CN110016458B - 一种发酵合成α-红没药醇的工程菌株及其构建方法 - Google Patents

一种发酵合成α-红没药醇的工程菌株及其构建方法 Download PDF

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CN110016458B
CN110016458B CN201910333500.XA CN201910333500A CN110016458B CN 110016458 B CN110016458 B CN 110016458B CN 201910333500 A CN201910333500 A CN 201910333500A CN 110016458 B CN110016458 B CN 110016458B
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康振
陈坚
堵国成
韦朝宝
王丽
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Abstract

本发明公开了一种发酵合成α‑红没药醇的工程菌株及其构建方法，属于生物工程技术领域。本发明以大肠杆菌和枯草芽孢杆菌为宿主，以枯草芽孢杆菌、大肠杆菌与酿酒酵母的三穿梭质粒pEBS为表达载体，在大肠杆菌和枯草芽孢杆菌引入将不同来源的BBS基因和不同来源的ispA基因进行不同组合的优化表达，再进行密码子优化、RBS优化、启动子优化等策略，实现了高效生物合成α‑红没药醇。本发明为微生物系统高效发酵制备(﹣)‑α‑红没药醇奠定了基础，适合于工业化生产应用。

Description

一种发酵合成α-红没药醇的工程菌株及其构建方法
技术领域
本发明涉及一种发酵合成α-红没药醇的工程菌株及其构建方法，属于生物工程技术领域。
背景技术
α-红没药醇，又称甜红没药醇、防风根醇，是自然界中存在较多的倍半萜化合物之一，有α-体和β-体两种结构。(﹣)-α-红没药醇主要存在于春黄菊精油中，含量达17％，其右旋体存在于胶杨精油以及某些苦槛蓝属和鼠尾草属的精油中。红没药醇具有降低皮肤炎症、抑菌抗刺激、活血止痛、愈合溃疡、溶解胆石等多种生物活性，因此它可以作为活性成分用于皮肤化妆品或抑制皮肤炎症，以保护和护理过敏性皮肤等，在医药行业中的用途较广。此外，红没药醇香气清淡愉快，也是一种稳定性较好的定香剂，在香料香精化妆品中的应用也日益受到重视。
红没药醇作为次级代谢产物，天然产量有限，若采用直接提取方法获取，不仅产量少而且会危及生态和环境安全，甚至造成濒危植物消失。红没药醇有复杂手性化学结构，若采用化学方法则化学合成难度高，纯度低，生物活性差。因此，利用生物构工程菌实现以廉价的碳源和培养基生产具有高附加值和广泛用途的红没药醇是一条最具有潜力的途径。
然而，现有的报道中，通过构建基因工程菌生产红没药醇的方法，其效果并不理想。如Gui Hwan Han等通过三个步骤来构建产红没药醇基因工程菌，(1)将德国洋甘菊(﹣)-α-红没药醇合成酶基因(MrBBS)引入大肠杆菌；(2)设计了外源性MVA途径以增加红没药醇前体库；(3)过表达ispA基因，该基因有效地提供(﹣)-α-红没药醇前体(FPP)。但该过程繁琐，需引入一整条外源途径，且产量仅为80mg/L。(Han G H,Kim S K,Yoon K S,etal.Fermentative production and direct extraction of(-)-α-bisabolol inmetabolically engineered Escherichia coli[J].Microbial Cell Factories,2016,15(1):185.)。
因此，提供一种高效生物合成红没药醇的方法，对于工业生产红没药醇具有重要的应用价值。
发明内容
本发明的第一个目的是提供一种发酵合成α-红没药醇的工程菌株，是以大肠杆菌或枯草芽孢杆菌为宿主，同时表达了BBS基因和ispA基因，所述ispA基因的氨基酸序列如SEQ ID NO.12-SEQ ID NO.14任一所示，所述BBS基因的氨基酸序列如SEQ ID NO.15或SEQID NO.16所示，或，表达了氨基酸序列如SEQ ID NO.17-SEQ ID NO.19任一所示的蛋白质。
在本发明的一种实施方式中，所述大肠杆菌包括大肠杆菌MG1655，大肠杆菌DH5α，大肠杆菌W3110或大肠杆菌BL21。
在本发明的一种实施方式中，所述枯草芽孢杆菌包括枯草芽孢杆菌168、枯草芽孢杆菌W600或枯草芽孢杆菌W800。
在本发明的一种实施方式中，所述ispA基因的核苷酸序列如SEQ ID NO.1-SEQ IDNO.3任一所示。
在本发明的一种实施方式中，所述BBS基因的核苷酸序列如SEQ ID NO.4或SEQ IDNO.5所示。
在本发明的一种实施方式中，所述工程菌株以pEBS为表达载体(构建方法见YangS,Liu Q,Zhang Y,et al.Construction and Characterization of Broad-spectrumPromoters for Synthetic Biology.[J].Acs Synthetic Biology,2017,7(1):287-291.)。
本发明的第二个目的是提供一种生产α-红没药醇的方法，应用了上述工程菌株进行发酵。
在本发明的一种实施方式中，将工程菌株接种于LB培养基中，置于200-220rpm，35-38℃培养10-12h，然后按5-10％的接种量转接于发酵培养基中，添加20-25％(v/v)的正十二烷，然后置于200-220rpm，35-38℃培养。培养50-70h
在本发明的一种实施方式中，所述发酵培养基组分为(g/L)：酵母粉2.5，蛋白胨5.0，Na2HPO4 6.78，KH2PO4 3.0，NaCl 0.5，NH4Cl 1.0，MgSO4·7H20 0.5，CaCl2 0.015，葡萄糖40；FeCl2·6H2O 0.013.5；MnCl2·4H2O，0.001；ZnCl2，0.0017；CuCl2·2H2O，0.00043。
本发明的第三个目的是提供上述工程菌在制备α-红没药醇或含α-红没药醇的产品中的应用。
本发明的有益效果：
本发明以大肠杆菌或枯草芽孢杆菌为宿主，以枯草芽孢杆菌、大肠杆菌与酿酒酵母的三穿梭质粒pEBS为表达载体，在大肠杆菌和枯草芽孢杆菌引入将不同来源的BBS基因和不同来源的ispA基因进行不同组合的优化表达，再进行密码子优化、RBS优化、启动子优化等策略，实现了高效生物合成α-红没药醇。本发明能够使大肠杆菌重组菌在摇瓶上培养60h本发明在大肠杆菌和枯草芽孢杆菌引入将不同来源的BBS基因和不同来源的ispA基因进行不同组合的优化表达，再进行密码子优化、RBS优化、启动子优化等策略，实现了高效生物合成α-红没药醇。本发明能够使大肠杆菌重组菌在摇瓶上培养60h后α-红没药醇积累量高达3.37g/L，枯草芽孢杆菌重组菌在摇瓶上培养60h后α-红没药醇积累量高达4.15g/L，在工业上具有潜在而广泛的应用价值。
附图说明
图1为不同来源的BBS基因和不同来源的ispA基因不同组合优化表达的构建示意图。
图2为异源表达不同来源BBS基因和ispA基因的大肠杆菌重组菌在摇瓶上培养60h后α-红没药醇积累量。
图3为异源表达不同来源BBS基因和ispA基因的枯草芽孢杆菌重组菌在摇瓶上培养60h后α-红没药醇积累量。
图4为优化BBS基因和ispA基因启动子的大肠杆菌、枯草芽孢杆菌重组菌在摇瓶上培养60h后α-红没药醇积累量。
具体实施方式
一、实施例涉及的核苷酸序列信息
(1)SEQ ID NO.1序列信息为来源于Bacillus subtilis subsp.subtilisstr.168的ispA基因核苷酸序列；
(2)SEQ ID NO.2序列信息为来源于Escherichia coli str.K-12substr.MG1655的ispA基因核苷酸序列；
(3)SEQ ID NO.3序列信息为来源于Enterococcus faecalis V583的ispA基因核苷酸序列；
(4)SEQ ID NO.4序列信息为来源于Streptomyces citricolor SC2的BBS基因核苷酸序列；
(5)SEQ ID NO.5序列信息为来源于Matricaria chamomilla var的BBS基因核苷酸序列；
(6)SEQ ID NO.6序列信息为组成型启动子Pbe(1)核苷酸序列；
(7)SEQ ID NO.7序列信息为组成型启动子Pbe(2)核苷酸序列；
(8)SEQ ID NO.8序列信息为组成型启动子Pbe(3)核苷酸序列。
二、表达系统的构建
分别以Bacillus subtilis subsp.subtilis str.168基因组、Escherichia colistr.K-12substr.MG1655基因组和Enterococcus faecalis V583基因组为模板，通过PCR扩增出不同来源的ispA DNA片段(分别如SEQ ID NO.1-SEQ ID NO.3所示)，使用的引物分别是bsA-R/bsA(sc)-F、bsA-R/bsA(mc)-F、ecA-R/ecA(sc)-F、ecA-R/ecA(mc)-F、efA-R/efA(sc)-F、efA-R/efA(mc)-F；分别以Streptomyces citricolor SC2基因组、Matricariachamomilla var基因组为模板，通过PCR扩增出不同来源的BBS DNA片段(分别如SEQ IDNO.4或SEQ ID NO.5所示)，使用的引物分别是scBBS-F/scBBS-R、mcBBS-F/mcBBS-R；将所得的不同来源ispA DNA片段、BBS DNA片段与pEBS(使用引物pEBS-F/pEBS-R扩增)骨架组装以产生pEBS-BBSscispAbs、pEBS-BBSmcispAbs、pEBS-BBSscispAec、pEBS-BBSmcispAec、pEBS-BBSscispAef和pEBS-BBSmcispAef。以pEBS-BBSmcispAbs为模板，分别使用引物Pbe(1)-F/Pbe(1)-R、Pbe(2)-F/Pbe(2)-R、Pbe(3)-F/Pbe(3)-R进行环化PCR扩增，再使用DpnⅠ消化后可得到pEBS-Pbe(1)-BBSmcispAbs、pEBS-Pbe(2)-BBSmcispAbs、pEBS-Pbe(2)-BBSmcispAbs，如图1所示。
三、重组菌的构建
分别向大肠杆菌MG1655，大肠杆菌DH5α，大肠杆菌W3110，大肠杆菌BL21转入重组质粒pEBS-BBSscispAbs、pEBS-BBSmcispAbs、pEBS-BBSscispAec、pEBS-BBSmcispAec、pEBS-BBSscispAef和pEBS-BBSmcispAef，得到大肠杆菌重组菌MSscAbs、MSmcAbs、MSscAec、MSmcAec、MSscAef、MSmcAef、DSscAbs、DSmcAbs、DSscAec、DSmcAec、DSscAef、DSmcAef、WSscAbs、WSmcAbs、WSscAec、WSmcAec、WSscAef、WSmcAef、BSscAbs、BSmcAbs、BSscAec、BSmcAec、BSscAef、BSmcAef。
分别向枯草芽孢杆菌168、枯草芽孢杆菌W600、枯草芽孢杆菌W800转入重组质粒pEBS-BBSscispAbs、pEBS-BBSmcispAbs、pEBS-BBSscispAec、pEBS-BBSmcispAec、pEBS-BBSscispAef和pEBS-BBSmcispAef，得到枯草芽孢杆菌重组菌168SscAbs、168SmcAbs、168SscAec、168SmcAec、168SscAef、168SmcAef、600SscAbs、600SmcAbs、600SscAec、600SmcAec、600SscAef、600SmcAef、800SscAbs、800SmcAbs、800SscAec、800SmcAec、800SscAef、800SmcAef。
分别向枯草芽孢杆菌168，大肠杆菌MG1655转入重组质粒pEBS-Pbe(1)-BBSmcispAbs、pEBS-Pbe(2)-BBSmcispAbs、pEBS-Pbe(3)-BBSmcispAbs，得到重组菌BSPbe(1)SA、BSPbe(2)SA、BSPbe(3)SA、ECPbe(1)SA、ECPbe(2)SA、ECPbe(3)SA。
四、样品的处理及气相色谱(GC)法分析α-红没药醇产量
取4mL发酵液，在12000r/min下离心10min，分离沉淀物，上清液和正十二烷的有机层。有机层直接进行(﹣)-α-红没药醇的GC分析，将分离的上清液与25％(v/v)n-混合十二烷，孵育30分钟，并以12000rpm离心10分钟。分离有机层并用于(﹣)-α-红没药醇的GC分析。沉淀用等体积去离子水25％悬浮后进行超声破碎，将裂解物用于通过上述方法提取和定量(﹣)-α-红没药醇。α-红没药醇产量为胞内和胞外麦角硫因含量之和。
GC分析条件:色谱柱为安捷伦HP-INNOWAX 133，柱流速保持在1mL/min，上样量1μL。柱温箱控温程序为：60℃保持2min，以10℃/min升温到300℃，并在300℃下保持10min。测量析出(﹣)-α-红没药醇峰面积。以(﹣)-α-红没药醇标准品标定各菌株产量。
五、实施例中涉及到的引物信息
引物信息如表1所示。
表1引物
Figure GDA0003740390410000051
Figure GDA0003740390410000061
实施例1 24株异源表达不同来源BBS基因和ispA基因的大肠杆菌重组菌的摇瓶培养及α-红没药醇含量测定
分别挑取上述构建的24株异源表达不同来源BBS基因和ispA基因的大肠杆菌重组菌株及对照菌(转化pEBS空质粒)单克隆接种于5mL LB培养基，根据需要添加终浓度为50μg/mL的卡那霉素，置于200rpm 37℃培养10-12h，然后按10％的接种量转接于250mL三角摇瓶，培养基为发酵培养基，装液量为20mL并添加的25％(v/v)的正十二烷。根据需有添加终浓度为50μg/mL的卡那霉素，然后置于220rpm 37℃培养。培养60h后进行样品的处理及GC分析(﹣)-α-红没药醇产量。结果如图2所示，重组菌都能实现异源合成(﹣)-α-红没药醇，其中重组菌MSmcAbs的(﹣)-α-红没药醇产量较高，达2.64g/L。
实施例2 18株异源表达不同来源BBS基因和ispA基因的枯草芽孢杆菌重组菌的摇瓶培养及α-红没药醇含量测定
分别挑取上述构建的18株异源表达不同来源BBS基因和ispA基因的枯草芽孢杆菌重组菌及对照菌(转化pEBS空质粒)单克隆接种于5mL LB培养基，根据需要添加终浓度为50μg/mL的卡那霉素，置于200rpm 37℃培养10-12h，然后按10％的接种量转接于250mL三角摇瓶，培养基为发酵培养基，装液量为20mL并添加的25％(v/v)的正十二烷。根据需有添加终浓度为50ug/mL的卡那霉素，然后置于220rpm 37℃培养。培养60h后进行样品的处理及GC分析(﹣)-α-红没药醇产量。结果如图3所示，重组菌都能实现异源合成(﹣)-α-红没药醇，其中重组菌168SmcAbs的(﹣)-α-红没药醇产量较高，达3.22g/L。
实施例3优化BBS基因和ispA基因启动子的大肠杆菌、枯草芽孢杆菌重组菌的摇瓶培养及α-红没药醇含量测定
分别挑取上述构建的优化BBS基因和ispA基因启动子的大肠杆菌、枯草芽孢杆菌重组菌及对照菌(转化pEBS空质粒)单克隆接种于5mL LB培养基，根据需要添加终浓度为50μg/mL的卡那霉素，置于200rpm 37℃培养10-12h，然后按10％的接种量转接于250mL三角摇瓶，培养基为发酵培养基，装液量为20mL并添加的25％(v/v)的正十二烷。根据需有添加终浓度为50ug/mL的卡那霉素，然后置于220rpm 37℃培养。培养60h后进行样品的处理及GC分析(﹣)-α-红没药醇产量。结果如图4所示，重组菌都能实现高效合成(﹣)-α-红没药醇，其中重组菌BSPbe(1)SA的(﹣)-α-红没药醇产量较高，达4.15g/L，重组菌ECPbe(1)SA的(﹣)-α-红没药醇产量也较高，达3.37g/L。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种发酵合成α- 红没药醇的工程菌株及其构建方法
<160> 40
<170> PatentIn version 3.3
<210> 1
<211> 942
<212> DNA
<213> 人工序列
<400> 1
gtgaactccc tcctccacgc cgcagagtta gcgccgaaga aacggaactg cagcccgcgc 60
agccccgagg agttcgaggc cgccgtcacc cgccacaccg cctgggcggt cggccgccac 120
ctcctcgcgc cgcaggacgt tccgcactac cggctcgcgc tgcccgacct gatcggccac 180
gcctaccccc gggcccgcgg gcccgaactc gacctgctgc tggacatcct cggctggttc 240
accatcctcg acgaccgctt cgacggaccg gtcggccacc gcccgaagga cgcccacgcc 300
ctgatcgacc cactgctcgg catcctccgg taccccgggc ccccggccat cgcgccggag 360
gacccgctcg tggccgcgtg gcgggacctc tggcaccgcc aggccgggcc gatgcccgac 420
acctggcgcc accgggccgc cgccgagtgg caggcctgcc tgaccacctt cctcgccgag 480
acccaccacc gggccggggg aaccactccg gacctccccg agacggcgct gctgcgccgc 540
cacgccagct gcctgtaccc gttcatgaac atgctggagc gggtgcgcgg caccgaggcc 600
ccggcgctgc tgctcgcgga gcccgccctg taccggctgc gcgcgtacac cgccgacgcg 660
gcaaccctca tcaacgacct ctgctcgctg caacgcgagg aaggcctgcc cgcggtccag 720
ttcaacatgg tgatgaccct tcagcgaacc cacgggctca gccggaacca ggcggtccag 780
gtggtccgca cccgggtgcg gcgcctgcgg gacgacagcg aggtgctccg cggccacctg 840
ctgcgccggc acccggccgc cggctggtac ctgaacggga cccgcgacat ggtcgacggc 900
ctccacgtct gggccggcac gtcgcgccgc taccacccat ga 942
<210> 2
<211> 1719
<212> DNA
<213> 人工序列
<400> 2
atgtcaactt tatcagtttc tactccttcc ttttcttcat ctccattgtc ttctgttaat 60
aagaatagca cgaagcaaca tgttactcgc aacagtgtca tcttccacga tagtatatgg 120
ggggatcaat ttcttgaata taaggagaaa ttcaatgtag ctactgagaa acagctaatc 180
gaggagctca aagaagaagt gagaaacgaa ctaatgataa gagcttgtaa tgaagcaagc 240
cgatatataa agcttataca actcattgat gtagttgaac gccttggcct agcctatcat 300
tttgaaaagg agatcgagga atccttgcaa catatctatg ttacatatgg ccataaatgg 360
accaactata acaacattga aagcctttcg ctgtggtttc gactgctacg acaaaatggc 420
ttcaacgtat catctgatat attcgagaac catatagatg agaagggaaa ctttcaggaa 480
tctttatgta atgatcctca agggatgctt gctttatacg aagcagcata tatgagggtg 540
gaaggagaaa taatactaga taaggcactc gagttcacca aactacacct tggcatcata 600
tccaatgatc cttcttgtga ctcttctcta agaacagaaa taaaacaagc tctaaagcag 660
ccgcttcgta gaaggttgcc aaggctagag gcggtgcgct acatagcaat ctaccaacaa 720
aaagcttctc acagtgaggt cttgttaaag cttgcaaagt tagacttcaa cgtgcttcaa 780
gaaatgcaca aagacgagct tagccaaatc tgcaaatggt ggaaagattt ggacattcga 840
aacaagttac catatgttcg agacagattg attgaaggct acttttggat attgggaatc 900
tatttcgagc ctcaacattc tcgtacaaga atgttcttaa tgaaaacatg catgtggtta 960
attgttttag atgatacatt tgataattat ggtacttatg aggaactcga gatatttaca 1020
caagctgtcg aaagatggtc cataacctgc ttggatgagc tgccagagta catgaaacta 1080
atatatcatg aacagtttcg tgttcaccaa gaaatggagg aatcacttga gaaggaggga 1140
aaagcatatc aaatccatta tattaaggag atggcgaaag agggcacacg cagcctttta 1200
ttagaagcca aatggttgaa agagggatac atgccaacat tagacgagta cctgtctaat 1260
tcactagtta cttgtggata tgcattgatg acagcaagat cttatgttgc ccgggatgac 1320
ggtatagtca ccgaggatgc ctttaaatgg gtggccacac atcctcctat tgtgaaagct 1380
gcatgtaaaa ttttaagact tatggatgat attgccaccc acaaggagga acaagaaaga 1440
ggccatattg cttcaagcat tgaatgctac cgaaaggaaa ctggtgcatc agaggaggaa 1500
gcatgcatgg atttcttaaa acaagtcgaa gatggttgga aggttataaa tcaggagtcg 1560
ctcatgccta cagatgtacc atttcctctc cttattcctg caatcaacct tgcgcgtgtg 1620
agtgatacct tatataaaga caatgatggc tacaatcatg ctgataaaga agtcattggt 1680
tacatcaaat cgctcttcgt tcaccctatg attgtctag 1719
<210> 3
<211> 891
<212> DNA
<213> 人工序列
<400> 3
gtgacaaata aattaacgag ctttctggcg gaccggaaaa aaacaattga aaatcagctt 60
tctgtctata cagaaaagct tgatatgccg gactcattaa agaaatctat gctatattct 120
ctacaggccg gcggaaagcg gttgcggcct ctgattgtac tggctgtttt aaatgcatat 180
ggaaaaagcg aaaaagacgg cattccggtg ggctgtgctg tcgaaatgat tcacacgtat 240
tcgttaattc atgatgatct tccatgcatg gatgatgacg atttgcgccg cgggaagccg 300
acaaaccata aagtgtttgg tgaagcgacg gcagtattag cgggtgacgg gctgctcaca 360
gaaagcttta agctgattac ctcccacgtg tcagacgagg tgtcagcaga aaagcgcctg 420
cggcttgtga atgaactgat ttcagcggca ggcaccgaag gcatggtcgg tgggcaagta 480
gctgatatgg aagcggaaaa ccgacaagtc acgcttgaag agctcgaatc cattcatgaa 540
cggaaaactg ccaagctcct tggcttttgt gtaatcgccg gtgctatttt ggctgatgcg 600
cctgaggaag acattgaaac actgcgtacc ttcagcagcc acattggaat cggatttcaa 660
atcagagacg atattttaga tttagaaggc agtgaagaga aaatcggcaa acgtgtcggc 720
tcggatacca caaatgacaa atcgacatac ccgtcgcttc tttcattgga aggggccaaa 780
cataaattgg atgttcatat aaaagaggcg aagcgattga tcggcggact ctctcttcaa 840
aaagaccttt tatatgagct ttgtgattta attgcggcaa gagatcacta a 891
<210> 4
<211> 900
<212> DNA
<213> 人工序列
<400> 4
atggactttc cgcagcaact cgaagcctgc gttaagcagg ccaaccaggc gctgagccgt 60
tttatcgccc cactgccctt tcagaacact cccgtggtcg aaaccatgca gtatggcgca 120
ttattaggtg gtaagcgcct gcgacctttc ctggtttatg ccaccggtca tatgttcggc 180
gttagcacaa acacgctgga cgcacccgct gccgccgttg agtgtatcca cgcttactca 240
ttaattcatg atgatttacc ggcaatggat gatgacgatc tgcgtcgcgg tttgccaacc 300
tgccatgtga agtttggcga agcaaacgcg attctcgctg gcgacgcttt acaaacgctg 360
gcgttctcga ttttaagcga tgccgatatg ccggaagtgt cggaccgcga cagaatttcg 420
atgatttctg aactggcgag cgccagtggt attgccggaa tgtgcggtgg tcaggcatta 480
gatttagacg cggaaggcaa acacgtacct ctggacgcgc ttgagcgtat tcatcgtcat 540
aaaaccggcg cattgattcg cgccgccgtt cgccttggtg cattaagcgc cggagataaa 600
ggacgtcgtg ctctgccggt actcgacaag tatgcagaga gcatcggcct tgccttccag 660
gttcaggatg acatcctgga tgtggtggga gatactgcaa cgttgggaaa acgccagggt 720
gccgaccagc aacttggtaa aagtacctac cctgcacttc tgggtcttga gcaagcccgg 780
aagaaagccc gggatctgat cgacgatgcc cgtcagtcgc tgaaacaact ggctgaacag 840
tcactcgata cctcggcact ggaagcgcta gcggactaca tcatccagcg taataaataa 900
<210> 5
<211> 882
<212> DNA
<213> 人工序列
<400> 5
atgacgaatt ttagtcaaca gcatttaccg ttggttgaaa aagtcatggt cgattttatc 60
gcagaatata ctgaaaatga gcgtttgaaa gaagcaatgc tctactcaat acacgcaggt 120
ggcaaacgat tacgcccgct attagtgtta acaacagtgg ccgcttttca aaaagagatg 180
gaaacacaag actatcaagt ggctgcctct ttagagatga ttcatacgta ttcattaatt 240
catgatgatt taccagcaat ggacgatgat gatttacgtc gtggcaaacc aaccaatcat 300
aaagtgtttg gtgaagcgac tgccatttta gcaggggacg gcttattaac aggtgcattt 360
cagttgcttt ctttgagtca attaggctta agtgaaaaag ttttactgat gcaacaactg 420
gcaaaggcag cggggaatca aggcatggtt tccggccaaa tgggtgatat tgaaggagaa 480
aaagtcagct tgactttaga agagttagcg gctgttcatg aaaagaaaac cggagcgcta 540
attgaattcg ccttaattgc tgggggcgta ttagccaatc aaacagaaga agtcattggt 600
ttactgacac agtttgcgca tcactatggc ttggcttttc aaattcgtga tgacttatta 660
gatgccacaa gtacagaagc agatttaggt aaaaaagtag gccgagatga agcgttgaat 720
aaaagtacgt atccagctct tctagggatt gctggcgcga aagatgcgct aacacatcaa 780
ttagcagaag gtagcgctgt tttagaaaaa atcaaagcaa atgttcccaa cttttcagaa 840
gaacatttag caaacttgtt aactcagtta caattgagat ag 882
<210> 6
<211> 60
<212> DNA
<213> 人工序列
<400> 6
agctagctca gtcctaggta ttgtgctagc aattgcagta ggcatgacaa aatggactca 60
<210> 7
<211> 60
<212> DNA
<213> 人工序列
<400> 7
ggctagctca gtcctaggta ctatgctagc aatgggctcg tgttgtacaa taaatgtagt 60
<210> 8
<211> 60
<212> DNA
<213> 人工序列
<400> 8
ggctagctca gtcctaggta tagtgctagc tctaagctag tgtattttgc gtttaatagt 60
<210> 9
<211> 2704
<212> DNA
<213> 人工序列
<400> 9
agctagctca gtcctaggta ttgtgctagc aattgcagta ggcatgacaa aatggactca 60
aagagaggaa tgtacacatg tcaactttat cagtttctac tccttccttt tcttcatctc 120
cattgtcttc tgttaataag aatagcacga agcaacatgt tactcgcaac agtgtcatct 180
tccacgatag tatatggggg gatcaatttc ttgaatataa ggagaaattc aatgtagcta 240
ctgagaaaca gctaatcgag gagctcaaag aagaagtgag aaacgaacta atgataagag 300
cttgtaatga agcaagccga tatataaagc ttatacaact cattgatgta gttgaacgcc 360
ttggcctagc ctatcatttt gaaaaggaga tcgaggaatc cttgcaacat atctatgtta 420
catatggcca taaatggacc aactataaca acattgaaag cctttcgctg tggtttcgac 480
tgctacgaca aaatggcttc aacgtatcat ctgatatatt cgagaaccat atagatgaga 540
agggaaactt tcaggaatct ttatgtaatg atcctcaagg gatgcttgct ttatacgaag 600
cagcatatat gagggtggaa ggagaaataa tactagataa ggcactcgag ttcaccaaac 660
tacaccttgg catcatatcc aatgatcctt cttgtgactc ttctctaaga acagaaataa 720
aacaagctct aaagcagccg cttcgtagaa ggttgccaag gctagaggcg gtgcgctaca 780
tagcaatcta ccaacaaaaa gcttctcaca gtgaggtctt gttaaagctt gcaaagttag 840
acttcaacgt gcttcaagaa atgcacaaag acgagcttag ccaaatctgc aaatggtgga 900
aagatttgga cattcgaaac aagttaccat atgttcgaga cagattgatt gaaggctact 960
tttggatatt gggaatctat ttcgagcctc aacattctcg tacaagaatg ttcttaatga 1020
aaacatgcat gtggttaatt gttttagatg atacatttga taattatggt acttatgagg 1080
aactcgagat atttacacaa gctgtcgaaa gatggtccat aacctgcttg gatgagctgc 1140
cagagtacat gaaactaata tatcatgaac agtttcgtgt tcaccaagaa atggaggaat 1200
cacttgagaa ggagggaaaa gcatatcaaa tccattatat taaggagatg gcgaaagagg 1260
gcacacgcag ccttttatta gaagccaaat ggttgaaaga gggatacatg ccaacattag 1320
acgagtacct gtctaattca ctagttactt gtggatatgc attgatgaca gcaagatctt 1380
atgttgcccg ggatgacggt atagtcaccg aggatgcctt taaatgggtg gccacacatc 1440
ctcctattgt gaaagctgca tgtaaaattt taagacttat ggatgatatt gccacccaca 1500
aggaggaaca agaaagaggc catattgctt caagcattga atgctaccga aaggaaactg 1560
gtgcatcaga ggaggaagca tgcatggatt tcttaaaaca agtcgaagat ggttggaagg 1620
ttataaatca ggagtcgctc atgcctacag atgtaccatt tcctctcctt attcctgcaa 1680
tcaaccttgc gcgtgtgagt gataccttat ataaagacaa tgatggctac aatcatgctg 1740
ataaagaagt cattggttac atcaaatcgc tcttcgttca ccctatgatt gtctagaaga 1800
gaggaatgta cacgtgacaa ataaattaac gagctttctg gcggaccgga aaaaaacaat 1860
tgaaaatcag ctttctgtct atacagaaaa gcttgatatg ccggactcat taaagaaatc 1920
tatgctatat tctctacagg ccggcggaaa gcggttgcgg cctctgattg tactggctgt 1980
tttaaatgca tatggaaaaa gcgaaaaaga cggcattccg gtgggctgtg ctgtcgaaat 2040
gattcacacg tattcgttaa ttcatgatga tcttccatgc atggatgatg acgatttgcg 2100
ccgcgggaag ccgacaaacc ataaagtgtt tggtgaagcg acggcagtat tagcgggtga 2160
cgggctgctc acagaaagct ttaagctgat tacctcccac gtgtcagacg aggtgtcagc 2220
agaaaagcgc ctgcggcttg tgaatgaact gatttcagcg gcaggcaccg aaggcatggt 2280
cggtgggcaa gtagctgata tggaagcgga aaaccgacaa gtcacgcttg aagagctcga 2340
atccattcat gaacggaaaa ctgccaagct ccttggcttt tgtgtaatcg ccggtgctat 2400
tttggctgat gcgcctgagg aagacattga aacactgcgt accttcagca gccacattgg 2460
aatcggattt caaatcagag acgatatttt agatttagaa ggcagtgaag agaaaatcgg 2520
caaacgtgtc ggctcggata ccacaaatga caaatcgaca tacccgtcgc ttctttcatt 2580
ggaaggggcc aaacataaat tggatgttca tataaaagag gcgaagcgat tgatcggcgg 2640
actctctctt caaaaagacc ttttatatga gctttgtgat ttaattgcgg caagagatca 2700
ctaa 2704
<210> 10
<211> 2704
<212> DNA
<213> 人工序列
<400> 10
ggctagctca gtcctaggta ctatgctagc aatgggctcg tgttgtacaa taaatgtagt 60
aagagaggaa tgtacacatg tcaactttat cagtttctac tccttccttt tcttcatctc 120
cattgtcttc tgttaataag aatagcacga agcaacatgt tactcgcaac agtgtcatct 180
tccacgatag tatatggggg gatcaatttc ttgaatataa ggagaaattc aatgtagcta 240
ctgagaaaca gctaatcgag gagctcaaag aagaagtgag aaacgaacta atgataagag 300
cttgtaatga agcaagccga tatataaagc ttatacaact cattgatgta gttgaacgcc 360
ttggcctagc ctatcatttt gaaaaggaga tcgaggaatc cttgcaacat atctatgtta 420
catatggcca taaatggacc aactataaca acattgaaag cctttcgctg tggtttcgac 480
tgctacgaca aaatggcttc aacgtatcat ctgatatatt cgagaaccat atagatgaga 540
agggaaactt tcaggaatct ttatgtaatg atcctcaagg gatgcttgct ttatacgaag 600
cagcatatat gagggtggaa ggagaaataa tactagataa ggcactcgag ttcaccaaac 660
tacaccttgg catcatatcc aatgatcctt cttgtgactc ttctctaaga acagaaataa 720
aacaagctct aaagcagccg cttcgtagaa ggttgccaag gctagaggcg gtgcgctaca 780
tagcaatcta ccaacaaaaa gcttctcaca gtgaggtctt gttaaagctt gcaaagttag 840
acttcaacgt gcttcaagaa atgcacaaag acgagcttag ccaaatctgc aaatggtgga 900
aagatttgga cattcgaaac aagttaccat atgttcgaga cagattgatt gaaggctact 960
tttggatatt gggaatctat ttcgagcctc aacattctcg tacaagaatg ttcttaatga 1020
aaacatgcat gtggttaatt gttttagatg atacatttga taattatggt acttatgagg 1080
aactcgagat atttacacaa gctgtcgaaa gatggtccat aacctgcttg gatgagctgc 1140
cagagtacat gaaactaata tatcatgaac agtttcgtgt tcaccaagaa atggaggaat 1200
cacttgagaa ggagggaaaa gcatatcaaa tccattatat taaggagatg gcgaaagagg 1260
gcacacgcag ccttttatta gaagccaaat ggttgaaaga gggatacatg ccaacattag 1320
acgagtacct gtctaattca ctagttactt gtggatatgc attgatgaca gcaagatctt 1380
atgttgcccg ggatgacggt atagtcaccg aggatgcctt taaatgggtg gccacacatc 1440
ctcctattgt gaaagctgca tgtaaaattt taagacttat ggatgatatt gccacccaca 1500
aggaggaaca agaaagaggc catattgctt caagcattga atgctaccga aaggaaactg 1560
gtgcatcaga ggaggaagca tgcatggatt tcttaaaaca agtcgaagat ggttggaagg 1620
ttataaatca ggagtcgctc atgcctacag atgtaccatt tcctctcctt attcctgcaa 1680
tcaaccttgc gcgtgtgagt gataccttat ataaagacaa tgatggctac aatcatgctg 1740
ataaagaagt cattggttac atcaaatcgc tcttcgttca ccctatgatt gtctagaaga 1800
gaggaatgta cacgtgacaa ataaattaac gagctttctg gcggaccgga aaaaaacaat 1860
tgaaaatcag ctttctgtct atacagaaaa gcttgatatg ccggactcat taaagaaatc 1920
tatgctatat tctctacagg ccggcggaaa gcggttgcgg cctctgattg tactggctgt 1980
tttaaatgca tatggaaaaa gcgaaaaaga cggcattccg gtgggctgtg ctgtcgaaat 2040
gattcacacg tattcgttaa ttcatgatga tcttccatgc atggatgatg acgatttgcg 2100
ccgcgggaag ccgacaaacc ataaagtgtt tggtgaagcg acggcagtat tagcgggtga 2160
cgggctgctc acagaaagct ttaagctgat tacctcccac gtgtcagacg aggtgtcagc 2220
agaaaagcgc ctgcggcttg tgaatgaact gatttcagcg gcaggcaccg aaggcatggt 2280
cggtgggcaa gtagctgata tggaagcgga aaaccgacaa gtcacgcttg aagagctcga 2340
atccattcat gaacggaaaa ctgccaagct ccttggcttt tgtgtaatcg ccggtgctat 2400
tttggctgat gcgcctgagg aagacattga aacactgcgt accttcagca gccacattgg 2460
aatcggattt caaatcagag acgatatttt agatttagaa ggcagtgaag agaaaatcgg 2520
caaacgtgtc ggctcggata ccacaaatga caaatcgaca tacccgtcgc ttctttcatt 2580
ggaaggggcc aaacataaat tggatgttca tataaaagag gcgaagcgat tgatcggcgg 2640
actctctctt caaaaagacc ttttatatga gctttgtgat ttaattgcgg caagagatca 2700
ctaa 2704
<210> 11
<211> 2704
<212> DNA
<213> 人工序列
<400> 11
ggctagctca gtcctaggta tagtgctagc tctaagctag tgtattttgc gtttaatagt 60
aagagaggaa tgtacacatg tcaactttat cagtttctac tccttccttt tcttcatctc 120
cattgtcttc tgttaataag aatagcacga agcaacatgt tactcgcaac agtgtcatct 180
tccacgatag tatatggggg gatcaatttc ttgaatataa ggagaaattc aatgtagcta 240
ctgagaaaca gctaatcgag gagctcaaag aagaagtgag aaacgaacta atgataagag 300
cttgtaatga agcaagccga tatataaagc ttatacaact cattgatgta gttgaacgcc 360
ttggcctagc ctatcatttt gaaaaggaga tcgaggaatc cttgcaacat atctatgtta 420
catatggcca taaatggacc aactataaca acattgaaag cctttcgctg tggtttcgac 480
tgctacgaca aaatggcttc aacgtatcat ctgatatatt cgagaaccat atagatgaga 540
agggaaactt tcaggaatct ttatgtaatg atcctcaagg gatgcttgct ttatacgaag 600
cagcatatat gagggtggaa ggagaaataa tactagataa ggcactcgag ttcaccaaac 660
tacaccttgg catcatatcc aatgatcctt cttgtgactc ttctctaaga acagaaataa 720
aacaagctct aaagcagccg cttcgtagaa ggttgccaag gctagaggcg gtgcgctaca 780
tagcaatcta ccaacaaaaa gcttctcaca gtgaggtctt gttaaagctt gcaaagttag 840
acttcaacgt gcttcaagaa atgcacaaag acgagcttag ccaaatctgc aaatggtgga 900
aagatttgga cattcgaaac aagttaccat atgttcgaga cagattgatt gaaggctact 960
tttggatatt gggaatctat ttcgagcctc aacattctcg tacaagaatg ttcttaatga 1020
aaacatgcat gtggttaatt gttttagatg atacatttga taattatggt acttatgagg 1080
aactcgagat atttacacaa gctgtcgaaa gatggtccat aacctgcttg gatgagctgc 1140
cagagtacat gaaactaata tatcatgaac agtttcgtgt tcaccaagaa atggaggaat 1200
cacttgagaa ggagggaaaa gcatatcaaa tccattatat taaggagatg gcgaaagagg 1260
gcacacgcag ccttttatta gaagccaaat ggttgaaaga gggatacatg ccaacattag 1320
acgagtacct gtctaattca ctagttactt gtggatatgc attgatgaca gcaagatctt 1380
atgttgcccg ggatgacggt atagtcaccg aggatgcctt taaatgggtg gccacacatc 1440
ctcctattgt gaaagctgca tgtaaaattt taagacttat ggatgatatt gccacccaca 1500
aggaggaaca agaaagaggc catattgctt caagcattga atgctaccga aaggaaactg 1560
gtgcatcaga ggaggaagca tgcatggatt tcttaaaaca agtcgaagat ggttggaagg 1620
ttataaatca ggagtcgctc atgcctacag atgtaccatt tcctctcctt attcctgcaa 1680
tcaaccttgc gcgtgtgagt gataccttat ataaagacaa tgatggctac aatcatgctg 1740
ataaagaagt cattggttac atcaaatcgc tcttcgttca ccctatgatt gtctagaaga 1800
gaggaatgta cacgtgacaa ataaattaac gagctttctg gcggaccgga aaaaaacaat 1860
tgaaaatcag ctttctgtct atacagaaaa gcttgatatg ccggactcat taaagaaatc 1920
tatgctatat tctctacagg ccggcggaaa gcggttgcgg cctctgattg tactggctgt 1980
tttaaatgca tatggaaaaa gcgaaaaaga cggcattccg gtgggctgtg ctgtcgaaat 2040
gattcacacg tattcgttaa ttcatgatga tcttccatgc atggatgatg acgatttgcg 2100
ccgcgggaag ccgacaaacc ataaagtgtt tggtgaagcg acggcagtat tagcgggtga 2160
cgggctgctc acagaaagct ttaagctgat tacctcccac gtgtcagacg aggtgtcagc 2220
agaaaagcgc ctgcggcttg tgaatgaact gatttcagcg gcaggcaccg aaggcatggt 2280
cggtgggcaa gtagctgata tggaagcgga aaaccgacaa gtcacgcttg aagagctcga 2340
atccattcat gaacggaaaa ctgccaagct ccttggcttt tgtgtaatcg ccggtgctat 2400
tttggctgat gcgcctgagg aagacattga aacactgcgt accttcagca gccacattgg 2460
aatcggattt caaatcagag acgatatttt agatttagaa ggcagtgaag agaaaatcgg 2520
caaacgtgtc ggctcggata ccacaaatga caaatcgaca tacccgtcgc ttctttcatt 2580
ggaaggggcc aaacataaat tggatgttca tataaaagag gcgaagcgat tgatcggcgg 2640
actctctctt caaaaagacc ttttatatga gctttgtgat ttaattgcgg caagagatca 2700
ctaa 2704
<210> 12
<211> 313
<212> PRT
<213> 人工序列
<400> 12
Val Asn Ser Leu Leu His Ala Ala Glu Leu Ala Pro Lys Lys Arg Asn
1 5 10 15
Cys Ser Pro Arg Ser Pro Glu Glu Phe Glu Ala Ala Val Thr Arg His
20 25 30
Thr Ala Trp Ala Val Gly Arg His Leu Leu Ala Pro Gln Asp Val Pro
35 40 45
His Tyr Arg Leu Ala Leu Pro Asp Leu Ile Gly His Ala Tyr Pro Arg
50 55 60
Ala Arg Gly Pro Glu Leu Asp Leu Leu Leu Asp Ile Leu Gly Trp Phe
65 70 75 80
Thr Ile Leu Asp Asp Arg Phe Asp Gly Pro Val Gly His Arg Pro Lys
85 90 95
Asp Ala His Ala Leu Ile Asp Pro Leu Leu Gly Ile Leu Arg Tyr Pro
100 105 110
Gly Pro Pro Ala Ile Ala Pro Glu Asp Pro Leu Val Ala Ala Trp Arg
115 120 125
Asp Leu Trp His Arg Gln Ala Gly Pro Met Pro Asp Thr Trp Arg His
130 135 140
Arg Ala Ala Ala Glu Trp Gln Ala Cys Leu Thr Thr Phe Leu Ala Glu
145 150 155 160
Thr His His Arg Ala Gly Gly Thr Thr Pro Asp Leu Pro Glu Thr Ala
165 170 175
Leu Leu Arg Arg His Ala Ser Cys Leu Tyr Pro Phe Met Asn Met Leu
180 185 190
Glu Arg Val Arg Gly Thr Glu Ala Pro Ala Leu Leu Leu Ala Glu Pro
195 200 205
Ala Leu Tyr Arg Leu Arg Ala Tyr Thr Ala Asp Ala Ala Thr Leu Ile
210 215 220
Asn Asp Leu Cys Ser Leu Gln Arg Glu Glu Gly Leu Pro Ala Val Gln
225 230 235 240
Phe Asn Met Val Met Thr Leu Gln Arg Thr His Gly Leu Ser Arg Asn
245 250 255
Gln Ala Val Gln Val Val Arg Thr Arg Val Arg Arg Leu Arg Asp Asp
260 265 270
Ser Glu Val Leu Arg Gly His Leu Leu Arg Arg His Pro Ala Ala Gly
275 280 285
Trp Tyr Leu Asn Gly Thr Arg Asp Met Val Asp Gly Leu His Val Trp
290 295 300
Ala Gly Thr Ser Arg Arg Tyr His Pro
305 310
<210> 13
<211> 572
<212> PRT
<213> 人工序列
<400> 13
Met Ser Thr Leu Ser Val Ser Thr Pro Ser Phe Ser Ser Ser Pro Leu
1 5 10 15
Ser Ser Val Asn Lys Asn Ser Thr Lys Gln His Val Thr Arg Asn Ser
20 25 30
Val Ile Phe His Asp Ser Ile Trp Gly Asp Gln Phe Leu Glu Tyr Lys
35 40 45
Glu Lys Phe Asn Val Ala Thr Glu Lys Gln Leu Ile Glu Glu Leu Lys
50 55 60
Glu Glu Val Arg Asn Glu Leu Met Ile Arg Ala Cys Asn Glu Ala Ser
65 70 75 80
Arg Tyr Ile Lys Leu Ile Gln Leu Ile Asp Val Val Glu Arg Leu Gly
85 90 95
Leu Ala Tyr His Phe Glu Lys Glu Ile Glu Glu Ser Leu Gln His Ile
100 105 110
Tyr Val Thr Tyr Gly His Lys Trp Thr Asn Tyr Asn Asn Ile Glu Ser
115 120 125
Leu Ser Leu Trp Phe Arg Leu Leu Arg Gln Asn Gly Phe Asn Val Ser
130 135 140
Ser Asp Ile Phe Glu Asn His Ile Asp Glu Lys Gly Asn Phe Gln Glu
145 150 155 160
Ser Leu Cys Asn Asp Pro Gln Gly Met Leu Ala Leu Tyr Glu Ala Ala
165 170 175
Tyr Met Arg Val Glu Gly Glu Ile Ile Leu Asp Lys Ala Leu Glu Phe
180 185 190
Thr Lys Leu His Leu Gly Ile Ile Ser Asn Asp Pro Ser Cys Asp Ser
195 200 205
Ser Leu Arg Thr Glu Ile Lys Gln Ala Leu Lys Gln Pro Leu Arg Arg
210 215 220
Arg Leu Pro Arg Leu Glu Ala Val Arg Tyr Ile Ala Ile Tyr Gln Gln
225 230 235 240
Lys Ala Ser His Ser Glu Val Leu Leu Lys Leu Ala Lys Leu Asp Phe
245 250 255
Asn Val Leu Gln Glu Met His Lys Asp Glu Leu Ser Gln Ile Cys Lys
260 265 270
Trp Trp Lys Asp Leu Asp Ile Arg Asn Lys Leu Pro Tyr Val Arg Asp
275 280 285
Arg Leu Ile Glu Gly Tyr Phe Trp Ile Leu Gly Ile Tyr Phe Glu Pro
290 295 300
Gln His Ser Arg Thr Arg Met Phe Leu Met Lys Thr Cys Met Trp Leu
305 310 315 320
Ile Val Leu Asp Asp Thr Phe Asp Asn Tyr Gly Thr Tyr Glu Glu Leu
325 330 335
Glu Ile Phe Thr Gln Ala Val Glu Arg Trp Ser Ile Thr Cys Leu Asp
340 345 350
Glu Leu Pro Glu Tyr Met Lys Leu Ile Tyr His Glu Gln Phe Arg Val
355 360 365
His Gln Glu Met Glu Glu Ser Leu Glu Lys Glu Gly Lys Ala Tyr Gln
370 375 380
Ile His Tyr Ile Lys Glu Met Ala Lys Glu Gly Thr Arg Ser Leu Leu
385 390 395 400
Leu Glu Ala Lys Trp Leu Lys Glu Gly Tyr Met Pro Thr Leu Asp Glu
405 410 415
Tyr Leu Ser Asn Ser Leu Val Thr Cys Gly Tyr Ala Leu Met Thr Ala
420 425 430
Arg Ser Tyr Val Ala Arg Asp Asp Gly Ile Val Thr Glu Asp Ala Phe
435 440 445
Lys Trp Val Ala Thr His Pro Pro Ile Val Lys Ala Ala Cys Lys Ile
450 455 460
Leu Arg Leu Met Asp Asp Ile Ala Thr His Lys Glu Glu Gln Glu Arg
465 470 475 480
Gly His Ile Ala Ser Ser Ile Glu Cys Tyr Arg Lys Glu Thr Gly Ala
485 490 495
Ser Glu Glu Glu Ala Cys Met Asp Phe Leu Lys Gln Val Glu Asp Gly
500 505 510
Trp Lys Val Ile Asn Gln Glu Ser Leu Met Pro Thr Asp Val Pro Phe
515 520 525
Pro Leu Leu Ile Pro Ala Ile Asn Leu Ala Arg Val Ser Asp Thr Leu
530 535 540
Tyr Lys Asp Asn Asp Gly Tyr Asn His Ala Asp Lys Glu Val Ile Gly
545 550 555 560
Tyr Ile Lys Ser Leu Phe Val His Pro Met Ile Val
565 570
<210> 14
<211> 296
<212> PRT
<213> 人工序列
<400> 14
Val Thr Asn Lys Leu Thr Ser Phe Leu Ala Asp Arg Lys Lys Thr Ile
1 5 10 15
Glu Asn Gln Leu Ser Val Tyr Thr Glu Lys Leu Asp Met Pro Asp Ser
20 25 30
Leu Lys Lys Ser Met Leu Tyr Ser Leu Gln Ala Gly Gly Lys Arg Leu
35 40 45
Arg Pro Leu Ile Val Leu Ala Val Leu Asn Ala Tyr Gly Lys Ser Glu
50 55 60
Lys Asp Gly Ile Pro Val Gly Cys Ala Val Glu Met Ile His Thr Tyr
65 70 75 80
Ser Leu Ile His Asp Asp Leu Pro Cys Met Asp Asp Asp Asp Leu Arg
85 90 95
Arg Gly Lys Pro Thr Asn His Lys Val Phe Gly Glu Ala Thr Ala Val
100 105 110
Leu Ala Gly Asp Gly Leu Leu Thr Glu Ser Phe Lys Leu Ile Thr Ser
115 120 125
His Val Ser Asp Glu Val Ser Ala Glu Lys Arg Leu Arg Leu Val Asn
130 135 140
Glu Leu Ile Ser Ala Ala Gly Thr Glu Gly Met Val Gly Gly Gln Val
145 150 155 160
Ala Asp Met Glu Ala Glu Asn Arg Gln Val Thr Leu Glu Glu Leu Glu
165 170 175
Ser Ile His Glu Arg Lys Thr Ala Lys Leu Leu Gly Phe Cys Val Ile
180 185 190
Ala Gly Ala Ile Leu Ala Asp Ala Pro Glu Glu Asp Ile Glu Thr Leu
195 200 205
Arg Thr Phe Ser Ser His Ile Gly Ile Gly Phe Gln Ile Arg Asp Asp
210 215 220
Ile Leu Asp Leu Glu Gly Ser Glu Glu Lys Ile Gly Lys Arg Val Gly
225 230 235 240
Ser Asp Thr Thr Asn Asp Lys Ser Thr Tyr Pro Ser Leu Leu Ser Leu
245 250 255
Glu Gly Ala Lys His Lys Leu Asp Val His Ile Lys Glu Ala Lys Arg
260 265 270
Leu Ile Gly Gly Leu Ser Leu Gln Lys Asp Leu Leu Tyr Glu Leu Cys
275 280 285
Asp Leu Ile Ala Ala Arg Asp His
290 295
<210> 15
<211> 299
<212> PRT
<213> 人工序列
<400> 15
Met Asp Phe Pro Gln Gln Leu Glu Ala Cys Val Lys Gln Ala Asn Gln
1 5 10 15
Ala Leu Ser Arg Phe Ile Ala Pro Leu Pro Phe Gln Asn Thr Pro Val
20 25 30
Val Glu Thr Met Gln Tyr Gly Ala Leu Leu Gly Gly Lys Arg Leu Arg
35 40 45
Pro Phe Leu Val Tyr Ala Thr Gly His Met Phe Gly Val Ser Thr Asn
50 55 60
Thr Leu Asp Ala Pro Ala Ala Ala Val Glu Cys Ile His Ala Tyr Ser
65 70 75 80
Leu Ile His Asp Asp Leu Pro Ala Met Asp Asp Asp Asp Leu Arg Arg
85 90 95
Gly Leu Pro Thr Cys His Val Lys Phe Gly Glu Ala Asn Ala Ile Leu
100 105 110
Ala Gly Asp Ala Leu Gln Thr Leu Ala Phe Ser Ile Leu Ser Asp Ala
115 120 125
Asp Met Pro Glu Val Ser Asp Arg Asp Arg Ile Ser Met Ile Ser Glu
130 135 140
Leu Ala Ser Ala Ser Gly Ile Ala Gly Met Cys Gly Gly Gln Ala Leu
145 150 155 160
Asp Leu Asp Ala Glu Gly Lys His Val Pro Leu Asp Ala Leu Glu Arg
165 170 175
Ile His Arg His Lys Thr Gly Ala Leu Ile Arg Ala Ala Val Arg Leu
180 185 190
Gly Ala Leu Ser Ala Gly Asp Lys Gly Arg Arg Ala Leu Pro Val Leu
195 200 205
Asp Lys Tyr Ala Glu Ser Ile Gly Leu Ala Phe Gln Val Gln Asp Asp
210 215 220
Ile Leu Asp Val Val Gly Asp Thr Ala Thr Leu Gly Lys Arg Gln Gly
225 230 235 240
Ala Asp Gln Gln Leu Gly Lys Ser Thr Tyr Pro Ala Leu Leu Gly Leu
245 250 255
Glu Gln Ala Arg Lys Lys Ala Arg Asp Leu Ile Asp Asp Ala Arg Gln
260 265 270
Ser Leu Lys Gln Leu Ala Glu Gln Ser Leu Asp Thr Ser Ala Leu Glu
275 280 285
Ala Leu Ala Asp Tyr Ile Ile Gln Arg Asn Lys
290 295
<210> 16
<211> 293
<212> PRT
<213> 人工序列
<400> 16
Met Thr Asn Phe Ser Gln Gln His Leu Pro Leu Val Glu Lys Val Met
1 5 10 15
Val Asp Phe Ile Ala Glu Tyr Thr Glu Asn Glu Arg Leu Lys Glu Ala
20 25 30
Met Leu Tyr Ser Ile His Ala Gly Gly Lys Arg Leu Arg Pro Leu Leu
35 40 45
Val Leu Thr Thr Val Ala Ala Phe Gln Lys Glu Met Glu Thr Gln Asp
50 55 60
Tyr Gln Val Ala Ala Ser Leu Glu Met Ile His Thr Tyr Ser Leu Ile
65 70 75 80
His Asp Asp Leu Pro Ala Met Asp Asp Asp Asp Leu Arg Arg Gly Lys
85 90 95
Pro Thr Asn His Lys Val Phe Gly Glu Ala Thr Ala Ile Leu Ala Gly
100 105 110
Asp Gly Leu Leu Thr Gly Ala Phe Gln Leu Leu Ser Leu Ser Gln Leu
115 120 125
Gly Leu Ser Glu Lys Val Leu Leu Met Gln Gln Leu Ala Lys Ala Ala
130 135 140
Gly Asn Gln Gly Met Val Ser Gly Gln Met Gly Asp Ile Glu Gly Glu
145 150 155 160
Lys Val Ser Leu Thr Leu Glu Glu Leu Ala Ala Val His Glu Lys Lys
165 170 175
Thr Gly Ala Leu Ile Glu Phe Ala Leu Ile Ala Gly Gly Val Leu Ala
180 185 190
Asn Gln Thr Glu Glu Val Ile Gly Leu Leu Thr Gln Phe Ala His His
195 200 205
Tyr Gly Leu Ala Phe Gln Ile Arg Asp Asp Leu Leu Asp Ala Thr Ser
210 215 220
Thr Glu Ala Asp Leu Gly Lys Lys Val Gly Arg Asp Glu Ala Leu Asn
225 230 235 240
Lys Ser Thr Tyr Pro Ala Leu Leu Gly Ile Ala Gly Ala Lys Asp Ala
245 250 255
Leu Thr His Gln Leu Ala Glu Gly Ser Ala Val Leu Glu Lys Ile Lys
260 265 270
Ala Asn Val Pro Asn Phe Ser Glu Glu His Leu Ala Asn Leu Leu Thr
275 280 285
Gln Leu Gln Leu Arg
290
<210> 17
<211> 836
<212> PRT
<213> 人工序列
<400> 17
Ser Leu Ser Pro Arg Tyr Cys Ala Ser Asn Cys Ser Arg His Asp Lys
1 5 10 15
Met Asp Ser Lys Arg Gly Met Tyr Thr Cys Gln Leu Tyr Gln Phe Leu
20 25 30
Leu Leu Pro Phe Leu His Leu His Cys Leu Leu Leu Ile Arg Ile Ala
35 40 45
Arg Ser Asn Met Leu Leu Ala Thr Val Ser Ser Ser Thr Ile Val Tyr
50 55 60
Gly Gly Ile Asn Phe Leu Asn Ile Arg Arg Asn Ser Met Leu Leu Arg
65 70 75 80
Asn Ser Ser Arg Ser Ser Lys Lys Lys Glu Thr Asn Glu Leu Val Met
85 90 95
Lys Gln Ala Asp Ile Ser Leu Tyr Asn Ser Leu Met Leu Asn Ala Leu
100 105 110
Ala Pro Ile Ile Leu Lys Arg Arg Ser Arg Asn Pro Cys Asn Ile Ser
115 120 125
Met Leu His Met Ala Ile Asn Gly Pro Thr Ile Thr Thr Leu Lys Ala
130 135 140
Phe Arg Cys Gly Phe Asp Cys Tyr Asp Lys Met Ala Ser Thr Tyr His
145 150 155 160
Leu Ile Tyr Ser Arg Thr Ile Met Arg Arg Glu Thr Phe Arg Asn Leu
165 170 175
Tyr Val Met Ile Leu Lys Gly Cys Leu Leu Tyr Thr Lys Gln His Ile
180 185 190
Gly Trp Lys Glu Lys Tyr Ile Arg His Ser Ser Ser Pro Asn Tyr Thr
195 200 205
Leu Ala Ser Tyr Pro Met Ile Leu Leu Val Thr Leu Leu Glu Gln Lys
210 215 220
Asn Lys Leu Ser Ser Arg Phe Val Glu Gly Cys Gln Gly Arg Arg Cys
225 230 235 240
Ala Thr Gln Ser Thr Asn Lys Lys Leu Leu Thr Val Arg Ser Cys Ser
245 250 255
Leu Gln Ser Thr Ser Thr Cys Phe Lys Lys Cys Thr Lys Thr Ser Leu
260 265 270
Ala Lys Ser Ala Asn Gly Gly Lys Ile Trp Thr Phe Glu Thr Ser Tyr
275 280 285
His Met Phe Glu Thr Asp Leu Lys Ala Thr Phe Gly Tyr Trp Glu Ser
290 295 300
Ile Ser Ser Leu Asn Ile Leu Val Gln Glu Cys Ser Lys His Ala Cys
305 310 315 320
Gly Leu Phe Met Ile His Leu Ile Ile Met Val Leu Met Arg Asn Ser
325 330 335
Arg Tyr Leu His Lys Leu Ser Lys Asp Gly Pro Pro Ala Trp Met Ser
340 345 350
Cys Gln Ser Thr Asn Tyr Ile Met Asn Ser Phe Val Phe Thr Lys Lys
355 360 365
Trp Arg Asn His Leu Arg Arg Arg Glu Lys His Ile Lys Ser Ile Ile
370 375 380
Leu Arg Arg Trp Arg Lys Arg Ala His Ala Ala Phe Tyr Lys Pro Asn
385 390 395 400
Gly Lys Arg Asp Thr Cys Gln His Thr Ser Thr Cys Leu Ile His Leu
405 410 415
Leu Val Asp Met His Gln Gln Asp Leu Met Leu Pro Gly Met Thr Val
420 425 430
Ser Pro Arg Met Pro Leu Asn Gly Trp Pro His Ile Leu Leu Leu Lys
435 440 445
Leu His Val Lys Phe Asp Leu Trp Met Ile Leu Pro Pro Thr Arg Arg
450 455 460
Asn Lys Lys Glu Ala Ile Leu Leu Gln Ala Leu Asn Ala Thr Glu Arg
465 470 475 480
Lys Leu Val His Gln Arg Arg Lys His Ala Trp Ile Ser Asn Lys Ser
485 490 495
Lys Met Val Gly Arg Leu Ile Arg Ser Arg Ser Cys Leu Gln Met Tyr
500 505 510
His Phe Leu Ser Leu Phe Leu Gln Ser Thr Leu Arg Val Val Ile Pro
515 520 525
Tyr Ile Lys Thr Met Met Ala Thr Ile Met Leu Ile Lys Lys Ser Leu
530 535 540
Val Thr Ser Asn Arg Ser Ser Phe Thr Leu Leu Ser Arg Arg Glu Glu
545 550 555 560
Cys Thr Arg Asp Lys Ile Asn Glu Leu Ser Gly Gly Pro Glu Lys Asn
565 570 575
Asn Lys Ser Ala Phe Cys Leu Tyr Arg Lys Ala Tyr Ala Gly Leu Ile
580 585 590
Lys Glu Ile Tyr Ala Ile Phe Ser Thr Gly Arg Arg Lys Ala Val Ala
595 600 605
Ala Ser Asp Cys Thr Gly Cys Phe Lys Cys Ile Trp Lys Lys Arg Lys
610 615 620
Arg Arg His Ser Gly Gly Leu Cys Cys Arg Asn Asp Ser His Val Phe
625 630 635 640
Val Asn Ser Ser Ser Met His Gly Arg Phe Ala Pro Arg Glu Ala Asp
645 650 655
Lys Pro Ser Val Trp Ser Asp Gly Ser Ile Ser Gly Arg Ala Ala His
660 665 670
Arg Lys Leu Ala Asp Tyr Leu Pro Arg Val Arg Arg Gly Val Ser Arg
675 680 685
Lys Ala Pro Ala Ala Cys Glu Thr Asp Phe Ser Gly Arg His Arg Arg
690 695 700
His Gly Arg Trp Ala Ser Ser Tyr Gly Ser Gly Lys Pro Thr Ser His
705 710 715 720
Ala Arg Ala Arg Ile His Ser Thr Glu Asn Cys Gln Ala Pro Trp Leu
725 730 735
Leu Cys Asn Arg Arg Cys Tyr Phe Gly Cys Ala Gly Arg His Asn Thr
740 745 750
Ala Tyr Leu Gln Gln Pro His Trp Asn Arg Ile Ser Asn Gln Arg Arg
755 760 765
Tyr Phe Arg Phe Arg Arg Gln Arg Glu Asn Arg Gln Thr Cys Arg Leu
770 775 780
Gly Tyr His Lys Gln Ile Asp Ile Pro Val Ala Ser Phe Ile Gly Arg
785 790 795 800
Gly Gln Thr Ile Gly Cys Ser Tyr Lys Arg Gly Glu Ala Ile Asp Arg
805 810 815
Arg Thr Leu Ser Ser Lys Arg Pro Phe Ile Ala Leu Phe Asn Cys Gly
820 825 830
Lys Arg Ser Leu
835
<210> 18
<211> 836
<212> PRT
<213> 人工序列
<400> 18
Gly Leu Ser Pro Arg Tyr Tyr Ala Ser Asn Gly Leu Val Leu Tyr Asn
1 5 10 15
Lys Cys Ser Lys Arg Gly Met Tyr Thr Cys Gln Leu Tyr Gln Phe Leu
20 25 30
Leu Leu Pro Phe Leu His Leu His Cys Leu Leu Leu Ile Arg Ile Ala
35 40 45
Arg Ser Asn Met Leu Leu Ala Thr Val Ser Ser Ser Thr Ile Val Tyr
50 55 60
Gly Gly Ile Asn Phe Leu Asn Ile Arg Arg Asn Ser Met Leu Leu Arg
65 70 75 80
Asn Ser Ser Arg Ser Ser Lys Lys Lys Glu Thr Asn Glu Leu Val Met
85 90 95
Lys Gln Ala Asp Ile Ser Leu Tyr Asn Ser Leu Met Leu Asn Ala Leu
100 105 110
Ala Pro Ile Ile Leu Lys Arg Arg Ser Arg Asn Pro Cys Asn Ile Ser
115 120 125
Met Leu His Met Ala Ile Asn Gly Pro Thr Ile Thr Thr Leu Lys Ala
130 135 140
Phe Arg Cys Gly Phe Asp Cys Tyr Asp Lys Met Ala Ser Thr Tyr His
145 150 155 160
Leu Ile Tyr Ser Arg Thr Ile Met Arg Arg Glu Thr Phe Arg Asn Leu
165 170 175
Tyr Val Met Ile Leu Lys Gly Cys Leu Leu Tyr Thr Lys Gln His Ile
180 185 190
Gly Trp Lys Glu Lys Tyr Ile Arg His Ser Ser Ser Pro Asn Tyr Thr
195 200 205
Leu Ala Ser Tyr Pro Met Ile Leu Leu Val Thr Leu Leu Glu Gln Lys
210 215 220
Asn Lys Leu Ser Ser Arg Phe Val Glu Gly Cys Gln Gly Arg Arg Cys
225 230 235 240
Ala Thr Gln Ser Thr Asn Lys Lys Leu Leu Thr Val Arg Ser Cys Ser
245 250 255
Leu Gln Ser Thr Ser Thr Cys Phe Lys Lys Cys Thr Lys Thr Ser Leu
260 265 270
Ala Lys Ser Ala Asn Gly Gly Lys Ile Trp Thr Phe Glu Thr Ser Tyr
275 280 285
His Met Phe Glu Thr Asp Leu Lys Ala Thr Phe Gly Tyr Trp Glu Ser
290 295 300
Ile Ser Ser Leu Asn Ile Leu Val Gln Glu Cys Ser Lys His Ala Cys
305 310 315 320
Gly Leu Phe Met Ile His Leu Ile Ile Met Val Leu Met Arg Asn Ser
325 330 335
Arg Tyr Leu His Lys Leu Ser Lys Asp Gly Pro Pro Ala Trp Met Ser
340 345 350
Cys Gln Ser Thr Asn Tyr Ile Met Asn Ser Phe Val Phe Thr Lys Lys
355 360 365
Trp Arg Asn His Leu Arg Arg Arg Glu Lys His Ile Lys Ser Ile Ile
370 375 380
Leu Arg Arg Trp Arg Lys Arg Ala His Ala Ala Phe Tyr Lys Pro Asn
385 390 395 400
Gly Lys Arg Asp Thr Cys Gln His Thr Ser Thr Cys Leu Ile His Leu
405 410 415
Leu Val Asp Met His Gln Gln Asp Leu Met Leu Pro Gly Met Thr Val
420 425 430
Ser Pro Arg Met Pro Leu Asn Gly Trp Pro His Ile Leu Leu Leu Lys
435 440 445
Leu His Val Lys Phe Asp Leu Trp Met Ile Leu Pro Pro Thr Arg Arg
450 455 460
Asn Lys Lys Glu Ala Ile Leu Leu Gln Ala Leu Asn Ala Thr Glu Arg
465 470 475 480
Lys Leu Val His Gln Arg Arg Lys His Ala Trp Ile Ser Asn Lys Ser
485 490 495
Lys Met Val Gly Arg Leu Ile Arg Ser Arg Ser Cys Leu Gln Met Tyr
500 505 510
His Phe Leu Ser Leu Phe Leu Gln Ser Thr Leu Arg Val Val Ile Pro
515 520 525
Tyr Ile Lys Thr Met Met Ala Thr Ile Met Leu Ile Lys Lys Ser Leu
530 535 540
Val Thr Ser Asn Arg Ser Ser Phe Thr Leu Leu Ser Arg Arg Glu Glu
545 550 555 560
Cys Thr Arg Asp Lys Ile Asn Glu Leu Ser Gly Gly Pro Glu Lys Asn
565 570 575
Asn Lys Ser Ala Phe Cys Leu Tyr Arg Lys Ala Tyr Ala Gly Leu Ile
580 585 590
Lys Glu Ile Tyr Ala Ile Phe Ser Thr Gly Arg Arg Lys Ala Val Ala
595 600 605
Ala Ser Asp Cys Thr Gly Cys Phe Lys Cys Ile Trp Lys Lys Arg Lys
610 615 620
Arg Arg His Ser Gly Gly Leu Cys Cys Arg Asn Asp Ser His Val Phe
625 630 635 640
Val Asn Ser Ser Ser Met His Gly Arg Phe Ala Pro Arg Glu Ala Asp
645 650 655
Lys Pro Ser Val Trp Ser Asp Gly Ser Ile Ser Gly Arg Ala Ala His
660 665 670
Arg Lys Leu Ala Asp Tyr Leu Pro Arg Val Arg Arg Gly Val Ser Arg
675 680 685
Lys Ala Pro Ala Ala Cys Glu Thr Asp Phe Ser Gly Arg His Arg Arg
690 695 700
His Gly Arg Trp Ala Ser Ser Tyr Gly Ser Gly Lys Pro Thr Ser His
705 710 715 720
Ala Arg Ala Arg Ile His Ser Thr Glu Asn Cys Gln Ala Pro Trp Leu
725 730 735
Leu Cys Asn Arg Arg Cys Tyr Phe Gly Cys Ala Gly Arg His Asn Thr
740 745 750
Ala Tyr Leu Gln Gln Pro His Trp Asn Arg Ile Ser Asn Gln Arg Arg
755 760 765
Tyr Phe Arg Phe Arg Arg Gln Arg Glu Asn Arg Gln Thr Cys Arg Leu
770 775 780
Gly Tyr His Lys Gln Ile Asp Ile Pro Val Ala Ser Phe Ile Gly Arg
785 790 795 800
Gly Gln Thr Ile Gly Cys Ser Tyr Lys Arg Gly Glu Ala Ile Asp Arg
805 810 815
Arg Thr Leu Ser Ser Lys Arg Pro Phe Ile Ala Leu Phe Asn Cys Gly
820 825 830
Lys Arg Ser Leu
835
<210> 19
<211> 836
<212> PRT
<213> 人工序列
<400> 19
Gly Leu Ser Pro Arg Tyr Ser Ala Ser Ser Lys Leu Val Tyr Phe Ala
1 5 10 15
Phe Asn Ser Lys Arg Gly Met Tyr Thr Cys Gln Leu Tyr Gln Phe Leu
20 25 30
Leu Leu Pro Phe Leu His Leu His Cys Leu Leu Leu Ile Arg Ile Ala
35 40 45
Arg Ser Asn Met Leu Leu Ala Thr Val Ser Ser Ser Thr Ile Val Tyr
50 55 60
Gly Gly Ile Asn Phe Leu Asn Ile Arg Arg Asn Ser Met Leu Leu Arg
65 70 75 80
Asn Ser Ser Arg Ser Ser Lys Lys Lys Glu Thr Asn Glu Leu Val Met
85 90 95
Lys Gln Ala Asp Ile Ser Leu Tyr Asn Ser Leu Met Leu Asn Ala Leu
100 105 110
Ala Pro Ile Ile Leu Lys Arg Arg Ser Arg Asn Pro Cys Asn Ile Ser
115 120 125
Met Leu His Met Ala Ile Asn Gly Pro Thr Ile Thr Thr Leu Lys Ala
130 135 140
Phe Arg Cys Gly Phe Asp Cys Tyr Asp Lys Met Ala Ser Thr Tyr His
145 150 155 160
Leu Ile Tyr Ser Arg Thr Ile Met Arg Arg Glu Thr Phe Arg Asn Leu
165 170 175
Tyr Val Met Ile Leu Lys Gly Cys Leu Leu Tyr Thr Lys Gln His Ile
180 185 190
Gly Trp Lys Glu Lys Tyr Ile Arg His Ser Ser Ser Pro Asn Tyr Thr
195 200 205
Leu Ala Ser Tyr Pro Met Ile Leu Leu Val Thr Leu Leu Glu Gln Lys
210 215 220
Asn Lys Leu Ser Ser Arg Phe Val Glu Gly Cys Gln Gly Arg Arg Cys
225 230 235 240
Ala Thr Gln Ser Thr Asn Lys Lys Leu Leu Thr Val Arg Ser Cys Ser
245 250 255
Leu Gln Ser Thr Ser Thr Cys Phe Lys Lys Cys Thr Lys Thr Ser Leu
260 265 270
Ala Lys Ser Ala Asn Gly Gly Lys Ile Trp Thr Phe Glu Thr Ser Tyr
275 280 285
His Met Phe Glu Thr Asp Leu Lys Ala Thr Phe Gly Tyr Trp Glu Ser
290 295 300
Ile Ser Ser Leu Asn Ile Leu Val Gln Glu Cys Ser Lys His Ala Cys
305 310 315 320
Gly Leu Phe Met Ile His Leu Ile Ile Met Val Leu Met Arg Asn Ser
325 330 335
Arg Tyr Leu His Lys Leu Ser Lys Asp Gly Pro Pro Ala Trp Met Ser
340 345 350
Cys Gln Ser Thr Asn Tyr Ile Met Asn Ser Phe Val Phe Thr Lys Lys
355 360 365
Trp Arg Asn His Leu Arg Arg Arg Glu Lys His Ile Lys Ser Ile Ile
370 375 380
Leu Arg Arg Trp Arg Lys Arg Ala His Ala Ala Phe Tyr Lys Pro Asn
385 390 395 400
Gly Lys Arg Asp Thr Cys Gln His Thr Ser Thr Cys Leu Ile His Leu
405 410 415
Leu Val Asp Met His Gln Gln Asp Leu Met Leu Pro Gly Met Thr Val
420 425 430
Ser Pro Arg Met Pro Leu Asn Gly Trp Pro His Ile Leu Leu Leu Lys
435 440 445
Leu His Val Lys Phe Asp Leu Trp Met Ile Leu Pro Pro Thr Arg Arg
450 455 460
Asn Lys Lys Glu Ala Ile Leu Leu Gln Ala Leu Asn Ala Thr Glu Arg
465 470 475 480
Lys Leu Val His Gln Arg Arg Lys His Ala Trp Ile Ser Asn Lys Ser
485 490 495
Lys Met Val Gly Arg Leu Ile Arg Ser Arg Ser Cys Leu Gln Met Tyr
500 505 510
His Phe Leu Ser Leu Phe Leu Gln Ser Thr Leu Arg Val Val Ile Pro
515 520 525
Tyr Ile Lys Thr Met Met Ala Thr Ile Met Leu Ile Lys Lys Ser Leu
530 535 540
Val Thr Ser Asn Arg Ser Ser Phe Thr Leu Leu Ser Arg Arg Glu Glu
545 550 555 560
Cys Thr Arg Asp Lys Ile Asn Glu Leu Ser Gly Gly Pro Glu Lys Asn
565 570 575
Asn Lys Ser Ala Phe Cys Leu Tyr Arg Lys Ala Tyr Ala Gly Leu Ile
580 585 590
Lys Glu Ile Tyr Ala Ile Phe Ser Thr Gly Arg Arg Lys Ala Val Ala
595 600 605
Ala Ser Asp Cys Thr Gly Cys Phe Lys Cys Ile Trp Lys Lys Arg Lys
610 615 620
Arg Arg His Ser Gly Gly Leu Cys Cys Arg Asn Asp Ser His Val Phe
625 630 635 640
Val Asn Ser Ser Ser Met His Gly Arg Phe Ala Pro Arg Glu Ala Asp
645 650 655
Lys Pro Ser Val Trp Ser Asp Gly Ser Ile Ser Gly Arg Ala Ala His
660 665 670
Arg Lys Leu Ala Asp Tyr Leu Pro Arg Val Arg Arg Gly Val Ser Arg
675 680 685
Lys Ala Pro Ala Ala Cys Glu Thr Asp Phe Ser Gly Arg His Arg Arg
690 695 700
His Gly Arg Trp Ala Ser Ser Tyr Gly Ser Gly Lys Pro Thr Ser His
705 710 715 720
Ala Arg Ala Arg Ile His Ser Thr Glu Asn Cys Gln Ala Pro Trp Leu
725 730 735
Leu Cys Asn Arg Arg Cys Tyr Phe Gly Cys Ala Gly Arg His Asn Thr
740 745 750
Ala Tyr Leu Gln Gln Pro His Trp Asn Arg Ile Ser Asn Gln Arg Arg
755 760 765
Tyr Phe Arg Phe Arg Arg Gln Arg Glu Asn Arg Gln Thr Cys Arg Leu
770 775 780
Gly Tyr His Lys Gln Ile Asp Ile Pro Val Ala Ser Phe Ile Gly Arg
785 790 795 800
Gly Gln Thr Ile Gly Cys Ser Tyr Lys Arg Gly Glu Ala Ile Asp Arg
805 810 815
Arg Thr Leu Ser Ser Lys Arg Pro Phe Ile Ala Leu Phe Asn Cys Gly
820 825 830
Lys Arg Ser Leu
835
<210> 20
<211> 36
<212> DNA
<213> 人工序列
<400> 20
gaaaaaaaat gggtaaggaa aagactcacg tttcga 36
<210> 21
<211> 36
<212> DNA
<213> 人工序列
<400> 21
gaaaaaaaat gggtaaggaa aagactcacg tttcga 36
<210> 22
<211> 59
<212> DNA
<213> 人工序列
<400> 22
gaaaaaagca tcgaaaaaaa agagaggaat gtacacatga actccctcct ccacgccgc 59
<210> 23
<211> 40
<212> DNA
<213> 人工序列
<400> 23
gtgtacattc ctctctttca tgggtggtag cggcgcgacg 40
<210> 24
<211> 59
<212> DNA
<213> 人工序列
<400> 24
gaaaaaagca tcgaaaaaaa agagaggaat gtacacatgt caactttatc agtttctac 59
<210> 25
<211> 40
<212> DNA
<213> 人工序列
<400> 25
gtgtacattc ctctcttcta gacaatcata gggtgaacga 40
<210> 26
<211> 59
<212> DNA
<213> 人工序列
<400> 26
tcgaaacgtg agtcttttcc ttacccattt tttttcttag tgatctcttg ccgcaatta 59
<210> 27
<211> 59
<212> DNA
<213> 人工序列
<400> 27
gcgccgctac cacccatgaa agagaggaat gtacacgtga caaataaatt aacgagctt 59
<210> 28
<211> 59
<212> DNA
<213> 人工序列
<400> 28
tcaccctatg attgtctaga agagaggaat gtacacgtga caaataaatt aacgagctt 59
<210> 29
<211> 59
<212> DNA
<213> 人工序列
<400> 29
tcgaaacgtg agtcttttcc ttacccattt tttttcttat ttattacgct ggatgatgt 59
<210> 30
<211> 59
<212> DNA
<213> 人工序列
<400> 30
gcgccgctac cacccatgaa agagaggaat gtacacatgg actttccgca gcaactcga 59
<210> 31
<211> 59
<212> DNA
<213> 人工序列
<400> 31
tcaccctatg attgtctaga agagaggaat gtacacatgg actttccgca gcaactcga 59
<210> 32
<211> 59
<212> DNA
<213> 人工序列
<400> 32
tcgaaacgtg agtcttttcc ttacccattt tttttcctat ctcaattgta actgagtta 59
<210> 33
<211> 59
<212> DNA
<213> 人工序列
<400> 33
gcgccgctac cacccatgaa agagaggaat gtacacatga cgaattttag tcaacagca 59
<210> 34
<211> 59
<212> DNA
<213> 人工序列
<400> 34
tcaccctatg attgtctaga agagaggaat gtacacatga cgaattttag tcaacagca 59
<210> 35
<211> 102
<212> DNA
<213> 人工序列
<400> 35
taagaacggt gctctcggat ccagctagct cagtcctagg tattgtgcta gcaattgcag 60
taggcatgac aaaatggact caaagagagg aatgtacaca tg 102
<210> 36
<211> 102
<212> DNA
<213> 人工序列
<400> 36
catgtgtaca ttcctctctt tgagtccatt ttgtcatgcc tactgcaatt gctagcacaa 60
tacctaggac tgagctagct ggatccgaga gcaccgttct ta 102
<210> 37
<211> 102
<212> DNA
<213> 人工序列
<400> 37
taagaacggt gctctcggat ccggctagct cagtcctagg tactatgcta gcaatgggct 60
cgtgttgtac aataaatgta gtaagagagg aatgtacaca tg 102
<210> 38
<211> 102
<212> DNA
<213> 人工序列
<400> 38
catgtgtaca ttcctctctt actacattta ttgtacaaca cgagcccatt gctagcatag 60
tacctaggac tgagctagcc ggatccgaga gcaccgttct ta 102
<210> 39
<211> 102
<212> DNA
<213> 人工序列
<400> 39
taagaacggt gctctcggat ccggctagct cagtcctagg tatagtgcta gctctaagct 60
agtgtatttt gcgtttaata gtaagagagg aatgtacaca tg 102
<210> 40
<211> 102
<212> DNA
<213> 人工序列
<400> 40
catgtgtaca ttcctctctt actattaaac gcaaaataca ctagcttaga gctagcacta 60
tacctaggac tgagctagcc ggatccgaga gcaccgttct ta 102

Claims (6)

1.一种发酵合成α-红没药醇的工程菌株，其特征在于，以大肠杆菌或枯草芽孢杆菌为宿主，同时表达了BBS基因和ispA基因，所述ispA基因表达氨基酸序列如SEQ ID NO.12-SEQID NO.14任一所示的蛋白，所述BBS基因表达氨基酸序列如SEQ ID NO.15或SEQ ID NO.16所示的蛋白，所述大肠杆菌为大肠杆菌MG1655，大肠杆菌DH5α，大肠杆菌W3110或大肠杆菌BL21；所述枯草芽孢杆菌为枯草芽孢杆菌168、枯草芽孢杆菌W600或枯草芽孢杆菌W800；所述工程菌株以pEBS为表达载体。
2.根据权利要求1所述的工程菌株，其特征在于，所述ispA基因的核苷酸序列如SEQ IDNO.1-SEQ ID NO.3任一所示。
3.根据权利要求1所述的工程菌株，其特征在于，所述BBS基因的核苷酸序列如SEQ IDNO.4或SEQ ID NO.5所示。
4.一种生产α-红没药醇的方法，其特征在于，应用权利要求1-3任一所述的工程菌株进行发酵。
5.如权利要求4所述的方法，其特征在于，将工程菌株接种于LB培养基中，置于200-220rpm，35-38℃培养10-12h，然后按5-10％的接种量转接于发酵培养基中，添加20-25％(v/v)的正十二烷，然后置于200-220rpm，35-38℃培养50-70h；所述发酵培养基组分为(g/L)：酵母粉2.5，蛋白胨5.0，Na2HPO4 6.78，KH2PO4 3.0，NaCl 0.5，NH4Cl 1.0，MgSO4·7H200.5，CaCl2 0.015，葡萄糖40；FeCl2·6H2O 0.013.5；MnCl2·4H2O，0.001；ZnCl2，0.0017；CuCl2·2H2O，0.00043。
6.权利要求1-3任一所述的工程菌株在制备α-红没药醇或含α-红没药醇的产品中的应用。
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