CN114250206B - 甲基转移酶突变体、重组载体、重组工程菌及其应用 - Google Patents
甲基转移酶突变体、重组载体、重组工程菌及其应用 Download PDFInfo
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- CN114250206B CN114250206B CN202111605490.4A CN202111605490A CN114250206B CN 114250206 B CN114250206 B CN 114250206B CN 202111605490 A CN202111605490 A CN 202111605490A CN 114250206 B CN114250206 B CN 114250206B
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Abstract
本申请涉及一种甲基转移酶突变体、重组载体、重组工程菌及其应用。该甲基转移酶突变体包括：(a)、由如SEQ ID No.2所示的氨基酸序列缺失、替代或增加一个或多个氨基酸组成的多肽；或，(b)、与如SEQ ID No.2所示的氨基酸序列组成的多肽具有至少90％同源性的多肽。上述甲基转移酶突变体在45℃下的半衰期在26min以上，高于野生型甲基转移酶的半衰期，适于在较高的温度下催化甲基化反应。
Description
技术领域
本申请涉及生物技术领域，特别是涉及一种甲基转移酶突变体、重组载体、重组工程菌及其应用。
背景技术
甲基转移酶是生物有机体内普遍存在的一种重要酶类，它能催化遗传物质DNA的甲基化，在基因表达及动物生长、发育中起着重要的调控作用；同时，也能催化多种生理过程中间产物的甲基化进而合成或降解生理活性物质。根据研究发现，人类的情绪和许多疾病的发生及植物的抗逆性都与甲基转移酶基因的表达有关。然而，市场上大多数甲基转移酶突变体的热稳定性较差，难以满足实际需求。
发明内容
基于此，有必提供一种热稳定性较好的甲基转移酶突变体、重组载体、重组工程菌及其应用。
一种甲基转移酶突变体，所述甲基转移酶突变体包括：
(a)、由如SEQ ID No.2所示的氨基酸序列缺失、替代或增加一个或多个氨基酸组成的多肽；或，
(b)、与如SEQ ID No.2所示的氨基酸序列组成的多肽具有至少90％同源性的多肽。
研究发现，包括由如SEQ ID No.2所示的氨基酸序列缺失、替代或增加一个或多个氨基酸得到的多肽；或，与如SEQ ID No.2所示的氨基酸序列组成的多肽具有至少90％同源性的多肽的甲基转移酶突变体具有较好的热稳定性。经试验验证，上述甲基转移酶突变体在45℃下的半衰期在26min以上，高于野生型甲基转移酶的半衰期，热稳定性较好，适于在较高的温度下催化甲基化反应，尤其是含有如SEQ ID No.2所示的氨基酸序列同时发生C185I突变、V218I突变S250A突变得到的多肽的甲基转移酶突变体在45℃下半衰期大约是野生型甲基转移酶的4倍，具有意料不到的效果。
其中一个实施例中，所述甲基转移酶突变体包括：由如SEQ ID No.2所示的氨基酸序列中第185位氨基酸、第218位氨基酸、第250位氨基酸及第317位氨基酸中的至少一个发生突变得到的多肽。
其中一个实施例中，所述甲基转移酶突变体包括：由如SEQ ID No.2所示的氨基酸序列发生C185I突变、V218I突变、S250A突变及A317S突变中至少一种得到的多肽。
其中一个实施例中，所述甲基转移酶突变体包括：由如SEQ ID No.2所示的氨基酸序列发生C185I突变、V218I突变、S250A突变及A317S突变中的任意一种得到的多肽。
其中一个实施例中，所述甲基转移酶突变体包括：由如SEQ ID No.2所示的氨基酸序列同时发生C185I突变、V218I突变、S250A突变及A317S突变中的任意两种得到的多肽。
其中一个实施例中，所述甲基转移酶突变体包括：由如SEQ ID No.2所示的氨基酸序列同时发生C185I突变、V218I突变、S250A突变及A317S突变中的任意三种得到的多肽。
其中一个实施例中，所述甲基转移酶突变体包括：由如SEQ ID No.2所示的氨基酸序列同时发生C185I突变、V218I突变、S250A突变及A317S突变得到的多肽。
其中一个实施例中，所述甲基转移酶突变体的氨基酸序列为如SEQ ID No.3～SEQID No.17所示的序列中的一种。
其中一个实施例中，所述甲基转移酶突变体的编码序列包括：
(a)、与如SEQ ID No.1所示的核苷酸序列组成的多核苷酸具有至少90％同源性的多核苷酸；或，
(b)、由如SEQ ID No.1所示的核苷酸序列缺失、替代或增加一个或多个碱基得到的多核苷酸。
其中一个实施例中，所述甲基转移酶突变体的核苷酸序列为如SEQ ID No.28～SEQ ID No.42所示的序列中的一种。
其中一个实施例中，所述甲基转移酶突变体为可溶性酶、固定化酶或者基因工程酶。
一种重组载体，含有上述甲基转移酶突变体的编码序列。
其中一个实施例中，包括如下步骤：采用第一扩增引物对对第一载体进行PCR扩增，得到重组载体，其中，所述第一载体含有如SEQ ID No.2所示的氨基酸序列对应的编码序列，所述第一扩增引物对含有所述甲基转移酶突变体的突变位点对应的核苷酸序列。
其中一个实施例中，所述第一扩增引物对选自如下引物对中的至少一对：核苷酸序列如SEQ ID No.20和SEQ ID No.21所示的引物对、核苷酸序列如SEQ ID No.22和SEQ IDNo.23所示的引物对、核苷酸序列如SEQ ID No.24和SEQ ID No.25所示的引物对、核苷酸序列如SEQ ID No.26和SEQ ID No.27所示的引物对。
一种重组工程菌，含上述重组载体。
上述甲基转移酶突变体、上述重组载体或者上述重组工程菌在催化甲基化反应中的应用。
附图说明
图1为氨基酸序列如SEQ ID No.2所示的甲基转移酶的蛋白模拟晶体结构示意图及突变点在晶体结构上的分布示意图。
具体实施方式
为使本申请的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例及附图对本申请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申请。但是本申请能够以很多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申请内涵的情况下做类似改进，因此本申请不受下面公开的具体实施的限制。
本申请一实施方式提供一种甲基转移酶突变体包括：
(a)、由如SEQ ID No.2所示的氨基酸序列缺失、替代或增加一个或多个氨基酸组成的多肽；或，
(b)、与如SEQ ID No.2所示的氨基酸序列组成的多肽具有至少90％同源性的多肽。
研究发现，包括由如SEQ ID No.2所示的氨基酸序列缺失、替代或增加一个或多个氨基酸得到的多肽；或，与如SEQ ID No.2所示的氨基酸序列组成的多肽具有至少90％同源性的多肽的甲基转移酶突变体具有较好的热稳定性。经试验验证，上述甲基转移酶突变体在45℃下的半衰期在26min以上，高于野生型甲基转移酶的半衰期，热稳定性较好，适于在较高的温度下催化甲基化反应，尤其是含有如SEQ ID No.2所示的氨基酸序列同时发生C185I突变、V218I突变S250A突变得到的多肽的甲基转移酶突变体在45℃下半衰期大约是野生型甲基转移酶的4倍，具有意料不到的效果。
如SEQ ID No.2所示的氨基酸序列为来源于Trypanosoma grayi的野生型甲基转移酶TGMT，命名为TGMT蛋白。编码该蛋白的核苷酸序列为SEQ ID No.1。
具体地，如SEQ ID No.2所示的氨基酸序列为MRRWGQSVTAVLLSLRVARSFTSFRTTRDVQMVSTIQVDECDEATDSTANVTGADKDAGDADESVVEMSEVEMQALPKTFRLSMPKESCLSTVERQQLTRMIFETHYAPEGAGKEDRAMWYQSALEVQWLEEWAFSDGEKDKDEARRAVKKAVLQRVDLHKPLAYIIGHQPFYGCNIKCSPPLLCPRPETEMWTHWYVSNYLNRSASTGGGQRSALRVLDMCCGTGCIGIAIATHVPHAEVVAVDIMDESVRAADENARLNGLDSRRYRVIKSDMFEAFLVKDSHNDSDVGRLNEEGVSGKRRLEEQHIGSFDVVVANPPYVLPEQYVNLPPGIKLWESKLALVGDAKREQQQYLYFQELCELGAAVLKPKTQRNTALVNAPNLIIEVGLQAERVASIMERSDLWEEVSVHLDYAQQPRWISANSTH。
在其中一个实施例中，甲基转移酶突变体包括：由如SEQ ID No.2所示的氨基酸序列中第185位氨基酸、第218位氨基酸、第250位氨基酸及第317位氨基酸中的至少一个发生突变得到的多肽。
本申请对氨基酸序列如SEQ ID No.2所示的野生型甲基转移酶进行结构分析及耐热性研究的过程中，发现第185位氨基酸(即C，半胱氨酸)、第218位氨基酸(即V，缬氨酸)、第250位氨基酸(即S，丝氨酸)、第317位氨基酸(即A，丙氨酸)、第409位氨基酸(即S，丝氨酸)对该酶的耐热性能影响较大，尤其是第185位氨基酸、第218位氨基酸、第250位氨基酸及第317位氨基酸对其酶耐热性能影响明显。
经进一步研究发现：甲基转移酶突变体，包括由如SEQ ID No.2所示的氨基酸序列发C185I突变、V218I突变、S250A突变及A317S突变中至少一种得到的多肽的，其在45℃下半衰期长于野生型甲基转移酶，热稳定性较好，适于在较高的温度下催化甲基化反应。
其中，甲基转移酶突变体包括：由如SEQ ID No.2所示的氨基酸序列发生C185I突变、V218I突变、S250A突变及A317S突变中的任意一种得到的多肽。
甲基转移酶突变体包括：由如SEQ ID No.2所示的氨基酸序列同时发生C185I突变、V218I突变、S250A突变及A317S突变中的任意两种得到的多肽。
甲基转移酶突变体包括：由如SEQ ID No.2所示的氨基酸序列同时发生C185I突变、V218I突变、S250A突变及A317S突变中的任意三种得到的多肽。
甲基转移酶突变体包括：由如SEQ ID No.2所示的氨基酸序列同时发生C185I突变、V218I突变、S250A突变及A317S突变得到的多肽。
在一个具体示例中，甲基转移酶突变体的氨基酸序列为如SEQ ID No.3～SEQ IDNo.17所示的序列中的一种。
其中一个实施例中，甲基转移酶突变体的编码序列包括：
(a)、与如SEQ ID No.1所示的核苷酸序列组成的多核苷酸具有至少90％同源性的多核苷酸；或，
(b)、由如SEQ ID No.1所示的核苷酸序列缺失、替代或增加一个或多个碱基得到的多核苷酸。
在一个具体示例中，甲基转移酶突变体的核苷酸序列为如SEQ ID No.28～SEQ IDNo.42所示的序列中的一种。
由于同一氨基酸可由几种不同的密码子来决定，故同一氨基酸可对应不同的核苷酸序列。因此本申请中的甲基转移酶突变体的氨基酸序列，包括由如SEQ ID No.1所示核苷酸序列进行1个或几个核苷酸取代而得到的密码子同义突变的核苷酸序列所编码得到。本领域技术人员可以根据本申请公开的甲基转移酶突变体的氨基酸序列，根据现有分子生物学技术，采用cDNA克隆和定点突变的方法或其他适合的方法获得本申请的甲基转移酶突变体，因此，编码上述甲基转移酶突变体的核苷酸序列并不仅限于如SEQ ID No.1所示的核苷酸序列。如果编码得到的蛋白与甲基转移酶突变体没有明显的功能差异，也包括在本申请的范围内。
此外，由于蛋白编码序列的多态性及变异，天然存在的蛋白质会出现基因突变，编码序列中碱基被缺失、替代或增加，或氨基酸的缺失、插入、取代或其它变异，从而导致蛋白质的氨基酸序列出现一个或多个氨基酸被缺失、替代或增加。因此，存在着一些生理和生物活性上基本等同于无变异蛋白质的蛋白。这些结构不同于相应的蛋白质，但与该蛋白质没有明显的功能差异的多肽或蛋白称为功能等同变异体。
功能等同的变异体同样适用于通过缺失、插入和突变等人工手段改变一个或多个密码子，从而向一种蛋白质的氨基酸序列中导入这类变异而制成的多肽。尽管这样能获得更多不同形式的变异体，但所得的变异体作为功能等同变异体的前提是其生理活性基本等同于原始无变异蛋白质的活性。
一般的，功能等同变异体的编码序列是同源的，因此，由至少一种改变(如蛋白质的编码序列中一个或多个碱基的缺失、插入或取代或者蛋白质的氨基酸序列中有一个或多个氨基酸缺失、插入或取代)所得的多肽或蛋白一般具有功能上等同于所述蛋白质的活性，因此，由上述核苷酸序列编码的到的多肽或上述氨基酸序列组成的多肽，如果编码得到的蛋白与甲基转移酶突变体没有明显的功能差异，也包括在本申请的范围内。
其中一个实施例中，甲基转移酶突变体为可溶性酶、固定化酶或者基因工程酶。
上述甲基转移酶突变体具有较高的热稳定性，适于在较高的温度下催化甲基化反应。
本申请一实施方式提供一种重组载体，含有上述甲基转移酶突变体的编码序列。
其中，重组载体为克隆载体或表达载体。
具体地，重组载体为含有上述甲基转移酶突变体的编码序列的pQE80L载体。需要说明的是，重组载体不限于为含有上述甲基转移酶突变体的编码序列的pQE80L载体，也可以将甲基转移酶突变体基因整合到其他载体上，例如可以为pET21b、pET22b、pET32a、pQE30等载体。
本申请一实施方式提供上述重组载体的制备方法，包括如下步骤：采用第一扩增引物对对第一载体进行PCR扩增，得到重组载体，其中，第一载体含有如SEQ ID No.2所示的氨基酸序列对应的编码序列。第一扩增引物对含有所述甲基转移酶突变体的突变位点对应的核苷酸序列。
其中，第一扩增引物对选自如下引物对中的至少一对：核苷酸序列如SEQ IDNo.20和SEQ ID No.21所示的引物对、核苷酸序列如SEQ ID No.22和SEQ ID No.23所示的引物对、核苷酸序列如SEQ ID No.24和SEQ ID No.25所示的引物对、核苷酸序列如SEQ IDNo.26和SEQ ID No.27所示的引物对。
需要说明的是，甲基转移酶突变体的单点突变体的构建中，以上述第一扩增引物对中的一对进行PCR扩增。甲基转移酶突变体的多点突变体的构建中，对一个突变位点进行单点突变后，再进行第二突变位点进行突变，依次叠加获得。具体地，按照以下方式进行：一个突变位点的扩增引物对扩增后，再用另一个突变位点的扩增引物对对含有在先突变的扩增产物进行扩增。
在其中一个实施例中，采用第一扩增引物对对第一载体进行PCR扩增的步骤之前，还包括构建第一载体的步骤。
其中，构建第一载体的步骤包括：采用序列如SEQ ID No.18～SEQ ID No.19所示的目的基因扩增引物对扩增目的基因，目的基因为如SEQ ID No.2所示的氨基酸序列对应的编码序列；将目的基因连接到空载体中，转化，提取阳性质粒，得到第一载体。具体地，如SEQ ID No.18所示的序列为：5’-CGCATATGATGCGGCGATGGGGTCAAT-3’(即上游引物，带下划线碱基为限制性内切酶NdeI识别位点)，如SEQ ID No.19所示的序列为：5’-TCAGCTCTCGAGTTAATGCGTGGAGTTGGCGG-3’(即下游引物，带下划线碱基为限制性内切酶XhoI识别位点)。
在其中一个实施例中，在采用第一扩增引物对对第一载体进行PCR扩增的步骤之前，还包括如下步骤：筛选甲基转移酶突变体的突变位点。
具体地，筛选甲基转移酶突变体的突变位点包括如下步骤：通过在NCBI数据库(http://www.ncbi.nlm.nih.gov/)中搜索甲基转移酶氨基酸序列(如SEQ ID No.2所示)，去除重复出现的相同序列后选取与目标蛋白序列一致性(identity)大于50％的蛋白质序列，然后进行多序列比对及Consensus sequence分析，并对野生型甲基转移酶进行结构分析及耐热性研究，发现第185位氨基酸(即C，半胱氨酸)、第218位氨基酸(即V，缬氨酸)、第250位氨基酸(即S，丝氨酸)、第317位氨基酸(即A，丙氨酸)、第409位氨基酸(即S，丝氨酸)对该酶的耐热性能影响较大，尤其是第185位氨基酸、第218位氨基酸、第250位氨基酸及第317位氨基酸对其酶耐热性能影响明显；同时根据蛋白模拟晶体结构进行突变位点分析和设计发现如SEQ ID No.2所示的氨基酸序列发生C185I、V218I、S250A、A317S中的至少一种得到的甲基转移酶突变体的热稳定性较高。
目前，在甲基转移酶的基因研究当中还未有关于生物信息学的研究报道，现今成熟的生物信息学技术必然为甲基转移酶的研究提供新的手段和方法，同时也将为甲基转移酶的功能研究提供新的领域。蛋白质工程是以蛋白质分子的结构规律及其生物功能的关系作为基础，通过化学、物理和分子生物学的手段进行基因修饰或基因合成，对现有蛋白质进行改造或制造一种新的蛋白质以满足人类对生产和生活的需求。理性设计是蛋白质工程改造中最常用的方法，利用计算机辅助分子模型结合定点突变，以实现蛋白质功能优化。Consensus Concept理论是基于同源蛋白的氨基酸序列信息，从进化的角度分析能改善蛋白功能的信息。本申请以Consensus Concept理论为指导思想，对甲基转移酶突变体家族序列进行整合分析，并结合生物信息学及晶体学方法辅助，进行突变位点分析和设计，获得具高热稳定性的甲基转移酶突变体。
上述重组载体的构建方法能够表达高稳定性的甲基转移酶突变体的重组载体。上述重组载体能够用于生产甲基转移酶突变体，以适于在较高的温度下催化甲基化反应。
本申请一实施方式提供一种重组工程菌含有上述实施方式的重组载体。
上述重组工程菌用于生产甲基转移酶突变体，以适于在较高的温度下催化甲基化反应；并且构建的高效表达的重组工程菌，具有其培养周期短、培养条件简单、目的蛋白产量高、纯化简单的优点。
进一步地，重组工程菌为含有上述实施方式的重组载体的大肠杆菌。可选地，重组工程菌为含有上述实施方式的重组载体的大肠杆菌BL21(DE3)。需要说明的是，重组工程菌不限于为含有上述实施方式的重组载体的大肠杆菌，也可以使用革兰氏阳性菌、革兰氏阴性菌、酵母和真菌等微生物宿主进行目的蛋白的表达。
以下为具体实施例部分。
实施例中采用试剂和仪器如非特别说明，均为本领域常规选择。实施例中未注明具体条件的实验方法，通常按照常规条件，例如文献、书本中所述的条件或者试剂盒生产厂家推荐的方法实现。实施例中所使用的试剂均为市售。
以下实施例中，如特别说明，基因来源：甲基转移酶来源于Trypanosoma grayi的野生型甲基转移酶TGMT，命名为TGMT蛋白，编码该蛋白的核苷酸序列为SEQ ID No.1，氨基酸序列为SEQ ID No.2。TGMT基因由苏州金唯智生物科技有限公司提供；PCR扩增酶为东洋纺提供的KOD高保真聚合酶。
实施例1
1、野生型甲基转移酶TGMT基因的克隆
将野生型甲基转移酶基因以大肠杆菌为宿主细胞进行密码子优化，得到优化的TGMT基因，其核苷酸序列为SEQ ID No.1，表达的氨基酸序列为SEQ ID No.2。
以SEQ ID No.1作为目的基因，采用上游扩增引物SEQ ID No.18和下游扩增引物SEQ ID No.19扩增目的基因。扩增条件为：在95℃下扩增2min，然后在56℃下扩增20sec、在72℃下扩增90sec，共30个循环，最后在72℃下扩增10min。
待反应结束，采用1.5％琼脂糖凝胶电泳检测PCR扩增产物，得到1.0kb的条带，其长度符合预期结果。按照捷瑞DNA胶回收试剂盒标准操作，回收、纯化该目的片段，使用限制性核酸内切酶XhoI和NdeI对该目的片段以及pET28a质粒进行双酶切，然后采用T4 DNA连接酶进行连接，将得到的连接产物转化至大肠杆菌BL21(DE3)感受态细胞中，将转化细胞涂布于含有100μg/mL卡那霉素的LB平板上，提取阳性克隆质粒，进行测序，结果显示克隆的甲基转移酶TGMT基因序列正确，且已正确接入pET28a质粒，得到重组质粒pET28a-TGMT。
2.TGMT蛋白的表达和纯化
将甘油管中的工程菌按体积比1％接种到含100μg/mL Kan的4mL LB培养基试管中，在37℃、220rpm条件下培养12h；取菌液4mL转接至含100μg/mL Kan的1L LB培养基摇瓶中，在37℃、220rpm条件下培养2.5h，使OD600达到0.6-0.8，加入1mM IPTG诱导剂，在25℃、200rpm条件下诱导培养14h。将发酵后收获的大肠杆菌菌体悬液超声破碎，再经过一步Ni-NTA亲和层析处理，得到纯度＞95％的TGMT蛋白，氨基酸序列为SEQ ID No.2。
实施例2
甲基转移酶突变体的构建、表达及纯化
本申请对野生型甲基转移酶进行结构分析及耐热性研究的过程中，发现第185位氨基酸(即C，半胱氨酸)、第218位氨基酸(即V，缬氨酸)、第250位氨基酸(即S，丝氨酸)、第317位氨基酸(即A，丙氨酸)、第409位氨基酸(即S，丝氨酸)对该酶的耐热性能影响较大，尤其是第185位氨基酸、第218位氨基酸、第250位氨基酸及第317位氨基酸对其酶耐热性能影响明显；同时根据蛋白模拟晶体结构进行突变位点分析和设计发现如SEQ ID No.2所示的氨基酸序列发生C185I、V218I、S250A、A317S中的至少一种得到的甲基转移酶突变体的热稳定性较高。该野生型甲基转移酶的蛋白模拟晶体结构示意图如图1所示。
1、TGMT蛋白单点突变体的构建
以实施例1得到的重组质粒pET28a-TGMT为模板，以带有突变位点的一对互补的寡核苷酸为扩增引物，用KOD高保真酶(由TakaRa公司提供)进行全质粒PCR扩增，获得具有特定突变位点的重组质粒；
其中，所采用的扩增引物对为：
(1)突变位点C185I：上游扩增引物的序列如SEQ ID No.20所示，下游扩增引物的序列如SEQ ID No.21所示。
(2)突变位点V218I：上游扩增引物的序列如SEQ ID No.22所示，下游扩增引物的序列如SEQ ID No.23所示。
(3)突变位点S250A：上游扩增引物的序列如SEQ ID No.24所示，下游扩增引物的序列如SEQ ID No.25所示。
(4)突变位点A317S：上游扩增引物的序列如SEQ ID No.26所示，下游扩增引物的序列如SEQ ID No.27所示。
扩增条件为：在95℃下扩增2min，然后在56℃下扩增20sec、在72℃下扩增90sec，共30个循环，最后在72℃下扩增10min；胶回收PCR扩增产物，采用DpnI酶(由Fermentas公司提供)在37℃条件下消化胶回收产物2h，降解初始模板；将消化产物转化至大肠杆菌BL21(DE3)感受态细胞中，涂布到含有100μg/mL卡那霉素LB琼脂平板上，37℃过夜培养，筛选阳性克隆，测序验证，得到含甲基转移酶单点突变体的重组菌。
2、TGMT蛋白组合突变体的构建
使用与单点突变体相似的构建方法，将稳定性提高的单点突变体累加组合，在SEQID No.2所示的氨基酸序列选择多个突变位点进行组合，如从上述4个突变位点中选择2～4个突变位点进行组合，分别得到不同的甲基转移酶组合突变体：
(1)选择2个突变位点进行组合，可构建6种热稳定性提高的甲基转移酶突变体及甲基转移酶组合突变体，其组合突变位点分别为：C185I/V218I、C185I/S250A、C185I/A317S、V218I/S250A、V218I/A317S、S250A/A317S；该6种热稳定性提高的甲基转移酶组合突变体的氨基酸序列分别如SEQ ID No.7、SEQ ID No.8、SEQ ID No.9、SEQ ID No.10、SEQ IDNo.11、SEQ ID No.12所示。
(2)选择3个突变位点进行组合，可构建4种热稳定性提高的甲基转移酶组合突变体，其组合突变位点分别为：C185I/V218I/S250A、C185I/V218I/A317S、V218I/S250A/A317S、C185I/S250A/A317S，该4种热稳定性提高的甲基转移酶组合突变体的氨基酸序列分别如SEQ ID No.13、SEQ ID No.14、SEQ ID No.15、SEQ ID No.16所示。
(3)选择4个突变位点进行组合，可构建1种热稳定性提高的甲基转移酶组合突变体，其组合突变位点分别为：C185I/V218I/S250A/A317S，该种热稳定性提高的甲基转移酶组合突变体的氨基酸序列如SEQ ID No.17所示。
其中，各甲基转移酶突变体的氨基酸序列和核苷酸序列如下：
突变位点为C185I的甲基转移酶突变体：氨基酸序列如SEQ ID No.3所示，核苷酸序列为SEQ ID No.28所示；
突变位点为V218I的甲基转移酶突变体：氨基酸序列如SEQ ID No.4所示，核苷酸序列为SEQ ID No.29所示；
突变位点为S250A的甲基转移酶突变体：氨基酸序列如SEQ ID No.5所示，核苷酸序列为SEQ ID No.30所示；
突变位点为A317S的甲基转移酶突变体：氨基酸序列如SEQ ID No.6所示，核苷酸序列为SEQ ID No.31所示；
突变位点为C185I/V218I的甲基转移酶突变体：氨基酸序列如SEQ ID No.7所示，核苷酸序列为SEQ ID No.32所示；
突变位点为C185I/S250A的甲基转移酶突变体：氨基酸序列如SEQ ID No.8所示，核苷酸序列为SEQ ID No.33所示；
突变位点为C185I/A317S的甲基转移酶突变体：氨基酸序列如SEQ ID No.9所示，核苷酸序列为SEQ ID No.34所示；
突变位点为V218I/S250A的甲基转移酶突变体：氨基酸序列如SEQ ID No.10所示，核苷酸序列为SEQ ID No.35所示；
突变位点为V218I/A317S的甲基转移酶突变体：氨基酸序列如SEQ ID No.11所示，核苷酸序列为SEQ ID No.36所示；
突变位点为S250A/A317S的甲基转移酶突变体：氨基酸序列如SEQ ID No.12所示，核苷酸序列为SEQ ID No.37所示；
突变位点为C185I/V218I/S250A的甲基转移酶突变体：氨基酸序列如SEQ IDNo.13所示，核苷酸序列为SEQ ID No.38所示；
突变位点为C185I/V218I/A317S的甲基转移酶突变体：氨基酸序列如SEQ IDNo.14所示，核苷酸序列为SEQ ID No.39所示；
突变位点为V218I/S250A/A317S的甲基转移酶突变体：氨基酸序列如SEQ IDNo.16所示，核苷酸序列为SEQ ID No.41所示；
突变位点为C185I/S250A/A317S的甲基转移酶突变体：氨基酸序列如SEQ IDNo.15所示，核苷酸序列为SEQ ID No.40所示；
突变位点为C185I/V218I/S250A/A317S的甲基转移酶突变体：氨基酸序列如SEQID No.17所示，核苷酸序列为SEQ ID No.42所示。
测试例1
甲基转移酶的酶学性质表征
将野生型甲基转移酶以及实施例2提供的多种甲基转移酶突变体进行热稳定性测试，按照常规甲基转移酶活力测定方法(参见文献DOI:10.1016/j.yexcr.2006.03.006)，具体为：
在一定温度下下孵育酶液，在不同处理时间取样，测定野生型甲基转移酶或甲基转移酶突变体残余活力百分比，以残余活力百分比的ln值对时间t(min)作图，直线的斜率为失活常数kinact，由t1/2＝ln2/kinact得到该种野生型甲基转移酶或甲基转移酶突变体在该温度下的半衰期。测定结果详见表1。
表1野生型甲基转移酶及其酶突变体的酶学性质表征
从表1可以看出，上述甲基转移酶突变体在45℃下的半衰期在26min以上，均长于野生型甲基转移酶在45℃下的半衰期，尤其是组合突变体表现出单点突变体热稳定性的叠加效果，特别是发生C185I/V218I/S250A突变得到的甲基转移酶突变体在45℃下的半衰期达105min，是野生型甲基转移酶的4倍，具有较好的热稳定性。
综上，本申请所提供的甲基转移酶突变体的热稳定性较好，适于在较高的温度下催化甲基化反应。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。
序列表
<110> 苏州瀚源新酶生物科技有限公司
<120> 甲基转移酶突变体、重组载体、重组工程菌及其应用
<160> 42
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tctgtccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg ggttttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgagagt gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttgc gaacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 2
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Cys Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Val Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ser Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ala Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 3
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Ile Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Val Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ser Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ala Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 4
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Cys Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Ile Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ser Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ala Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 5
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Cys Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Val Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ala Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ala Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 6
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Cys Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Val Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ser Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ser Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 7
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Ile Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Ile Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ser Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ala Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 8
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Ile Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Val Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ala Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ala Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 9
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Ile Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Val Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ser Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ser Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 10
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Cys Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Ile Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ala Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ala Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 11
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Cys Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Ile Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ser Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ser Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 12
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Cys Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Val Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ala Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ser Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 13
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Ile Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Ile Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ala Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ala Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 14
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Ile Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Ile Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ser Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ser Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 15
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Ile Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Val Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ala Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ser Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 16
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Cys Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Ile Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ala Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ser Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 17
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Met Arg Arg Trp Gly Gln Ser Val Thr Ala Val Leu Leu Ser Leu Arg
1 5 10 15
Val Ala Arg Ser Phe Thr Ser Phe Arg Thr Thr Arg Asp Val Gln Met
20 25 30
Val Ser Thr Ile Gln Val Asp Glu Cys Asp Glu Ala Thr Asp Ser Thr
35 40 45
Ala Asn Val Thr Gly Ala Asp Lys Asp Ala Gly Asp Ala Asp Glu Ser
50 55 60
Val Val Glu Met Ser Glu Val Glu Met Gln Ala Leu Pro Lys Thr Phe
65 70 75 80
Arg Leu Ser Met Pro Lys Glu Ser Cys Leu Ser Thr Val Glu Arg Gln
85 90 95
Gln Leu Thr Arg Met Ile Phe Glu Thr His Tyr Ala Pro Glu Gly Ala
100 105 110
Gly Lys Glu Asp Arg Ala Met Trp Tyr Gln Ser Ala Leu Glu Val Gln
115 120 125
Trp Leu Glu Glu Trp Ala Phe Ser Asp Gly Glu Lys Asp Lys Asp Glu
130 135 140
Ala Arg Arg Ala Val Lys Lys Ala Val Leu Gln Arg Val Asp Leu His
145 150 155 160
Lys Pro Leu Ala Tyr Ile Ile Gly His Gln Pro Phe Tyr Gly Cys Asn
165 170 175
Ile Lys Cys Ser Pro Pro Leu Leu Ile Pro Arg Pro Glu Thr Glu Met
180 185 190
Trp Thr His Trp Tyr Val Ser Asn Tyr Leu Asn Arg Ser Ala Ser Thr
195 200 205
Gly Gly Gly Gln Arg Ser Ala Leu Arg Ile Leu Asp Met Cys Cys Gly
210 215 220
Thr Gly Cys Ile Gly Ile Ala Ile Ala Thr His Val Pro His Ala Glu
225 230 235 240
Val Val Ala Val Asp Ile Met Asp Glu Ala Val Arg Ala Ala Asp Glu
245 250 255
Asn Ala Arg Leu Asn Gly Leu Asp Ser Arg Arg Tyr Arg Val Ile Lys
260 265 270
Ser Asp Met Phe Glu Ala Phe Leu Val Lys Asp Ser His Asn Asp Ser
275 280 285
Asp Val Gly Arg Leu Asn Glu Glu Gly Val Ser Gly Lys Arg Arg Leu
290 295 300
Glu Glu Gln His Ile Gly Ser Phe Asp Val Val Val Ser Asn Pro Pro
305 310 315 320
Tyr Val Leu Pro Glu Gln Tyr Val Asn Leu Pro Pro Gly Ile Lys Leu
325 330 335
Trp Glu Ser Lys Leu Ala Leu Val Gly Asp Ala Lys Arg Glu Gln Gln
340 345 350
Gln Tyr Leu Tyr Phe Gln Glu Leu Cys Glu Leu Gly Ala Ala Val Leu
355 360 365
Lys Pro Lys Thr Gln Arg Asn Thr Ala Leu Val Asn Ala Pro Asn Leu
370 375 380
Ile Ile Glu Val Gly Leu Gln Ala Glu Arg Val Ala Ser Ile Met Glu
385 390 395 400
Arg Ser Asp Leu Trp Glu Glu Val Ser Val His Leu Asp Tyr Ala Gln
405 410 415
Gln Pro Arg Trp Ile Ser Ala Asn Ser Thr His
420 425
<210> 18
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cgcatatgat gcggcgatgg ggtcaat 27
<210> 19
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
tcagctctcg agttaatgcg tggagttggc gg 32
<210> 20
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
cgcatatgac cactcctcat tccgcgacca ga 32
<210> 21
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
tcagctctcg agggtcgcgg aatgaggagt ggtgga 36
<210> 22
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
cgcatatgcg cttcggattt tggacatgtg ct 32
<210> 23
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tcagctctcg agcatgtcca aaatccgaag cgccga 36
<210> 24
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
cgcatatgat ggatgaggct gtgagggcgg ctgatgagaa 40
<210> 25
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tcagctctcg agatcagccg ccctcacagc ctcatccata atgt 44
<210> 26
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
cgcatatgac gttgttgtta gtaacccacc gtacgtcttg cct 43
<210> 27
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
tcagctctcg aggacgtacg gtgggttact aacaacaacg tcaaacg 47
<210> 28
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tcattccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg ggttttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgagagt gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttgc gaacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 29
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tctgtccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg gattttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgagagt gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttgc gaacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 30
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tctgtccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg ggttttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgaggct gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttgc gaacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 31
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tctgtccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg ggttttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgagagt gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttag taacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 32
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tcattccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg gattttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgagagt gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttgc gaacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 33
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tcattccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg ggttttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgaggct gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttgc gaacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 34
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tcattccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg ggttttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgagagt gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttag taacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 35
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tctgtccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg gattttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgaggct gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttgc gaacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 36
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tctgtccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg gattttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgagagt gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttag taacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 37
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tctgtccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg ggttttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgaggct gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttag taacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 38
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tcattccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg gattttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgaggct gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttgc gaacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 39
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tcattccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg gattttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgagagt gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttag taacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 40
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tcattccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg ggttttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgaggct gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttag taacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 41
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tctgtccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg gattttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgaggct gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttag taacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
<210> 42
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
atgcggcgat ggggtcaatc agtcaccgcg gtgttactct ctctgcgcgt ggcccgctcc 60
tttacatctt tccgtacgac gcgtgatgta cagatggtct ctacaatcca agttgacgag 120
tgtgacgaag cgacagactc cacagcaaat gtcactgggg ctgacaagga tgcgggtgat 180
gcagatgaat ctgttgtcga gatgagtgag gtggagatgc aggcgcttcc caagacattt 240
cggctgtcga tgccgaagga atcgtgcctc tcaacggtgg agcggcagca gctcacacgg 300
atgatcttcg agacgcacta cgcgccggag ggtgcaggaa aggaggaccg cgcaatgtgg 360
taccagagcg cgttggaggt gcagtggctt gaggaatggg cattcagtga cggagagaag 420
gacaaggatg aggctcgtcg cgctgtcaag aaggcggtgc tgcaacgtgt tgatttacac 480
aagccactcg cctatattat cgggcatcag ccgttttacg gttgcaacat taagtgttct 540
ccaccactcc tcattccgcg accagagacg gagatgtgga cccattggta tgtcagcaac 600
tacctgaatc gttcggcatc aacgggagga ggacagaggt cggcgcttcg gattttggac 660
atgtgctgcg gcaccggttg catcggaatt gccatcgcca cgcatgtgcc gcacgcagaa 720
gttgtcgcgg ttgacattat ggatgaggct gtgagggcgg ctgatgagaa tgcaaggcta 780
aacggtctgg actcgagacg ttatcgtgtt attaagagcg acatgtttga ggcgttcctt 840
gtgaaggata gtcataatga cagtgatgtt ggaagactga acgaagaggg tgtcagtgga 900
aagagaagac tagaagaaca acacattggc tcgtttgacg ttgttgttag taacccaccg 960
tacgtcttgc ctgaacagta cgtcaacctt ccaccaggca ttaaactgtg ggagtcaaag 1020
ctcgccttgg tgggtgacgc gaagcgggag cagcagcagt atctctactt ccaggagctc 1080
tgtgagctgg gggcagcggt gctaaagccg aagacccaac gcaacacggc cctcgtgaat 1140
gcgccgaatc tcatcattga ggtgggtctg caggcggagc gcgttgcgtc catcatggag 1200
cgcagtgatc tgtgggagga ggtgagtgtg cacctcgact acgcgcagca gccgcggtgg 1260
atctccgcca actccacgca ttaa 1284
Claims (5)
1.一种甲基转移酶突变体，其特征在于，所述甲基转移酶突变体的氨基酸序列为如SEQID No.3～SEQ ID No.15、SEQ ID No.17所示的序列中的一种。
2.根据权利要求1所述的甲基转移酶突变体，其特征在于，编码所述甲基转移酶突变体的核苷酸序列为如SEQ ID No.28～SEQ ID No.40、SEQ ID No.42所示的序列中的一种。
3.一种重组载体，其特征在于，含有权利要求1-2任一项所述的甲基转移酶突变体的编码序列。
4.一种重组工程菌，其特征在于，含有权利要求3所述的重组载体。
5.权利要求1-4任一项所述的甲基转移酶突变体、权利要求3所述的重组载体或者权利要求4所述的重组工程菌在催化甲基化反应中的应用。
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| JP2000093184A (ja) * | 1998-09-22 | 2000-04-04 | Keiichi Hiramatsu | 新規酵素及び該酵素をコードするポリヌクレオチド |
| CN101743016A (zh) * | 2007-07-13 | 2010-06-16 | 传染性疾病研究院 | 预防、治疗和诊断利什曼病的利什曼原虫固醇24-c-甲基转移酶组合物 |
| CN112359032A (zh) * | 2020-11-11 | 2021-02-12 | 中国科学院苏州生物医学工程技术研究所 | 突变型酯酶及其应用、重组载体及其制备方法和应用、重组工程菌及其应用 |
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| CN101743016A (zh) * | 2007-07-13 | 2010-06-16 | 传染性疾病研究院 | 预防、治疗和诊断利什曼病的利什曼原虫固醇24-c-甲基转移酶组合物 |
| CN112359032A (zh) * | 2020-11-11 | 2021-02-12 | 中国科学院苏州生物医学工程技术研究所 | 突变型酯酶及其应用、重组载体及其制备方法和应用、重组工程菌及其应用 |
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