CN112980753B

CN112980753B - 用于外源蛋白分泌的糖苷水解酶融合表达系统

Info

Publication number: CN112980753B
Application number: CN201911282970.4A
Authority: CN
Inventors: 游淳; 石婷; 刘珊
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2019-12-13
Filing date: 2019-12-13
Publication date: 2023-12-19
Anticipated expiration: 2039-12-13
Also published as: CN112980753A

Abstract

本发明提供一种能改善外源蛋白在枯草芽孢杆菌中的分泌生产的融合表达系统，所示融合表达系统包含一种与外源蛋白融合的天然糖苷水解酶。本发明还提供利用所述融合表达系统生产外源蛋白的方法。

Description

用于外源蛋白分泌的糖苷水解酶融合表达系统

技术领域

本发明属于生物技术和基因工程技术领域，具体地涉及一种基于糖苷水解酶的枯草芽孢杆菌(Bacillus subtilis)融合表达系统。本发明还涉及利用所述融合表达系统在枯草芽孢杆菌中分泌生产外源蛋白的方法。

发明背景

蛋白质的重组生产是现代生物技术和酶工业应用中的重要技术之一。与胞内生产相比，由于分泌过程有利于蛋白质在低还原环境下折叠、可溶性表达及生物活性保持，同时分泌蛋白易于纯化，简化了下游操作步骤，所以分泌表达是一种理想的外源蛋白生产方式。枯草芽孢杆菌是一般认为安全(Generally Recognized As Safe,GRAS)的食品级微生物，具有较好的分泌能力、易于培养、不产生内毒素、遗传操作相对简单和发酵工艺成熟等优点，被认为是理想的外源蛋白表达宿主菌。目前为止许多来源于不同生物的外源蛋白已经在B.subtilis中实现了分泌表达，但分泌量低这一瓶颈仍未得到解决。其中，外源大蛋白，例如分子量大于80kDa的大蛋白的分泌尤为困难。目前，除芽孢杆菌属的两个普鲁兰酶以较高水平分泌外，仅极少数80kDa以上的外源蛋白在枯草芽孢杆菌中成功分泌，且分泌量非常低，仅有几十毫克每升。因此，亟需有效改善外源蛋白在枯草芽孢杆菌中的分泌生产的方法。

发明内容

本发明人发现将编码来源于枯草芽孢杆菌的糖苷水解酶家族5葡聚糖内切酶(B.subtilis native glycoside hydrolase family 5endoglucanase，BsCel5)的DNA片段与编码目的蛋白的第二个DNA片段融合在一个读码框中时，所编码的融合蛋白的分泌水平明显提高。本发明人还发现，BsCel5能够有效改善外源大蛋白，例如分子量大于80kDa的外源蛋白的分泌。因此，本发明的第一个方面提供基于糖苷水解酶的融合表达系统。

所述融合表达系统包含能够编码至少以下元件的DNA序列，所述元件包括信号肽、糖苷水解酶和外源蛋白，并且依上述顺序排列融合在一个开放读码框内。

所述糖苷水解酶包含如SEQ ID NO.2所示的氨基酸序列或与其具有至少70％同一性的氨基酸序列。所述氨基酸序列包含纤维素结合模块(CBM)，其能够有效结合纤维素，从而便于融合蛋白的回收。优选地，所述糖苷水解酶为枯草芽孢杆菌天然糖苷水解酶家族5葡聚糖内切酶BsCel5，其序列如SEQ ID NO.2所示。编码BsCel5的DNA序列在SEQ ID NO.1中示出。

任选地，糖苷水解酶和外源蛋白之间可插入接头序列、用于蛋白酶剪切的靶位点或内含肽。

任选地，信号肽和糖苷水解酶之间可插入前肽序列以提高融合蛋白的分泌水平。

本领域技术人员可以理解，本领域已知的可在枯草芽孢杆菌中起作用的各种信号肽均可用于本发明，包括但不限于，来源于枯草芽孢杆菌的蛋白酶nprB信号肽、来源于枯草芽孢杆菌的α淀粉酶amyE信号肽和来源于地衣芽孢杆菌(B.licheniformis)的amyL信号肽。优选地，本发明中使用的信号肽为来源于枯草芽孢杆菌的蛋白酶nprB信号肽。

本领域技术人员可以理解，本领域已知的各种枯草芽孢杆菌菌株均可用作本发明的融合表达系统中的宿主。优选地，所述枯草芽孢杆菌宿主为敲除蛋白酶的枯草芽孢杆菌菌株，包括但不限于WB800、WB600、SCK6、1A751。更优选地，所述枯草芽孢杆菌宿主为敲除蛋白酶和α淀粉酶的菌株。

可将糖苷水解酶蛋白序列最小化至某种程度而仍维持其在融合蛋白表达中使用时的有利特性。这种对DNA序列以及因此所编码的蛋白质序列的最小化可通过分子生物学领域已知的任何方法实现。

本发明的第二个方面提供利用所述融合表达系统在枯草芽孢杆菌中分泌生产外源蛋白的方法。所述方法包括下述步骤：

(1)构建合适的枯草芽孢杆菌细胞，其包含能够编码至少以下元件的DNA序列，所述元件包括信号肽、糖苷水解酶和外源蛋白，并且依上述顺序排列融合在一个开放读码框内；

(2)在适于生长和分泌的条件下培养步骤(1)中所构建的细胞；和

(3)回收所述蛋白。

其中，信号肽和糖苷水解酶如上文对于融合表达系统所描述的。

优选地，步骤(3)包括使用固体纤维素例如再生无定形纤维素(Regeneratedamorphous cellulose，RAC)回收所述蛋白。

任选地，糖苷水解酶和外源蛋白之间可插入用于蛋白酶剪切的靶位点，并且所述分泌生产外源蛋白的方法包括在所述靶位点切割融合蛋白。

任选地，糖苷水解酶和外源蛋白之间可插入内含肽序列，并且所述分泌生产外源蛋白的方法包括在合适的条件下断裂内含肽。

本发明的有益效果为：

(1)通过利用本发明的融合表达系统或方法，有可能以较高产量产生所需外源蛋白，尤其是分子量大于80kDa的外源蛋白，从而使生产更经济；

(2)本发明的融合表达系统或方法中使用的糖苷水解酶包含纤维素结合模块，使得融合蛋白能够特异性结合固体纤维素，因此融合蛋白的回收可通过简单的固体/液体分离进行，有利于降低生产成本。

附图说明

图1A和B分别为重组质粒pNWP43N-StIA和pNWP43N-BsCel5-StIA的图谱。其中P₄₃、RBS、SP_nprB、Bscel5、StIA和term分别代表P43启动子、核糖体结合位点、来源于枯草芽孢杆菌168的蛋白酶NprB的信号肽编码序列、BsCel5基因、来源于硫化叶菌(Sulfolobustokodaii)的异淀粉酶基因和BsCel5的终止子。箭头显示这些基因的转录方向。

图2为两种表达方式下StIA分泌的SDS-PAGE比较。泳道1，蛋白marker；泳道2，阴性对照(pNWP43N)表达上清；泳道3，包含pNWP43N-StIA的枯草芽孢杆菌菌株的培养上清；泳道4，包含pNWP43N-BsCel5-StIA的枯草芽孢杆菌菌株的培养上清。

图3为回收的IA的SDS-PAGE电泳图。泳道1，蛋白marker；泳道2，包含pNWP43N-BsCel5-intein-StIA的枯草芽孢杆菌菌株的培养上清；泳道3，使用RAC回收的BsCel5-intein-StIA融合蛋白；泳道4：内含肽断裂后回收的StIA。

具体实施方式

为更进一步阐述本发明所采取的技术手段及其效果，以下通过具体实施例来进一步说明本发明的技术方案。但是应理解所述实施例仅是范例性的，不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改或替换均落入本发明的保护范围。

实施例1：pNWP43N-StIA和pNWP43N-BsCel5-StIA重组质粒的构建

编码异淀粉酶(GenBank登录号：BAB65940.1)的DNA片段使用下列引物从硫化叶菌的基因组扩增：

pNWP3N-StIA-IF：

5′-GTAACACATGCCTCAGCTGCAATGGTGTTCAGCCATAAAGATCGTC-3′

pNWP43N-StIA-IR：

5′-CACAACGCAAACCTCCTATTAGATGTTAATATTCAATGCGACGATAAACC-3′

pNWP43N载体线性骨架使用下列引物以pNWP43N质粒为模板扩增，其包含P43启动子和来源于枯草芽孢杆菌168的蛋白酶NprB的信号肽：

pNWP43N-StIA-VF：

5′-GGTTTATCGTCGCATTGAATATTAACATCTAATAGGAGGTTTGCGTTGTG-3′

pNWP43N-StIA-VR：

5′-GACGATCTTTATGGCTGAACACCATTGCAGCTGAGGCATGTGTTAC-3′

所有引物均由苏州金唯智生物科技有限公司合成。基因的PCR条件为98℃变性2min，按如下参数循环30次：98℃变性15s，58℃退火15s，72℃延伸1min，最后72℃延伸5min。PCR反应所得到的产物分别用1％的琼脂糖凝胶电泳分析结果。经凝胶成像系统成像确认片段大小正确后，采用DNA纯化回收试剂盒(天根生化科技有限公司，中国)回收目的片段。

pNWP43N-StIA重组质粒的构建使用POE-PCR进行。POE-PCR体系如下：纯化后的pNWP3N线性骨架，200ng；纯化后的StIA片段131ng；2×PrimeSTAR MAX DNA Polymerase(大连宝生物，中国)，25μL，加水补足50μL。POE-PCR条件为98℃变性2min，按如下参数循环30次：98℃变性15s，58℃退火15s，72℃延伸3min，最后72℃延伸5min。POE-PCR产物用转化入枯草芽孢杆菌SCK6/△amyE感受态，感受态制作和转化过程如Zhang XZ,Zhang Y-HP.2011.Simple,fast and high-efficiency transformation system for directedevolution of cellulase in Bacillus subtilis.Microb Biotechnol 4(1):98-105中所描述的。挑选转化子进行菌落PCR及双酶切鉴定，选择2-3个阳性转化子送测序进一步验证，测序结果显示成功获得pNWP3N-StIA重组质粒。

编码成熟糖苷水解酶BsCel5(GenBank登录号：CAA82317，30-499氨基酸)的DNA片段使用下列引物从枯草芽孢杆菌168的基因组扩增：

pNWP43N-BsCel5-StIA-IF：

5′-GTAACACATGCCTCAGCTGCAGCAGGGACAAAAACGCCAGTAGCCA-3′

pNWP43N-BsCel5-StIA-IR：

5′-GACGATCTTTATGGCTGAACACCATATTTGGTTCTGTTCCCCAAATCAGT-3′

pNWP43N-IA载体线性骨架使用下列引物以pNWP43N-IA质粒为模板扩增：

pNWP43N-BsCel5-StIA-VF：

5′-ACTGATTTGGGGAACAGAACCAAATATGGTGTTCAGCCATAAAGATCGTC-3′

pNWP43N-BsCel5-StIA-VR：

5′-TGGCTACTGGCGTTTTTGTCCCTGCTGCAGCTGAGGCATGTGTTAC-3′

pNWP43N-BsCel5-StIA的构建如上文对于pNWP43N-StIA所述。在pNWP43N-BsCel5-StIA中，NprB信号肽、BsCel5、StIA依次融合在一个开放读码框中。编码BsCel5-StIA的开放读码框的总DNA序列示于SEQ ID NO.3，其衍生的氨基酸序列见SEQ ID NO.4。在SEQ IDNO.4中的氨基酸具有以下特征：

a.a.1-29：枯草芽孢杆菌的NprB的信号肽；

a.a.30-499：枯草芽孢杆菌的糖苷水解酶家族5葡聚糖内切酶

a.a.500-1215：硫化叶菌的异淀粉酶

实施例2：BsCel5-StIA融合蛋白的表达和检测

将编码BsCel5-StIA融合蛋白的SCK6/△amyE菌株和两个对照菌株：包含pNWP43N-StIA重组质粒的SCK6/△amyE菌株和包含pNWP43N空质粒的SCK6/△amyE菌株在SR培养基(1.5％蛋白胨,2.5％酵母提取物和0.3％K₂HPO₄)中以30℃和250rpm培养48h。将40μL无细胞上清与10μL 5×SDS上样缓冲液混合，100℃煮5min后取20μL上样至12％SDS-PAGE。120V恒压处理～1-1.5h，待溴酚蓝指示剂条带离开凝胶后停止电泳。考马斯亮蓝染色检测蛋白分泌情况。如图2中所示，单独表达时使用SDS-PAGE检测不到StIA的分泌，而使用基于BsCel5的融合表达系统上清中可以观察到明显的分子量大小对应于BsCel5-StIA(135kDa)的蛋白条带。

使用碘法以玉米支链淀粉为底物分析上述三个菌株的培养上清中的异淀粉酶活性。酶活测定体系包含0.35％(wt/v)玉米支链淀粉、40mM醋酸钠缓冲液(pH 5.5)、0.5mMMgCl₂和适量浓缩后的上清，总体积500μL。50℃孵育30min后，取50μL反应混合物，与50μL0.01M I₂-0.1M KI溶液混合，然后用蒸馏水稀释至1mL，立即测量在610nm处的吸光值。1U酶活定义为在上述测定条件下使得610nm处的吸光值在1h内增加0.1所需要的酶量。在同样的条件下测定比酶活以确定蛋白的分泌量。表1显示，在含有BsCel5-StIA融合蛋白的SCK6/△amyE菌株中融合蛋白的分泌量相对于仅编码的SCK6/△amyE菌株有6.5倍的显著增加。上述结果表明，显然基于BsCel5的融合表达系统可有效显著增加诸如StIA这样的外源大蛋白的分泌。

表1

实施例3：使用BsCel5 N端30个氨基酸促进StIA分泌

使用引物N30-StIA-IF和pNWP43N-StIA-IR扩增IA片段，使用引物pNWP43N-StIA-VF和N30-StIA-VR以pNWP43N-BsCel5-IA为模板扩增包含BsCel5 N端30个氨基酸的编码序列的线性载体骨架。

所使用的引物序列如下：

N30-StIA-IF：

5′-CCGAGACGGTAAAGCGGTACAGCTGATGGTGTTCAGCCATAAAGATCGTC-3′

N30-StIA-VR：

5′-GACGATCTTTATGGCTGAACACCATCAGCTGTACCGCTTTACCGTCTCGG-3′

然后，使用如上所述的POE-PCR方法构建pNWP43N-N30-StIA重组质粒，其中NprB信号肽、BsCel5 N端30个氨基酸、StIA依次融合在一个开放读码框中。编码融合蛋白的开放读码框的总DNA序列示于SEQ ID NO.5，其衍生的氨基酸序列见SEQ ID NO.6。在SEQ ID NO.6中的氨基酸具有以下特征：

a.a.1-29：枯草芽孢杆菌的NprB的信号肽；

a.a.30-59：枯草芽孢杆菌的糖苷水解酶家族5葡聚糖内切酶的N端30个氨基酸残基

a.a.60-775：硫化叶菌的异淀粉酶

将编码N30-StIA融合蛋白的SCK6/△amyE菌株在SR培养基中以30℃和250rpm培养48h。上清中的异淀粉酶酶活为1.63U/mL，融合蛋白的分泌量为8.57mg/L，相对于仅编码的SCK6/△amyE菌株提高5.3倍。

实施例4：BsCel5-IA融合蛋白的回收和内含肽切割

使用POE-PCR将内含肽编码序列插入BsCel5与StIA之间，获得pNWP43N-BsCel5-intein-StIA重组质粒，其中NprB信号肽、BsCel5、内含肽、StIA依次融合在一个开放读码框中。编码融合蛋白的开放读码框的总DNA序列示于SEQ ID NO.7，其衍生的氨基酸序列见SEQID NO.8。在SEQ ID NO.8中的氨基酸具有以下特征：

a.a.1-29：枯草芽孢杆菌的NprB的信号肽；

a.a.30-499：枯草芽孢杆菌的糖苷水解酶家族5葡聚糖内切酶

a.a.500-678：内含肽

a.a.679-1394：硫化叶菌的异淀粉酶

将编码N30-StIA融合蛋白的SCK6/△amyE菌株在SR培养基中以30℃和250rpm培养48h。离心去除细胞。向1L上清中加入～30mg RAC，冰上吸附15-30min后，4℃ 5000×g离心10min。将RAC沉淀用30mL 50mM Tris-HCl缓冲液(pH 8.0)洗涤3次以去除非特异性吸附的杂蛋白。最后，将RAC沉淀重悬在5mL 50mM HEPES缓冲液(pH 6.5)中，40℃孵育8h以诱导内含肽自切割，切割的StIA释放入液相中。

取5μL内含肽自切割之前的RAC悬浮液样品和内含肽切割后的上清液样品进行SDS-PAGE分析，图3显示，BsCel5-内含肽-StIA融合蛋白通过RAC亲和吸附有效回收，并且在适当条件下内含肽有效断裂释放StIA蛋白。

序列表

<110> 中国科学院天津工业生物技术研究所

<120> 用于外源蛋白分泌的糖苷水解酶融合表达系统

<130> 1

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1410

<212> DNA

<213> 人工序列()

<400> 1

gcagggacaa aaacgccagt agccaagaat ggccagctta gcataaaagg tacacagctc 60

gttaaccgag acggtaaagc ggtacagctg aaggggatca gttcacacgg attgcaatgg 120

tatggagaat atgtcaataa agacagctta aaatggctga gagatgattg gggtatcacc 180

gttttccgtg cagcgatgta tacggcagat ggcggttata ttgacaaccc gtccgtgaaa 240

aataaagtaa aagaagcggt tgaagcggca aaagagcttg ggatatatgt catcattgac 300

tggcatatct taaatgacgg taatccaaac caaaataaag agaaggcaaa agaattcttc 360

aaggaaatgt caagccttta cggaaacacg ccaaacgtca tttatgaaat tgcaaacgaa 420

ccaaacggtg atgtgaactg gaagcgtgat attaaaccat atgcggaaga agtgatttca 480

gttatccgca aaaatgatcc agacaacatc atcattgtcg gaaccggtac atggagccag 540

gatgtgaatg atgctgccga tgaccagcta aaagatgcaa acgttatgta cgcacttcat 600

ttttatgccg gcacacacgg ccaattttta cgggataaag caaactatgc actcagcaaa 660

ggagcaccta tttttgtgac agagtgggga acaagcgacg cgtctggcaa tggcggtgta 720

ttccttgatc aatcgaggga atggctgaaa tatctcgaca gcaagaccat tagctgggtg 780

aactggaatc tttctgataa gcaggaatca tcctcagctt taaagccggg ggcatctaaa 840

acaggcggct ggcggttgtc agatttatct gcttcaggaa cattcgttag agaaaacatt 900

ctcggcacca aagattcgac gaaggacatt cctgaaacgc catcaaaaga taaacccaca 960

caggaaaatg gtatttctgt acagtacaga gcaggggatg ggagtatgaa cagcaaccaa 1020

atccgtccgc agcttcaaat aaaaaataac ggcaatacca cggttgattt aaaagatgtc 1080

actgcccgtt actggtataa agcgaaaaac aaaggccaaa actttgactg tgactacgcg 1140

cagattggat gcggcaatgt gacacacaag tttgtgacgt tgcataaacc aaagcaaggt 1200

gcagatacct atctggaact tggatttaaa aacggaacgt tggcaccggg agcaagcaca 1260

gggaatattc agctccgtct tcacaatgat gactggagca attatgcaca aagcggcgat 1320

tattcctttt tcaaatcaaa tacgtttaaa acaacgaaaa aaatcacatt atatgatcaa 1380

ggaaaactga tttggggaac agaaccaaat 1410

<210> 2

<211> 499

<212> PRT

<213> 人工序列()

<400> 2

Met Arg Asn Ser Thr Lys Thr Ser Leu Leu Leu Ala Gly Leu Cys Thr

1 5 10 15

Ala Ala Gln Met Val Phe Val Thr His Ala Ser Ala Ala Ala Gly Thr

20 25 30

Lys Thr Pro Val Ala Lys Asn Gly Gln Leu Ser Ile Lys Gly Thr Gln

35 40 45

Leu Val Asn Arg Asp Gly Lys Ala Val Gln Leu Lys Gly Ile Ser Ser

50 55 60

His Gly Leu Gln Trp Tyr Gly Glu Tyr Val Asn Lys Asp Ser Leu Lys

65 70 75 80

Trp Leu Arg Asp Asp Trp Gly Ile Thr Val Phe Arg Ala Ala Met Tyr

85 90 95

Thr Ala Asp Gly Gly Tyr Ile Asp Asn Pro Ser Val Lys Asn Lys Val

100 105 110

Lys Glu Ala Val Glu Ala Ala Lys Glu Leu Gly Ile Tyr Val Ile Ile

115 120 125

Asp Trp His Ile Leu Asn Asp Gly Asn Pro Asn Gln Asn Lys Glu Lys

130 135 140

Ala Lys Glu Phe Phe Lys Glu Met Ser Ser Leu Tyr Gly Asn Thr Pro

145 150 155 160

Asn Val Ile Tyr Glu Ile Ala Asn Glu Pro Asn Gly Asp Val Asn Trp

165 170 175

Lys Arg Asp Ile Lys Pro Tyr Ala Glu Glu Val Ile Ser Val Ile Arg

180 185 190

Lys Asn Asp Pro Asp Asn Ile Ile Ile Val Gly Thr Gly Thr Trp Ser

195 200 205

Gln Asp Val Asn Asp Ala Ala Asp Asp Gln Leu Lys Asp Ala Asn Val

210 215 220

Met Tyr Ala Leu His Phe Tyr Ala Gly Thr His Gly Gln Phe Leu Arg

225 230 235 240

Asp Lys Ala Asn Tyr Ala Leu Ser Lys Gly Ala Pro Ile Phe Val Thr

245 250 255

Glu Trp Gly Thr Ser Asp Ala Ser Gly Asn Gly Gly Val Phe Leu Asp

260 265 270

Gln Ser Arg Glu Trp Leu Lys Tyr Leu Asp Ser Lys Thr Ile Ser Trp

275 280 285

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

290 295 300

Pro Gly Ala Ser Lys Thr Gly Gly Trp Arg Leu Ser Asp Leu Ser Ala

305 310 315 320

Ser Gly Thr Phe Val Arg Glu Asn Ile Leu Gly Thr Lys Asp Ser Thr

325 330 335

Lys Asp Ile Pro Glu Thr Pro Ser Lys Asp Lys Pro Thr Gln Glu Asn

340 345 350

Gly Ile Ser Val Gln Tyr Arg Ala Gly Asp Gly Ser Met Asn Ser Asn

355 360 365

Gln Ile Arg Pro Gln Leu Gln Ile Lys Asn Asn Gly Asn Thr Thr Val

370 375 380

Asp Leu Lys Asp Val Thr Ala Arg Tyr Trp Tyr Lys Ala Lys Asn Lys

385 390 395 400

Gly Gln Asn Phe Asp Cys Asp Tyr Ala Gln Ile Gly Cys Gly Asn Val

405 410 415

Thr His Lys Phe Val Thr Leu His Lys Pro Lys Gln Gly Ala Asp Thr

420 425 430

Tyr Leu Glu Leu Gly Phe Lys Asn Gly Thr Leu Ala Pro Gly Ala Ser

435 440 445

Thr Gly Asn Ile Gln Leu Arg Leu His Asn Asp Asp Trp Ser Asn Tyr

450 455 460

Ala Gln Ser Gly Asp Tyr Ser Phe Phe Lys Ser Asn Thr Phe Lys Thr

465 470 475 480

Thr Lys Lys Ile Thr Leu Tyr Asp Gln Gly Lys Leu Ile Trp Gly Thr

485 490 495

Glu Pro Asn

<210> 3

<211> 3648

<212> DNA

<213> 人工序列()

<400> 3

atgcgcaact cgaccaagac atctctatta ctggccggct tatgcacagc ggcccaaatg 60

gtttttgtaa cacatgcctc agctgcagca gggacaaaaa cgccagtagc caagaatggc 120

cagcttagca taaaaggtac acagctcgtt aaccgagacg gtaaagcggt acagctgaag 180

gggatcagtt cacacggatt gcaatggtat ggagaatatg tcaataaaga cagcttaaaa 240

tggctgagag atgattgggg tatcaccgtt ttccgtgcag cgatgtatac ggcagatggc 300

ggttatattg acaacccgtc cgtgaaaaat aaagtaaaag aagcggttga agcggcaaaa 360

gagcttggga tatatgtcat cattgactgg catatcttaa atgacggtaa tccaaaccaa 420

aataaagaga aggcaaaaga attcttcaag gaaatgtcaa gcctttacgg aaacacgcca 480

aacgtcattt atgaaattgc aaacgaacca aacggtgatg tgaactggaa gcgtgatatt 540

aaaccatatg cggaagaagt gatttcagtt atccgcaaaa atgatccaga caacatcatc 600

attgtcggaa ccggtacatg gagccaggat gtgaatgatg ctgccgatga ccagctaaaa 660

gatgcaaacg ttatgtacgc acttcatttt tatgccggca cacacggcca atttttacgg 720

gataaagcaa actatgcact cagcaaagga gcacctattt ttgtgacaga gtggggaaca 780

agcgacgcgt ctggcaatgg cggtgtattc cttgatcaat cgagggaatg gctgaaatat 840

ctcgacagca agaccattag ctgggtgaac tggaatcttt ctgataagca ggaatcatcc 900

tcagctttaa agccgggggc atctaaaaca ggcggctggc ggttgtcaga tttatctgct 960

tcaggaacat tcgttagaga aaacattctc ggcaccaaag attcgacgaa ggacattcct 1020

gaaacgccat caaaagataa acccacacag gaaaatggta tttctgtaca gtacagagca 1080

ggggatggga gtatgaacag caaccaaatc cgtccgcagc ttcaaataaa aaataacggc 1140

aataccacgg ttgatttaaa agatgtcact gcccgttact ggtataaagc gaaaaacaaa 1200

ggccaaaact ttgactgtga ctacgcgcag attggatgcg gcaatgtgac acacaagttt 1260

gtgacgttgc ataaaccaaa gcaaggtgca gatacctatc tggaacttgg atttaaaaac 1320

ggaacgttgg caccgggagc aagcacaggg aatattcagc tccgtcttca caatgatgac 1380

tggagcaatt atgcacaaag cggcgattat tcctttttca aatcaaatac gtttaaaaca 1440

acgaaaaaaa tcacattata tgatcaagga aaactgattt ggggaacaga accaaatatg 1500

gttttttcac acaaggatag accattaaga ccaggagagc catatcctct tggagctaat 1560

tgggaagaag aagatgatgg tgtgaacttc tctatctttt cggaaaatgc gactaaggtt 1620

gaacttttaa tttactcccc tactaatcag aaatatccta aagaagttat cgaggttaag 1680

cagagatctg gtgatatttg gcacgtcttt gtcccaggtt tgggaccggg tacactttac 1740

gcatatagaa tttatggtcc ttataagcca gatcaaggtt taagatttaa tcctaataag 1800

gttctaattg atccttatgc taaggctata aatgggacat taaactggaa tgatgctgtt 1860

tttggttata agataggcga ttctaaccag gatttgtcct ttgatgatag gccagatgat 1920

gaatttattc ctaagggtgt tgttattaat ccttattttg agtgggatga tgatcacttt 1980

tttaggagaa agaagatacc attaaaggat actattattt atgaagttca tgttaaaggt 2040

tttactaaat taagacctga tttaccagaa aatattagag gtacttataa aggatttgcc 2100

tctagacaga tgatcgaata tttgaaagat ttgggggtaa ctacagtcga gataatgcca 2160

gtacagcagt ttgttgatga taggtttcta gtagagaagg gattaaggaa ttactgggga 2220

tataatccca taaattattt ttcacctgaa tgtagatatt cctcttctgg ctgtatgggt 2280

gaacaagtta acgagtttaa ggagatggtt aatgagctgc acaacgctgg cttcgaggta 2340

attattgatg ttgtttataa ccatactgcg gaagggaatc atttaggtcc tactctttca 2400

ttcagaggta tagataattt ggcttattac atgttagttc cagataataa gagatattat 2460

ttagacttta ctggaactgg aaacacctta aatctgagtc atccgagggt attgcaaatg 2520

gttctggata gtcttagata ttgggtttta gagatgcatg ttgacggttt taggtttgat 2580

ttagctgctg ccctagctag acaattatac agtgtaaata tgctttcaac tttctttgtt 2640

gcaattcagc aagatcccgt tctttctcaa gttaagttaa tagcggaacc ttgggatgtt 2700

ggtccagggg gatatcaggt tggtaatttt ccatatttgt gggccgaatg gaacggtaag 2760

tatagagata ctataaggag attttggaga ggtgaggcga tcccctatga ggagttggct 2820

aataggctta tgggttctcc agatttatat gctggaaata ataagactcc tttcgctagt 2880

ataaattata taacttctca tgatggtttt actttagagg atttagttag ttataatcaa 2940

aagcataatg aagctaacgg ttttaataat caagatggca tgaacgagaa ttatagttgg 3000

aattgtggag ttgagggaga gactaatgat gctaatgtta ttcaatgtag agagaaacaa 3060

aaaaggaatt ttatcataac actttttgta agtcaagggg ttccaatgat tttaggtggc 3120

gatgagctaa gtagaacaca aagaggaaat aacaatgctt tttgccaaga taacgaaata 3180

agttggttta attggaatct tgatgagagg aaacagaggt ttcatgattt tgttaggagt 3240

atgatttatt tctatagagc tcatccaata tttagaagag aaagatactt tcaaggtaag 3300

aaattacatg gtatgccatt aaaggatgtc acttttctaa aaccagatgg aaatgaagct 3360

gacgaacaaa catggaagtc accaactaat tttattgcat atattttaga gggtagtgtt 3420

attgatgaag taaatgatag gggtgagaga atagctgacg attctttctt aatcatcctt 3480

aatggttcac caaataatat taagttcaaa ttcccgcaag gtaaatggag tttagttgtt 3540

tcttcatatt tgagagaact tagagatgac gagagagttg ttgatggtgg caaggaactg 3600

gaaattgagg gaaggaccgc aatggtatat aggaggattg aatattag 3648

<210> 4

<211> 1215

<212> PRT

<213> 人工序列()

<400> 4

Met Arg Asn Ser Thr Lys Thr Ser Leu Leu Leu Ala Gly Leu Cys Thr

1 5 10 15

Ala Ala Gln Met Val Phe Val Thr His Ala Ser Ala Ala Ala Gly Thr

20 25 30

Lys Thr Pro Val Ala Lys Asn Gly Gln Leu Ser Ile Lys Gly Thr Gln

35 40 45

Leu Val Asn Arg Asp Gly Lys Ala Val Gln Leu Lys Gly Ile Ser Ser

50 55 60

His Gly Leu Gln Trp Tyr Gly Glu Tyr Val Asn Lys Asp Ser Leu Lys

65 70 75 80

Trp Leu Arg Asp Asp Trp Gly Ile Thr Val Phe Arg Ala Ala Met Tyr

85 90 95

Thr Ala Asp Gly Gly Tyr Ile Asp Asn Pro Ser Val Lys Asn Lys Val

100 105 110

Lys Glu Ala Val Glu Ala Ala Lys Glu Leu Gly Ile Tyr Val Ile Ile

115 120 125

Asp Trp His Ile Leu Asn Asp Gly Asn Pro Asn Gln Asn Lys Glu Lys

130 135 140

Ala Lys Glu Phe Phe Lys Glu Met Ser Ser Leu Tyr Gly Asn Thr Pro

145 150 155 160

Asn Val Ile Tyr Glu Ile Ala Asn Glu Pro Asn Gly Asp Val Asn Trp

165 170 175

Lys Arg Asp Ile Lys Pro Tyr Ala Glu Glu Val Ile Ser Val Ile Arg

180 185 190

Lys Asn Asp Pro Asp Asn Ile Ile Ile Val Gly Thr Gly Thr Trp Ser

195 200 205

Gln Asp Val Asn Asp Ala Ala Asp Asp Gln Leu Lys Asp Ala Asn Val

210 215 220

Met Tyr Ala Leu His Phe Tyr Ala Gly Thr His Gly Gln Phe Leu Arg

225 230 235 240

Asp Lys Ala Asn Tyr Ala Leu Ser Lys Gly Ala Pro Ile Phe Val Thr

245 250 255

Glu Trp Gly Thr Ser Asp Ala Ser Gly Asn Gly Gly Val Phe Leu Asp

260 265 270

Gln Ser Arg Glu Trp Leu Lys Tyr Leu Asp Ser Lys Thr Ile Ser Trp

275 280 285

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

290 295 300

Pro Gly Ala Ser Lys Thr Gly Gly Trp Arg Leu Ser Asp Leu Ser Ala

305 310 315 320

Ser Gly Thr Phe Val Arg Glu Asn Ile Leu Gly Thr Lys Asp Ser Thr

325 330 335

Lys Asp Ile Pro Glu Thr Pro Ser Lys Asp Lys Pro Thr Gln Glu Asn

340 345 350

Gly Ile Ser Val Gln Tyr Arg Ala Gly Asp Gly Ser Met Asn Ser Asn

355 360 365

Gln Ile Arg Pro Gln Leu Gln Ile Lys Asn Asn Gly Asn Thr Thr Val

370 375 380

Asp Leu Lys Asp Val Thr Ala Arg Tyr Trp Tyr Lys Ala Lys Asn Lys

385 390 395 400

Gly Gln Asn Phe Asp Cys Asp Tyr Ala Gln Ile Gly Cys Gly Asn Val

405 410 415

Thr His Lys Phe Val Thr Leu His Lys Pro Lys Gln Gly Ala Asp Thr

420 425 430

Tyr Leu Glu Leu Gly Phe Lys Asn Gly Thr Leu Ala Pro Gly Ala Ser

435 440 445

Thr Gly Asn Ile Gln Leu Arg Leu His Asn Asp Asp Trp Ser Asn Tyr

450 455 460

Ala Gln Ser Gly Asp Tyr Ser Phe Phe Lys Ser Asn Thr Phe Lys Thr

465 470 475 480

Thr Lys Lys Ile Thr Leu Tyr Asp Gln Gly Lys Leu Ile Trp Gly Thr

485 490 495

Glu Pro Asn Met Val Phe Ser His Lys Asp Arg Pro Leu Arg Pro Gly

500 505 510

Glu Pro Tyr Pro Leu Gly Ala Asn Trp Glu Glu Glu Asp Asp Gly Val

515 520 525

Asn Phe Ser Ile Phe Ser Glu Asn Ala Thr Lys Val Glu Leu Leu Ile

530 535 540

Tyr Ser Pro Thr Asn Gln Lys Tyr Pro Lys Glu Val Ile Glu Val Lys

545 550 555 560

Gln Arg Ser Gly Asp Ile Trp His Val Phe Val Pro Gly Leu Gly Pro

565 570 575

Gly Thr Leu Tyr Ala Tyr Arg Ile Tyr Gly Pro Tyr Lys Pro Asp Gln

580 585 590

Gly Leu Arg Phe Asn Pro Asn Lys Val Leu Ile Asp Pro Tyr Ala Lys

595 600 605

Ala Ile Asn Gly Thr Leu Asn Trp Asn Asp Ala Val Phe Gly Tyr Lys

610 615 620

Ile Gly Asp Ser Asn Gln Asp Leu Ser Phe Asp Asp Arg Pro Asp Asp

625 630 635 640

Glu Phe Ile Pro Lys Gly Val Val Ile Asn Pro Tyr Phe Glu Trp Asp

645 650 655

Asp Asp His Phe Phe Arg Arg Lys Lys Ile Pro Leu Lys Asp Thr Ile

660 665 670

Ile Tyr Glu Val His Val Lys Gly Phe Thr Lys Leu Arg Pro Asp Leu

675 680 685

Pro Glu Asn Ile Arg Gly Thr Tyr Lys Gly Phe Ala Ser Arg Gln Met

690 695 700

Ile Glu Tyr Leu Lys Asp Leu Gly Val Thr Thr Val Glu Ile Met Pro

705 710 715 720

Val Gln Gln Phe Val Asp Asp Arg Phe Leu Val Glu Lys Gly Leu Arg

725 730 735

Asn Tyr Trp Gly Tyr Asn Pro Ile Asn Tyr Phe Ser Pro Glu Cys Arg

740 745 750

Tyr Ser Ser Ser Gly Cys Met Gly Glu Gln Val Asn Glu Phe Lys Glu

755 760 765

Met Val Asn Glu Leu His Asn Ala Gly Phe Glu Val Ile Ile Asp Val

770 775 780

Val Tyr Asn His Thr Ala Glu Gly Asn His Leu Gly Pro Thr Leu Ser

785 790 795 800

Phe Arg Gly Ile Asp Asn Leu Ala Tyr Tyr Met Leu Val Pro Asp Asn

805 810 815

Lys Arg Tyr Tyr Leu Asp Phe Thr Gly Thr Gly Asn Thr Leu Asn Leu

820 825 830

Ser His Pro Arg Val Leu Gln Met Val Leu Asp Ser Leu Arg Tyr Trp

835 840 845

Val Leu Glu Met His Val Asp Gly Phe Arg Phe Asp Leu Ala Ala Ala

850 855 860

Leu Ala Arg Gln Leu Tyr Ser Val Asn Met Leu Ser Thr Phe Phe Val

865 870 875 880

Ala Ile Gln Gln Asp Pro Val Leu Ser Gln Val Lys Leu Ile Ala Glu

885 890 895

Pro Trp Asp Val Gly Pro Gly Gly Tyr Gln Val Gly Asn Phe Pro Tyr

900 905 910

Leu Trp Ala Glu Trp Asn Gly Lys Tyr Arg Asp Thr Ile Arg Arg Phe

915 920 925

Trp Arg Gly Glu Ala Ile Pro Tyr Glu Glu Leu Ala Asn Arg Leu Met

930 935 940

Gly Ser Pro Asp Leu Tyr Ala Gly Asn Asn Lys Thr Pro Phe Ala Ser

945 950 955 960

Ile Asn Tyr Ile Thr Ser His Asp Gly Phe Thr Leu Glu Asp Leu Val

965 970 975

Ser Tyr Asn Gln Lys His Asn Glu Ala Asn Gly Phe Asn Asn Gln Asp

980 985 990

Gly Met Asn Glu Asn Tyr Ser Trp Asn Cys Gly Val Glu Gly Glu Thr

995 1000 1005

Asn Asp Ala Asn Val Ile Gln Cys Arg Glu Lys Gln Lys Arg Asn Phe

1010 1015 1020

Ile Ile Thr Leu Phe Val Ser Gln Gly Val Pro Met Ile Leu Gly Gly

1025 1030 1035 1040

Asp Glu Leu Ser Arg Thr Gln Arg Gly Asn Asn Asn Ala Phe Cys Gln

1045 1050 1055

Asp Asn Glu Ile Ser Trp Phe Asn Trp Asn Leu Asp Glu Arg Lys Gln

1060 1065 1070

Arg Phe His Asp Phe Val Arg Ser Met Ile Tyr Phe Tyr Arg Ala His

1075 1080 1085

Pro Ile Phe Arg Arg Glu Arg Tyr Phe Gln Gly Lys Lys Leu His Gly

1090 1095 1100

Met Pro Leu Lys Asp Val Thr Phe Leu Lys Pro Asp Gly Asn Glu Ala

1105 1110 1115 1120

Asp Glu Gln Thr Trp Lys Ser Pro Thr Asn Phe Ile Ala Tyr Ile Leu

1125 1130 1135

Glu Gly Ser Val Ile Asp Glu Val Asn Asp Arg Gly Glu Arg Ile Ala

1140 1145 1150

Asp Asp Ser Phe Leu Ile Ile Leu Asn Gly Ser Pro Asn Asn Ile Lys

1155 1160 1165

Phe Lys Phe Pro Gln Gly Lys Trp Ser Leu Val Val Ser Ser Tyr Leu

1170 1175 1180

Arg Glu Leu Arg Asp Asp Glu Arg Val Val Asp Gly Gly Lys Glu Leu

1185 1190 1195 1200

Glu Ile Glu Gly Arg Thr Ala Met Val Tyr Arg Arg Ile Glu Tyr

1205 1210 1215

<210> 5

<211> 2328

<212> DNA

<213> 人工序列()

<400> 5

atgcgcaact cgaccaagac atctctatta ctggccggct tatgcacagc ggcccaaatg 60

gtttttgtaa cacatgcctc agctgcagca gggacaaaaa cgccagtagc caagaatggc 120

cagcttagca taaaaggtac acagctcgtt aaccgagacg gtaaagcggt acagctgatg 180

gttttttcac acaaggatag accattaaga ccaggagagc catatcctct tggagctaat 240

tgggaagaag aagatgatgg tgtgaacttc tctatctttt cggaaaatgc gactaaggtt 300

gaacttttaa tttactcccc tactaatcag aaatatccta aagaagttat cgaggttaag 360

cagagatctg gtgatatttg gcacgtcttt gtcccaggtt tgggaccggg tacactttac 420

gcatatagaa tttatggtcc ttataagcca gatcaaggtt taagatttaa tcctaataag 480

gttctaattg atccttatgc taaggctata aatgggacat taaactggaa tgatgctgtt 540

tttggttata agataggcga ttctaaccag gatttgtcct ttgatgatag gccagatgat 600

gaatttattc ctaagggtgt tgttattaat ccttattttg agtgggatga tgatcacttt 660

tttaggagaa agaagatacc attaaaggat actattattt atgaagttca tgttaaaggt 720

tttactaaat taagacctga tttaccagaa aatattagag gtacttataa aggatttgcc 780

tctagacaga tgatcgaata tttgaaagat ttgggggtaa ctacagtcga gataatgcca 840

gtacagcagt ttgttgatga taggtttcta gtagagaagg gattaaggaa ttactgggga 900

tataatccca taaattattt ttcacctgaa tgtagatatt cctcttctgg ctgtatgggt 960

gaacaagtta acgagtttaa ggagatggtt aatgagctgc acaacgctgg cttcgaggta 1020

attattgatg ttgtttataa ccatactgcg gaagggaatc atttaggtcc tactctttca 1080

ttcagaggta tagataattt ggcttattac atgttagttc cagataataa gagatattat 1140

ttagacttta ctggaactgg aaacacctta aatctgagtc atccgagggt attgcaaatg 1200

gttctggata gtcttagata ttgggtttta gagatgcatg ttgacggttt taggtttgat 1260

ttagctgctg ccctagctag acaattatac agtgtaaata tgctttcaac tttctttgtt 1320

gcaattcagc aagatcccgt tctttctcaa gttaagttaa tagcggaacc ttgggatgtt 1380

ggtccagggg gatatcaggt tggtaatttt ccatatttgt gggccgaatg gaacggtaag 1440

tatagagata ctataaggag attttggaga ggtgaggcga tcccctatga ggagttggct 1500

aataggctta tgggttctcc agatttatat gctggaaata ataagactcc tttcgctagt 1560

ataaattata taacttctca tgatggtttt actttagagg atttagttag ttataatcaa 1620

aagcataatg aagctaacgg ttttaataat caagatggca tgaacgagaa ttatagttgg 1680

aattgtggag ttgagggaga gactaatgat gctaatgtta ttcaatgtag agagaaacaa 1740

aaaaggaatt ttatcataac actttttgta agtcaagggg ttccaatgat tttaggtggc 1800

gatgagctaa gtagaacaca aagaggaaat aacaatgctt tttgccaaga taacgaaata 1860

agttggttta attggaatct tgatgagagg aaacagaggt ttcatgattt tgttaggagt 1920

atgatttatt tctatagagc tcatccaata tttagaagag aaagatactt tcaaggtaag 1980

aaattacatg gtatgccatt aaaggatgtc acttttctaa aaccagatgg aaatgaagct 2040

gacgaacaaa catggaagtc accaactaat tttattgcat atattttaga gggtagtgtt 2100

attgatgaag taaatgatag gggtgagaga atagctgacg attctttctt aatcatcctt 2160

aatggttcac caaataatat taagttcaaa ttcccgcaag gtaaatggag tttagttgtt 2220

tcttcatatt tgagagaact tagagatgac gagagagttg ttgatggtgg caaggaactg 2280

gaaattgagg gaaggaccgc aatggtatat aggaggattg aatattag 2328

<210> 6

<211> 775

<212> PRT

<213> 人工序列()

<400> 6

Met Arg Asn Ser Thr Lys Thr Ser Leu Leu Leu Ala Gly Leu Cys Thr

1 5 10 15

Ala Ala Gln Met Val Phe Val Thr His Ala Ser Ala Ala Ala Gly Thr

20 25 30

Lys Thr Pro Val Ala Lys Asn Gly Gln Leu Ser Ile Lys Gly Thr Gln

35 40 45

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

50 55 60

Lys Asp Arg Pro Leu Arg Pro Gly Glu Pro Tyr Pro Leu Gly Ala Asn

65 70 75 80

Trp Glu Glu Glu Asp Asp Gly Val Asn Phe Ser Ile Phe Ser Glu Asn

85 90 95

Ala Thr Lys Val Glu Leu Leu Ile Tyr Ser Pro Thr Asn Gln Lys Tyr

100 105 110

Pro Lys Glu Val Ile Glu Val Lys Gln Arg Ser Gly Asp Ile Trp His

115 120 125

Val Phe Val Pro Gly Leu Gly Pro Gly Thr Leu Tyr Ala Tyr Arg Ile

130 135 140

Tyr Gly Pro Tyr Lys Pro Asp Gln Gly Leu Arg Phe Asn Pro Asn Lys

145 150 155 160

Val Leu Ile Asp Pro Tyr Ala Lys Ala Ile Asn Gly Thr Leu Asn Trp

165 170 175

Asn Asp Ala Val Phe Gly Tyr Lys Ile Gly Asp Ser Asn Gln Asp Leu

180 185 190

Ser Phe Asp Asp Arg Pro Asp Asp Glu Phe Ile Pro Lys Gly Val Val

195 200 205

Ile Asn Pro Tyr Phe Glu Trp Asp Asp Asp His Phe Phe Arg Arg Lys

210 215 220

Lys Ile Pro Leu Lys Asp Thr Ile Ile Tyr Glu Val His Val Lys Gly

225 230 235 240

Phe Thr Lys Leu Arg Pro Asp Leu Pro Glu Asn Ile Arg Gly Thr Tyr

245 250 255

Lys Gly Phe Ala Ser Arg Gln Met Ile Glu Tyr Leu Lys Asp Leu Gly

260 265 270

Val Thr Thr Val Glu Ile Met Pro Val Gln Gln Phe Val Asp Asp Arg

275 280 285

Phe Leu Val Glu Lys Gly Leu Arg Asn Tyr Trp Gly Tyr Asn Pro Ile

290 295 300

Asn Tyr Phe Ser Pro Glu Cys Arg Tyr Ser Ser Ser Gly Cys Met Gly

305 310 315 320

Glu Gln Val Asn Glu Phe Lys Glu Met Val Asn Glu Leu His Asn Ala

325 330 335

Gly Phe Glu Val Ile Ile Asp Val Val Tyr Asn His Thr Ala Glu Gly

340 345 350

Asn His Leu Gly Pro Thr Leu Ser Phe Arg Gly Ile Asp Asn Leu Ala

355 360 365

Tyr Tyr Met Leu Val Pro Asp Asn Lys Arg Tyr Tyr Leu Asp Phe Thr

370 375 380

Gly Thr Gly Asn Thr Leu Asn Leu Ser His Pro Arg Val Leu Gln Met

385 390 395 400

Val Leu Asp Ser Leu Arg Tyr Trp Val Leu Glu Met His Val Asp Gly

405 410 415

Phe Arg Phe Asp Leu Ala Ala Ala Leu Ala Arg Gln Leu Tyr Ser Val

420 425 430

Asn Met Leu Ser Thr Phe Phe Val Ala Ile Gln Gln Asp Pro Val Leu

435 440 445

Ser Gln Val Lys Leu Ile Ala Glu Pro Trp Asp Val Gly Pro Gly Gly

450 455 460

Tyr Gln Val Gly Asn Phe Pro Tyr Leu Trp Ala Glu Trp Asn Gly Lys

465 470 475 480

Tyr Arg Asp Thr Ile Arg Arg Phe Trp Arg Gly Glu Ala Ile Pro Tyr

485 490 495

Glu Glu Leu Ala Asn Arg Leu Met Gly Ser Pro Asp Leu Tyr Ala Gly

500 505 510

Asn Asn Lys Thr Pro Phe Ala Ser Ile Asn Tyr Ile Thr Ser His Asp

515 520 525

Gly Phe Thr Leu Glu Asp Leu Val Ser Tyr Asn Gln Lys His Asn Glu

530 535 540

Ala Asn Gly Phe Asn Asn Gln Asp Gly Met Asn Glu Asn Tyr Ser Trp

545 550 555 560

Asn Cys Gly Val Glu Gly Glu Thr Asn Asp Ala Asn Val Ile Gln Cys

565 570 575

Arg Glu Lys Gln Lys Arg Asn Phe Ile Ile Thr Leu Phe Val Ser Gln

580 585 590

Gly Val Pro Met Ile Leu Gly Gly Asp Glu Leu Ser Arg Thr Gln Arg

595 600 605

Gly Asn Asn Asn Ala Phe Cys Gln Asp Asn Glu Ile Ser Trp Phe Asn

610 615 620

Trp Asn Leu Asp Glu Arg Lys Gln Arg Phe His Asp Phe Val Arg Ser

625 630 635 640

Met Ile Tyr Phe Tyr Arg Ala His Pro Ile Phe Arg Arg Glu Arg Tyr

645 650 655

Phe Gln Gly Lys Lys Leu His Gly Met Pro Leu Lys Asp Val Thr Phe

660 665 670

Leu Lys Pro Asp Gly Asn Glu Ala Asp Glu Gln Thr Trp Lys Ser Pro

675 680 685

Thr Asn Phe Ile Ala Tyr Ile Leu Glu Gly Ser Val Ile Asp Glu Val

690 695 700

Asn Asp Arg Gly Glu Arg Ile Ala Asp Asp Ser Phe Leu Ile Ile Leu

705 710 715 720

Asn Gly Ser Pro Asn Asn Ile Lys Phe Lys Phe Pro Gln Gly Lys Trp

725 730 735

Ser Leu Val Val Ser Ser Tyr Leu Arg Glu Leu Arg Asp Asp Glu Arg

740 745 750

Val Val Asp Gly Gly Lys Glu Leu Glu Ile Glu Gly Arg Thr Ala Met

755 760 765

Val Tyr Arg Arg Ile Glu Tyr

770 775

<210> 7

<211> 4185

<212> DNA

<213> 人工序列()

<400> 7

atgcgcaact cgaccaagac atctctatta ctggccggct tatgcacagc ggcccaaatg 60

gtttttgtaa cacatgcctc agctgcagca gggacaaaaa cgccagtagc caagaatggc 120

cagcttagca taaaaggtac acagctcgtt aaccgagacg gtaaagcggt acagctgaag 180

gggatcagtt cacacggatt gcaatggtat ggagaatatg tcaataaaga cagcttaaaa 240

tggctgagag atgattgggg tatcaccgtt ttccgtgcag cgatgtatac ggcagatggc 300

ggttatattg acaacccgtc cgtgaaaaat aaagtaaaag aagcggttga agcggcaaaa 360

gagcttggga tatatgtcat cattgactgg catatcttaa atgacggtaa tccaaaccaa 420

aataaagaga aggcaaaaga attcttcaag gaaatgtcaa gcctttacgg aaacacgcca 480

aacgtcattt atgaaattgc aaacgaacca aacggtgatg tgaactggaa gcgtgatatt 540

aaaccatatg cggaagaagt gatttcagtt atccgcaaaa atgatccaga caacatcatc 600

attgtcggaa ccggtacatg gagccaggat gtgaatgatg ctgccgatga ccagctaaaa 660

gatgcaaacg ttatgtacgc acttcatttt tatgccggca cacacggcca atttttacgg 720

gataaagcaa actatgcact cagcaaagga gcacctattt ttgtgacaga gtggggaaca 780

agcgacgcgt ctggcaatgg cggtgtattc cttgatcaat cgagggaatg gctgaaatat 840

ctcgacagca agaccattag ctgggtgaac tggaatcttt ctgataagca ggaatcatcc 900

tcagctttaa agccgggggc atctaaaaca ggcggctggc ggttgtcaga tttatctgct 960

tcaggaacat tcgttagaga aaacattctc ggcaccaaag attcgacgaa ggacattcct 1020

gaaacgccat caaaagataa acccacacag gaaaatggta tttctgtaca gtacagagca 1080

ggggatggga gtatgaacag caaccaaatc cgtccgcagc ttcaaataaa aaataacggc 1140

aataccacgg ttgatttaaa agatgtcact gcccgttact ggtataaagc gaaaaacaaa 1200

ggccaaaact ttgactgtga ctacgcgcag attggatgcg gcaatgtgac acacaagttt 1260

gtgacgttgc ataaaccaaa gcaaggtgca gatacctatc tggaacttgg atttaaaaac 1320

ggaacgttgg caccgggagc aagcacaggg aatattcagc tccgtcttca caatgatgac 1380

tggagcaatt atgcacaaag cggcgattat tcctttttca aatcaaatac gtttaaaaca 1440

acgaaaaaaa tcacattata tgatcaagga aaactgattt ggggaacaga accaaatcct 1500

aacaacggta acaacggtct cgaactgcgc gagtccggag ctatctctgg cgatagtctg 1560

atcagcctgg ctagcacagg aaaaagagtt tctattaaag atttgttaga tgaaaaagat 1620

tttgaaatat gggcaattaa tgaacagacg atgaagctag aatcagctaa agttagtcgt 1680

gtattttgta ctggcaaaaa gctagtttat attctaaaaa ctcgactagg tagaactatc 1740

aaggcaacag caaatcatag atttttaact attgatggtt ggaaaagatt agatgagcta 1800

tctttaaaag agcatattgc tctaccccgt aaactagaaa gctcctcttt acaattgtca 1860

ccagaaatag aaaagttgtc tcagagtgat atttactggg actccatcgt ttctattacg 1920

gagactggag tcgaagaggt ttttgatttg actgtgccag gaccacataa ctttgtcgcg 1980

aatgacatca ttgtacacaa cggaagaaga gccatgggcg gccgcgaatt cctcatggtt 2040

ttttcacaca aggatagacc attaagacca ggagagccat atcctcttgg agctaattgg 2100

gaagaagaag atgatggtgt gaacttctct atcttttcgg aaaatgcgac taaggttgaa 2160

cttttaattt actcccctac taatcagaaa tatcctaaag aagttatcga ggttaagcag 2220

agatctggtg atatttggca cgtctttgtc ccaggtttgg gaccgggtac actttacgca 2280

tatagaattt atggtcctta taagccagat caaggtttaa gatttaatcc taataaggtt 2340

ctaattgatc cttatgctaa ggctataaat gggacattaa actggaatga tgctgttttt 2400

ggttataaga taggcgattc taaccaggat ttgtcctttg atgataggcc agatgatgaa 2460

tttattccta agggtgttgt tattaatcct tattttgagt gggatgatga tcactttttt 2520

aggagaaaga agataccatt aaaggatact attatttatg aagttcatgt taaaggtttt 2580

actaaattaa gacctgattt accagaaaat attagaggta cttataaagg atttgcctct 2640

agacagatga tcgaatattt gaaagatttg ggggtaacta cagtcgagat aatgccagta 2700

cagcagtttg ttgatgatag gtttctagta gagaagggat taaggaatta ctggggatat 2760

aatcccataa attatttttc acctgaatgt agatattcct cttctggctg tatgggtgaa 2820

caagttaacg agtttaagga gatggttaat gagctgcaca acgctggctt cgaggtaatt 2880

attgatgttg tttataacca tactgcggaa gggaatcatt taggtcctac tctttcattc 2940

agaggtatag ataatttggc ttattacatg ttagttccag ataataagag atattattta 3000

gactttactg gaactggaaa caccttaaat ctgagtcatc cgagggtatt gcaaatggtt 3060

ctggatagtc ttagatattg ggttttagag atgcatgttg acggttttag gtttgattta 3120

gctgctgccc tagctagaca attatacagt gtaaatatgc tttcaacttt ctttgttgca 3180

attcagcaag atcccgttct ttctcaagtt aagttaatag cggaaccttg ggatgttggt 3240

ccagggggat atcaggttgg taattttcca tatttgtggg ccgaatggaa cggtaagtat 3300

agagatacta taaggagatt ttggagaggt gaggcgatcc cctatgagga gttggctaat 3360

aggcttatgg gttctccaga tttatatgct ggaaataata agactccttt cgctagtata 3420

aattatataa cttctcatga tggttttact ttagaggatt tagttagtta taatcaaaag 3480

cataatgaag ctaacggttt taataatcaa gatggcatga acgagaatta tagttggaat 3540

tgtggagttg agggagagac taatgatgct aatgttattc aatgtagaga gaaacaaaaa 3600

aggaatttta tcataacact ttttgtaagt caaggggttc caatgatttt aggtggcgat 3660

gagctaagta gaacacaaag aggaaataac aatgcttttt gccaagataa cgaaataagt 3720

tggtttaatt ggaatcttga tgagaggaaa cagaggtttc atgattttgt taggagtatg 3780

atttatttct atagagctca tccaatattt agaagagaaa gatactttca aggtaagaaa 3840

ttacatggta tgccattaaa ggatgtcact tttctaaaac cagatggaaa tgaagctgac 3900

gaacaaacat ggaagtcacc aactaatttt attgcatata ttttagaggg tagtgttatt 3960

gatgaagtaa atgatagggg tgagagaata gctgacgatt ctttcttaat catccttaat 4020

ggttcaccaa ataatattaa gttcaaattc ccgcaaggta aatggagttt agttgtttct 4080

tcatatttga gagaacttag agatgacgag agagttgttg atggtggcaa ggaactggaa 4140

attgagggaa ggaccgcaat ggtatatagg aggattgaat attag 4185

<210> 8

<211> 1394

<212> PRT

<213> 人工序列()

<400> 8

Met Arg Asn Ser Thr Lys Thr Ser Leu Leu Leu Ala Gly Leu Cys Thr

1 5 10 15

Ala Ala Gln Met Val Phe Val Thr His Ala Ser Ala Ala Ala Gly Thr

20 25 30

Lys Thr Pro Val Ala Lys Asn Gly Gln Leu Ser Ile Lys Gly Thr Gln

35 40 45

Leu Val Asn Arg Asp Gly Lys Ala Val Gln Leu Lys Gly Ile Ser Ser

50 55 60

His Gly Leu Gln Trp Tyr Gly Glu Tyr Val Asn Lys Asp Ser Leu Lys

65 70 75 80

Trp Leu Arg Asp Asp Trp Gly Ile Thr Val Phe Arg Ala Ala Met Tyr

85 90 95

Thr Ala Asp Gly Gly Tyr Ile Asp Asn Pro Ser Val Lys Asn Lys Val

100 105 110

Lys Glu Ala Val Glu Ala Ala Lys Glu Leu Gly Ile Tyr Val Ile Ile

115 120 125

Asp Trp His Ile Leu Asn Asp Gly Asn Pro Asn Gln Asn Lys Glu Lys

130 135 140

Ala Lys Glu Phe Phe Lys Glu Met Ser Ser Leu Tyr Gly Asn Thr Pro

145 150 155 160

Asn Val Ile Tyr Glu Ile Ala Asn Glu Pro Asn Gly Asp Val Asn Trp

165 170 175

Lys Arg Asp Ile Lys Pro Tyr Ala Glu Glu Val Ile Ser Val Ile Arg

180 185 190

Lys Asn Asp Pro Asp Asn Ile Ile Ile Val Gly Thr Gly Thr Trp Ser

195 200 205

Gln Asp Val Asn Asp Ala Ala Asp Asp Gln Leu Lys Asp Ala Asn Val

210 215 220

Met Tyr Ala Leu His Phe Tyr Ala Gly Thr His Gly Gln Phe Leu Arg

225 230 235 240

Asp Lys Ala Asn Tyr Ala Leu Ser Lys Gly Ala Pro Ile Phe Val Thr

245 250 255

Glu Trp Gly Thr Ser Asp Ala Ser Gly Asn Gly Gly Val Phe Leu Asp

260 265 270

Gln Ser Arg Glu Trp Leu Lys Tyr Leu Asp Ser Lys Thr Ile Ser Trp

275 280 285

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

290 295 300

Pro Gly Ala Ser Lys Thr Gly Gly Trp Arg Leu Ser Asp Leu Ser Ala

305 310 315 320

Ser Gly Thr Phe Val Arg Glu Asn Ile Leu Gly Thr Lys Asp Ser Thr

325 330 335

Lys Asp Ile Pro Glu Thr Pro Ser Lys Asp Lys Pro Thr Gln Glu Asn

340 345 350

Gly Ile Ser Val Gln Tyr Arg Ala Gly Asp Gly Ser Met Asn Ser Asn

355 360 365

Gln Ile Arg Pro Gln Leu Gln Ile Lys Asn Asn Gly Asn Thr Thr Val

370 375 380

Asp Leu Lys Asp Val Thr Ala Arg Tyr Trp Tyr Lys Ala Lys Asn Lys

385 390 395 400

Gly Gln Asn Phe Asp Cys Asp Tyr Ala Gln Ile Gly Cys Gly Asn Val

405 410 415

Thr His Lys Phe Val Thr Leu His Lys Pro Lys Gln Gly Ala Asp Thr

420 425 430

Tyr Leu Glu Leu Gly Phe Lys Asn Gly Thr Leu Ala Pro Gly Ala Ser

435 440 445

Thr Gly Asn Ile Gln Leu Arg Leu His Asn Asp Asp Trp Ser Asn Tyr

450 455 460

Ala Gln Ser Gly Asp Tyr Ser Phe Phe Lys Ser Asn Thr Phe Lys Thr

465 470 475 480

Thr Lys Lys Ile Thr Leu Tyr Asp Gln Gly Lys Leu Ile Trp Gly Thr

485 490 495

Glu Pro Asn Pro Asn Asn Gly Asn Asn Gly Leu Glu Leu Arg Glu Ser

500 505 510

Gly Ala Ile Ser Gly Asp Ser Leu Ile Ser Leu Ala Ser Thr Gly Lys

515 520 525

Arg Val Ser Ile Lys Asp Leu Leu Asp Glu Lys Asp Phe Glu Ile Trp

530 535 540

Ala Ile Asn Glu Gln Thr Met Lys Leu Glu Ser Ala Lys Val Ser Arg

545 550 555 560

Val Phe Cys Thr Gly Lys Lys Leu Val Tyr Ile Leu Lys Thr Arg Leu

565 570 575

Gly Arg Thr Ile Lys Ala Thr Ala Asn His Arg Phe Leu Thr Ile Asp

580 585 590

Gly Trp Lys Arg Leu Asp Glu Leu Ser Leu Lys Glu His Ile Ala Leu

595 600 605

Pro Arg Lys Leu Glu Ser Ser Ser Leu Gln Leu Ser Pro Glu Ile Glu

610 615 620

Lys Leu Ser Gln Ser Asp Ile Tyr Trp Asp Ser Ile Val Ser Ile Thr

625 630 635 640

Glu Thr Gly Val Glu Glu Val Phe Asp Leu Thr Val Pro Gly Pro His

645 650 655

Asn Phe Val Ala Asn Asp Ile Ile Val His Asn Gly Arg Arg Ala Met

660 665 670

Gly Gly Arg Glu Phe Leu Met Val Phe Ser His Lys Asp Arg Pro Leu

675 680 685

Arg Pro Gly Glu Pro Tyr Pro Leu Gly Ala Asn Trp Glu Glu Glu Asp

690 695 700

Asp Gly Val Asn Phe Ser Ile Phe Ser Glu Asn Ala Thr Lys Val Glu

705 710 715 720

Leu Leu Ile Tyr Ser Pro Thr Asn Gln Lys Tyr Pro Lys Glu Val Ile

725 730 735

Glu Val Lys Gln Arg Ser Gly Asp Ile Trp His Val Phe Val Pro Gly

740 745 750

Leu Gly Pro Gly Thr Leu Tyr Ala Tyr Arg Ile Tyr Gly Pro Tyr Lys

755 760 765

Pro Asp Gln Gly Leu Arg Phe Asn Pro Asn Lys Val Leu Ile Asp Pro

770 775 780

Tyr Ala Lys Ala Ile Asn Gly Thr Leu Asn Trp Asn Asp Ala Val Phe

785 790 795 800

Gly Tyr Lys Ile Gly Asp Ser Asn Gln Asp Leu Ser Phe Asp Asp Arg

805 810 815

Pro Asp Asp Glu Phe Ile Pro Lys Gly Val Val Ile Asn Pro Tyr Phe

820 825 830

Glu Trp Asp Asp Asp His Phe Phe Arg Arg Lys Lys Ile Pro Leu Lys

835 840 845

Asp Thr Ile Ile Tyr Glu Val His Val Lys Gly Phe Thr Lys Leu Arg

850 855 860

Pro Asp Leu Pro Glu Asn Ile Arg Gly Thr Tyr Lys Gly Phe Ala Ser

865 870 875 880

Arg Gln Met Ile Glu Tyr Leu Lys Asp Leu Gly Val Thr Thr Val Glu

885 890 895

Ile Met Pro Val Gln Gln Phe Val Asp Asp Arg Phe Leu Val Glu Lys

900 905 910

Gly Leu Arg Asn Tyr Trp Gly Tyr Asn Pro Ile Asn Tyr Phe Ser Pro

915 920 925

Glu Cys Arg Tyr Ser Ser Ser Gly Cys Met Gly Glu Gln Val Asn Glu

930 935 940

Phe Lys Glu Met Val Asn Glu Leu His Asn Ala Gly Phe Glu Val Ile

945 950 955 960

Ile Asp Val Val Tyr Asn His Thr Ala Glu Gly Asn His Leu Gly Pro

965 970 975

Thr Leu Ser Phe Arg Gly Ile Asp Asn Leu Ala Tyr Tyr Met Leu Val

980 985 990

Pro Asp Asn Lys Arg Tyr Tyr Leu Asp Phe Thr Gly Thr Gly Asn Thr

995 1000 1005

Leu Asn Leu Ser His Pro Arg Val Leu Gln Met Val Leu Asp Ser Leu

1010 1015 1020

Arg Tyr Trp Val Leu Glu Met His Val Asp Gly Phe Arg Phe Asp Leu

1025 1030 1035 1040

Ala Ala Ala Leu Ala Arg Gln Leu Tyr Ser Val Asn Met Leu Ser Thr

1045 1050 1055

Phe Phe Val Ala Ile Gln Gln Asp Pro Val Leu Ser Gln Val Lys Leu

1060 1065 1070

Ile Ala Glu Pro Trp Asp Val Gly Pro Gly Gly Tyr Gln Val Gly Asn

1075 1080 1085

Phe Pro Tyr Leu Trp Ala Glu Trp Asn Gly Lys Tyr Arg Asp Thr Ile

1090 1095 1100

Arg Arg Phe Trp Arg Gly Glu Ala Ile Pro Tyr Glu Glu Leu Ala Asn

1105 1110 1115 1120

Arg Leu Met Gly Ser Pro Asp Leu Tyr Ala Gly Asn Asn Lys Thr Pro

1125 1130 1135

Phe Ala Ser Ile Asn Tyr Ile Thr Ser His Asp Gly Phe Thr Leu Glu

1140 1145 1150

Asp Leu Val Ser Tyr Asn Gln Lys His Asn Glu Ala Asn Gly Phe Asn

1155 1160 1165

Asn Gln Asp Gly Met Asn Glu Asn Tyr Ser Trp Asn Cys Gly Val Glu

1170 1175 1180

Gly Glu Thr Asn Asp Ala Asn Val Ile Gln Cys Arg Glu Lys Gln Lys

1185 1190 1195 1200

Arg Asn Phe Ile Ile Thr Leu Phe Val Ser Gln Gly Val Pro Met Ile

1205 1210 1215

Leu Gly Gly Asp Glu Leu Ser Arg Thr Gln Arg Gly Asn Asn Asn Ala

1220 1225 1230

Phe Cys Gln Asp Asn Glu Ile Ser Trp Phe Asn Trp Asn Leu Asp Glu

1235 1240 1245

Arg Lys Gln Arg Phe His Asp Phe Val Arg Ser Met Ile Tyr Phe Tyr

1250 1255 1260

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

1265 1270 1275 1280

Leu His Gly Met Pro Leu Lys Asp Val Thr Phe Leu Lys Pro Asp Gly

1285 1290 1295

Asn Glu Ala Asp Glu Gln Thr Trp Lys Ser Pro Thr Asn Phe Ile Ala

1300 1305 1310

Tyr Ile Leu Glu Gly Ser Val Ile Asp Glu Val Asn Asp Arg Gly Glu

1315 1320 1325

Arg Ile Ala Asp Asp Ser Phe Leu Ile Ile Leu Asn Gly Ser Pro Asn

1330 1335 1340

Asn Ile Lys Phe Lys Phe Pro Gln Gly Lys Trp Ser Leu Val Val Ser

1345 1350 1355 1360

Ser Tyr Leu Arg Glu Leu Arg Asp Asp Glu Arg Val Val Asp Gly Gly

1365 1370 1375

Lys Glu Leu Glu Ile Glu Gly Arg Thr Ala Met Val Tyr Arg Arg Ile

1380 1385 1390

Glu Tyr

Claims

1.一种枯草芽孢杆菌融合表达系统，其特征在于：包含能够编码至少以下元件的DNA序列，所述元件包括信号肽、糖苷水解酶或其部分和外源蛋白，并且依上述顺序排列融合在一个开放读码框内，其中所述糖苷水解酶为如SEQ ID NO .2所示的氨基酸序列或如SEQ IDNO .2所示的前30个氨基酸序列，所述糖苷水解酶或其部分和外源蛋白之间具有接头序列、用于蛋白酶剪切的靶位点或内含肽插入。

2.使用权利要求1所述的融合表达系统分泌生产外源蛋白的方法，其特征在于：所述方法包括步骤如下：

(1)构建合适的枯草芽孢杆菌细胞，其包含能够编码至少以下元件的DNA序列，所述元件包括信号肽、糖苷水解酶或其部分和外源蛋白，并且依上述顺序排列融合在一个开放读码框内，所述糖苷水解酶或其部分和外源蛋白之间具有内含肽序列，并且所述分泌生产外源蛋白的方法包括在合适的条件下断裂内含肽以释放外源蛋白，所述糖苷水解酶或其部分和外源蛋白之间具有用于蛋白酶剪切的靶位点，并且所述分泌生产外源蛋白的方法包括在所述靶位点切割融合蛋白；

(3) 使用再生无定形纤维素回收所述蛋白。