单磷酸酯激酶IPK)、mts(丁二烯合成酶MTS)、isps(丁二烯合成酶ISPS)、thim(巴豆醇激酶THK)、erg12(巴豆醇激酶MK)、thik(巴豆醇激酶TK)。
在本发明中，所述甲基杆菌优选包括扭脱甲基杆菌AM1、扭脱甲基杆菌PA1或耐辐射甲基杆菌。本发明对所述扭脱甲基杆菌AM1、扭脱甲基杆菌PA1或耐辐射甲基杆菌的来源没有特殊的限定，采用本领域技术人员熟知的相应菌种市售机构进行购买即可。在本发明中，所述扭脱甲基杆菌AM1优选来源于青岛农业大学，杨松课题组，所述扭脱甲基杆菌PA1(Methylobacterium extorquens PA1)和耐辐射甲基杆菌(Methylobacterium radiotolerans)优选从德国微生物和菌种保藏中心(DSMZ)(http://www.dsmz.de/)购买。
在本发明中，所述丁二烯合成酶MTS的氨基酸序列如SEQ ID NO.1所示；所述丁二烯合成酶ISPS的氨基酸序列如SEQ ID NO.2所示。
在本发明中，所述巴豆醇单磷酸酯激酶IPK的氨基酸序列如SEQ ID NO.3所示。
在本发明中，所述巴豆醇激酶THK的氨基酸序列如SEQ ID NO.4所示；所述巴豆醇激酶MK的氨基酸序列如SEQ ID NO.5所示；所述巴豆醇激酶TK的氨基酸序列如SEQ ID NO.6所示。在本发明中，所述巴豆醇激酶THK优选进行基因突变，突变后的巴豆醇激酶的氨基酸序列更优选如SEQ ID NO.12所示。
在本发明中，所述辅酶A还原酶FAR来自Marinobacter manganoxydans或Hahella chejuensis。在本发明中，来自Marinobactermanganoxydans的辅酶A还原酶FAR的氨基酸序列如SEQ ID NO.7所示；来自Hahella chejuensis的辅酶A还原酶(FAR)的氨基酸序列如SEQ ID NO.8所示的序列；丁醛脱氢酶BLD的氨基酸序列如SEQ ID NO.9所示；丁醇脱氢酶BDH的氨基酸序列如SEQ ID NO.10所示；醛醇脱氢酶ADHE2的氨基酸序列如SEQ ID NO.11所示的序列。
本发明还提供了一种基于上述技术方案所述丁二烯生产菌的丁二烯生产方法，包括以下步骤：以甲醇为原料对上述技术方案所述丁二烯生产菌进行培养。本发明对所述丁二烯生产菌的培养条件没有特殊的限定，采用本领域技术人员熟知的常规甲基杆菌属培养条件即可，如25～40℃，培养40～120小时收集得到丁二烯。本发明对所述培养基没有特殊的限定，优选采用Hypho培养基或NB培养基，所述NB培养基包括蛋白胨10g/L、牛肉膏3g/L和氯化钠5g/L。在本发明中，所述Hypho培养基的配制方法优选如下：大量元素(2X)：大量元素A：5.06g/LK2HPO4，5.17g/LNaH2PO4；大量元素B：0.4095g/LMgSO4·7H2O，1g/L(NH4)2SO4；若配制固体培养基，则在大量元素B中，加入琼脂30g/L。单独灭菌，使用时按照1：1比例混匀。微量元素：微量元素A和微量元素B分开储存，配置100mL 1000X微量元素。微量元素A：1gNa2EDTA，0.1g FeSO4·7H20，用1MNaOH调pH至4；微量元素B：0.14g CaCl2·2H2O，0.1g MnCl2·4H2O，0.02g Na2MoO4·2H2O，0.03g CuSO4·5H2O，0.32g CoCl2·6H2O，0.44g ZnSO4·7H2O，用HCl调pH到1-2。微量元素需要过滤除菌，不能高压灭菌，配好之后放在4℃保存，最长可使用两周。使用时加入1X的微量元素A和微量元素B，甲醇的最终浓度为125mM。本发明优选采用固相微萃取纤维(SPME fiber)对菌体培养液顶空的丁二烯气体进行萃取取样。
本发明利用甲醇为碳源的甲基杆菌作为研究对象，利用代谢工程、合成生物学等手段，在其中外源引入丁二烯合成途径，可以实现由甲醇合成丁二烯的工业化构想。本发明直接以甲醇为碳源，由于甲醇的生物毒性，在发酵生产的前期，降低了除菌操作的成本，在运行过程中，能够大大地降低了培养基染菌的风险，保证产品质量，减少倒罐、停产的发生，对于工业发酵具有极大的经济价值。此外，在后期的分离环节中，由于甲基杆菌培养所使用的培养基是加入甲醇的非糖基培养基，相比于成分复杂的糖基培养基，分离成本大大降低。本发明利用具有竞争力的价格和供应充足的原料甲醇，通过微生物代谢，直接合成丁二烯产品，并且整个生产过程条件温和、污染小，发酵中不易染菌。
本发明在生产菌构建生产过程中，涉及对各中间产物和丁二烯的浓度的检测，通过所述检测能够实现高产菌的筛选。本发明对所述中间产物和丁二烯浓度的检测方法没有特殊的限定，采用本领域技术人员熟知的检测方法即可。具体地：
本发明所述巴豆醇浓度检测方法优选包括以下步骤：
样品运用安捷伦5975B/6890N气相质谱检测，色谱柱为HP-5MS(30m×0.25mm×0.25um film；Restek,Bellefonte,PA,USA)。使用超高压纯氮作为载气，流速为1mL/min，上样体积为1μL，使安捷伦7890自动进样，无分流模式自动进样。进样口和传输管的温度设为280℃。温度梯度为：60℃保持0.25分钟，以5℃/min速度增加到280℃，保持10min；离子源和四级杆的温度分别设为250℃和150℃。色谱图用安捷伦数据分析软件进行分析。
本发明所述巴豆醇单磷酸酯和巴豆醇焦磷酸酯浓度检测优选包括以下步骤：
(1)质谱条件：液相质谱(LC-QQQ-MS)用于靶向检测中心代谢途径的小分子代谢物，采用多反应监控模式设置母离子和子离子。ESI设置条件为:毛细管电压：4000V，碰撞电压：135V，气体温度300℃，喷雾器35psig，气体流速为10L/min。LC-QExactive-MS仪器用于对未知化合物进行MS/MS检测，设置条件如下：喷雾电压设置为+3.2KV，鞘气压力为35arb，辅助气压力为10arb，毛细管温度为320℃，加热器温度为350℃。
(2)色谱条件：
样品在Advance Bio Glycan色谱柱(150×2.1mm,1.8μm；USA)中分离。流动相A为乙腈/水(5:95v/v)，含20mM醋酸铵和40mM氨水。流动相B由乙腈/水(95:5v/v)组成。洗脱梯度为:0～2分钟，80％B；2–5分钟，80％–60％B；5–6分钟，60％–5％B；6–8分钟，5％B；8–8.01分钟，5％–80％B；8.01–12分钟，80％B，总共的洗脱时间为12分钟，流速为0.3mL/min，上样体积为3μL。数据处理均使用QualitativeAnalysis B.06.00软件
本发明所述丁二烯浓度检测优选包括以下步骤：
(1)丁二烯标准样制备方法
丁二烯标品浓度为2mg/mL，分别上样体积为0，0.02，0.05，0.10，0.14，0.2，0.3μL制得丁二烯标准样
(2)固相微萃取(SPME)纤维萃取取样
由于丁二烯在培养条件下基本完全存在于气相，所以在本文中采用固相微萃取纤维(SPME fiber)对实施例中得到的酶反应液或菌体培养液顶空的丁二烯气体进行萃取取样。具体的方法为:将样品瓶置于37℃培养箱反应后，再将SPME fiber穿破样品瓶的橡胶塞进行萃取取样5min。注意萃取时间对分析结果有较大的影响，所以应该严格控制萃取的时间。
(3)丁二烯浓度检测
用SPME f'iber萃取样品后，应立即将fiber插入气象色谱仪(GC-7900，中国)进样口中进行检测。色谱分析方法为:以氦气作为载气，流速为6.2mL/min；色谱柱采用Rt-AluminaBOND/Na2SO4(50m×0.32mm×5.0μm)，初始柱温40℃，以30℃/min的速度升温至130℃，保持10min，停止检测；FID检测器温度200℃；进样口温度200℃。此色谱条件下丁二烯的保留时间约在8.3-8.6min之间。用上述方法制得的丁二烯标准样品的含量的标准曲线如图6所示。
下面结合具体实施例对本发明所述的一种丁二烯生产菌及其生产丁二烯的方法做进一步详细的介绍，本发明的技术方案包括但不限于以下实施例。
实施例1
丁二烯合成途径包括4步反应(如图2所示)：
1)以巴豆酰辅酶A为前体物，本发明辅酶A还原酶(辅酶A还原酶Far或丁醛脱氢酶-丁醇脱氢酶BLD-BDH或醛醇脱氢酶ADHE2)将巴豆酰辅酶A还原成巴豆醇；
2)巴豆醇被巴豆醇激酶(THK或MK或TK)磷酸化成巴豆醇单磷酸酯；
3)巴豆醇单磷酸酯激酶IPK催化巴豆醇单磷酸酯磷酸化生成巴豆醇焦磷酸酯；
4)丁二烯合成酶MTS或ISPS催化巴豆醇焦磷酸酯生成丁二烯。
(1)针对第1步反应，根据底物巴豆酰辅酶A的结构特点和酶催化反应机理，本发明分别筛选来自糖丁酸梭状芽胞杆菌(Clostridium saccharoperbutylacetonicum)的bld-bdh，来自丙酮丁醇梭菌(Clostridium acetobutylicum)的adhE2，来自深海锰氧化菌(Marinobactermanganoxydans)的Mmfar，来自河氏菌(Hahella chejuensis)的Hcfar作为候选基因，在含有助溶标签TRX的pET32M中构建含以上基因的重组质粒(助溶标签在N端)，在大肠杆菌(E.coliBL21-Z)中诱导表达、分离纯化获得四个重组酶，即丁醛脱氢酶-丁醇脱氢酶(butyraldehyde dehydrogenase-butanol dehydrogenase,BLD-BDH)，醛醇脱氢酶(butyraldehyde and butanol dehydrogenase,ADHE2)，两个脂酰辅酶A还原酶(Fatty Acyl-CoAReductase,FAR)；设计体外酶活反应体系，GC-MS直接比较分析产物巴豆醇的生成量，发现来源于深海锰氧化菌(M.manganoxydans)的脂酰辅酶A还原酶FAR具有最高的催化效力；酶动力学研究进一步表征FAR催化底物巴豆醇的Km和kcat值分别是5.48±1.27mM和0.05±0.009s-1，3h其转化合成巴豆醇的转化率是1％，而催化本源底物十六烷酰辅酶A(palmitoyl-CoA)的Km是4.0±0.3μM，Vmax是197±8nmol/min/mg；为了解析该双功能酶的催化巴豆酰辅酶A效力偏低的原因，分别以巴豆酰辅酶A和巴豆醛作为底物，酶活分析发现FAR催化巴豆酰辅酶A合成巴豆醛是主要限速反应，而巴豆醛合成巴豆醇的反应速率较快，这为后续改造提高FAR催化能力，提供重要信息基础。
(2)针对第2步反应，根据与巴豆醇分子结构相似性和酶催化反应机理，选择来源于大肠杆菌(Escherichia coli.K-12)的thim基因编码的羟乙基噻唑激酶(hydroxyethylthiazole kinase)，肠道沙门氏菌(Salmonella enterica)thik基因编码的硫胺素激酶(thiamine kinase)和酿酒酵母(Saccharomycescerevisiae)erg12基因编码的甲羟戊酸激酶(mevalonate kinase)作为候选酶，对候选基因在甲基细菌中过表达并进行1mM巴豆醇底物饲喂，检测细胞内巴豆醇单磷酸酯的积累，发现来源于大肠杆菌的基因thim编码的羟乙基噻唑激酶催化巴豆醇生成巴豆醇单磷酸酯的产量最高，此外转化空质粒pCM80的重组子也产生少量巴豆醇单磷酸酯，体外检测甲基细菌基因组上gck的重组酶活性，证明该酶可能具有一定催化混乱性；体外酶活动力学分析酶THK，该激酶对于巴豆醇的催化效力Km值7.23±0.79mM，kcat值是1.24±0.26s-1，6h时间内巴豆醇到巴豆醇单磷酸酯的转化率是22.4％。
为进一步提高该酶催化效力，通过酶定向进化策略，高通量筛选获得酶催化效率提高的菌株，具体筛选策略(图3)如下：1)首先对基因thim进行易错PCR，获得该基因的易错突变库；2)构建突变重组子库；3)在48孔板上培养突变菌株，指数初期加200μM浓度的巴豆醇培养至指数后期，取上清液加入高锰酸钾作为颜色指示剂，上清液中被酶THK催化后剩余的巴豆醇与高锰酸钾发生氧化还原反应，产生颜色变化；4)用酶标仪检测其OD，进行高通量筛选OD值最高(即高锰酸钾反应量最小)的菌株；5)将获得的酶催化活性提高的突变序列重组酶进行体外酶活检测，确定其酶的催化活性的提高。
高通量筛选的结果：通过分析2141个突变株，筛选获得1个正突变株，其THK的氨基酸序列在82位处M(甲硫氨酸)变成V(缬氨酸)，突变序列重组酶体外酶活研究证明生成巴豆醇单磷酸酯的转化率提高10倍，利用同源建模解析其催化效力提高的可能机制是：M82与M28是两个临近的甲硫氨酸，具有强疏水性相互作用和明显的范德华力，这个作用限制了与底物作相用的分支环的柔性，并且对该分支环具有的反向拉力使底物孔道变大，不利于小底物分子的结合催化，而M82V这一突变消除了强疏水性相互作用和明显的范德华力，促进了与底物分子的结合，提高了酶催化效率(图4)。
M82V这一突变消除了强疏水性相互作用和明显的范德华力，以该突变酶作为出发模板，一方面进一步通过易错PCR和高通量筛选策略开展定向进化研究，另一方面对突变点进行定点饱和突变，研究是否可能进一步提高催化效率。
(3)针对第3步反应，根据与巴豆醇单磷酸酯结构的相似性和酶催化反应机理，选择来自古细菌(Methanocaldococcus jannaschii，Methanocaldococcus thermautotrophicus,Thermoplasma acidophilum)的异戊烯基磷酸激酶(isopentenylphosphatekinase，IPK)，该酶能够催化异戊烯基磷酸酯(isopentenyl phosphate，IP)磷酸化生成异戊烯焦磷酸酯(isopentenyl diphosphate，IPP)，以质粒pET32M作为骨架构建重组质粒pET32M-ipk，在大肠杆菌(Escherichia coliBL21-Z)中表达、纯化，以巴豆醇单磷酸酯作为底物，体外建立级联反应(图5)，即：巴豆醇单磷酸酯在纯化的异戊烯基磷酸激酶催化下和ATP反应生成巴豆醇焦磷酸酯和ADP，ADP和磷酸烯醇式丙酮酸(PEP)在丙酮酸激酶的催化下生成ATP和丙酮酸，丙酮酸和NADH在乳酸脱氢酶的催化下生成乳酸和NAD+，通过340nm下检测NADH的下降表征IPK催化巴豆醇单磷酸酯的能力，酶动力学分析显示其催化巴豆醇单磷酸酯的Km和kcat值分别是1.28±0.5mM和127.94±30.34s-1，同时我们通过LC-MS检测是否确实有巴豆醇焦磷酸酯的生成，进而验证该催化反应产物的正确性，体外酶活结果在6h时间内巴豆醇单磷酸酯生成巴豆醇焦磷酸酯的转化率是57.2％。
(4)针对第4步反应，烯萜合成酶(即丁二烯合成酶)能够催化作用于焦磷酸酯键，去除焦磷酸，同时发生环化、氢离子迁移等重排，生成各种烯萜化合物，前期文献报道异戊烯焦磷酸IPP生成异戊二烯是在来源于银白杨(Populus alba)，山葛(Pueraria montana)物种的异戊二烯合成酶(isoprene synthase)的催化下产生，另外有文献报道来源于蓝桉树(Eucalyptusglobulus)物种的单烯萜合成酶(monotepene synthase)能催化香叶基焦磷酸酯生成烯萜醇类的芳樟醇(linalool)，考虑分子结构相似度，我们首选ISPS和MTS作为候选酶。在含有助溶标签TRX的pET32M中构建含mts基因的重组质粒(助溶标签在N端)，大肠杆菌培养和优化诱导条件后，但始终是包涵体蛋白，进而采用含有信号肽片段22个氨基酸长度的pET25b质粒(助溶标签在N端)，仍然无法获得可溶性蛋白，最终我们选择利用含有SUMO标签的pET28a构建重组质粒，发现在0.5mM IPTG，20℃，220rpm的诱导条件下，有一定量可溶性蛋白出现，Westenblotting分析确定该蛋白是目的条带，成功在3L菌液中纯化出5.4mg的MTS，体外酶活结果在6h时间内巴豆醇焦磷酸酯生成丁二烯的转化率是0.06％。
实施例2
承接实施例1的实验操作，从巴豆酰辅酶A到丁二烯体外一锅反应的构建。
选择每步催化效率最高的酶构建一锅反应体系，分别是FAR(来自Marinobactermanganoxydans)、THK、IPK和MTS这四种酶，首先分别优化了四个酶反应的pH，pH梯度是6、6.5、7、7.5、8、8.5和9，发现pH在8.0时多数酶催化生成各自目标产物最高。优化了四个酶反应的温度，温度梯度为：29.5℃、32℃、34.5℃、37℃、39.5℃、42℃和44.5℃。发现在39.5℃多数酶催化生成目标产物最高。
在最优温度是39.5℃和pH是8.0的条件下，优化了各个酶在一锅反应中的最优酶量，酶量梯度为0.25mg/mL、0.5mg/mL、1mg/mL和1.5mg/mL。发现FAR在1mg/mL的浓度时，催化产生的目标产物已经达到了较高值。THK、IPK和MTS的分别是1mg/mL、0.5mg/mL和1.5mg/mL。
在以上优化的条件下，加入4mM的巴豆酰辅酶A，反应6h后测得丁二烯产量为2.63ng/mL，在反应3h时再补加一次各反应所需的酶，丁二烯产量提高至4.15ng/mL。
(6)将这四步反应所需的基因之间用RBS连接，构建在pCM80质粒上，电转入扭脱甲基杆菌AM1，在AM1体内过表达这四步反应所需的基因，培养基加入123mM的甲醇，培养至至指数期收集细胞。破壁获得粗酶液，然后用粗酶液外加4mM巴豆酰辅酶A，6h测得丁二烯产量为1.03ng/mL。
实施例3
在扭脱甲基杆菌AM1(Methylobacterium extorquens AM1)表达far+thim+ipk+mts这四个基因，每两个基因间用RBS连接。构建BamHI酶切位点+far+RBS+thim+RBS+ipk+RBS+mts+SacI酶切位点的目的基因片段，同时对该目的基因片段和pCM80进行分步酶切，将目的基因和质粒骨架通过酶切位点BamHI和SacI连接后，及得到质粒pYJ。然后点转入扭脱甲基杆菌AM1中，构建得菌株M.extorquensAM1-pYJ。接种菌株AM1-pYJ制得种子液，然后进行小瓶培养，具体的小瓶培养条件为在50mL厌氧小瓶中以10mL带有10ug/mL四环素的Hypho培养基在30℃，200rpm的摇床避光密封培养70h。接种量的标准为以OD600＝0.8的种子液接种1mL，上述培养的菌体生长状况用吸光度OD600来表征；丁二烯产量用单位体积菌液，单位OD600值的菌体产生的丁二烯质量来表征(ng/m1/OD600)。丁二烯产量的测定方法参照专利实验方法中描述的方法。经过70h培养后，构建的菌株AM1-pYJ的丁二烯产量为1.7ng/m1/OD600。
Hypho培养基：
大量元素(2X)：大量元素A：5.06g/LK2HPO4，5.17g/LNaH2PO4；大量元素B：0.4095g/LMgSO4·7H2O，1g/L(NH4)2SO4；若配制固体培养基，则在大量元素B中，加入琼脂30g/L。单独灭菌，使用时按照1：1比例混匀。
微量元素：微量元素A和微量元素B分开储存，配置100mL 1000X微量元素。微量元素A：1gNa2EDTA，0.1gFeSO4·7H20，用1MNaOH调pH至4；微量元素B：0.14g CaCl2·2H2O，0.1g MnCl2·4H2O，0.02gNa2MoO4·2H2O，0.03g CuSO4·5H2O，0.32g CoCl2·6H2O，0.44g ZnSO4·7H2O，用HCl调pH到1-2。微量元素需要过滤除菌，切记不能高压灭菌，配好之后放在4℃保存，最长可使用两周。使用时加入1X的微量元素A和微量元素B，甲醇的最终浓度为125mM。
实施例4
(1)在扭脱甲基杆菌PA1(Methylobacterium extorquens PA1)表达far+thim+ipk+mts这四个基因，扭脱甲基杆菌PA1的培养基、培养方法、相关基因操作和相关菌种的构建方法同实施例4的甲基扭脱杆菌AM1相同。采用实施例3中构建的pYJ质粒，将pYJ质粒用电击转化法导入到扭脱甲基杆菌PA1中，并筛选得到带有pYJ质粒的扭脱甲基杆菌PA1，PA1-pYJ。具体的构建方法和培养方法与实施例3所述的方法相同。经过70h培养后，PA1-pYJ的丁二烯产量为1.2ng/ml/OD600。
实施例5
(2)耐辐射甲基杆菌(Methylobacterium radiotolerans)表达far+thim+ipk+mts这四个基因，耐辐射甲基杆菌的培养基、培养方法、相关基因操作和相关菌种的构建方法同实施例3的甲基扭脱杆菌AM1相同。采用实施例3中构建的pYJ质粒，将pYJ质粒用电击转化法导入到耐辐射甲基杆菌中，并筛选得到带有pYJ质粒的耐辐射甲基杆菌，MR-pYJ。具体的构建方法和培养方法与实施例3所述的方法相同。经过70h培养后，MR-pYJ的丁二烯产量为0.9ng/ml/OD600。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
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<110> 青岛农业大学
<120> 一种丁二烯生产菌及其生产丁二烯的方法
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<170> SIPOSequenceListing 1.0
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435 440 445
Pro Ser Ile Arg Lys Glu Glu Leu Glu Ser Leu Glu His Tyr His Asp
450 455 460
Leu Leu Arg Leu Pro Ser Leu Ile Phe Arg Leu Thr Asn Asp Ile Ala
465 470 475 480
Ser Ser Ser Ala Glu Leu Glu Arg Gly Glu Thr Thr Asn Ser Ile Arg
485 490 495
Cys Phe Met Gln Glu Lys Gly Ile Ser Glu Leu Glu Ala Arg Glu Cys
500 505 510
Val Lys Glu Glu Ile Asp Thr Ala Trp Lys Lys Met Asn Lys Tyr Met
515 520 525
Val Asp Arg Ser Thr Phe Asn Gln Ser Phe Val Arg Met Thr Tyr Asn
530 535 540
Leu Ala Arg Met Ala His Cys Val Tyr Gln Asp Gly Asp Ala Ile Gly
545 550 555 560
Ser Pro Asp Asp Leu Ser Trp Asn Arg Val His Ser Leu Ile Ile Lys
565 570 575
Pro Ile Ser Pro Ala Ala
580
<210> 2
<211> 608
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Thr Asn Leu Leu Cys Leu Ser Asn Lys Leu Ser Ser Pro Thr
1 5 10 15
Pro Thr Pro Ser Thr Arg Phe Pro Gln Ser Lys Asn Phe Ile Thr Gln
20 25 30
Lys Thr Ser Leu Ala Asn Pro Lys Pro Trp Arg Val Ile Cys Ala Thr
35 40 45
Ser Ser Gln Phe Thr Gln Ile Thr Glu His Asn Ser Arg Arg Ser Ala
50 55 60
Asn Tyr Gln Pro Asn Leu Trp Asn Phe Glu Phe Leu Gln Ser Leu Glu
65 70 75 80
Asn Asp Leu Lys Val Glu Lys Leu Glu Glu Lys Ala Thr Lys Leu Glu
85 90 95
Glu Glu Val Arg Cys Met Ile Asn Arg Val Asp Thr Gln Pro Leu Ser
100 105 110
Leu Leu Glu Leu Ile Asp Asp Val Gln Arg Leu Gly Leu Thr Tyr Lys
115 120 125
Phe Glu Lys Asp Ile Ile Lys Ala Leu Glu Asn Ile Val Leu Leu Asp
130 135 140
Glu Asn Lys Lys Asn Lys Ser Asp Leu His Ala Thr Ala Leu Ser Phe
145 150 155 160
Arg Leu Leu Arg Gln His Gly Phe Glu Val Ser Gln Asp Val Phe Glu
165 170 175
Arg Phe Lys Asp Lys Glu Gly Gly Phe Ser Gly Glu Leu Lys Gly Asp
180 185 190
Val Gln Gly Leu Leu Ser Leu Tyr Glu Ala Ser Tyr Leu Gly Phe Glu
195 200 205
Gly Glu Asn Leu Leu Glu Glu Ala Arg Thr Phe Ser Ile Thr His Leu
210 215 220
Lys Asn Asn Leu Lys Glu Gly Ile Asn Thr Lys Val Ala Glu Gln Val
225 230 235 240
Ser His Ala Leu Glu Leu Pro Tyr His Gln Arg Leu His Arg Leu Glu
245 250 255
Ala Arg Trp Phe Leu Asp Lys Tyr Glu Pro Lys Glu Pro His His Gln
260 265 270
Leu Leu Leu Glu Leu Ala Lys Leu Asp Phe Asn Met Val Gln Thr Leu
275 280 285
His Gln Lys Glu Leu Gln Asp Leu Ser Arg Trp Trp Thr Glu Met Gly
290 295 300
Leu Ala Ser Lys Leu Asp Phe Val Arg Asp Arg Leu Met Glu Val Tyr
305 310 315 320
Phe Trp Ala Leu Gly Met Ala Pro Asp Pro Gln Phe Gly Glu Cys Arg
325 330 335
Lys Ala Val Thr Lys Met Phe Gly Leu Val Thr Ile Ile Asp Asp Val
340 345 350
Tyr Asp Val Tyr Gly Thr Leu Asp Glu Leu Gln Leu Phe Thr Asp Ala
355 360 365
Val Glu Arg Trp Asp Val Asn Ala Ile Asn Thr Leu Pro Asp Tyr Met
370 375 380
Lys Leu Cys Phe Leu Ala Leu Tyr Asn Thr Val Asn Asp Thr Ser Tyr
385 390 395 400
Ser Ile Leu Lys Glu Lys Gly His Asn Asn Leu Ser Tyr Leu Thr Lys
405 410 415
Ser Trp Arg Glu Leu Cys Lys Ala Phe Leu Gln Glu Ala Lys Trp Ser
420 425 430
Asn Asn Lys Ile Ile Pro Ala Phe Ser Lys Tyr Leu Glu Asn Ala Ser
435 440 445
Val Ser Ser Ser Gly Val Ala Leu Leu Ala Pro Ser Tyr Phe Ser Val
450 455 460
Cys Gln Gln Gln Glu Asp Ile Ser Asp His Ala Leu Arg Ser Leu Thr
465 470 475 480
Asp Phe His Gly Leu Val Arg Ser Ser Cys Val Ile Phe Arg Leu Cys
485 490 495
Asn Asp Leu Ala Thr Ser Ala Ala Glu Leu Glu Arg Gly Glu Thr Thr
500 505 510
Asn Ser Ile Ile Ser Tyr Met His Glu Asn Asp Gly Thr Ser Glu Glu
515 520 525
Gln Ala Arg Glu Glu Leu Arg Lys Leu Ile Asp Ala Glu Trp Lys Lys
530 535 540
Met Asn Arg Glu Arg Val Ser Asp Ser Thr Leu Leu Pro Lys Ala Phe
545 550 555 560
Met Glu Ile Ala Val Asn Met Ala Arg Val Ser His Cys Thr Tyr Gln
565 570 575
Tyr Gly Asp Gly Leu Gly Arg Pro Asp Tyr Ala Thr Glu Asn Arg Ile
580 585 590
Lys Leu Leu Leu Ile Asp Pro Phe Pro Ile Asn Gln Leu Met Tyr Val
595 600 605
<210> 3
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ile Ile Leu Lys Leu Gly Gly Ser Val Ile Thr Arg Lys Asp Ser
1 5 10 15
Glu Glu Pro Ala Ile Asp Arg Asp Asn Leu Glu Arg Ile Ala Ser Glu
20 25 30
Ile Gly Asn Ala Ser Pro Ser Ser Leu Met Ile Val His Gly Ala Gly
35 40 45
Ser Phe Gly His Pro Phe Ala Gly Glu Tyr Arg Ile Gly Ser Glu Ile
50 55 60
Glu Asn Glu Glu Asp Leu Arg Arg Arg Arg Phe Gly Phe Ala Leu Thr
65 70 75 80
Gln Asn Trp Val Lys Lys Leu Asn Ser His Val Cys Asp Ala Leu Leu
85 90 95
Ala Glu Gly Ile Pro Ala Val Ser Met Gln Pro Ser Ala Phe Ile Arg
100 105 110
Ala His Ala Gly Arg Ile Ser His Ala Asp Ile Ser Leu Ile Arg Ser
115 120 125
Tyr Leu Glu Glu Gly Met Val Pro Val Val Tyr Gly Asp Val Val Leu
130 135 140
Asp Ser Asp Arg Arg Leu Lys Phe Ser Val Ile Ser Gly Asp Gln Leu
145 150 155 160
Ile Asn His Phe Ser Leu Arg Leu Met Pro Glu Arg Val Ile Leu Gly
165 170 175
Thr Asp Val Asp Gly Val Tyr Thr Arg Asn Pro Lys Lys His Pro Asp
180 185 190
Ala Arg Leu Leu Asp Val Ile Gly Ser Leu Asp Asp Leu Glu Ser Leu
195 200 205
Asp Gly Thr Leu Asn Thr Asp Val Thr Gly Gly Met Val Gly Lys Ile
210 215 220
Arg Glu Leu Leu Leu Leu Ala Glu Lys Gly Val Glu Ser Glu Ile Ile
225 230 235 240
Asn Ala Ala Val Pro Gly Asn Ile Glu Arg Ala Leu Leu Gly Glu Glu
245 250 255
Val Arg Gly Thr Arg Ile Thr Gly Lys His
260 265
<210> 4
<211> 262
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Gln Val Asp Leu Leu Gly Ser Ala Gln Ser Ala His Ala Leu His
1 5 10 15
Leu Phe His Gln His Ser Pro Leu Val His Cys Met Thr Asn Asp Val
20 25 30
Val Gln Thr Phe Thr Ala Asn Thr Leu Leu Ala Leu Gly Ala Ser Pro
35 40 45
Ala Met Val Ile Glu Thr Glu Glu Ala Ser Gln Phe Ala Ala Ile Ala
50 55 60
Ser Ala Leu Leu Ile Asn Val Gly Thr Leu Thr Gln Pro Arg Ala Gln
65 70 75 80
Ala Met Arg Ala Ala Val Glu Gln Ala Lys Ser Ser Gln Thr Pro Trp
85 90 95
Thr Leu Asp Pro Val Ala Val Gly Ala Leu Asp Tyr Arg Arg His Phe
100 105 110
Cys His Glu Leu Leu Ser Phe Lys Pro Ala Ala Ile Arg Gly Asn Ala
115 120 125
Ser Glu Ile Met Ala Leu Ala Gly Ile Ala Asn Gly Gly Arg Gly Val
130 135 140
Asp Thr Thr Asp Ala Ala Ala Asn Ala Ile Pro Ala Ala Gln Thr Leu
145 150 155 160
Ala Arg Glu Thr Gly Ala Ile Val Val Val Thr Gly Glu Met Asp Tyr
165 170 175
Val Thr Asp Gly His Arg Ile Ile Gly Ile His Gly Gly Asp Pro Leu
180 185 190
Met Thr Lys Val Val Gly Thr Gly Cys Ala Leu Ser Ala Val Val Ala
195 200 205
Ala Cys Cys Ala Leu Pro Gly Asp Thr Leu Glu Asn Val Ala Ser Ala
210 215 220
Cys His Trp Met Lys Gln Ala Gly Glu Arg Ala Val Ala Arg Ser Glu
225 230 235 240
Gly Pro Gly Ser Phe Val Pro His Phe Leu Asp Ala Leu Trp Gln Leu
245 250 255
Thr Gln Glu Val Gln Ala
260
<210> 5
<211> 443
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ser Leu Pro Phe Leu Thr Ser Ala Pro Gly Lys Val Ile Ile Phe
1 5 10 15
Gly Glu His Ser Ala Val Tyr Asn Lys Pro Ala Val Ala Ala Ser Val
20 25 30
Ser Ala Leu Arg Thr Tyr Leu Leu Ile Ser Glu Ser Ser Ala Pro Asp
35 40 45
Thr Ile Glu Leu Asp Phe Pro Asp Ile Ser Phe Asn His Lys Trp Ser
50 55 60
Ile Asn Asp Phe Asn Ala Ile Thr Glu Asp Gln Val Asn Ser Gln Lys
65 70 75 80
Leu Ala Lys Ala Gln Gln Ala Thr Asp Gly Leu Ser Gln Glu Leu Val
85 90 95
Ser Leu Leu Asp Pro Leu Leu Ala Gln Leu Ser Glu Ser Phe His Tyr
100 105 110
His Ala Ala Phe Cys Phe Leu Tyr Met Phe Val Cys Leu Cys Pro His
115 120 125
Ala Lys Asn Ile Lys Phe Ser Leu Lys Ser Thr Leu Pro Ile Gly Ala
130 135 140
Gly Leu Gly Ser Ser Ala Ser Ile Ser Val Ser Leu Ala Leu Ala Met
145 150 155 160
Ala Tyr Leu Gly Gly Leu Ile Gly Ser Asn Asp Leu Glu Lys Leu Ser
165 170 175
Glu Asn Asp Lys His Ile Val Asn Gln Trp Ala Phe Ile Gly Glu Lys
180 185 190
Cys Ile His Gly Thr Pro Ser Gly Ile Asp Asn Ala Val Ala Thr Tyr
195 200 205
Gly Asn Ala Leu Leu Phe Glu Lys Asp Ser His Asn Gly Thr Ile Asn
210 215 220
Thr Asn Asn Phe Lys Phe Leu Asp Asp Phe Pro Ala Ile Pro Met Ile
225 230 235 240
Leu Thr Tyr Thr Arg Ile Pro Arg Ser Thr Lys Asp Leu Val Ala Arg
245 250 255
Val Arg Val Leu Val Thr Glu Lys Phe Pro Glu Val Met Lys Pro Ile
260 265 270
Leu Asp Ala Met Gly Glu Cys Ala Leu Gln Gly Leu Glu Ile Met Thr
275 280 285
Lys Leu Ser Lys Cys Lys Gly Thr Asp Asp Glu Ala Val Glu Thr Asn
290 295 300
Asn Glu Leu Tyr Glu Gln Leu Leu Glu Leu Ile Arg Ile Asn His Gly
305 310 315 320
Leu Leu Val Ser Ile Gly Val Ser His Pro Gly Leu Glu Leu Ile Lys
325 330 335
Asn Leu Ser Asp Asp Leu Arg Ile Gly Ser Thr Lys Leu Thr Gly Ala
340 345 350
Gly Gly Gly Gly Cys Ser Leu Thr Leu Leu Arg Arg Asp Ile Thr Gln
355 360 365
Glu Gln Ile Asp Ser Phe Lys Lys Lys Leu Gln Asp Asp Phe Ser Tyr
370 375 380
Glu Thr Phe Glu Thr Asp Leu Gly Gly Thr Gly Cys Cys Leu Leu Ser
385 390 395 400
Ala Lys Asn Leu Asn Lys Asp Leu Lys Ile Lys Ser Leu Val Phe Gln
405 410 415
Leu Phe Glu Asn Lys Thr Thr Thr Lys Gln Gln Ile Asp Asp Leu Leu
420 425 430
Leu Pro Gly Asn Thr Asn Leu Pro Trp Thr Ser
435 440
<210> 6
<211> 274
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Val Arg Ser Asn Asn Asn Asn Pro Leu Thr Arg Asp Glu Ile Leu Ser
1 5 10 15
Arg Tyr Phe Pro Gln Tyr Arg Pro Ala Val Ala Thr Ser Gln Gly Leu
20 25 30
Ser Gly Gly Ser Cys Ile Ile Ala His Asp Thr His Arg Val Val Leu
35 40 45
Arg Arg His His Asp Pro Asp Ala Pro Pro Ala His Phe Leu Arg His
50 55 60
Tyr Arg Ala Leu Ser Gln Leu Pro Ala Ser Leu Ala Pro Arg Ala Leu
65 70 75 80
Phe Tyr Thr Pro Gly Trp Met Ala Val Glu Tyr Leu His Gly Val Val
85 90 95
Asn Ser Ala Leu Pro Asp Ala Asp Glu Leu Ala Ala Leu Leu Tyr His
100 105 110
Leu His Gln Gln Pro Arg Phe Gly Trp Arg Ile Ala Leu Ser Pro Leu
115 120 125
Leu Ala Gln Tyr Trp Ser Cys Cys Asp Pro Ala Arg Arg Thr Pro Phe
130 135 140
Trp Leu Arg Arg Leu Lys Gln Leu Gln Lys Asn Gly Glu Pro Arg Pro
145 150 155 160
Leu Arg Leu Ala Pro Leu His Met Asp Val His Gly Asp Asn Ile Val
165 170 175
Leu Thr Ser Ala Gly Leu Arg Leu Ile Asp Trp Glu Tyr Ala Gly Asp
180 185 190
Gly Asp Ile Ala Leu Glu Leu Ala Ala Val Trp Val Glu Asp Glu Arg
195 200 205
Gln His Arg Gln Leu Ala Asp Ala Tyr Ala Ala Arg Ala Arg Ile Asp
210 215 220
Ala Arg Gln Leu Trp Arg Gln Ile Arg Leu Trp His Pro Trp Val Ile
225 230 235 240
Met Leu Lys Ala Gly Trp Phe Glu Tyr Arg Trp Arg Gln Thr Gly Glu
245 250 255
Gln Gln Phe Ile Arg Leu Ala Asp Glu Thr Trp Arg Gln Leu Arg Met
260 265 270
Lys Gly
<210> 7
<211> 661
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Asn Tyr Phe Leu Thr Gly Gly Thr Gly Phe Ile Gly Arg Phe Leu
1 5 10 15
Val Glu Lys Leu Leu Ala Arg Gly Gly Thr Val His Val Leu Val Arg
20 25 30
Glu Gln Ser Gln Asp Lys Leu Asp Lys Leu Arg Glu Arg Trp Gly Ala
35 40 45
Asp Glu Thr Gln Val Lys Ala Val Ile Gly Asp Leu Thr Ser Lys Asn
50 55 60
Leu Gly Ile Asp Ala Lys Thr Met Lys Ala Leu Lys Gly Lys Ile Asp
65 70 75 80
His Phe Phe His Leu Ala Ala Val Tyr Asp Met Gly Ala Asp Glu Glu
85 90 95
Ala Gln Gln Ala Thr Asn Ile Glu Gly Thr Arg Ala Ala Val Asn Ala
100 105 110
Ala Glu Ala Met Gly Ala Lys His Phe His His Val Ser Ser Ile Ala
115 120 125
Ala Ala Gly Leu Phe Lys Gly Ile Phe Arg Glu Asp Met Phe Glu Glu
130 135 140
Ala Glu Lys Leu Asp His Pro Tyr Leu Arg Thr Lys His Glu Ser Glu
145 150 155 160
Lys Val Val Arg Glu Glu Cys Lys Val Pro Phe Arg Ile Tyr Arg Pro
165 170 175
Gly Met Val Ile Gly His Thr Ala Thr Gly Glu Met Asp Lys Val Asp
180 185 190
Gly Pro Tyr Tyr Phe Phe Lys Met Ile Gln Lys Ile Arg His Ala Leu
195 200 205
Pro Gln Trp Val Pro Thr Ile Gly Val Glu Gly Gly Arg Leu Asn Ile
210 215 220
Val Pro Val Asp Phe Val Val Asn Ala Met Asp His Ile Ala His Leu
225 230 235 240
Glu Gly Glu Asp Gly Lys Cys Phe His Leu Val Asp Thr Asp Pro Tyr
245 250 255
Lys Val Gly Glu Ile Leu Asn Ile Phe Ser Glu Ala Gly His Ala Pro
260 265 270
Arg Met Gly Met Arg Ile Asp Ser Arg Met Phe Gly Phe Ile Pro Pro
275 280 285
Phe Ile Arg Gln Ser Leu Lys Asn Leu Pro Pro Val Lys Arg Leu Thr
290 295 300
Ser Ala Ile Leu Asp Asp Met Gly Ile Pro Pro Ser Val Met Ser Phe
305 310 315 320
Ile Asn Tyr Pro Thr Arg Phe Asp Ala Arg Glu Thr Glu Arg Val Leu
325 330 335
Lys Gly Thr Gly Ile Glu Val Pro Arg Leu Pro Asp Tyr Ala Pro Val
340 345 350
Ile Trp Asp Tyr Trp Glu Arg Asn Leu Asp Pro Asp Leu Phe Lys Asp
355 360 365
Arg Thr Leu Lys Gly Thr Val Glu Gly Arg Val Cys Val Val Thr Gly
370 375 380
Ala Thr Ser Gly Ile Gly Leu Ala Thr Ala Gln Lys Leu Ala Asp Ala
385 390 395 400
Gly Ala Ile Leu Val Ile Gly Ala Arg Lys Leu Glu Arg Leu Lys Glu
405 410 415
Val Ala Ala Glu Leu Glu Ser Arg Gly Ala Ser Val His Ala Tyr Pro
420 425 430
Cys Asp Phe Ser Asp Met Asp Ala Cys Asp Glu Phe Val Lys Thr Val
435 440 445
Leu Asp Asn His Gly Gln Val Asp Val Leu Val Asn Asn Ala Gly Arg
450 455 460
Ser Ile Arg Arg Ser Leu Asp Leu Ser Phe Asp Arg Phe His Asp Phe
465 470 475 480
Glu Arg Thr Met Gln Leu Asn Tyr Phe Gly Ser Val Arg Leu Ile Met
485 490 495
Gly Phe Ala Pro Lys Met Leu Glu Asn Arg Arg Gly His Val Val Asn
500 505 510
Ile Ser Ser Ile Gly Val Leu Thr Asn Ala Pro Arg Phe Ser Ala Tyr
515 520 525
Val Ala Ser Lys Ser Ala Leu Asp Ala Phe Ser Arg Cys Ala Ala Ser
530 535 540
Glu Trp Ser Asp Arg Asn Val Thr Phe Thr Thr Ile Asn Met Pro Leu
545 550 555 560
Val Lys Thr Pro Met Ile Ala Pro Thr Lys Ile Tyr Asp Ser Val Pro
565 570 575
Thr Leu Thr Pro Asp Glu Ala Ala Thr Met Val Ala Asp Ala Ile Val
580 585 590
Tyr Arg Pro Lys Arg Ile Ala Thr Arg Leu Gly Ile Phe Ala Gln Val
595 600 605
Leu His Ala Leu Ala Pro Lys Met Ala Glu Ile Val Met Asn Thr Gly
610 615 620
Tyr Arg Met Phe Pro Asp Ser Pro Ala Ala Ala Gly Ser Arg Ser Gly
625 630 635 640
Glu Lys Pro Lys Val Ser Ser Glu Gln Val Ala Phe Ala Ala Ile Met
645 650 655
Arg Gly Ile Tyr Trp
660
<210> 8
<211> 661
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Asn Tyr Phe Val Thr Gly Gly Thr Gly Phe Ile Gly Arg Phe Leu
1 5 10 15
Val Pro Lys Leu Leu Lys Arg Gly Gly Thr Val Tyr Leu Leu Val Arg
20 25 30
Glu Ala Ser Leu Pro Lys Leu Asp Glu Leu Arg Glu Arg Trp Asn Ala
35 40 45
Ser Asp Glu Gln Val Val Gly Val Val Gly Asp Leu Ala Gln Pro Met
50 55 60
Leu Gly Val Ser Glu Lys Asp Ala Ala Met Leu Arg Gly Lys Val Gly
65 70 75 80
His Phe Phe His Leu Ala Ala Ile Tyr Asp Met Gln Ala Ser Ala Glu
85 90 95
Ser Gln Glu Gln Ala Asn Ile Glu Gly Thr Arg Asn Ala Val Lys Leu
100 105 110
Ala Asp Ser Leu Lys Ala Ala Cys Phe His His Val Ser Ser Ile Ala
115 120 125
Ala Ala Gly Leu Tyr Arg Gly Ile Phe Arg Glu Asp Met Phe Glu Glu
130 135 140
Ala Glu Lys Leu Asp Asn Pro Tyr Leu Arg Thr Lys His Glu Ser Glu
145 150 155 160
Lys Val Val Arg Glu Glu Cys Gln Thr Pro Trp Arg Val Tyr Arg Pro
165 170 175
Gly Met Val Val Gly His Ser Lys Thr Gly Glu Ile Asp Lys Ile Asp
180 185 190
Gly Pro Tyr Tyr Phe Phe Lys Leu Ile Gln Lys Leu Arg Ser Ala Leu
195 200 205
Pro Gln Trp Met Pro Thr Val Gly Leu Glu Gly Gly Arg Ile Asn Ile
210 215 220
Val Pro Val Asp Phe Val Val Asp Ala Met Asp His Ile Ala His Ala
225 230 235 240
Glu Gly Glu Asp Gly Lys Cys Phe His Leu Thr Asp Pro Asp Pro Tyr
245 250 255
Lys Val Gly Glu Ile Leu Asn Ile Phe Ala Glu Ala Gly His Ala Pro
260 265 270
Lys Met Ala Met Arg Ile Asp Ala Arg Met Phe Gly Phe Ile Pro Pro
275 280 285
Met Ile Arg Gln Gly Ile Ala Arg Leu Pro Pro Val Gln Arg Met Lys
290 295 300
Asn Ala Val Leu Asn Asp Leu Gly Ile Pro Asp Glu Val Met Ser Phe
305 310 315 320
Ile Asn Tyr Pro Thr Arg Phe Asp Asn Arg Glu Thr Glu Arg Leu Leu
325 330 335
Lys Gly Thr Ala Ile Ala Val Pro Arg Leu Gln Asp Tyr Ser Pro Ala
340 345 350
Ile Trp Asp Tyr Trp Glu Arg His Leu Asp Pro Asp Leu His Lys Asp
355 360 365
Arg Thr Leu Arg Gly Ala Val Glu Gly Arg Val Cys Val Ile Thr Gly
370 375 380
Ala Thr Ser Gly Ile Gly Leu Ser Ala Ala Arg Lys Leu Ala Glu Ala
385 390 395 400
Gly Ala Lys Val Val Ile Ala Ala Arg Thr Leu Glu Lys Leu Gln Glu
405 410 415
Val Lys Lys Glu Leu Glu Glu Leu Gly Gly Glu Val Tyr Glu Tyr Ser
420 425 430
Val Asp Leu Ser Asp Leu Glu Asp Cys Asp Arg Phe Val Ala Asn Val
435 440 445
Leu Lys Asp Leu Gly His Val Asp Val Leu Val Asn Asn Ala Gly Arg
450 455 460
Ser Ile Arg Arg Ser Ile Gln His Ala Phe Asp Arg Phe His Asp Phe
465 470 475 480
Glu Arg Thr Met Gln Leu Asn Tyr Phe Gly Ser Leu Arg Leu Ile Met
485 490 495
Gly Phe Ala Pro Ser Met Leu Glu Arg Arg Arg Gly His Ile Val Asn
500 505 510
Ile Ser Ser Ile Gly Val Leu Thr Asn Ala Pro Arg Phe Ser Ala Tyr
515 520 525
Val Ala Ser Lys Ala Ala Leu Asp Ala Phe Ser Arg Cys Ala Ala Ala
530 535 540
Glu Phe Ser Asp Lys Asn Val Thr Phe Thr Thr Ile Asn Met Pro Leu
545 550 555 560
Val Arg Thr Pro Met Ile Ser Pro Thr Lys Ile Tyr Asp Ser Val Pro
565 570 575
Thr Leu Thr Pro Glu Glu Ala Ala Asp Leu Val Ala Glu Ala Ile Ile
580 585 590
His Arg Pro Lys Arg Ile Ala Thr Arg Leu Gly Val Phe Ala Gln Val
595 600 605
Leu His Ser Met Ala Pro Lys Phe Ser Glu Ile Ile Met Asn Thr Gly
610 615 620
Phe Lys Met Phe Pro Asp Ser Ser Ala Ala Thr Gly Gly Lys Asp Gly
625 630 635 640
Glu Lys Pro Lys Val Ser Thr Glu Gln Val Ala Phe Ala Ala Ile Met
645 650 655
Arg Gly Ile His Trp
660
<210> 9
<211> 468
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Ile Lys Asp Thr Leu Val Ser Ile Thr Lys Asp Leu Lys Leu Lys
1 5 10 15
Thr Asn Val Glu Asn Ala Asn Leu Lys Asn Tyr Lys Asp Asp Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Asn Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Ile
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Thr Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Asp Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Ser Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Ser Val Lys
340 345 350
Cys Ile Ile Cys Glu Val Ser Ala Arg His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Ile Glu Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 10
<211> 382
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Glu Asn Phe Arg Phe Asn Ala Tyr Thr Glu Met Leu Phe Gly Lys
1 5 10 15
Gly Gln Ile Glu Lys Leu Pro Glu Val Leu Lys Arg Tyr Gly Lys Asn
20 25 30
Ile Leu Leu Ala Tyr Gly Gly Gly Ser Ile Lys Lys Asn Gly Leu Tyr
35 40 45
Asp Thr Ile Gln Lys Leu Leu Lys Asp Phe Asn Ile Val Glu Leu Ser
50 55 60
Gly Ile Glu Pro Asn Pro Arg Ile Glu Thr Val Arg Arg Gly Val Glu
65 70 75 80
Leu Cys Arg Lys Asn Lys Val Asp Val Ile Leu Ala Val Gly Gly Gly
85 90 95
Ser Thr Ile Asp Cys Ser Lys Val Ile Gly Ala Gly Tyr Tyr Tyr Ala
100 105 110
Gly Asp Ala Trp Asp Leu Val Lys Asn Pro Ala Lys Ile Gly Glu Val
115 120 125
Leu Pro Ile Val Thr Val Leu Thr Met Ala Ala Thr Gly Ser Glu Met
130 135 140
Asn Arg Asn Ala Val Ile Ser Lys Met Asp Thr Asn Glu Lys Leu Gly
145 150 155 160
Thr Gly Ser Pro Lys Met Ile Pro Gln Thr Ser Ile Leu Asp Pro Glu
165 170 175
Tyr Leu Tyr Thr Leu Pro Ala Ile Gln Thr Ala Ala Gly Cys Ala Asp
180 185 190
Ile Met Ser His Ile Phe Glu Gln Tyr Phe Asn Lys Thr Thr Asp Ala
195 200 205
Phe Val Gln Asp Lys Phe Ala Glu Gly Leu Leu Gln Thr Cys Ile Lys
210 215 220
Tyr Cys Pro Val Ala Leu Lys Glu Pro Lys Asn Tyr Glu Ala Arg Ala
225 230 235 240
Asn Ile Met Trp Ala Ser Ser Met Ala Leu Asn Gly Leu Leu Gly Ser
245 250 255
Gly Lys Ala Gly Ala Trp Thr Cys His Pro Ile Glu His Glu Leu Ser
260 265 270
Ala Phe Tyr Asp Ile Thr His Gly Val Gly Leu Ala Ile Leu Thr Pro
275 280 285
Ser Trp Met Arg Tyr Ile Leu Ser Asp Val Thr Val Asp Lys Phe Val
290 295 300
Asn Val Trp His Leu Glu Gln Lys Glu Asp Lys Phe Ala Leu Ala Asn
305 310 315 320
Glu Ala Ile Asp Ala Thr Glu Lys Phe Phe Lys Ala Cys Gly Ile Pro
325 330 335
Met Thr Leu Thr Glu Leu Gly Ile Asp Lys Ala Asn Phe Glu Lys Met
340 345 350
Ala Lys Ala Ala Val Glu His Gly Ala Leu Glu Tyr Ala Tyr Val Ser
355 360 365
Leu Asn Ala Glu Asp Val Tyr Lys Ile Leu Glu Met Ser Leu
370 375 380
<210> 11
<211> 858
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Lys Val Thr Asn Gln Lys Glu Leu Lys Gln Lys Leu Asn Glu Leu
1 5 10 15
Arg Glu Ala Gln Lys Lys Phe Ala Thr Tyr Thr Gln Glu Gln Val Asp
20 25 30
Lys Ile Phe Lys Gln Cys Ala Ile Ala Ala Ala Lys Glu Arg Ile Asn
35 40 45
Leu Ala Lys Leu Ala Val Glu Glu Thr Gly Ile Gly Leu Val Glu Asp
50 55 60
Lys Ile Ile Lys Asn His Phe Ala Ala Glu Tyr Ile Tyr Asn Lys Tyr
65 70 75 80
Lys Asn Glu Lys Thr Cys Gly Ile Ile Asp His Asp Asp Ser Leu Gly
85 90 95
Ile Thr Lys Val Ala Glu Pro Ile Gly Ile Val Ala Ala Ile Val Pro
100 105 110
Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu
115 120 125
Lys Thr Arg Asn Ala Ile Phe Phe Ser Pro His Pro Arg Ala Lys Lys
130 135 140
Ser Thr Ile Ala Ala Ala Lys Leu Ile Leu Asp Ala Ala Val Lys Ala
145 150 155 160
Gly Ala Pro Lys Asn Ile Ile Gly Trp Ile Asp Glu Pro Ser Ile Glu
165 170 175
Leu Ser Gln Asp Leu Met Ser Glu Ala Asp Ile Ile Leu Ala Thr Gly
180 185 190
Gly Pro Ser Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro Ala Ile
195 200 205
Gly Val Gly Ala Gly Asn Thr Pro Ala Ile Ile Asp Glu Ser Ala Asp
210 215 220
Ile Asp Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr Asp Asn
225 230 235 240
Gly Val Ile Cys Ala Ser Glu Gln Ser Ile Leu Val Met Asn Ser Ile
245 250 255
Tyr Glu Lys Val Lys Glu Glu Phe Val Lys Arg Gly Ser Tyr Ile Leu
260 265 270
Asn Gln Asn Glu Ile Ala Lys Ile Lys Glu Thr Met Phe Lys Asn Gly
275 280 285
Ala Ile Asn Ala Asp Ile Val Gly Lys Ser Ala Tyr Ile Ile Ala Lys
290 295 300
Met Ala Gly Ile Glu Val Pro Gln Thr Thr Lys Ile Leu Ile Gly Glu
305 310 315 320
Val Gln Ser Val Glu Lys Ser Glu Leu Phe Ser His Glu Lys Leu Ser
325 330 335
Pro Val Leu Ala Met Tyr Lys Val Lys Asp Phe Asp Glu Ala Leu Lys
340 345 350
Lys Ala Gln Arg Leu Ile Glu Leu Gly Gly Ser Gly His Thr Ser Ser
355 360 365
Leu Tyr Ile Asp Ser Gln Asn Asn Lys Asp Lys Val Lys Glu Phe Gly
370 375 380
Leu Ala Met Lys Thr Ser Arg Thr Phe Ile Asn Met Pro Ser Ser Gln
385 390 395 400
Gly Ala Ser Gly Asp Leu Tyr Asn Phe Ala Ile Ala Pro Ser Phe Thr
405 410 415
Leu Gly Cys Gly Thr Trp Gly Gly Asn Ser Val Ser Gln Asn Val Glu
420 425 430
Pro Lys His Leu Leu Asn Ile Lys Ser Val Ala Glu Arg Arg Glu Asn
435 440 445
Met Leu Trp Phe Lys Val Pro Gln Lys Ile Tyr Phe Lys Tyr Gly Cys
450 455 460
Leu Arg Phe Ala Leu Lys Glu Leu Lys Asp Met Asn Lys Lys Arg Ala
465 470 475 480
Phe Ile Val Thr Asp Lys Asp Leu Phe Lys Leu Gly Tyr Val Asn Lys
485 490 495
Ile Thr Lys Val Leu Asp Glu Ile Asp Ile Lys Tyr Ser Ile Phe Thr
500 505 510
Asp Ile Lys Ser Asp Pro Thr Ile Asp Ser Val Lys Lys Gly Ala Lys
515 520 525
Glu Met Leu Asn Phe Glu Pro Asp Thr Ile Ile Ser Ile Gly Gly Gly
530 535 540
Ser Pro Met Asp Ala Ala Lys Val Met His Leu Leu Tyr Glu Tyr Pro
545 550 555 560
Glu Ala Glu Ile Glu Asn Leu Ala Ile Asn Phe Met Asp Ile Arg Lys
565 570 575
Arg Ile Cys Asn Phe Pro Lys Leu Gly Thr Lys Ala Ile Ser Val Ala
580 585 590
Ile Pro Thr Thr Ala Gly Thr Gly Ser Glu Ala Thr Pro Phe Ala Val
595 600 605
Ile Thr Asn Asp Glu Thr Gly Met Lys Tyr Pro Leu Thr Ser Tyr Glu
610 615 620
Leu Thr Pro Asn Met Ala Ile Ile Asp Thr Glu Leu Met Leu Asn Met
625 630 635 640
Pro Arg Lys Leu Thr Ala Ala Thr Gly Ile Asp Ala Leu Val His Ala
645 650 655
Ile Glu Ala Tyr Val Ser Val Met Ala Thr Asp Tyr Thr Asp Glu Leu
660 665 670
Ala Leu Arg Ala Ile Lys Met Ile Phe Lys Tyr Leu Pro Arg Ala Tyr
675 680 685
Lys Asn Gly Thr Asn Asp Ile Glu Ala Arg Glu Lys Met Ala His Ala
690 695 700
Ser Asn Ile Ala Gly Met Ala Phe Ala Asn Ala Phe Leu Gly Val Cys
705 710 715 720
His Ser Met Ala His Lys Leu Gly Ala Met His His Val Pro His Gly
725 730 735
Ile Ala Cys Ala Val Leu Ile Glu Glu Val Ile Lys Tyr Asn Ala Thr
740 745 750
Asp Cys Pro Thr Lys Gln Thr Ala Phe Pro Gln Tyr Lys Ser Pro Asn
755 760 765
Ala Lys Arg Lys Tyr Ala Glu Ile Ala Glu Tyr Leu Asn Leu Lys Gly
770 775 780
Thr Ser Asp Thr Glu Lys Val Thr Ala Leu Ile Glu Ala Ile Ser Lys
785 790 795 800
Leu Lys Ile Asp Leu Ser Ile Pro Gln Asn Ile Ser Ala Ala Gly Ile
805 810 815
Asn Lys Lys Asp Phe Tyr Asn Thr Leu Asp Lys Met Ser Glu Leu Ala
820 825 830
Phe Asp Asp Gln Cys Thr Thr Ala Asn Pro Arg Tyr Pro Leu Ile Ser
835 840 845
Glu Leu Lys Asp Ile Tyr Ile Lys Ser Phe
850 855
<210> 12
<211> 262
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Gln Val Asp Leu Leu Gly Ser Ala Gln Ser Ala His Ala Leu His
1 5 10 15
Leu Phe His Gln His Ser Pro Leu Val His Cys Met Thr Asn Asp Val
20 25 30
Val Gln Thr Phe Thr Ala Asn Thr Leu Leu Ala Leu Gly Ala Ser Pro
35 40 45
Ala Met Val Ile Glu Thr Glu Glu Ala Ser Gln Phe Ala Ala Ile Ala
50 55 60
Ser Ala Leu Leu Ile Asn Val Gly Thr Leu Thr Gln Pro Arg Ala Gln
65 70 75 80
Ala Val Arg Ala Ala Val Glu Gln Ala Lys Ser Ser Gln Thr Pro Trp
85 90 95
Thr Leu Asp Pro Val Ala Val Gly Ala Leu Asp Tyr Arg Arg His Phe
100 105 110
Cys His Glu Leu Leu Ser Phe Lys Pro Ala Ala Ile Arg Gly Asn Ala
115 120 125
Ser Glu Ile Met Ala Leu Ala Gly Ile Ala Asn Gly Gly Arg Gly Val
130 135 140
Asp Thr Thr Asp Ala Ala Ala Asn Ala Ile Pro Ala Ala Gln Thr Leu
145 150 155 160
Ala Arg Glu Thr Gly Ala Ile Val Val Val Thr Gly Glu Met Asp Tyr
165 170 175
Val Thr Asp Gly His Arg Ile Ile Gly Ile His Gly Gly Asp Pro Leu
180 185 190
Met Thr Lys Val Val Gly Thr Gly Cys Ala Leu Ser Ala Val Val Ala
195 200 205
Ala Cys Cys Ala Leu Pro Gly Asp Thr Leu Glu Asn Val Ala Ser Ala
210 215 220
Cys His Trp Met Lys Gln Ala Gly Glu Arg Ala Val Ala Arg Ser Glu
225 230 235 240
Gly Pro Gly Ser Phe Val Pro His Phe Leu Asp Ala Leu Trp Gln Leu
245 250 255
Thr Gln Glu Val Gln Ala
260