CN116262916A - 一种重组微生物及其构建方法与应用 - Google Patents
一种重组微生物及其构建方法与应用 Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种重组微生物及其构建方法与应用，本发明所提供的重组微生物涉及磷酸烯醇式丙酮酸羧化酶、二氢硫辛酰胺乙酰转移酶的突变体。具体地，本发明所提供的重组微生物中含有编码基因ppc和/或aceF的突变体；更具体地，重组微生物中ppc基因编码的磷酸烯醇式丙酮酸羧化酶的第186位氨基酸和/或aceF基因编码的二氢硫辛酰胺乙酰转移酶的第77位氨基酸发生突变。本发明通过对磷酸烯醇式丙酮酸羧化酶，和/或二氢硫辛酰胺乙酰转移酶进行修饰，使得微生物能够高效地生成缬氨酸等支链氨基酸或其衍生物。
Description
技术领域
本发明涉及生物工程技术领域，具体涉及一种重组微生物及其构建方法与应用。
背景技术
L-缬氨酸(L-valine)，化学名称为L-α-氨基异戊酸，分子式为C5H11NO2，相对分子质量为117.15。L-缬氨酸呈白色结晶或结晶性粉末，无臭，味苦，在水中溶解度：25℃为88.5g/L，50℃为96.2g/L，不溶于冷乙醇、乙醚、丙酮。L-缬氨酸等电点为5.96，熔点315℃。
L-缬氨酸是人体八种必需氨基酸之一，又是三种支链氨基酸(包括缬氨酸、亮氨酸、异亮氨酸)之一，因其特殊的结构和功能，在人类生命代谢中具有特别重要的地位。L-缬氨酸可以广泛应用于医药工业、食品工业和饲料工业等。医药工业中，L-缬氨酸可作氨基酸输液、综合氨基酸制剂的主要成分，可治疗肝功能衰竭、中枢神经系统功能紊乱。食品工业中，L-缬氨酸可用作食品添加剂、营养增补液及风味剂等。L-缬氨酸也可用作氨基酸功能量饮料与运动员饮料，有形成肌肉、强化肝功能、减轻肌肉疲劳等作用。饲料工业中，对动物的乳腺组织分泌乳汁有重要的促进作用。
目前L-缬氨酸的生产方法有三种：提取法、化学合成法、微生物发酵法。提取法和化学合成法由于原料来源受限制、生产成本高、污染环境，难以实现工业化生产。微生物发酵法生产L-缬氨酸具有原料成本低、反应条件温和、容易实现大规模生产等优点，是目前生产L-缬氨酸最主要的方法。但目前L-缬氨酸的菌种的发酵性能仍较差、L-缬氨酸的转化率仍较低，仍不能满足大规模工业化生产的需求。
发明内容
本发明的目的是提供一种高效率生产缬氨酸的新微生物，以及采用所述微生物生产缬氨酸的方法。
本发明通过修饰谷氨酸棒杆菌的磷酸烯醇式丙酮酸羧化酶，和/或二氢硫辛酰胺乙酰转移酶，使得谷氨酸棒杆菌能够高效地生成缬氨酸。
第一方面，本发明提供磷酸烯醇式丙酮酸羧化酶的突变体，所述磷酸烯醇式丙酮酸羧化酶突变体是，由ppc基因编码的磷酸烯醇式丙酮酸羧化酶的第186位氨基酸由甘氨酸突变为丙氨酸、缬氨酸或精氨酸。
磷酸烯醇式丙酮酸羧化酶(PEPC)，由ppc基因编码，催化磷酸烯醇式丙酮酸(PEP)生成草酰乙酸(OAA)。
在本发明提供的突变体中，磷酸烯醇式丙酮酸羧化酶突变体的氨基酸序列如SEQID No.10-12。
ppc基因编码的磷酸烯醇式丙酮酸羧化酶是丙酮酸合同的关键酶，增强磷酸烯醇式丙酮酸羧化酶的活性，有助于提高缬氨酸的前体物丙酮酸在胞内的水平，间接提高丙酮酸到缬氨酸的代谢流，从而实现缬氨酸的高产。本发明通过对ppc基因修饰，将磷酸烯醇式丙酮酸羧化酶的第186位氨基酸由甘氨酸(G)突变为丙氨酸(A)、缬氨酸(V)或精氨酸(R)，实现了对磷酸烯醇式丙酮酸羧化酶的突变，使得所述微生物产缬氨酸的能力与未修饰的菌株相比增强，最终提高了缬氨酸的产量。
第二方面，本发明提供，二氢硫辛酰胺乙酰转移酶的突变体，所述二氢硫辛酰胺乙酰转移酶突变体是，由aceF基因编码的二氢硫辛酰胺乙酰转移酶的第77位氨基酸由甘氨酸突变为天冬氨酸、缬氨酸或丙氨酸。
二氢硫辛酰胺乙酰转移酶(E2p)，由aceF基因编码，是丙酮酸脱氢酶多酶复合物(PDHC)的一个亚基。
PDHC是一组限速酶，由丙酮酸脱氢酶(E1p)、二氢硫辛酰胺乙酰转移酶(E2p)、二氢硫辛酰胺脱氢酶(E3p)3个亚基组成。在谷氨酸棒杆菌中，PDHC催化糖酵解过程产生的丙酮酸不可逆地氧化脱羧转化成乙酰辅酶A，将糖的有氧氧化与三羧酸循环和氧化磷酸化连接起来，是三羧酸循环的关键酶。
PDHC的亚基E2p/E3p与α-酮戊二酸脱氢酶复合体(ODHC)的亚基相同，即aceF基因与sucB基因的产物相同(均为二氢硫辛酰胺乙酰转移酶)，lpdA基因与lpd基因编码的产物相同(均为二氢硫辛酰胺脱氢酶)。
在本发明提供的突变体中，二氢硫辛酰胺乙酰转移酶的突变体的氨基酸序列如SEQ ID No.14-16所示。
丙酮酸脱氢酶多酶复合物(PDHC)是丙酮酸进入三羧酸循环的关键酶，降低PDHC的活性，有助于提高缬氨酸的前体物丙酮酸在胞内的水平，间接提高丙酮酸到缬氨酸的代谢流，从而实现缬氨酸的高产。本发明通过对aceF基因修饰，将二氢硫辛酰胺乙酰转移酶的第77位氨基酸由甘氨酸(G)突变为天冬氨酸(D)、缬氨酸(V)或丙氨酸(A)，实现了对丙酮酸脱氢酶多酶复合物的亚基(二氢硫辛酰胺乙酰转移酶)的突变，使得所述微生物产缬氨酸的能力与未修饰的菌株相比增强，最终提高了缬氨酸的产量。
第三方面，本发明提供编码上述突变体的核苷酸序列，编码磷酸烯醇式丙酮酸羧化酶突变体的核苷酸序列如SEQ ID No.2-4所示；编码二氢硫辛酰胺乙酰转移酶突变体的核苷酸序列如SEQ ID No.6-8所示。
第四方面，本发明请求保护含有上述核苷酸序列的生物材料，所述生物材料为表达盒、载体或宿主细胞。
第五方面，本发明提供一种重组微生物，所述重组微生物中含有磷酸烯醇式丙酮酸羧化酶突变体和/或二氢硫辛酰胺乙酰转移酶突变体。
在本发明提供的重组微生物中，所述磷酸烯醇式丙酮酸羧化酶突变体是，由ppc基因编码的磷酸烯醇式丙酮酸羧化酶的第186位氨基酸由甘氨酸突变为丙氨酸、缬氨酸或精氨酸；所述二氢硫辛酰胺乙酰转移酶突变体是，由aceF基因编码的二氢硫辛酰胺乙酰转移酶的第77位氨基酸由甘氨酸突变为天冬氨酸、缬氨酸或丙氨酸。
本发明提供的重组微生物为谷氨酸棒杆菌、北京棒杆菌或黄色短杆菌。
具体地，本发明提供三种类型的棒杆菌，第一种棒杆菌细胞内由ppc基因编码的磷酸烯醇式丙酮酸羧化酶的第186位氨基酸由甘氨酸(G)突变为丙氨酸(A)、缬氨酸(V)或精氨酸(R)，具体的，对应的碱基由GGT突变为GCT、GTT或CGT。
第二种棒杆菌，其细胞内由aceF基因编码的二氢硫辛酰胺乙酰转移酶的第77位氨基酸由甘氨酸(G)突变为天冬氨酸(D)、缬氨酸(V)或丙氨酸(A)，具体的，对应的碱基序列由GGC突变为GAC、GTC或GCC。
第三种棒杆菌为上述2种突变体的叠加，即其细胞内由ppc基因编码的磷酸烯醇式丙酮酸羧化酶的第186位氨基酸由甘氨酸(G)突变为丙氨酸(A)、缬氨酸(V)或精氨酸(R)，同时，其细胞内由aceF基因编码的二氢硫辛酰胺乙酰转移酶的第77位氨基酸由甘氨酸(G)突变为天冬氨酸(D)、缬氨酸(V)或丙氨酸(A)。
本发明还请求保护，上述的突变体或上述的核苷酸序列或上述的生物材料或上述的重组微生物在提高支链氨基酸或其衍生物产量中的应用。
第六方面，本发明提供ppc基因编码的磷酸烯醇式丙酮酸羧化酶突变体的构建方法，所用引物的序列如SEQ ID No.17-26。
以及，aceF基因编码的二氢硫辛酰胺乙酰转移酶突变体的构建方法，所用引物的序列如SEQ ID No.27-36。
本发明的有益效果在于：
(1)在微生物中，将磷酸烯醇式丙酮酸羧化酶的第186位氨基酸由甘氨酸(G)突变为丙氨酸(A)、缬氨酸(V)或精氨酸(R)，或二氢硫辛酰胺乙酰转移酶的第77位氨基酸由甘氨酸(G)突变为天冬氨酸(D)、缬氨酸(V)或丙氨酸(A)后，缬氨酸产量均有提升，尤以磷酸烯醇式丙酮酸羧化酶的第186位氨基酸由甘氨酸(G)突变为精氨酸(R)叠加二氢硫辛酰胺乙酰转移酶的第77位氨基酸由甘氨酸(G)突变为天冬氨酸(D)效果最好。
(2)本发明提供的磷酸烯醇式丙酮酸羧化酶突变菌株2-PPCG186A、2-PPCG186V、2-PPCG186R的L-缬氨酸产量和转化率较出发菌株显著提高，且仍能够保持较好的生长性能；二氢硫辛酰胺乙酰转移酶突变菌株2-aceFG77D、2-aceFG77V、2-aceFG77A的L-缬氨酸产量和转化率较出发菌株显著提高，生长略有下降，但仍能够保持较好的生长性能。磷酸烯醇式丙酮酸羧化酶和二氢硫辛酰胺乙酰转移酶双突变菌株2-PPCG186R-aceFG77D表现最为突出，缬氨酸产量为8.9g/L，比出发菌株产量提高3.0g/L，提高了50.8％。
(3)本发明提供的磷酸烯醇式丙酮酸羧化酶、二氢硫辛酰胺乙酰转移酶的突变体可应用于谷氨酸棒杆菌，北京棒杆菌或黄色短杆菌等，含有突变体的菌株可用于生产缬氨酸、异亮氨酸、亮氨酸等支链氨基酸或其衍生物。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例中涉及的引物名称及序列如表1所示。
表1 引物
实施例1构建磷酸烯醇式丙酮酸羧化酶突变菌株2-PPCG186A、2-PPCG186V、2-PPCG186R
本发明的出发菌株MHZ-1012-2为谷氨酸棒杆菌，其构建方法详见专利文件CN201611250330.1，该菌株于2016年11月30日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏中心地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏编号为CGMCC No.13406。
以MHZ-1012-2为出发菌株，将MHZ-1012-2中ppc基因突变为编码SEQ ID NO.2、3、4所示的磷酸烯醇式丙酮酸羧化酶突变体的基因，依次构建磷酸烯醇式丙酮酸羧化酶突变菌株2-PPCG186A、2-PPCG186V、2-PPCG186R。具体构建方法如下。
1、质粒pK18mobsacB-PPCG186A的构建
利用Phusion超保真聚合酶(New England BioLabs)，以出发菌株MHZ-1012-2的基因组为模板，以ppcG186A-UP-1F/ppcG186A-UP-1R为引物，制备重组片段UP-1，以ppcG186A-DN-2F/ppcG186A-DN-2R为引物，制备重组片段DN-1；以质粒pk18-mob-sacB为模板，以ppcG186A-pk18-3F/ppcG186A-pk18-3R为引物获得片段pk18-1，经琼脂糖凝胶回收试剂盒(天根)纯化，随后按照吉普森组装试剂盒配置体系进行反应，反应体系表2所示。
表2 吉普森组装反应体系
| 组分 | UP-1 | DN-1 | pk18-1 | CE Buffer | CE Exnase | 无菌水 |
| 体积/μL | 1 | 1 | 2 | 4 | 2 | 10 |
将配制的反应体系于37℃反应30min，吸取10μL转化Trans1T1感受态细胞(TransGen Biotech)，挑取单克隆，通过菌落PCR鉴定插入的片段正确，进一步酶切鉴定得到片段插入pK18mobsacB的阳性克隆，最后将质粒送至金唯智生物科技有限公司进行测序，将所得到的测序正确的质粒命名为pK18mobsacB-ppcG186A。
2、质粒pK18mobsacB-ppcG186V、pK18mobsacB-ppcG186R的构建
按上述1相同的方法，将引物ppcG186A-UP-1R分别替换为ppcG186V-UP-1R、ppcG186R-UP-1R，将引物ppcG186A-DN-2F分别替换为ppcG186V-DN-2F、ppcG186R-DN-2F，将所构建的质粒分别命名为pK18mobsacB-ppcG186V、pK18mobsacB-ppcG186R。
3、磷酸烯醇式丙酮酸羧化酶突变菌株2-PPCG186A、2-PPCG186V、2-PPCG186R的构建
将上述1、2所述方法构建获得的重组质粒pK18mobsacB-ppcG186A、pK18mobsacB-ppcG186V、pK18mobsacB-ppcG186R分别转入出发菌株MHZ-1012-2中，在含有15mg/L的卡那霉素的选择培养基上选择交换重组子。培养的温度为30℃，倒置培养。将筛选获得的转化子过夜培养于普通液体脑心浸液培养基中，培养温度为30℃，回转摇床220rpm振荡培养。此培养过程中，转化子发生第二次重组，通过基因交换将载体序列从基因组中除去。将培养物做连续梯度稀释(10-2连续稀释至10-4)，稀释液涂布在含有10％蔗糖的普通固体脑心浸液培养基上，33℃静置培养48h。对在此培养基上长出的转化子进行鉴定。通过PCR扩增目的序列，核苷酸测序分析，获得目的突变菌株，分别命名为2-PPCG186A、2-PPCG186V、2-PPCG186R。
实施例2构建二氢硫辛酰胺乙酰转移酶突变菌株2-aceFG77D、2-aceFG77V、2-aceFG77A
以MHZ-1012-2为出发菌株，将MHZ-1012-2中aceF基因突变为编码SEQ ID NO.6、7、8任一项所示的二氢硫辛酰胺乙酰转移酶突变体的基因，依次构建二氢硫辛酰胺乙酰转移酶突变菌株2-aceFG77D、2-aceFG77V、2-aceFG77A。具体构建方法如下。
1、质粒pK18mobsacB-aceFG77D、pK18mobsacB-aceFG77V、pK18mobsacB-aceFG77A的构建
按上述实施例1中质粒pK18mobsacB-ppcG186A的构建相同的方法，引物依次进行替换，从而完成构建，将所构建的质粒分别命名为pK18mobsacB-aceFG77D、pK18mobsacB-aceFG77V、pK18mobsacB-aceFG77A。
2、二氢硫辛酰胺乙酰转移酶突变菌株2-aceFG77D、2-aceFG77V、2-aceFG77A的构建
将上述1所述方法构建获得的重组质粒pK18mobsacB-aceFG77D、pK18mobsacB-aceFG77V、pK18mobsacB-aceFG77A分别转入出发菌株MHZ-1012-2中，在含有15mg/L的卡那霉素的选择培养基上选择交换重组子。培养的温度为30℃，倒置培养。将筛选获得的转化子过夜培养于普通液体脑心浸液培养基中，培养温度为30℃，回转摇床220rpm振荡培养。此培养过程中，转化子发生第二次重组，通过基因交换将载体序列从基因组中除去。将培养物做连续梯度稀释(10-2连续稀释至10-4)，稀释液涂布在含有10％蔗糖的普通固体脑心浸液培养基上，33℃静置培养48h。对在此培养基上长出的转化子进行鉴定。通过PCR扩增目的序列，核苷酸测序分析，获得目的突变菌株，分别命名为2-aceFG77D、2-aceFG77V、2-aceFG77A。
实施例3构建磷酸烯醇式丙酮酸羧化酶和二氢硫辛酰胺乙酰转移酶双突变菌株2-PPCG186R-aceFG77D
将上述实施例2所述方法构建获得的重组质粒pK18mobsacB-aceFG77D转入上述实施例1所述的方法构建获得的重组菌2-PPCG186R中，在含有15mg/L的卡那霉素的选择培养基上选择交换重组子。培养的温度为30℃，倒置培养。将筛选获得的转化子过夜培养于普通液体脑心浸液培养基中，培养温度为30℃，回转摇床220rpm振荡培养。此培养过程中，转化子发生第二次重组，通过基因交换将载体序列从基因组中除去。将培养物做连续梯度稀释(10-2连续稀释至10-4)，稀释液涂布在含有10％蔗糖的普通固体脑心浸液培养基上，33℃静置培养48h。对在此培养基上长出的转化子进行鉴定。通过PCR扩增目的序列，核苷酸测序分析，获得目的突变菌株命名为2-PPCG186R-aceFG77D。
实施例4菌株发酵生产L-缬氨酸
对实施例1-3构建的突变菌株进行发酵验证，具体方法如下：
1、培养基
种子培养基：豆粕抽提物15g/L，葡萄糖20g/L，硫酸铵7g/L，硫酸镁0.5g/L，磷酸二氢钾1g/L，磷酸氢二钾1g/L，尿素2g/L，余量为水，pH 7.2。
发酵培养基：豆粕抽提物15g/L，葡萄糖60g/L，硫酸铵20g/L，硫酸镁0.5g/L，磷酸二氢钾1g/L，磷酸氢二钾1g/L，尿素2g/L，碳酸钙40g/L，VB3 15mg/L，VH 50μg/L，VB1·HCl100μg/L，余量为水，pH 7.2。
2、摇瓶发酵生产L-缬氨酸
(1)种子培养：挑取斜面种子1环接至装有50mL种子培养基的500mL三角瓶中，30℃、220r/min振荡培养10-12h；
(2)发酵培养：将5mL种子液接种至装有50mL发酵培养基的500mL三角瓶中，30℃、220r/min振荡培养48h；
(3)取1mL发酵液离心(12000rpm，2min)，收集上清液，用HPLC检测酵液中的L-缬氨酸、L-亮氨酸。同时采用分光光度法检测发酵液在562nm下的OD值，结果如表3所示。
表3 菌株生长和缬氨酸产量检测结果
注：*表示与出发菌株相比具有显著差异。
表3结果显示，出发菌株MHZ-1012-2的L-缬氨酸产量仅为5.9g/L，磷酸烯醇式丙酮酸羧化酶突变菌株2-PPCG186A、2-PPCG186V、2-PPCG186R的L-缬氨酸产量和转化率较出发菌株显著提高，且仍能够保持较好的生长性能；二氢硫辛酰胺乙酰转移酶突变菌株2-aceFG77D、2-aceFG77V、2-aceFG77A的L-缬氨酸产量和转化率较出发菌株显著提高，生长略有下降，但仍能够保持较好的生长性能。磷酸烯醇式丙酮酸羧化酶和二氢硫辛酰胺乙酰转移酶双突变菌株2-PPCG186R-aceFG77D表现最为突出，缬氨酸产量为8.9g/L，比出发菌株产量提高3.0g/L，提高了50.8％。
由此可见，本发明提供的磷酸烯醇式丙酮酸羧化酶和/或二氢硫辛酰胺乙酰转移酶突变体，及磷酸烯醇式丙酮酸羧化酶和/或二氢硫辛酰胺乙酰转移酶突变菌株，对目标产物缬氨酸的产量提升具有显著的促进作用。该磷酸烯醇式丙酮酸羧化酶和/或二氢硫辛酰胺乙酰转移酶突变体及其重组微生物为产缬氨酸、亮氨酸、异亮氨酸及以其为前体的衍生物的生产菌株的构建提供借鉴。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 廊坊梅花生物技术开发有限公司
<120> 一种重组微生物及其构建方法与应用
<130> KHP211123187.6
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2760
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgactgatt ttttacgcga tgacatcagg ttcctcggtc aaatcctcgg tgaggtaatt 60
gcggaacaag aaggccagga ggtttatgaa ctggtcgaac aagcgcgcct gacttctttt 120
gatatcgcca agggcaacgc cgaaatggat agcctggttc aggttttcga cggcattact 180
ccagccaagg caacaccgat tgctcgcgca ttttcccact tcgctctgct ggctaacctg 240
gcggaagacc tctacgatga agagcttcgt gaacaggctc tcgatgcagg cgacacccct 300
ccggacagca ctcttgatgc cacctggctg aaactcaatg agggcaatgt tggcgcagaa 360
gctgtggccg atgtgctgcg caatgctgag gtggcgccgg ttctgactgc gcacccaact 420
gagactcgcc gccgcactgt ttttgatgcg caaaagtgga tcaccaccca catgcgtgaa 480
cgccacgctt tgcagtctgc ggagcctacc gctcgtacgc aaagcaagtt ggatgagatc 540
gagaagaaca tccgcggtcg catcaccatt ttgtggcaga ccgcgttgat tcgtgtggcc 600
cgcccacgta tcgaggacga gatcgaagta gggctgcgct actacaagct gagccttttg 660
gaagagattc cacgtatcaa ccgtgatgtg gctgttgagc ttcgtgagcg tttcggcgag 720
gatgttcctt tgaagcccgt ggtcaagcca ggttcctgga ttggtggaga ccacgacggt 780
aacccttatg tcaccgcgga aacagttgag tattccactc accgcgctgc ggaaaccgtg 840
ctcaagtact atgcacgcca gctgcattcc ctcgagcatg agctcagcct gtcggaccgc 900
atgaataagg tcaccccgca gctgcttgcg ctggcagatg ccgggcacaa cgacgtgcca 960
agccgcgtgg atgagcctta tcgacgcgcc gtccatggcg ttcgcggacg tatcctcgcg 1020
acgacggccg agctgatcgg cgaggacgcc gttgagggcg tgtggttcaa ggtctttact 1080
ccatacgcat ctccggaaga attcttaaac gatgcgttga ccattgatca ttctctgcgt 1140
gaatccaatg acgttctcat tgccgatgat cgtttgtctg tgctgatttc tgccatcgag 1200
agctttggat tcaaccttta cgcactggat ctgcgccaaa actccgaaag ctacgaggac 1260
gtcctcaccg agcttttcga acgcgcccaa gtcaccgcaa actaccgcga gctgtctgaa 1320
gcagagaagc ttgaggtgct gctgaaggaa ctgcgcagcc ctcgtccgct gatcccgcac 1380
ggttcagatg aatacagcga ggtcaccgac cgcgagctcg gcatcttccg caccgcgtcg 1440
gaggctgtta agaaattcgg gccacggatg gtgcctcact gcatcatctc catggcatca 1500
tcggtcaccg atgtgctcga gccgatggta ttgctcaagg aattcggcct cattgcagcc 1560
aacggcgaca acccacgcgg caccgtcgat gtcatcccac tgttcgaaac catcgaagat 1620
ctccaggccg gcgccggaat cctcgacgaa ctgtggaaaa ttgatcttta ccgcaactac 1680
ctcctgcagc gcgacaacgt ccaggaagtc atgctcggtt actccgattc caacaaggat 1740
ggcggatatt tctccgcaaa ctgggcgctt tacgacgcgg aactgcagct cgtcgaacta 1800
tgccgatcag ccggggtcaa gcttcgcctg ttccacggcc gtggtggcac cgtcggccgc 1860
ggtggcggac cttcctacga cgcgattctt gcccagccca ggggggctgt ccaaggttcc 1920
gtgcgcatca ccgagcaggg cgagatcatc tccgctaagt acggcaaccc cgaaaccgcg 1980
cgccgaaacc tcgaagctct ggtctcagca acgcttgagg catcgcttct cgacgtctcc 2040
gaactcaccg atcaccaacg cgcgtacgac atcatgagtg agatctctga gctcagcttg 2100
aagaagtacg cctccttggt gcacgaggat caaggcttca tcgattactt cacccagtcc 2160
acgccgctgc aggagattgg atccctcaac atcggatcca ggccttcctc acgcaagcag 2220
acctcctcgg tggaagattt gcgagcaatc ccgtgggtgc tcagttggtc ccagtctcgt 2280
gtcatgctgc cgggctggtt tggtgtcggc accgcacttg agcaatggat tggcgaaggg 2340
gagcaggcca cccagcgcat tgccgagcta caaacactca acgagtcctg gccatttttc 2400
acctcagtgt tggataacat ggctcaggtg atgtccaagg cagagctgcg tttggcaaag 2460
ctctacgcag acctgatccc agatagggaa gtagctgagc gcgtttatgc cgtcatccgc 2520
gaggaatact tcctgaccaa gaagatgttc tgcgtaatca ccggttctga tgatctgctt 2580
gatgacaacc cgcttctcgc acgatccgtc cagcgccgat acccctacct gcttccactc 2640
aacgtgatcc aggtagagat gatgcgacgc taccgaaaag gcgaccaaag cgagcaagta 2700
tcccgcaaca tccagctgac catgaacggt ctttccactg cactgcgcaa ctctggctag 2760
<210> 2
<211> 2760
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgactgatt ttttacgcga tgacatcagg ttcctcggtc aaatcctcgg tgaggtaatt 60
gcggaacaag aaggccagga ggtttatgaa ctggtcgaac aagcgcgcct gacttctttt 120
gatatcgcca agggcaacgc cgaaatggat agcctggttc aggttttcga cggcattact 180
ccagccaagg caacaccgat tgctcgcgca ttttcccact tcgctctgct ggctaacctg 240
gcggaagacc tctacgatga agagcttcgt gaacaggctc tcgatgcagg cgacacccct 300
ccggacagca ctcttgatgc cacctggctg aaactcaatg agggcaatgt tggcgcagaa 360
gctgtggccg atgtgctgcg caatgctgag gtggcgccgg ttctgactgc gcacccaact 420
gagactcgcc gccgcactgt ttttgatgcg caaaagtgga tcaccaccca catgcgtgaa 480
cgccacgctt tgcagtctgc ggagcctacc gctcgtacgc aaagcaagtt ggatgagatc 540
gagaagaaca tccgcgctcg catcaccatt ttgtggcaga ccgcgttgat tcgtgtggcc 600
cgcccacgta tcgaggacga gatcgaagta gggctgcgct actacaagct gagccttttg 660
gaagagattc cacgtatcaa ccgtgatgtg gctgttgagc ttcgtgagcg tttcggcgag 720
gatgttcctt tgaagcccgt ggtcaagcca ggttcctgga ttggtggaga ccacgacggt 780
aacccttatg tcaccgcgga aacagttgag tattccactc accgcgctgc ggaaaccgtg 840
ctcaagtact atgcacgcca gctgcattcc ctcgagcatg agctcagcct gtcggaccgc 900
atgaataagg tcaccccgca gctgcttgcg ctggcagatg ccgggcacaa cgacgtgcca 960
agccgcgtgg atgagcctta tcgacgcgcc gtccatggcg ttcgcggacg tatcctcgcg 1020
acgacggccg agctgatcgg cgaggacgcc gttgagggcg tgtggttcaa ggtctttact 1080
ccatacgcat ctccggaaga attcttaaac gatgcgttga ccattgatca ttctctgcgt 1140
gaatccaatg acgttctcat tgccgatgat cgtttgtctg tgctgatttc tgccatcgag 1200
agctttggat tcaaccttta cgcactggat ctgcgccaaa actccgaaag ctacgaggac 1260
gtcctcaccg agcttttcga acgcgcccaa gtcaccgcaa actaccgcga gctgtctgaa 1320
gcagagaagc ttgaggtgct gctgaaggaa ctgcgcagcc ctcgtccgct gatcccgcac 1380
ggttcagatg aatacagcga ggtcaccgac cgcgagctcg gcatcttccg caccgcgtcg 1440
gaggctgtta agaaattcgg gccacggatg gtgcctcact gcatcatctc catggcatca 1500
tcggtcaccg atgtgctcga gccgatggta ttgctcaagg aattcggcct cattgcagcc 1560
aacggcgaca acccacgcgg caccgtcgat gtcatcccac tgttcgaaac catcgaagat 1620
ctccaggccg gcgccggaat cctcgacgaa ctgtggaaaa ttgatcttta ccgcaactac 1680
ctcctgcagc gcgacaacgt ccaggaagtc atgctcggtt actccgattc caacaaggat 1740
ggcggatatt tctccgcaaa ctgggcgctt tacgacgcgg aactgcagct cgtcgaacta 1800
tgccgatcag ccggggtcaa gcttcgcctg ttccacggcc gtggtggcac cgtcggccgc 1860
ggtggcggac cttcctacga cgcgattctt gcccagccca ggggggctgt ccaaggttcc 1920
gtgcgcatca ccgagcaggg cgagatcatc tccgctaagt acggcaaccc cgaaaccgcg 1980
cgccgaaacc tcgaagctct ggtctcagca acgcttgagg catcgcttct cgacgtctcc 2040
gaactcaccg atcaccaacg cgcgtacgac atcatgagtg agatctctga gctcagcttg 2100
aagaagtacg cctccttggt gcacgaggat caaggcttca tcgattactt cacccagtcc 2160
acgccgctgc aggagattgg atccctcaac atcggatcca ggccttcctc acgcaagcag 2220
acctcctcgg tggaagattt gcgagcaatc ccgtgggtgc tcagttggtc ccagtctcgt 2280
gtcatgctgc cgggctggtt tggtgtcggc accgcacttg agcaatggat tggcgaaggg 2340
gagcaggcca cccagcgcat tgccgagcta caaacactca acgagtcctg gccatttttc 2400
acctcagtgt tggataacat ggctcaggtg atgtccaagg cagagctgcg tttggcaaag 2460
ctctacgcag acctgatccc agatagggaa gtagctgagc gcgtttatgc cgtcatccgc 2520
gaggaatact tcctgaccaa gaagatgttc tgcgtaatca ccggttctga tgatctgctt 2580
gatgacaacc cgcttctcgc acgatccgtc cagcgccgat acccctacct gcttccactc 2640
aacgtgatcc aggtagagat gatgcgacgc taccgaaaag gcgaccaaag cgagcaagta 2700
tcccgcaaca tccagctgac catgaacggt ctttccactg cactgcgcaa ctctggctag 2760
<210> 3
<211> 2760
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgactgatt ttttacgcga tgacatcagg ttcctcggtc aaatcctcgg tgaggtaatt 60
gcggaacaag aaggccagga ggtttatgaa ctggtcgaac aagcgcgcct gacttctttt 120
gatatcgcca agggcaacgc cgaaatggat agcctggttc aggttttcga cggcattact 180
ccagccaagg caacaccgat tgctcgcgca ttttcccact tcgctctgct ggctaacctg 240
gcggaagacc tctacgatga agagcttcgt gaacaggctc tcgatgcagg cgacacccct 300
ccggacagca ctcttgatgc cacctggctg aaactcaatg agggcaatgt tggcgcagaa 360
gctgtggccg atgtgctgcg caatgctgag gtggcgccgg ttctgactgc gcacccaact 420
gagactcgcc gccgcactgt ttttgatgcg caaaagtgga tcaccaccca catgcgtgaa 480
cgccacgctt tgcagtctgc ggagcctacc gctcgtacgc aaagcaagtt ggatgagatc 540
gagaagaaca tccgcgttcg catcaccatt ttgtggcaga ccgcgttgat tcgtgtggcc 600
cgcccacgta tcgaggacga gatcgaagta gggctgcgct actacaagct gagccttttg 660
gaagagattc cacgtatcaa ccgtgatgtg gctgttgagc ttcgtgagcg tttcggcgag 720
gatgttcctt tgaagcccgt ggtcaagcca ggttcctgga ttggtggaga ccacgacggt 780
aacccttatg tcaccgcgga aacagttgag tattccactc accgcgctgc ggaaaccgtg 840
ctcaagtact atgcacgcca gctgcattcc ctcgagcatg agctcagcct gtcggaccgc 900
atgaataagg tcaccccgca gctgcttgcg ctggcagatg ccgggcacaa cgacgtgcca 960
agccgcgtgg atgagcctta tcgacgcgcc gtccatggcg ttcgcggacg tatcctcgcg 1020
acgacggccg agctgatcgg cgaggacgcc gttgagggcg tgtggttcaa ggtctttact 1080
ccatacgcat ctccggaaga attcttaaac gatgcgttga ccattgatca ttctctgcgt 1140
gaatccaatg acgttctcat tgccgatgat cgtttgtctg tgctgatttc tgccatcgag 1200
agctttggat tcaaccttta cgcactggat ctgcgccaaa actccgaaag ctacgaggac 1260
gtcctcaccg agcttttcga acgcgcccaa gtcaccgcaa actaccgcga gctgtctgaa 1320
gcagagaagc ttgaggtgct gctgaaggaa ctgcgcagcc ctcgtccgct gatcccgcac 1380
ggttcagatg aatacagcga ggtcaccgac cgcgagctcg gcatcttccg caccgcgtcg 1440
gaggctgtta agaaattcgg gccacggatg gtgcctcact gcatcatctc catggcatca 1500
tcggtcaccg atgtgctcga gccgatggta ttgctcaagg aattcggcct cattgcagcc 1560
aacggcgaca acccacgcgg caccgtcgat gtcatcccac tgttcgaaac catcgaagat 1620
ctccaggccg gcgccggaat cctcgacgaa ctgtggaaaa ttgatcttta ccgcaactac 1680
ctcctgcagc gcgacaacgt ccaggaagtc atgctcggtt actccgattc caacaaggat 1740
ggcggatatt tctccgcaaa ctgggcgctt tacgacgcgg aactgcagct cgtcgaacta 1800
tgccgatcag ccggggtcaa gcttcgcctg ttccacggcc gtggtggcac cgtcggccgc 1860
ggtggcggac cttcctacga cgcgattctt gcccagccca ggggggctgt ccaaggttcc 1920
gtgcgcatca ccgagcaggg cgagatcatc tccgctaagt acggcaaccc cgaaaccgcg 1980
cgccgaaacc tcgaagctct ggtctcagca acgcttgagg catcgcttct cgacgtctcc 2040
gaactcaccg atcaccaacg cgcgtacgac atcatgagtg agatctctga gctcagcttg 2100
aagaagtacg cctccttggt gcacgaggat caaggcttca tcgattactt cacccagtcc 2160
acgccgctgc aggagattgg atccctcaac atcggatcca ggccttcctc acgcaagcag 2220
acctcctcgg tggaagattt gcgagcaatc ccgtgggtgc tcagttggtc ccagtctcgt 2280
gtcatgctgc cgggctggtt tggtgtcggc accgcacttg agcaatggat tggcgaaggg 2340
gagcaggcca cccagcgcat tgccgagcta caaacactca acgagtcctg gccatttttc 2400
acctcagtgt tggataacat ggctcaggtg atgtccaagg cagagctgcg tttggcaaag 2460
ctctacgcag acctgatccc agatagggaa gtagctgagc gcgtttatgc cgtcatccgc 2520
gaggaatact tcctgaccaa gaagatgttc tgcgtaatca ccggttctga tgatctgctt 2580
gatgacaacc cgcttctcgc acgatccgtc cagcgccgat acccctacct gcttccactc 2640
aacgtgatcc aggtagagat gatgcgacgc taccgaaaag gcgaccaaag cgagcaagta 2700
tcccgcaaca tccagctgac catgaacggt ctttccactg cactgcgcaa ctctggctag 2760
<210> 4
<211> 2760
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgactgatt ttttacgcga tgacatcagg ttcctcggtc aaatcctcgg tgaggtaatt 60
gcggaacaag aaggccagga ggtttatgaa ctggtcgaac aagcgcgcct gacttctttt 120
gatatcgcca agggcaacgc cgaaatggat agcctggttc aggttttcga cggcattact 180
ccagccaagg caacaccgat tgctcgcgca ttttcccact tcgctctgct ggctaacctg 240
gcggaagacc tctacgatga agagcttcgt gaacaggctc tcgatgcagg cgacacccct 300
ccggacagca ctcttgatgc cacctggctg aaactcaatg agggcaatgt tggcgcagaa 360
gctgtggccg atgtgctgcg caatgctgag gtggcgccgg ttctgactgc gcacccaact 420
gagactcgcc gccgcactgt ttttgatgcg caaaagtgga tcaccaccca catgcgtgaa 480
cgccacgctt tgcagtctgc ggagcctacc gctcgtacgc aaagcaagtt ggatgagatc 540
gagaagaaca tccgccgtcg catcaccatt ttgtggcaga ccgcgttgat tcgtgtggcc 600
cgcccacgta tcgaggacga gatcgaagta gggctgcgct actacaagct gagccttttg 660
gaagagattc cacgtatcaa ccgtgatgtg gctgttgagc ttcgtgagcg tttcggcgag 720
gatgttcctt tgaagcccgt ggtcaagcca ggttcctgga ttggtggaga ccacgacggt 780
aacccttatg tcaccgcgga aacagttgag tattccactc accgcgctgc ggaaaccgtg 840
ctcaagtact atgcacgcca gctgcattcc ctcgagcatg agctcagcct gtcggaccgc 900
atgaataagg tcaccccgca gctgcttgcg ctggcagatg ccgggcacaa cgacgtgcca 960
agccgcgtgg atgagcctta tcgacgcgcc gtccatggcg ttcgcggacg tatcctcgcg 1020
acgacggccg agctgatcgg cgaggacgcc gttgagggcg tgtggttcaa ggtctttact 1080
ccatacgcat ctccggaaga attcttaaac gatgcgttga ccattgatca ttctctgcgt 1140
gaatccaatg acgttctcat tgccgatgat cgtttgtctg tgctgatttc tgccatcgag 1200
agctttggat tcaaccttta cgcactggat ctgcgccaaa actccgaaag ctacgaggac 1260
gtcctcaccg agcttttcga acgcgcccaa gtcaccgcaa actaccgcga gctgtctgaa 1320
gcagagaagc ttgaggtgct gctgaaggaa ctgcgcagcc ctcgtccgct gatcccgcac 1380
ggttcagatg aatacagcga ggtcaccgac cgcgagctcg gcatcttccg caccgcgtcg 1440
gaggctgtta agaaattcgg gccacggatg gtgcctcact gcatcatctc catggcatca 1500
tcggtcaccg atgtgctcga gccgatggta ttgctcaagg aattcggcct cattgcagcc 1560
aacggcgaca acccacgcgg caccgtcgat gtcatcccac tgttcgaaac catcgaagat 1620
ctccaggccg gcgccggaat cctcgacgaa ctgtggaaaa ttgatcttta ccgcaactac 1680
ctcctgcagc gcgacaacgt ccaggaagtc atgctcggtt actccgattc caacaaggat 1740
ggcggatatt tctccgcaaa ctgggcgctt tacgacgcgg aactgcagct cgtcgaacta 1800
tgccgatcag ccggggtcaa gcttcgcctg ttccacggcc gtggtggcac cgtcggccgc 1860
ggtggcggac cttcctacga cgcgattctt gcccagccca ggggggctgt ccaaggttcc 1920
gtgcgcatca ccgagcaggg cgagatcatc tccgctaagt acggcaaccc cgaaaccgcg 1980
cgccgaaacc tcgaagctct ggtctcagca acgcttgagg catcgcttct cgacgtctcc 2040
gaactcaccg atcaccaacg cgcgtacgac atcatgagtg agatctctga gctcagcttg 2100
aagaagtacg cctccttggt gcacgaggat caaggcttca tcgattactt cacccagtcc 2160
acgccgctgc aggagattgg atccctcaac atcggatcca ggccttcctc acgcaagcag 2220
acctcctcgg tggaagattt gcgagcaatc ccgtgggtgc tcagttggtc ccagtctcgt 2280
gtcatgctgc cgggctggtt tggtgtcggc accgcacttg agcaatggat tggcgaaggg 2340
gagcaggcca cccagcgcat tgccgagcta caaacactca acgagtcctg gccatttttc 2400
acctcagtgt tggataacat ggctcaggtg atgtccaagg cagagctgcg tttggcaaag 2460
ctctacgcag acctgatccc agatagggaa gtagctgagc gcgtttatgc cgtcatccgc 2520
gaggaatact tcctgaccaa gaagatgttc tgcgtaatca ccggttctga tgatctgctt 2580
gatgacaacc cgcttctcgc acgatccgtc cagcgccgat acccctacct gcttccactc 2640
aacgtgatcc aggtagagat gatgcgacgc taccgaaaag gcgaccaaag cgagcaagta 2700
tcccgcaaca tccagctgac catgaacggt ctttccactg cactgcgcaa ctctggctag 2760
<210> 5
<211> 2013
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggcgttct ccgtagagat gcccgagctg ggcgaatcag taaccgaagg cacgatcacc 60
cagtggttga agtctgttgg tgacactgtt gaggtagatg agccgttgct cgaggtctca 120
actgacaagg tcgacaccga gattccctct cctgtcgccg gtgtcatcct agagattaag 180
gctgaagagg atgacaccgt cgacgtcggc ggtgtcattg caataatcgg cgatgctgat 240
gagactcctg ccaacgaagc tcctgccgac gaggcaccag ctcctgccga agaggaagaa 300
ccagttaagg aagagccaaa gaaggaggca gctcctgaag ctccagcagc aactggcgcc 360
gcaaccgatg tggaaatgcc agaactcggc gaatccgtca ccgaaggcac cattacccag 420
tggctcaagg ctgtcggcga caccgtcgaa gtagacgaac cacttcttga ggtctccacc 480
gacaaggtcg acaccgaaat cccatctcca gtagcaggca ccatcgtgga gatccttgca 540
gacgaagacg acaccgtcga cgtcggcgca gtcatcgccc gcatcggtga cgcaaacgca 600
gctgcagcac ctgccgaaga ggaagcagca cctgccgaag aggaagagcc agttaaggaa 660
gagccaaaga aggaggcagc tcctgaagct ccagcagcaa ctggcgcagc aaccgatgtg 720
gaaatgccag aactcggcga atccgtcacc gaaggcacca ttacccagtg gctcaaggct 780
gtcggcgaca ccgtcgaagt agacgaacca cttcttgagg tctccaccga caaggtcgac 840
accgaaatcc catccccagt agcaggcacc atcgtggaga tccttgcaga cgaagacgac 900
accgtcgacg tcggcgcagt catcgcccgc atcggtgacg caaacgcagc tgcagcacct 960
ggcgaagaga aagcagcacc tgccgaagag gaagagccaa agcaggaaga gccaaagaag 1020
gaagagccaa agaaggaagc agctactaca cctgctgcgg catccgcaac tgtgtccgct 1080
tctggcgaca acgttccata cgtcacccca ctggtgcgca agcttgctga aaagcacggc 1140
gttgacttga acaccgtgac cggtaccggt atcggtggcc gtatccgcaa gcaggatgtt 1200
ttggctgctg cgaacggcga ggctgcacct gctgaggctg ctgctcctgt ttccgcttgg 1260
tccactaagt ctgttgatcc tgagaaggct aagctccgtg gtaccactca gaaggtcaac 1320
cgcatccgtg agatcaccgc gatgaagacc gtcgaggctc tgcagatttc tgctcagctc 1380
acccagctgc acgaggtcga tatgactcgc gttgctgagc tgcgtaagaa gaacaagccc 1440
gcgttcatcg agaagcacgg tgtgaacctc acttacctgc cattcttcgt gaaggcagtt 1500
gtcgaggctt tggtttccca tccaaacgtc aacgcgtctt acaacgcgaa gaccaaggag 1560
atgacctacc actcctccgt taacctctcc atcgctgttg ataccccagc tggtctgttg 1620
accccagtca ttcacgatgc tcaggatctc tccatcccag agatcgcaaa ggcaattgtt 1680
gacctggctg atcgttcacg caacaacaag ctgaagccaa acgatctgtc cggtggcacc 1740
ttcaccatca ccaacattgg ttctgaaggc gcactgtctg ataccccaat cctggttcca 1800
ccacaggctg gcatcttggg caccggcgcg atcgtgaagc gtccagttgt catcaccgag 1860
gatggaattg attccatcgc gatccgtcag atggtcttcc tgccactgac ctacgaccac 1920
caggttgtag atggcgcaga tgctggtcgc ttcctgacca ccatcaagga ccgccttgag 1980
accgctaact tcgaaggcga tctgcagctc taa 2013
<210> 6
<211> 2013
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atggcgttct ccgtagagat gcccgagctg ggcgaatcag taaccgaagg cacgatcacc 60
cagtggttga agtctgttgg tgacactgtt gaggtagatg agccgttgct cgaggtctca 120
actgacaagg tcgacaccga gattccctct cctgtcgccg gtgtcatcct agagattaag 180
gctgaagagg atgacaccgt cgacgtcggc ggtgtcattg caataatcga cgatgctgat 240
gagactcctg ccaacgaagc tcctgccgac gaggcaccag ctcctgccga agaggaagaa 300
ccagttaagg aagagccaaa gaaggaggca gctcctgaag ctccagcagc aactggcgcc 360
gcaaccgatg tggaaatgcc agaactcggc gaatccgtca ccgaaggcac cattacccag 420
tggctcaagg ctgtcggcga caccgtcgaa gtagacgaac cacttcttga ggtctccacc 480
gacaaggtcg acaccgaaat cccatctcca gtagcaggca ccatcgtgga gatccttgca 540
gacgaagacg acaccgtcga cgtcggcgca gtcatcgccc gcatcggtga cgcaaacgca 600
gctgcagcac ctgccgaaga ggaagcagca cctgccgaag aggaagagcc agttaaggaa 660
gagccaaaga aggaggcagc tcctgaagct ccagcagcaa ctggcgcagc aaccgatgtg 720
gaaatgccag aactcggcga atccgtcacc gaaggcacca ttacccagtg gctcaaggct 780
gtcggcgaca ccgtcgaagt agacgaacca cttcttgagg tctccaccga caaggtcgac 840
accgaaatcc catccccagt agcaggcacc atcgtggaga tccttgcaga cgaagacgac 900
accgtcgacg tcggcgcagt catcgcccgc atcggtgacg caaacgcagc tgcagcacct 960
ggcgaagaga aagcagcacc tgccgaagag gaagagccaa agcaggaaga gccaaagaag 1020
gaagagccaa agaaggaagc agctactaca cctgctgcgg catccgcaac tgtgtccgct 1080
tctggcgaca acgttccata cgtcacccca ctggtgcgca agcttgctga aaagcacggc 1140
gttgacttga acaccgtgac cggtaccggt atcggtggcc gtatccgcaa gcaggatgtt 1200
ttggctgctg cgaacggcga ggctgcacct gctgaggctg ctgctcctgt ttccgcttgg 1260
tccactaagt ctgttgatcc tgagaaggct aagctccgtg gtaccactca gaaggtcaac 1320
cgcatccgtg agatcaccgc gatgaagacc gtcgaggctc tgcagatttc tgctcagctc 1380
acccagctgc acgaggtcga tatgactcgc gttgctgagc tgcgtaagaa gaacaagccc 1440
gcgttcatcg agaagcacgg tgtgaacctc acttacctgc cattcttcgt gaaggcagtt 1500
gtcgaggctt tggtttccca tccaaacgtc aacgcgtctt acaacgcgaa gaccaaggag 1560
atgacctacc actcctccgt taacctctcc atcgctgttg ataccccagc tggtctgttg 1620
accccagtca ttcacgatgc tcaggatctc tccatcccag agatcgcaaa ggcaattgtt 1680
gacctggctg atcgttcacg caacaacaag ctgaagccaa acgatctgtc cggtggcacc 1740
ttcaccatca ccaacattgg ttctgaaggc gcactgtctg ataccccaat cctggttcca 1800
ccacaggctg gcatcttggg caccggcgcg atcgtgaagc gtccagttgt catcaccgag 1860
gatggaattg attccatcgc gatccgtcag atggtcttcc tgccactgac ctacgaccac 1920
caggttgtag atggcgcaga tgctggtcgc ttcctgacca ccatcaagga ccgccttgag 1980
accgctaact tcgaaggcga tctgcagctc taa 2013
<210> 7
<211> 2013
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggcgttct ccgtagagat gcccgagctg ggcgaatcag taaccgaagg cacgatcacc 60
cagtggttga agtctgttgg tgacactgtt gaggtagatg agccgttgct cgaggtctca 120
actgacaagg tcgacaccga gattccctct cctgtcgccg gtgtcatcct agagattaag 180
gctgaagagg atgacaccgt cgacgtcggc ggtgtcattg caataatcgt cgatgctgat 240
gagactcctg ccaacgaagc tcctgccgac gaggcaccag ctcctgccga agaggaagaa 300
ccagttaagg aagagccaaa gaaggaggca gctcctgaag ctccagcagc aactggcgcc 360
gcaaccgatg tggaaatgcc agaactcggc gaatccgtca ccgaaggcac cattacccag 420
tggctcaagg ctgtcggcga caccgtcgaa gtagacgaac cacttcttga ggtctccacc 480
gacaaggtcg acaccgaaat cccatctcca gtagcaggca ccatcgtgga gatccttgca 540
gacgaagacg acaccgtcga cgtcggcgca gtcatcgccc gcatcggtga cgcaaacgca 600
gctgcagcac ctgccgaaga ggaagcagca cctgccgaag aggaagagcc agttaaggaa 660
gagccaaaga aggaggcagc tcctgaagct ccagcagcaa ctggcgcagc aaccgatgtg 720
gaaatgccag aactcggcga atccgtcacc gaaggcacca ttacccagtg gctcaaggct 780
gtcggcgaca ccgtcgaagt agacgaacca cttcttgagg tctccaccga caaggtcgac 840
accgaaatcc catccccagt agcaggcacc atcgtggaga tccttgcaga cgaagacgac 900
accgtcgacg tcggcgcagt catcgcccgc atcggtgacg caaacgcagc tgcagcacct 960
ggcgaagaga aagcagcacc tgccgaagag gaagagccaa agcaggaaga gccaaagaag 1020
gaagagccaa agaaggaagc agctactaca cctgctgcgg catccgcaac tgtgtccgct 1080
tctggcgaca acgttccata cgtcacccca ctggtgcgca agcttgctga aaagcacggc 1140
gttgacttga acaccgtgac cggtaccggt atcggtggcc gtatccgcaa gcaggatgtt 1200
ttggctgctg cgaacggcga ggctgcacct gctgaggctg ctgctcctgt ttccgcttgg 1260
tccactaagt ctgttgatcc tgagaaggct aagctccgtg gtaccactca gaaggtcaac 1320
cgcatccgtg agatcaccgc gatgaagacc gtcgaggctc tgcagatttc tgctcagctc 1380
acccagctgc acgaggtcga tatgactcgc gttgctgagc tgcgtaagaa gaacaagccc 1440
gcgttcatcg agaagcacgg tgtgaacctc acttacctgc cattcttcgt gaaggcagtt 1500
gtcgaggctt tggtttccca tccaaacgtc aacgcgtctt acaacgcgaa gaccaaggag 1560
atgacctacc actcctccgt taacctctcc atcgctgttg ataccccagc tggtctgttg 1620
accccagtca ttcacgatgc tcaggatctc tccatcccag agatcgcaaa ggcaattgtt 1680
gacctggctg atcgttcacg caacaacaag ctgaagccaa acgatctgtc cggtggcacc 1740
ttcaccatca ccaacattgg ttctgaaggc gcactgtctg ataccccaat cctggttcca 1800
ccacaggctg gcatcttggg caccggcgcg atcgtgaagc gtccagttgt catcaccgag 1860
gatggaattg attccatcgc gatccgtcag atggtcttcc tgccactgac ctacgaccac 1920
caggttgtag atggcgcaga tgctggtcgc ttcctgacca ccatcaagga ccgccttgag 1980
accgctaact tcgaaggcga tctgcagctc taa 2013
<210> 8
<211> 2013
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggcgttct ccgtagagat gcccgagctg ggcgaatcag taaccgaagg cacgatcacc 60
cagtggttga agtctgttgg tgacactgtt gaggtagatg agccgttgct cgaggtctca 120
actgacaagg tcgacaccga gattccctct cctgtcgccg gtgtcatcct agagattaag 180
gctgaagagg atgacaccgt cgacgtcggc ggtgtcattg caataatcgc cgatgctgat 240
gagactcctg ccaacgaagc tcctgccgac gaggcaccag ctcctgccga agaggaagaa 300
ccagttaagg aagagccaaa gaaggaggca gctcctgaag ctccagcagc aactggcgcc 360
gcaaccgatg tggaaatgcc agaactcggc gaatccgtca ccgaaggcac cattacccag 420
tggctcaagg ctgtcggcga caccgtcgaa gtagacgaac cacttcttga ggtctccacc 480
gacaaggtcg acaccgaaat cccatctcca gtagcaggca ccatcgtgga gatccttgca 540
gacgaagacg acaccgtcga cgtcggcgca gtcatcgccc gcatcggtga cgcaaacgca 600
gctgcagcac ctgccgaaga ggaagcagca cctgccgaag aggaagagcc agttaaggaa 660
gagccaaaga aggaggcagc tcctgaagct ccagcagcaa ctggcgcagc aaccgatgtg 720
gaaatgccag aactcggcga atccgtcacc gaaggcacca ttacccagtg gctcaaggct 780
gtcggcgaca ccgtcgaagt agacgaacca cttcttgagg tctccaccga caaggtcgac 840
accgaaatcc catccccagt agcaggcacc atcgtggaga tccttgcaga cgaagacgac 900
accgtcgacg tcggcgcagt catcgcccgc atcggtgacg caaacgcagc tgcagcacct 960
ggcgaagaga aagcagcacc tgccgaagag gaagagccaa agcaggaaga gccaaagaag 1020
gaagagccaa agaaggaagc agctactaca cctgctgcgg catccgcaac tgtgtccgct 1080
tctggcgaca acgttccata cgtcacccca ctggtgcgca agcttgctga aaagcacggc 1140
gttgacttga acaccgtgac cggtaccggt atcggtggcc gtatccgcaa gcaggatgtt 1200
ttggctgctg cgaacggcga ggctgcacct gctgaggctg ctgctcctgt ttccgcttgg 1260
tccactaagt ctgttgatcc tgagaaggct aagctccgtg gtaccactca gaaggtcaac 1320
cgcatccgtg agatcaccgc gatgaagacc gtcgaggctc tgcagatttc tgctcagctc 1380
acccagctgc acgaggtcga tatgactcgc gttgctgagc tgcgtaagaa gaacaagccc 1440
gcgttcatcg agaagcacgg tgtgaacctc acttacctgc cattcttcgt gaaggcagtt 1500
gtcgaggctt tggtttccca tccaaacgtc aacgcgtctt acaacgcgaa gaccaaggag 1560
atgacctacc actcctccgt taacctctcc atcgctgttg ataccccagc tggtctgttg 1620
accccagtca ttcacgatgc tcaggatctc tccatcccag agatcgcaaa ggcaattgtt 1680
gacctggctg atcgttcacg caacaacaag ctgaagccaa acgatctgtc cggtggcacc 1740
ttcaccatca ccaacattgg ttctgaaggc gcactgtctg ataccccaat cctggttcca 1800
ccacaggctg gcatcttggg caccggcgcg atcgtgaagc gtccagttgt catcaccgag 1860
gatggaattg attccatcgc gatccgtcag atggtcttcc tgccactgac ctacgaccac 1920
caggttgtag atggcgcaga tgctggtcgc ttcctgacca ccatcaagga ccgccttgag 1980
accgctaact tcgaaggcga tctgcagctc taa 2013
<210> 9
<211> 919
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Thr Asp Phe Leu Arg Asp Asp Ile Arg Phe Leu Gly Gln Ile Leu
1 5 10 15
Gly Glu Val Ile Ala Glu Gln Glu Gly Gln Glu Val Tyr Glu Leu Val
20 25 30
Glu Gln Ala Arg Leu Thr Ser Phe Asp Ile Ala Lys Gly Asn Ala Glu
35 40 45
Met Asp Ser Leu Val Gln Val Phe Asp Gly Ile Thr Pro Ala Lys Ala
50 55 60
Thr Pro Ile Ala Arg Ala Phe Ser His Phe Ala Leu Leu Ala Asn Leu
65 70 75 80
Ala Glu Asp Leu Tyr Asp Glu Glu Leu Arg Glu Gln Ala Leu Asp Ala
85 90 95
Gly Asp Thr Pro Pro Asp Ser Thr Leu Asp Ala Thr Trp Leu Lys Leu
100 105 110
Asn Glu Gly Asn Val Gly Ala Glu Ala Val Ala Asp Val Leu Arg Asn
115 120 125
Ala Glu Val Ala Pro Val Leu Thr Ala His Pro Thr Glu Thr Arg Arg
130 135 140
Arg Thr Val Phe Asp Ala Gln Lys Trp Ile Thr Thr His Met Arg Glu
145 150 155 160
Arg His Ala Leu Gln Ser Ala Glu Pro Thr Ala Arg Thr Gln Ser Lys
165 170 175
Leu Asp Glu Ile Glu Lys Asn Ile Arg Gly Arg Ile Thr Ile Leu Trp
180 185 190
Gln Thr Ala Leu Ile Arg Val Ala Arg Pro Arg Ile Glu Asp Glu Ile
195 200 205
Glu Val Gly Leu Arg Tyr Tyr Lys Leu Ser Leu Leu Glu Glu Ile Pro
210 215 220
Arg Ile Asn Arg Asp Val Ala Val Glu Leu Arg Glu Arg Phe Gly Glu
225 230 235 240
Asp Val Pro Leu Lys Pro Val Val Lys Pro Gly Ser Trp Ile Gly Gly
245 250 255
Asp His Asp Gly Asn Pro Tyr Val Thr Ala Glu Thr Val Glu Tyr Ser
260 265 270
Thr His Arg Ala Ala Glu Thr Val Leu Lys Tyr Tyr Ala Arg Gln Leu
275 280 285
His Ser Leu Glu His Glu Leu Ser Leu Ser Asp Arg Met Asn Lys Val
290 295 300
Thr Pro Gln Leu Leu Ala Leu Ala Asp Ala Gly His Asn Asp Val Pro
305 310 315 320
Ser Arg Val Asp Glu Pro Tyr Arg Arg Ala Val His Gly Val Arg Gly
325 330 335
Arg Ile Leu Ala Thr Thr Ala Glu Leu Ile Gly Glu Asp Ala Val Glu
340 345 350
Gly Val Trp Phe Lys Val Phe Thr Pro Tyr Ala Ser Pro Glu Glu Phe
355 360 365
Leu Asn Asp Ala Leu Thr Ile Asp His Ser Leu Arg Glu Ser Asn Asp
370 375 380
Val Leu Ile Ala Asp Asp Arg Leu Ser Val Leu Ile Ser Ala Ile Glu
385 390 395 400
Ser Phe Gly Phe Asn Leu Tyr Ala Leu Asp Leu Arg Gln Asn Ser Glu
405 410 415
Ser Tyr Glu Asp Val Leu Thr Glu Leu Phe Glu Arg Ala Gln Val Thr
420 425 430
Ala Asn Tyr Arg Glu Leu Ser Glu Ala Glu Lys Leu Glu Val Leu Leu
435 440 445
Lys Glu Leu Arg Ser Pro Arg Pro Leu Ile Pro His Gly Ser Asp Glu
450 455 460
Tyr Ser Glu Val Thr Asp Arg Glu Leu Gly Ile Phe Arg Thr Ala Ser
465 470 475 480
Glu Ala Val Lys Lys Phe Gly Pro Arg Met Val Pro His Cys Ile Ile
485 490 495
Ser Met Ala Ser Ser Val Thr Asp Val Leu Glu Pro Met Val Leu Leu
500 505 510
Lys Glu Phe Gly Leu Ile Ala Ala Asn Gly Asp Asn Pro Arg Gly Thr
515 520 525
Val Asp Val Ile Pro Leu Phe Glu Thr Ile Glu Asp Leu Gln Ala Gly
530 535 540
Ala Gly Ile Leu Asp Glu Leu Trp Lys Ile Asp Leu Tyr Arg Asn Tyr
545 550 555 560
Leu Leu Gln Arg Asp Asn Val Gln Glu Val Met Leu Gly Tyr Ser Asp
565 570 575
Ser Asn Lys Asp Gly Gly Tyr Phe Ser Ala Asn Trp Ala Leu Tyr Asp
580 585 590
Ala Glu Leu Gln Leu Val Glu Leu Cys Arg Ser Ala Gly Val Lys Leu
595 600 605
Arg Leu Phe His Gly Arg Gly Gly Thr Val Gly Arg Gly Gly Gly Pro
610 615 620
Ser Tyr Asp Ala Ile Leu Ala Gln Pro Arg Gly Ala Val Gln Gly Ser
625 630 635 640
Val Arg Ile Thr Glu Gln Gly Glu Ile Ile Ser Ala Lys Tyr Gly Asn
645 650 655
Pro Glu Thr Ala Arg Arg Asn Leu Glu Ala Leu Val Ser Ala Thr Leu
660 665 670
Glu Ala Ser Leu Leu Asp Val Ser Glu Leu Thr Asp His Gln Arg Ala
675 680 685
Tyr Asp Ile Met Ser Glu Ile Ser Glu Leu Ser Leu Lys Lys Tyr Ala
690 695 700
Ser Leu Val His Glu Asp Gln Gly Phe Ile Asp Tyr Phe Thr Gln Ser
705 710 715 720
Thr Pro Leu Gln Glu Ile Gly Ser Leu Asn Ile Gly Ser Arg Pro Ser
725 730 735
Ser Arg Lys Gln Thr Ser Ser Val Glu Asp Leu Arg Ala Ile Pro Trp
740 745 750
Val Leu Ser Trp Ser Gln Ser Arg Val Met Leu Pro Gly Trp Phe Gly
755 760 765
Val Gly Thr Ala Leu Glu Gln Trp Ile Gly Glu Gly Glu Gln Ala Thr
770 775 780
Gln Arg Ile Ala Glu Leu Gln Thr Leu Asn Glu Ser Trp Pro Phe Phe
785 790 795 800
Thr Ser Val Leu Asp Asn Met Ala Gln Val Met Ser Lys Ala Glu Leu
805 810 815
Arg Leu Ala Lys Leu Tyr Ala Asp Leu Ile Pro Asp Arg Glu Val Ala
820 825 830
Glu Arg Val Tyr Ala Val Ile Arg Glu Glu Tyr Phe Leu Thr Lys Lys
835 840 845
Met Phe Cys Val Ile Thr Gly Ser Asp Asp Leu Leu Asp Asp Asn Pro
850 855 860
Leu Leu Ala Arg Ser Val Gln Arg Arg Tyr Pro Tyr Leu Leu Pro Leu
865 870 875 880
Asn Val Ile Gln Val Glu Met Met Arg Arg Tyr Arg Lys Gly Asp Gln
885 890 895
Ser Glu Gln Val Ser Arg Asn Ile Gln Leu Thr Met Asn Gly Leu Ser
900 905 910
Thr Ala Leu Arg Asn Ser Gly
915
<210> 10
<211> 919
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Thr Asp Phe Leu Arg Asp Asp Ile Arg Phe Leu Gly Gln Ile Leu
1 5 10 15
Gly Glu Val Ile Ala Glu Gln Glu Gly Gln Glu Val Tyr Glu Leu Val
20 25 30
Glu Gln Ala Arg Leu Thr Ser Phe Asp Ile Ala Lys Gly Asn Ala Glu
35 40 45
Met Asp Ser Leu Val Gln Val Phe Asp Gly Ile Thr Pro Ala Lys Ala
50 55 60
Thr Pro Ile Ala Arg Ala Phe Ser His Phe Ala Leu Leu Ala Asn Leu
65 70 75 80
Ala Glu Asp Leu Tyr Asp Glu Glu Leu Arg Glu Gln Ala Leu Asp Ala
85 90 95
Gly Asp Thr Pro Pro Asp Ser Thr Leu Asp Ala Thr Trp Leu Lys Leu
100 105 110
Asn Glu Gly Asn Val Gly Ala Glu Ala Val Ala Asp Val Leu Arg Asn
115 120 125
Ala Glu Val Ala Pro Val Leu Thr Ala His Pro Thr Glu Thr Arg Arg
130 135 140
Arg Thr Val Phe Asp Ala Gln Lys Trp Ile Thr Thr His Met Arg Glu
145 150 155 160
Arg His Ala Leu Gln Ser Ala Glu Pro Thr Ala Arg Thr Gln Ser Lys
165 170 175
Leu Asp Glu Ile Glu Lys Asn Ile Arg Ala Arg Ile Thr Ile Leu Trp
180 185 190
Gln Thr Ala Leu Ile Arg Val Ala Arg Pro Arg Ile Glu Asp Glu Ile
195 200 205
Glu Val Gly Leu Arg Tyr Tyr Lys Leu Ser Leu Leu Glu Glu Ile Pro
210 215 220
Arg Ile Asn Arg Asp Val Ala Val Glu Leu Arg Glu Arg Phe Gly Glu
225 230 235 240
Asp Val Pro Leu Lys Pro Val Val Lys Pro Gly Ser Trp Ile Gly Gly
245 250 255
Asp His Asp Gly Asn Pro Tyr Val Thr Ala Glu Thr Val Glu Tyr Ser
260 265 270
Thr His Arg Ala Ala Glu Thr Val Leu Lys Tyr Tyr Ala Arg Gln Leu
275 280 285
His Ser Leu Glu His Glu Leu Ser Leu Ser Asp Arg Met Asn Lys Val
290 295 300
Thr Pro Gln Leu Leu Ala Leu Ala Asp Ala Gly His Asn Asp Val Pro
305 310 315 320
Ser Arg Val Asp Glu Pro Tyr Arg Arg Ala Val His Gly Val Arg Gly
325 330 335
Arg Ile Leu Ala Thr Thr Ala Glu Leu Ile Gly Glu Asp Ala Val Glu
340 345 350
Gly Val Trp Phe Lys Val Phe Thr Pro Tyr Ala Ser Pro Glu Glu Phe
355 360 365
Leu Asn Asp Ala Leu Thr Ile Asp His Ser Leu Arg Glu Ser Asn Asp
370 375 380
Val Leu Ile Ala Asp Asp Arg Leu Ser Val Leu Ile Ser Ala Ile Glu
385 390 395 400
Ser Phe Gly Phe Asn Leu Tyr Ala Leu Asp Leu Arg Gln Asn Ser Glu
405 410 415
Ser Tyr Glu Asp Val Leu Thr Glu Leu Phe Glu Arg Ala Gln Val Thr
420 425 430
Ala Asn Tyr Arg Glu Leu Ser Glu Ala Glu Lys Leu Glu Val Leu Leu
435 440 445
Lys Glu Leu Arg Ser Pro Arg Pro Leu Ile Pro His Gly Ser Asp Glu
450 455 460
Tyr Ser Glu Val Thr Asp Arg Glu Leu Gly Ile Phe Arg Thr Ala Ser
465 470 475 480
Glu Ala Val Lys Lys Phe Gly Pro Arg Met Val Pro His Cys Ile Ile
485 490 495
Ser Met Ala Ser Ser Val Thr Asp Val Leu Glu Pro Met Val Leu Leu
500 505 510
Lys Glu Phe Gly Leu Ile Ala Ala Asn Gly Asp Asn Pro Arg Gly Thr
515 520 525
Val Asp Val Ile Pro Leu Phe Glu Thr Ile Glu Asp Leu Gln Ala Gly
530 535 540
Ala Gly Ile Leu Asp Glu Leu Trp Lys Ile Asp Leu Tyr Arg Asn Tyr
545 550 555 560
Leu Leu Gln Arg Asp Asn Val Gln Glu Val Met Leu Gly Tyr Ser Asp
565 570 575
Ser Asn Lys Asp Gly Gly Tyr Phe Ser Ala Asn Trp Ala Leu Tyr Asp
580 585 590
Ala Glu Leu Gln Leu Val Glu Leu Cys Arg Ser Ala Gly Val Lys Leu
595 600 605
Arg Leu Phe His Gly Arg Gly Gly Thr Val Gly Arg Gly Gly Gly Pro
610 615 620
Ser Tyr Asp Ala Ile Leu Ala Gln Pro Arg Gly Ala Val Gln Gly Ser
625 630 635 640
Val Arg Ile Thr Glu Gln Gly Glu Ile Ile Ser Ala Lys Tyr Gly Asn
645 650 655
Pro Glu Thr Ala Arg Arg Asn Leu Glu Ala Leu Val Ser Ala Thr Leu
660 665 670
Glu Ala Ser Leu Leu Asp Val Ser Glu Leu Thr Asp His Gln Arg Ala
675 680 685
Tyr Asp Ile Met Ser Glu Ile Ser Glu Leu Ser Leu Lys Lys Tyr Ala
690 695 700
Ser Leu Val His Glu Asp Gln Gly Phe Ile Asp Tyr Phe Thr Gln Ser
705 710 715 720
Thr Pro Leu Gln Glu Ile Gly Ser Leu Asn Ile Gly Ser Arg Pro Ser
725 730 735
Ser Arg Lys Gln Thr Ser Ser Val Glu Asp Leu Arg Ala Ile Pro Trp
740 745 750
Val Leu Ser Trp Ser Gln Ser Arg Val Met Leu Pro Gly Trp Phe Gly
755 760 765
Val Gly Thr Ala Leu Glu Gln Trp Ile Gly Glu Gly Glu Gln Ala Thr
770 775 780
Gln Arg Ile Ala Glu Leu Gln Thr Leu Asn Glu Ser Trp Pro Phe Phe
785 790 795 800
Thr Ser Val Leu Asp Asn Met Ala Gln Val Met Ser Lys Ala Glu Leu
805 810 815
Arg Leu Ala Lys Leu Tyr Ala Asp Leu Ile Pro Asp Arg Glu Val Ala
820 825 830
Glu Arg Val Tyr Ala Val Ile Arg Glu Glu Tyr Phe Leu Thr Lys Lys
835 840 845
Met Phe Cys Val Ile Thr Gly Ser Asp Asp Leu Leu Asp Asp Asn Pro
850 855 860
Leu Leu Ala Arg Ser Val Gln Arg Arg Tyr Pro Tyr Leu Leu Pro Leu
865 870 875 880
Asn Val Ile Gln Val Glu Met Met Arg Arg Tyr Arg Lys Gly Asp Gln
885 890 895
Ser Glu Gln Val Ser Arg Asn Ile Gln Leu Thr Met Asn Gly Leu Ser
900 905 910
Thr Ala Leu Arg Asn Ser Gly
915
<210> 11
<211> 919
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Thr Asp Phe Leu Arg Asp Asp Ile Arg Phe Leu Gly Gln Ile Leu
1 5 10 15
Gly Glu Val Ile Ala Glu Gln Glu Gly Gln Glu Val Tyr Glu Leu Val
20 25 30
Glu Gln Ala Arg Leu Thr Ser Phe Asp Ile Ala Lys Gly Asn Ala Glu
35 40 45
Met Asp Ser Leu Val Gln Val Phe Asp Gly Ile Thr Pro Ala Lys Ala
50 55 60
Thr Pro Ile Ala Arg Ala Phe Ser His Phe Ala Leu Leu Ala Asn Leu
65 70 75 80
Ala Glu Asp Leu Tyr Asp Glu Glu Leu Arg Glu Gln Ala Leu Asp Ala
85 90 95
Gly Asp Thr Pro Pro Asp Ser Thr Leu Asp Ala Thr Trp Leu Lys Leu
100 105 110
Asn Glu Gly Asn Val Gly Ala Glu Ala Val Ala Asp Val Leu Arg Asn
115 120 125
Ala Glu Val Ala Pro Val Leu Thr Ala His Pro Thr Glu Thr Arg Arg
130 135 140
Arg Thr Val Phe Asp Ala Gln Lys Trp Ile Thr Thr His Met Arg Glu
145 150 155 160
Arg His Ala Leu Gln Ser Ala Glu Pro Thr Ala Arg Thr Gln Ser Lys
165 170 175
Leu Asp Glu Ile Glu Lys Asn Ile Arg Val Arg Ile Thr Ile Leu Trp
180 185 190
Gln Thr Ala Leu Ile Arg Val Ala Arg Pro Arg Ile Glu Asp Glu Ile
195 200 205
Glu Val Gly Leu Arg Tyr Tyr Lys Leu Ser Leu Leu Glu Glu Ile Pro
210 215 220
Arg Ile Asn Arg Asp Val Ala Val Glu Leu Arg Glu Arg Phe Gly Glu
225 230 235 240
Asp Val Pro Leu Lys Pro Val Val Lys Pro Gly Ser Trp Ile Gly Gly
245 250 255
Asp His Asp Gly Asn Pro Tyr Val Thr Ala Glu Thr Val Glu Tyr Ser
260 265 270
Thr His Arg Ala Ala Glu Thr Val Leu Lys Tyr Tyr Ala Arg Gln Leu
275 280 285
His Ser Leu Glu His Glu Leu Ser Leu Ser Asp Arg Met Asn Lys Val
290 295 300
Thr Pro Gln Leu Leu Ala Leu Ala Asp Ala Gly His Asn Asp Val Pro
305 310 315 320
Ser Arg Val Asp Glu Pro Tyr Arg Arg Ala Val His Gly Val Arg Gly
325 330 335
Arg Ile Leu Ala Thr Thr Ala Glu Leu Ile Gly Glu Asp Ala Val Glu
340 345 350
Gly Val Trp Phe Lys Val Phe Thr Pro Tyr Ala Ser Pro Glu Glu Phe
355 360 365
Leu Asn Asp Ala Leu Thr Ile Asp His Ser Leu Arg Glu Ser Asn Asp
370 375 380
Val Leu Ile Ala Asp Asp Arg Leu Ser Val Leu Ile Ser Ala Ile Glu
385 390 395 400
Ser Phe Gly Phe Asn Leu Tyr Ala Leu Asp Leu Arg Gln Asn Ser Glu
405 410 415
Ser Tyr Glu Asp Val Leu Thr Glu Leu Phe Glu Arg Ala Gln Val Thr
420 425 430
Ala Asn Tyr Arg Glu Leu Ser Glu Ala Glu Lys Leu Glu Val Leu Leu
435 440 445
Lys Glu Leu Arg Ser Pro Arg Pro Leu Ile Pro His Gly Ser Asp Glu
450 455 460
Tyr Ser Glu Val Thr Asp Arg Glu Leu Gly Ile Phe Arg Thr Ala Ser
465 470 475 480
Glu Ala Val Lys Lys Phe Gly Pro Arg Met Val Pro His Cys Ile Ile
485 490 495
Ser Met Ala Ser Ser Val Thr Asp Val Leu Glu Pro Met Val Leu Leu
500 505 510
Lys Glu Phe Gly Leu Ile Ala Ala Asn Gly Asp Asn Pro Arg Gly Thr
515 520 525
Val Asp Val Ile Pro Leu Phe Glu Thr Ile Glu Asp Leu Gln Ala Gly
530 535 540
Ala Gly Ile Leu Asp Glu Leu Trp Lys Ile Asp Leu Tyr Arg Asn Tyr
545 550 555 560
Leu Leu Gln Arg Asp Asn Val Gln Glu Val Met Leu Gly Tyr Ser Asp
565 570 575
Ser Asn Lys Asp Gly Gly Tyr Phe Ser Ala Asn Trp Ala Leu Tyr Asp
580 585 590
Ala Glu Leu Gln Leu Val Glu Leu Cys Arg Ser Ala Gly Val Lys Leu
595 600 605
Arg Leu Phe His Gly Arg Gly Gly Thr Val Gly Arg Gly Gly Gly Pro
610 615 620
Ser Tyr Asp Ala Ile Leu Ala Gln Pro Arg Gly Ala Val Gln Gly Ser
625 630 635 640
Val Arg Ile Thr Glu Gln Gly Glu Ile Ile Ser Ala Lys Tyr Gly Asn
645 650 655
Pro Glu Thr Ala Arg Arg Asn Leu Glu Ala Leu Val Ser Ala Thr Leu
660 665 670
Glu Ala Ser Leu Leu Asp Val Ser Glu Leu Thr Asp His Gln Arg Ala
675 680 685
Tyr Asp Ile Met Ser Glu Ile Ser Glu Leu Ser Leu Lys Lys Tyr Ala
690 695 700
Ser Leu Val His Glu Asp Gln Gly Phe Ile Asp Tyr Phe Thr Gln Ser
705 710 715 720
Thr Pro Leu Gln Glu Ile Gly Ser Leu Asn Ile Gly Ser Arg Pro Ser
725 730 735
Ser Arg Lys Gln Thr Ser Ser Val Glu Asp Leu Arg Ala Ile Pro Trp
740 745 750
Val Leu Ser Trp Ser Gln Ser Arg Val Met Leu Pro Gly Trp Phe Gly
755 760 765
Val Gly Thr Ala Leu Glu Gln Trp Ile Gly Glu Gly Glu Gln Ala Thr
770 775 780
Gln Arg Ile Ala Glu Leu Gln Thr Leu Asn Glu Ser Trp Pro Phe Phe
785 790 795 800
Thr Ser Val Leu Asp Asn Met Ala Gln Val Met Ser Lys Ala Glu Leu
805 810 815
Arg Leu Ala Lys Leu Tyr Ala Asp Leu Ile Pro Asp Arg Glu Val Ala
820 825 830
Glu Arg Val Tyr Ala Val Ile Arg Glu Glu Tyr Phe Leu Thr Lys Lys
835 840 845
Met Phe Cys Val Ile Thr Gly Ser Asp Asp Leu Leu Asp Asp Asn Pro
850 855 860
Leu Leu Ala Arg Ser Val Gln Arg Arg Tyr Pro Tyr Leu Leu Pro Leu
865 870 875 880
Asn Val Ile Gln Val Glu Met Met Arg Arg Tyr Arg Lys Gly Asp Gln
885 890 895
Ser Glu Gln Val Ser Arg Asn Ile Gln Leu Thr Met Asn Gly Leu Ser
900 905 910
Thr Ala Leu Arg Asn Ser Gly
915
<210> 12
<211> 919
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Thr Asp Phe Leu Arg Asp Asp Ile Arg Phe Leu Gly Gln Ile Leu
1 5 10 15
Gly Glu Val Ile Ala Glu Gln Glu Gly Gln Glu Val Tyr Glu Leu Val
20 25 30
Glu Gln Ala Arg Leu Thr Ser Phe Asp Ile Ala Lys Gly Asn Ala Glu
35 40 45
Met Asp Ser Leu Val Gln Val Phe Asp Gly Ile Thr Pro Ala Lys Ala
50 55 60
Thr Pro Ile Ala Arg Ala Phe Ser His Phe Ala Leu Leu Ala Asn Leu
65 70 75 80
Ala Glu Asp Leu Tyr Asp Glu Glu Leu Arg Glu Gln Ala Leu Asp Ala
85 90 95
Gly Asp Thr Pro Pro Asp Ser Thr Leu Asp Ala Thr Trp Leu Lys Leu
100 105 110
Asn Glu Gly Asn Val Gly Ala Glu Ala Val Ala Asp Val Leu Arg Asn
115 120 125
Ala Glu Val Ala Pro Val Leu Thr Ala His Pro Thr Glu Thr Arg Arg
130 135 140
Arg Thr Val Phe Asp Ala Gln Lys Trp Ile Thr Thr His Met Arg Glu
145 150 155 160
Arg His Ala Leu Gln Ser Ala Glu Pro Thr Ala Arg Thr Gln Ser Lys
165 170 175
Leu Asp Glu Ile Glu Lys Asn Ile Arg Arg Arg Ile Thr Ile Leu Trp
180 185 190
Gln Thr Ala Leu Ile Arg Val Ala Arg Pro Arg Ile Glu Asp Glu Ile
195 200 205
Glu Val Gly Leu Arg Tyr Tyr Lys Leu Ser Leu Leu Glu Glu Ile Pro
210 215 220
Arg Ile Asn Arg Asp Val Ala Val Glu Leu Arg Glu Arg Phe Gly Glu
225 230 235 240
Asp Val Pro Leu Lys Pro Val Val Lys Pro Gly Ser Trp Ile Gly Gly
245 250 255
Asp His Asp Gly Asn Pro Tyr Val Thr Ala Glu Thr Val Glu Tyr Ser
260 265 270
Thr His Arg Ala Ala Glu Thr Val Leu Lys Tyr Tyr Ala Arg Gln Leu
275 280 285
His Ser Leu Glu His Glu Leu Ser Leu Ser Asp Arg Met Asn Lys Val
290 295 300
Thr Pro Gln Leu Leu Ala Leu Ala Asp Ala Gly His Asn Asp Val Pro
305 310 315 320
Ser Arg Val Asp Glu Pro Tyr Arg Arg Ala Val His Gly Val Arg Gly
325 330 335
Arg Ile Leu Ala Thr Thr Ala Glu Leu Ile Gly Glu Asp Ala Val Glu
340 345 350
Gly Val Trp Phe Lys Val Phe Thr Pro Tyr Ala Ser Pro Glu Glu Phe
355 360 365
Leu Asn Asp Ala Leu Thr Ile Asp His Ser Leu Arg Glu Ser Asn Asp
370 375 380
Val Leu Ile Ala Asp Asp Arg Leu Ser Val Leu Ile Ser Ala Ile Glu
385 390 395 400
Ser Phe Gly Phe Asn Leu Tyr Ala Leu Asp Leu Arg Gln Asn Ser Glu
405 410 415
Ser Tyr Glu Asp Val Leu Thr Glu Leu Phe Glu Arg Ala Gln Val Thr
420 425 430
Ala Asn Tyr Arg Glu Leu Ser Glu Ala Glu Lys Leu Glu Val Leu Leu
435 440 445
Lys Glu Leu Arg Ser Pro Arg Pro Leu Ile Pro His Gly Ser Asp Glu
450 455 460
Tyr Ser Glu Val Thr Asp Arg Glu Leu Gly Ile Phe Arg Thr Ala Ser
465 470 475 480
Glu Ala Val Lys Lys Phe Gly Pro Arg Met Val Pro His Cys Ile Ile
485 490 495
Ser Met Ala Ser Ser Val Thr Asp Val Leu Glu Pro Met Val Leu Leu
500 505 510
Lys Glu Phe Gly Leu Ile Ala Ala Asn Gly Asp Asn Pro Arg Gly Thr
515 520 525
Val Asp Val Ile Pro Leu Phe Glu Thr Ile Glu Asp Leu Gln Ala Gly
530 535 540
Ala Gly Ile Leu Asp Glu Leu Trp Lys Ile Asp Leu Tyr Arg Asn Tyr
545 550 555 560
Leu Leu Gln Arg Asp Asn Val Gln Glu Val Met Leu Gly Tyr Ser Asp
565 570 575
Ser Asn Lys Asp Gly Gly Tyr Phe Ser Ala Asn Trp Ala Leu Tyr Asp
580 585 590
Ala Glu Leu Gln Leu Val Glu Leu Cys Arg Ser Ala Gly Val Lys Leu
595 600 605
Arg Leu Phe His Gly Arg Gly Gly Thr Val Gly Arg Gly Gly Gly Pro
610 615 620
Ser Tyr Asp Ala Ile Leu Ala Gln Pro Arg Gly Ala Val Gln Gly Ser
625 630 635 640
Val Arg Ile Thr Glu Gln Gly Glu Ile Ile Ser Ala Lys Tyr Gly Asn
645 650 655
Pro Glu Thr Ala Arg Arg Asn Leu Glu Ala Leu Val Ser Ala Thr Leu
660 665 670
Glu Ala Ser Leu Leu Asp Val Ser Glu Leu Thr Asp His Gln Arg Ala
675 680 685
Tyr Asp Ile Met Ser Glu Ile Ser Glu Leu Ser Leu Lys Lys Tyr Ala
690 695 700
Ser Leu Val His Glu Asp Gln Gly Phe Ile Asp Tyr Phe Thr Gln Ser
705 710 715 720
Thr Pro Leu Gln Glu Ile Gly Ser Leu Asn Ile Gly Ser Arg Pro Ser
725 730 735
Ser Arg Lys Gln Thr Ser Ser Val Glu Asp Leu Arg Ala Ile Pro Trp
740 745 750
Val Leu Ser Trp Ser Gln Ser Arg Val Met Leu Pro Gly Trp Phe Gly
755 760 765
Val Gly Thr Ala Leu Glu Gln Trp Ile Gly Glu Gly Glu Gln Ala Thr
770 775 780
Gln Arg Ile Ala Glu Leu Gln Thr Leu Asn Glu Ser Trp Pro Phe Phe
785 790 795 800
Thr Ser Val Leu Asp Asn Met Ala Gln Val Met Ser Lys Ala Glu Leu
805 810 815
Arg Leu Ala Lys Leu Tyr Ala Asp Leu Ile Pro Asp Arg Glu Val Ala
820 825 830
Glu Arg Val Tyr Ala Val Ile Arg Glu Glu Tyr Phe Leu Thr Lys Lys
835 840 845
Met Phe Cys Val Ile Thr Gly Ser Asp Asp Leu Leu Asp Asp Asn Pro
850 855 860
Leu Leu Ala Arg Ser Val Gln Arg Arg Tyr Pro Tyr Leu Leu Pro Leu
865 870 875 880
Asn Val Ile Gln Val Glu Met Met Arg Arg Tyr Arg Lys Gly Asp Gln
885 890 895
Ser Glu Gln Val Ser Arg Asn Ile Gln Leu Thr Met Asn Gly Leu Ser
900 905 910
Thr Ala Leu Arg Asn Ser Gly
915
<210> 13
<211> 670
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Ala Phe Ser Val Glu Met Pro Glu Leu Gly Glu Ser Val Thr Glu
1 5 10 15
Gly Thr Ile Thr Gln Trp Leu Lys Ser Val Gly Asp Thr Val Glu Val
20 25 30
Asp Glu Pro Leu Leu Glu Val Ser Thr Asp Lys Val Asp Thr Glu Ile
35 40 45
Pro Ser Pro Val Ala Gly Val Ile Leu Glu Ile Lys Ala Glu Glu Asp
50 55 60
Asp Thr Val Asp Val Gly Gly Val Ile Ala Ile Ile Gly Asp Ala Asp
65 70 75 80
Glu Thr Pro Ala Asn Glu Ala Pro Ala Asp Glu Ala Pro Ala Pro Ala
85 90 95
Glu Glu Glu Glu Pro Val Lys Glu Glu Pro Lys Lys Glu Ala Ala Pro
100 105 110
Glu Ala Pro Ala Ala Thr Gly Ala Ala Thr Asp Val Glu Met Pro Glu
115 120 125
Leu Gly Glu Ser Val Thr Glu Gly Thr Ile Thr Gln Trp Leu Lys Ala
130 135 140
Val Gly Asp Thr Val Glu Val Asp Glu Pro Leu Leu Glu Val Ser Thr
145 150 155 160
Asp Lys Val Asp Thr Glu Ile Pro Ser Pro Val Ala Gly Thr Ile Val
165 170 175
Glu Ile Leu Ala Asp Glu Asp Asp Thr Val Asp Val Gly Ala Val Ile
180 185 190
Ala Arg Ile Gly Asp Ala Asn Ala Ala Ala Ala Pro Ala Glu Glu Glu
195 200 205
Ala Ala Pro Ala Glu Glu Glu Glu Pro Val Lys Glu Glu Pro Lys Lys
210 215 220
Glu Ala Ala Pro Glu Ala Pro Ala Ala Thr Gly Ala Ala Thr Asp Val
225 230 235 240
Glu Met Pro Glu Leu Gly Glu Ser Val Thr Glu Gly Thr Ile Thr Gln
245 250 255
Trp Leu Lys Ala Val Gly Asp Thr Val Glu Val Asp Glu Pro Leu Leu
260 265 270
Glu Val Ser Thr Asp Lys Val Asp Thr Glu Ile Pro Ser Pro Val Ala
275 280 285
Gly Thr Ile Val Glu Ile Leu Ala Asp Glu Asp Asp Thr Val Asp Val
290 295 300
Gly Ala Val Ile Ala Arg Ile Gly Asp Ala Asn Ala Ala Ala Ala Pro
305 310 315 320
Gly Glu Glu Lys Ala Ala Pro Ala Glu Glu Glu Glu Pro Lys Gln Glu
325 330 335
Glu Pro Lys Lys Glu Glu Pro Lys Lys Glu Ala Ala Thr Thr Pro Ala
340 345 350
Ala Ala Ser Ala Thr Val Ser Ala Ser Gly Asp Asn Val Pro Tyr Val
355 360 365
Thr Pro Leu Val Arg Lys Leu Ala Glu Lys His Gly Val Asp Leu Asn
370 375 380
Thr Val Thr Gly Thr Gly Ile Gly Gly Arg Ile Arg Lys Gln Asp Val
385 390 395 400
Leu Ala Ala Ala Asn Gly Glu Ala Ala Pro Ala Glu Ala Ala Ala Pro
405 410 415
Val Ser Ala Trp Ser Thr Lys Ser Val Asp Pro Glu Lys Ala Lys Leu
420 425 430
Arg Gly Thr Thr Gln Lys Val Asn Arg Ile Arg Glu Ile Thr Ala Met
435 440 445
Lys Thr Val Glu Ala Leu Gln Ile Ser Ala Gln Leu Thr Gln Leu His
450 455 460
Glu Val Asp Met Thr Arg Val Ala Glu Leu Arg Lys Lys Asn Lys Pro
465 470 475 480
Ala Phe Ile Glu Lys His Gly Val Asn Leu Thr Tyr Leu Pro Phe Phe
485 490 495
Val Lys Ala Val Val Glu Ala Leu Val Ser His Pro Asn Val Asn Ala
500 505 510
Ser Tyr Asn Ala Lys Thr Lys Glu Met Thr Tyr His Ser Ser Val Asn
515 520 525
Leu Ser Ile Ala Val Asp Thr Pro Ala Gly Leu Leu Thr Pro Val Ile
530 535 540
His Asp Ala Gln Asp Leu Ser Ile Pro Glu Ile Ala Lys Ala Ile Val
545 550 555 560
Asp Leu Ala Asp Arg Ser Arg Asn Asn Lys Leu Lys Pro Asn Asp Leu
565 570 575
Ser Gly Gly Thr Phe Thr Ile Thr Asn Ile Gly Ser Glu Gly Ala Leu
580 585 590
Ser Asp Thr Pro Ile Leu Val Pro Pro Gln Ala Gly Ile Leu Gly Thr
595 600 605
Gly Ala Ile Val Lys Arg Pro Val Val Ile Thr Glu Asp Gly Ile Asp
610 615 620
Ser Ile Ala Ile Arg Gln Met Val Phe Leu Pro Leu Thr Tyr Asp His
625 630 635 640
Gln Val Val Asp Gly Ala Asp Ala Gly Arg Phe Leu Thr Thr Ile Lys
645 650 655
Asp Arg Leu Glu Thr Ala Asn Phe Glu Gly Asp Leu Gln Leu
660 665 670
<210> 14
<211> 670
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Met Ala Phe Ser Val Glu Met Pro Glu Leu Gly Glu Ser Val Thr Glu
1 5 10 15
Gly Thr Ile Thr Gln Trp Leu Lys Ser Val Gly Asp Thr Val Glu Val
20 25 30
Asp Glu Pro Leu Leu Glu Val Ser Thr Asp Lys Val Asp Thr Glu Ile
35 40 45
Pro Ser Pro Val Ala Gly Val Ile Leu Glu Ile Lys Ala Glu Glu Asp
50 55 60
Asp Thr Val Asp Val Gly Gly Val Ile Ala Ile Ile Asp Asp Ala Asp
65 70 75 80
Glu Thr Pro Ala Asn Glu Ala Pro Ala Asp Glu Ala Pro Ala Pro Ala
85 90 95
Glu Glu Glu Glu Pro Val Lys Glu Glu Pro Lys Lys Glu Ala Ala Pro
100 105 110
Glu Ala Pro Ala Ala Thr Gly Ala Ala Thr Asp Val Glu Met Pro Glu
115 120 125
Leu Gly Glu Ser Val Thr Glu Gly Thr Ile Thr Gln Trp Leu Lys Ala
130 135 140
Val Gly Asp Thr Val Glu Val Asp Glu Pro Leu Leu Glu Val Ser Thr
145 150 155 160
Asp Lys Val Asp Thr Glu Ile Pro Ser Pro Val Ala Gly Thr Ile Val
165 170 175
Glu Ile Leu Ala Asp Glu Asp Asp Thr Val Asp Val Gly Ala Val Ile
180 185 190
Ala Arg Ile Gly Asp Ala Asn Ala Ala Ala Ala Pro Ala Glu Glu Glu
195 200 205
Ala Ala Pro Ala Glu Glu Glu Glu Pro Val Lys Glu Glu Pro Lys Lys
210 215 220
Glu Ala Ala Pro Glu Ala Pro Ala Ala Thr Gly Ala Ala Thr Asp Val
225 230 235 240
Glu Met Pro Glu Leu Gly Glu Ser Val Thr Glu Gly Thr Ile Thr Gln
245 250 255
Trp Leu Lys Ala Val Gly Asp Thr Val Glu Val Asp Glu Pro Leu Leu
260 265 270
Glu Val Ser Thr Asp Lys Val Asp Thr Glu Ile Pro Ser Pro Val Ala
275 280 285
Gly Thr Ile Val Glu Ile Leu Ala Asp Glu Asp Asp Thr Val Asp Val
290 295 300
Gly Ala Val Ile Ala Arg Ile Gly Asp Ala Asn Ala Ala Ala Ala Pro
305 310 315 320
Gly Glu Glu Lys Ala Ala Pro Ala Glu Glu Glu Glu Pro Lys Gln Glu
325 330 335
Glu Pro Lys Lys Glu Glu Pro Lys Lys Glu Ala Ala Thr Thr Pro Ala
340 345 350
Ala Ala Ser Ala Thr Val Ser Ala Ser Gly Asp Asn Val Pro Tyr Val
355 360 365
Thr Pro Leu Val Arg Lys Leu Ala Glu Lys His Gly Val Asp Leu Asn
370 375 380
Thr Val Thr Gly Thr Gly Ile Gly Gly Arg Ile Arg Lys Gln Asp Val
385 390 395 400
Leu Ala Ala Ala Asn Gly Glu Ala Ala Pro Ala Glu Ala Ala Ala Pro
405 410 415
Val Ser Ala Trp Ser Thr Lys Ser Val Asp Pro Glu Lys Ala Lys Leu
420 425 430
Arg Gly Thr Thr Gln Lys Val Asn Arg Ile Arg Glu Ile Thr Ala Met
435 440 445
Lys Thr Val Glu Ala Leu Gln Ile Ser Ala Gln Leu Thr Gln Leu His
450 455 460
Glu Val Asp Met Thr Arg Val Ala Glu Leu Arg Lys Lys Asn Lys Pro
465 470 475 480
Ala Phe Ile Glu Lys His Gly Val Asn Leu Thr Tyr Leu Pro Phe Phe
485 490 495
Val Lys Ala Val Val Glu Ala Leu Val Ser His Pro Asn Val Asn Ala
500 505 510
Ser Tyr Asn Ala Lys Thr Lys Glu Met Thr Tyr His Ser Ser Val Asn
515 520 525
Leu Ser Ile Ala Val Asp Thr Pro Ala Gly Leu Leu Thr Pro Val Ile
530 535 540
His Asp Ala Gln Asp Leu Ser Ile Pro Glu Ile Ala Lys Ala Ile Val
545 550 555 560
Asp Leu Ala Asp Arg Ser Arg Asn Asn Lys Leu Lys Pro Asn Asp Leu
565 570 575
Ser Gly Gly Thr Phe Thr Ile Thr Asn Ile Gly Ser Glu Gly Ala Leu
580 585 590
Ser Asp Thr Pro Ile Leu Val Pro Pro Gln Ala Gly Ile Leu Gly Thr
595 600 605
Gly Ala Ile Val Lys Arg Pro Val Val Ile Thr Glu Asp Gly Ile Asp
610 615 620
Ser Ile Ala Ile Arg Gln Met Val Phe Leu Pro Leu Thr Tyr Asp His
625 630 635 640
Gln Val Val Asp Gly Ala Asp Ala Gly Arg Phe Leu Thr Thr Ile Lys
645 650 655
Asp Arg Leu Glu Thr Ala Asn Phe Glu Gly Asp Leu Gln Leu
660 665 670
<210> 15
<211> 670
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Ala Phe Ser Val Glu Met Pro Glu Leu Gly Glu Ser Val Thr Glu
1 5 10 15
Gly Thr Ile Thr Gln Trp Leu Lys Ser Val Gly Asp Thr Val Glu Val
20 25 30
Asp Glu Pro Leu Leu Glu Val Ser Thr Asp Lys Val Asp Thr Glu Ile
35 40 45
Pro Ser Pro Val Ala Gly Val Ile Leu Glu Ile Lys Ala Glu Glu Asp
50 55 60
Asp Thr Val Asp Val Gly Gly Val Ile Ala Ile Ile Val Asp Ala Asp
65 70 75 80
Glu Thr Pro Ala Asn Glu Ala Pro Ala Asp Glu Ala Pro Ala Pro Ala
85 90 95
Glu Glu Glu Glu Pro Val Lys Glu Glu Pro Lys Lys Glu Ala Ala Pro
100 105 110
Glu Ala Pro Ala Ala Thr Gly Ala Ala Thr Asp Val Glu Met Pro Glu
115 120 125
Leu Gly Glu Ser Val Thr Glu Gly Thr Ile Thr Gln Trp Leu Lys Ala
130 135 140
Val Gly Asp Thr Val Glu Val Asp Glu Pro Leu Leu Glu Val Ser Thr
145 150 155 160
Asp Lys Val Asp Thr Glu Ile Pro Ser Pro Val Ala Gly Thr Ile Val
165 170 175
Glu Ile Leu Ala Asp Glu Asp Asp Thr Val Asp Val Gly Ala Val Ile
180 185 190
Ala Arg Ile Gly Asp Ala Asn Ala Ala Ala Ala Pro Ala Glu Glu Glu
195 200 205
Ala Ala Pro Ala Glu Glu Glu Glu Pro Val Lys Glu Glu Pro Lys Lys
210 215 220
Glu Ala Ala Pro Glu Ala Pro Ala Ala Thr Gly Ala Ala Thr Asp Val
225 230 235 240
Glu Met Pro Glu Leu Gly Glu Ser Val Thr Glu Gly Thr Ile Thr Gln
245 250 255
Trp Leu Lys Ala Val Gly Asp Thr Val Glu Val Asp Glu Pro Leu Leu
260 265 270
Glu Val Ser Thr Asp Lys Val Asp Thr Glu Ile Pro Ser Pro Val Ala
275 280 285
Gly Thr Ile Val Glu Ile Leu Ala Asp Glu Asp Asp Thr Val Asp Val
290 295 300
Gly Ala Val Ile Ala Arg Ile Gly Asp Ala Asn Ala Ala Ala Ala Pro
305 310 315 320
Gly Glu Glu Lys Ala Ala Pro Ala Glu Glu Glu Glu Pro Lys Gln Glu
325 330 335
Glu Pro Lys Lys Glu Glu Pro Lys Lys Glu Ala Ala Thr Thr Pro Ala
340 345 350
Ala Ala Ser Ala Thr Val Ser Ala Ser Gly Asp Asn Val Pro Tyr Val
355 360 365
Thr Pro Leu Val Arg Lys Leu Ala Glu Lys His Gly Val Asp Leu Asn
370 375 380
Thr Val Thr Gly Thr Gly Ile Gly Gly Arg Ile Arg Lys Gln Asp Val
385 390 395 400
Leu Ala Ala Ala Asn Gly Glu Ala Ala Pro Ala Glu Ala Ala Ala Pro
405 410 415
Val Ser Ala Trp Ser Thr Lys Ser Val Asp Pro Glu Lys Ala Lys Leu
420 425 430
Arg Gly Thr Thr Gln Lys Val Asn Arg Ile Arg Glu Ile Thr Ala Met
435 440 445
Lys Thr Val Glu Ala Leu Gln Ile Ser Ala Gln Leu Thr Gln Leu His
450 455 460
Glu Val Asp Met Thr Arg Val Ala Glu Leu Arg Lys Lys Asn Lys Pro
465 470 475 480
Ala Phe Ile Glu Lys His Gly Val Asn Leu Thr Tyr Leu Pro Phe Phe
485 490 495
Val Lys Ala Val Val Glu Ala Leu Val Ser His Pro Asn Val Asn Ala
500 505 510
Ser Tyr Asn Ala Lys Thr Lys Glu Met Thr Tyr His Ser Ser Val Asn
515 520 525
Leu Ser Ile Ala Val Asp Thr Pro Ala Gly Leu Leu Thr Pro Val Ile
530 535 540
His Asp Ala Gln Asp Leu Ser Ile Pro Glu Ile Ala Lys Ala Ile Val
545 550 555 560
Asp Leu Ala Asp Arg Ser Arg Asn Asn Lys Leu Lys Pro Asn Asp Leu
565 570 575
Ser Gly Gly Thr Phe Thr Ile Thr Asn Ile Gly Ser Glu Gly Ala Leu
580 585 590
Ser Asp Thr Pro Ile Leu Val Pro Pro Gln Ala Gly Ile Leu Gly Thr
595 600 605
Gly Ala Ile Val Lys Arg Pro Val Val Ile Thr Glu Asp Gly Ile Asp
610 615 620
Ser Ile Ala Ile Arg Gln Met Val Phe Leu Pro Leu Thr Tyr Asp His
625 630 635 640
Gln Val Val Asp Gly Ala Asp Ala Gly Arg Phe Leu Thr Thr Ile Lys
645 650 655
Asp Arg Leu Glu Thr Ala Asn Phe Glu Gly Asp Leu Gln Leu
660 665 670
<210> 16
<211> 670
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Met Ala Phe Ser Val Glu Met Pro Glu Leu Gly Glu Ser Val Thr Glu
1 5 10 15
Gly Thr Ile Thr Gln Trp Leu Lys Ser Val Gly Asp Thr Val Glu Val
20 25 30
Asp Glu Pro Leu Leu Glu Val Ser Thr Asp Lys Val Asp Thr Glu Ile
35 40 45
Pro Ser Pro Val Ala Gly Val Ile Leu Glu Ile Lys Ala Glu Glu Asp
50 55 60
Asp Thr Val Asp Val Gly Gly Val Ile Ala Ile Ile Ala Asp Ala Asp
65 70 75 80
Glu Thr Pro Ala Asn Glu Ala Pro Ala Asp Glu Ala Pro Ala Pro Ala
85 90 95
Glu Glu Glu Glu Pro Val Lys Glu Glu Pro Lys Lys Glu Ala Ala Pro
100 105 110
Glu Ala Pro Ala Ala Thr Gly Ala Ala Thr Asp Val Glu Met Pro Glu
115 120 125
Leu Gly Glu Ser Val Thr Glu Gly Thr Ile Thr Gln Trp Leu Lys Ala
130 135 140
Val Gly Asp Thr Val Glu Val Asp Glu Pro Leu Leu Glu Val Ser Thr
145 150 155 160
Asp Lys Val Asp Thr Glu Ile Pro Ser Pro Val Ala Gly Thr Ile Val
165 170 175
Glu Ile Leu Ala Asp Glu Asp Asp Thr Val Asp Val Gly Ala Val Ile
180 185 190
Ala Arg Ile Gly Asp Ala Asn Ala Ala Ala Ala Pro Ala Glu Glu Glu
195 200 205
Ala Ala Pro Ala Glu Glu Glu Glu Pro Val Lys Glu Glu Pro Lys Lys
210 215 220
Glu Ala Ala Pro Glu Ala Pro Ala Ala Thr Gly Ala Ala Thr Asp Val
225 230 235 240
Glu Met Pro Glu Leu Gly Glu Ser Val Thr Glu Gly Thr Ile Thr Gln
245 250 255
Trp Leu Lys Ala Val Gly Asp Thr Val Glu Val Asp Glu Pro Leu Leu
260 265 270
Glu Val Ser Thr Asp Lys Val Asp Thr Glu Ile Pro Ser Pro Val Ala
275 280 285
Gly Thr Ile Val Glu Ile Leu Ala Asp Glu Asp Asp Thr Val Asp Val
290 295 300
Gly Ala Val Ile Ala Arg Ile Gly Asp Ala Asn Ala Ala Ala Ala Pro
305 310 315 320
Gly Glu Glu Lys Ala Ala Pro Ala Glu Glu Glu Glu Pro Lys Gln Glu
325 330 335
Glu Pro Lys Lys Glu Glu Pro Lys Lys Glu Ala Ala Thr Thr Pro Ala
340 345 350
Ala Ala Ser Ala Thr Val Ser Ala Ser Gly Asp Asn Val Pro Tyr Val
355 360 365
Thr Pro Leu Val Arg Lys Leu Ala Glu Lys His Gly Val Asp Leu Asn
370 375 380
Thr Val Thr Gly Thr Gly Ile Gly Gly Arg Ile Arg Lys Gln Asp Val
385 390 395 400
Leu Ala Ala Ala Asn Gly Glu Ala Ala Pro Ala Glu Ala Ala Ala Pro
405 410 415
Val Ser Ala Trp Ser Thr Lys Ser Val Asp Pro Glu Lys Ala Lys Leu
420 425 430
Arg Gly Thr Thr Gln Lys Val Asn Arg Ile Arg Glu Ile Thr Ala Met
435 440 445
Lys Thr Val Glu Ala Leu Gln Ile Ser Ala Gln Leu Thr Gln Leu His
450 455 460
Glu Val Asp Met Thr Arg Val Ala Glu Leu Arg Lys Lys Asn Lys Pro
465 470 475 480
Ala Phe Ile Glu Lys His Gly Val Asn Leu Thr Tyr Leu Pro Phe Phe
485 490 495
Val Lys Ala Val Val Glu Ala Leu Val Ser His Pro Asn Val Asn Ala
500 505 510
Ser Tyr Asn Ala Lys Thr Lys Glu Met Thr Tyr His Ser Ser Val Asn
515 520 525
Leu Ser Ile Ala Val Asp Thr Pro Ala Gly Leu Leu Thr Pro Val Ile
530 535 540
His Asp Ala Gln Asp Leu Ser Ile Pro Glu Ile Ala Lys Ala Ile Val
545 550 555 560
Asp Leu Ala Asp Arg Ser Arg Asn Asn Lys Leu Lys Pro Asn Asp Leu
565 570 575
Ser Gly Gly Thr Phe Thr Ile Thr Asn Ile Gly Ser Glu Gly Ala Leu
580 585 590
Ser Asp Thr Pro Ile Leu Val Pro Pro Gln Ala Gly Ile Leu Gly Thr
595 600 605
Gly Ala Ile Val Lys Arg Pro Val Val Ile Thr Glu Asp Gly Ile Asp
610 615 620
Ser Ile Ala Ile Arg Gln Met Val Phe Leu Pro Leu Thr Tyr Asp His
625 630 635 640
Gln Val Val Asp Gly Ala Asp Ala Gly Arg Phe Leu Thr Thr Ile Lys
645 650 655
Asp Arg Leu Glu Thr Ala Asn Phe Glu Gly Asp Leu Gln Leu
660 665 670
<210> 17
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
acaacgtcgt gactgggaaa acccgatgac atcaggttcc tcggtc 46
<210> 18
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gtctgccaca aaatggtgat gcgagcgcgg atgttcttct cgatc 45
<210> 19
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gatcgagaag aacatccgcg ctcgcatcac cattttgtgg cagac 45
<210> 20
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
cgtaatcatg tcatagctgt ttccacacgc cctcaacggc gtcct 45
<210> 21
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gaccgaggaa cctgatgtca tcgggttttc ccagtcacga cgttgt 46
<210> 22
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
aggacgccgt tgagggcgtg tggaaacagc tatgacatga ttacg 45
<210> 23
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gtctgccaca aaatggtgat gcgaacgcgg atgttcttct cgatc 45
<210> 24
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gatcgagaag aacatccgcg ttcgcatcac cattttgtgg cagac 45
<210> 25
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gtctgccaca aaatggtgat gcgacggcgg atgttcttct cgatc 45
<210> 26
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gatcgagaag aacatccgcc gtcgcatcac cattttgtgg cagac 45
<210> 27
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
acaacgtcgt gactgggaaa accctggcgt tctccgtaga gatgc 45
<210> 28
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
ggcaggagtc tcatcagcat cgtcgattat tgcaatgaca ccgc 44
<210> 29
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
gcggtgtcat tgcaataatc gacgatgctg atgagactcc tgcc 44
<210> 30
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
cgtaatcatg tcatagctgt ttccgtagta gctgcttcct tctttg 46
<210> 31
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gcatctctac ggagaacgcc agggttttcc cagtcacgac gttgt 45
<210> 32
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
caaagaagga agcagctact acggaaacag ctatgacatg attacg 46
<210> 33
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
ggcaggagtc tcatcagcat cgacgattat tgcaatgaca ccgc 44
<210> 34
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
gcggtgtcat tgcaataatc gtcgatgctg atgagactcc tgcc 44
<210> 35
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
ggcaggagtc tcatcagcat cggcgattat tgcaatgaca ccgc 44
<210> 36
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
gcggtgtcat tgcaataatc gccgatgctg atgagactcc tgcc 44
Claims (10)
1.磷酸烯醇式丙酮酸羧化酶的突变体，其特征在于，所述突变体是，由ppc基因编码的磷酸烯醇式丙酮酸羧化酶的第186位氨基酸由甘氨酸突变为丙氨酸、缬氨酸或精氨酸。
2.根据权利要求1所述的突变体，其特征在于，磷酸烯醇式丙酮酸羧化酶突变体的氨基酸序列如SEQ ID No.10-12。
3.二氢硫辛酰胺乙酰转移酶的突变体，其特征在于，所述突变体是，由aceF基因编码的二氢硫辛酰胺乙酰转移酶的第77位氨基酸由甘氨酸突变为天冬氨酸、缬氨酸或丙氨酸。
4.根据权利要求3所述的突变体，其特征在于，二氢硫辛酰胺乙酰转移酶的突变体的氨基酸序列如SEQ ID No.14-16所示。
5.编码权利要求1-4任一项所述突变体的核苷酸序列，其特征在于，编码磷酸烯醇式丙酮酸羧化酶突变体的核苷酸序列如SEQ ID No.2-4所示；编码二氢硫辛酰胺乙酰转移酶突变体的核苷酸序列如SEQ ID No.6-8所示。
6.含有权利要求5所述核苷酸序列的生物材料，其特征在于，所述生物材料为表达盒、载体或宿主细胞。
7.一种重组微生物，其特征在于，所述重组微生物中含有磷酸烯醇式丙酮酸羧化酶突变体和/或二氢硫辛酰胺乙酰转移酶突变体。
8.根据权利要求7所述的重组微生物，其特征在于，所述磷酸烯醇式丙酮酸羧化酶突变体是，由ppc基因编码的磷酸烯醇式丙酮酸羧化酶的第186位氨基酸由甘氨酸突变为丙氨酸、缬氨酸或精氨酸；所述二氢硫辛酰胺乙酰转移酶突变体是，由aceF基因编码的二氢硫辛酰胺乙酰转移酶的第77位氨基酸由甘氨酸突变为天冬氨酸、缬氨酸或丙氨酸。
9.根据权利要求7-8任一项所述的重组微生物，其特征在于，所述重组微生物为谷氨酸棒杆菌、北京棒杆菌或黄色短杆菌。
10.权利要求1-4任一项所述的突变体或权利要求5所述的核苷酸序列或权利要求6所述的生物材料或权利要求7-9任一项所述的重组微生物在提高支链氨基酸或其衍生物产量中的应用。
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