CN106916797B - 一种高活性漆酶突变体蛋白及其制备方法 - Google Patents

一种高活性漆酶突变体蛋白及其制备方法 Download PDF

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CN106916797B
CN106916797B CN201710106590.XA CN201710106590A CN106916797B CN 106916797 B CN106916797 B CN 106916797B CN 201710106590 A CN201710106590 A CN 201710106590A CN 106916797 B CN106916797 B CN 106916797B
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王刚刚
谢天
刘忠川
冯红
李雷
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Chengdu Institute of Biology of CAS
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Abstract

本发明涉及医药学技术领域，具体涉及一种高活性漆酶突变体蛋白，其突变体是由野生型漆酶的基因突变得到，所述突变体为CH6‑CotA，CH6‑F207Y，CH6‑V403T，CH6‑P455S，CH6‑TS或CH6‑TSY中的任意一种。本发明以野生型漆酶NH6‑CotA为模板，扩增CotA基因，然后将CotA基因和pET22b载体分别双酶切，酶切产物通过连接、转化和测序构建His标签在C端的突变体CH6‑CotA菌株，然后再通过定点突变和组合突变得到高活性漆酶蛋白的突变体。与野生型漆酶相比，本发明构建的漆酶突变体的活性得到显著提升，从而为漆酶更加广泛地应用于工业生产中奠定了基础。

Description

一种高活性漆酶突变体蛋白及其制备方法
技术领域
本发明属于医药学技术领域，尤其涉及一种高活性漆酶突变体蛋白及其制备方法。
背景技术
漆酶(EC 1.10.3.2)是一种多铜氧化酶，其是从底物吸收电子，传递给氧气生成水，具有环保和高效的特点。漆酶底物范围很广，包括邻苯二酚、对苯二酚、多酚、甲氧基替代酚、氮羟基化合物、苯胺和呈现还原性的有机化合物，因环保高效且具有广泛的底物谱，所以漆酶在很多领域都有巨大的应用潜力，如生物修复、生物合成、生成检测和生物质能源等等。尽管如此，野生型漆酶存在活性低的问题，因此，对野生型漆酶进行改造以筛选高活性漆酶突变体具有重要意义。
发明内容
本发明的首要目的是提供一种高活性漆酶突变体蛋白。
为了实现上述目的，本发明采用了以下技术方案：一种高活性漆酶突变体蛋白，其突变体是由野生型漆酶的基因突变得到，所述突变体为CH6-CotA，CH6-F207Y， CH6-V403T，CH6-P455S，CH6-TS或CH6-TSY中的任意一种；CH6-CotA、CH6-F207Y、CH6-V403T、CH6-P455S、CH6-TS、CH6-TSY的核苷酸序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ IDNO.4、SEQ ID NO.5、SEQ ID NO.6所示。
具体的，CH6-CotA、CH6-F207Y、CH6-V403T、CH6-P455S、CH6-TS、CH6-TSY的氨基酸序列分别如SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20所示。
本发明的另外一个目的是提供一种如上所述的高活性漆酶突变体蛋白的制备方法，其步骤如下：
a）引物设计：CH6-CotA的正、反向引物的核苷酸序列分别如SEQ ID NO.7、SEQ IDNO.8所示；CH6-F207Y的正、反向引物的核苷酸序列分别如SEQ ID NO.9、SEQ ID NO.10所示；CH6-V403T的正、反向引物的核苷酸序列分别如SEQ ID NO.11、SEQ ID NO.12所示；CH6-P455S的正、反向引物的核苷酸序列分别如SEQ ID NO.13、SEQ ID NO.14所示；
b）DpnⅠ酶切消化：分别用各对正、反向引物对pET22b/CotA质粒（CotA基因来自枯草芽孢杆菌Bacillus subtilis 168，经克隆后连接到pET22b载体上）进行扩增，将扩增产物用DpnⅠ酶在35-40℃下酶切4.5-5.5h，扩增条件：94℃，5min；94℃，1min；68℃，30s；72℃,1min，循环30次；72℃，5min；4℃保温；
c）退火、纯化回收：将酶切产物混合，72℃反应10 min后室温退火，将退火后的产物用试剂盒（Universal DNA Purification Kit，全式金生物技术公司）纯化回收；
d）转化、测序：将回收产物转化到感受态DH5α大肠杆菌中，平板培养，挑取单菌落测序，得测序正确的高活性漆酶衍生物的突变体CH6-CotA菌株、CH6-F207Y菌株、CH6-V403T菌株、CH6-P455S菌株；
e）以CH6-P455S的质粒为模板，SEQ ID NO.11、SEQ ID NO.12所示的核苷酸序列为正、反向引物对模板进行扩增，扩增产物按照步骤b）的条件DpnⅠ酶切消化，之后重复步骤c）、d），得双突变的突变体CH6-TS 菌株；
f）以CH6-TS的质粒为模板，SEQ ID NO.9、SEQ ID NO.10所示的核苷酸序列为正、反向引物对模板进行扩增，扩增产物按照步骤b）的条件DpnⅠ酶切消化，之后重复步骤c）、d），得三突变的突变体CH6-TSY菌株。
实际上，以上突变体的构建原理是：以野生型漆酶NH6-CotA为模板，扩增CotA基因（CotA是芽孢杆菌属的具有漆酶活性一种蛋白），然后将CotA基因和pET22b载体分别双酶切，酶切产物通过连接、转化和测序构建His标签在C端的突变体CH6-CotA菌株，然后再通过定点突变和组合突变得到高活性漆酶蛋白的突变体CH6-CotA菌株、CH6-F207Y菌株、CH6-V403T菌株、CH6-P455S菌株、CH6-TS菌株和CH6-TSY菌株。
采用上述技术方案产生的有益效果在于：本发明通过DpnⅠ法构建突变体，最终获得CotA的突变体CH6-CotA、CH6-F207Y、CH6-V403T、CH6-P455S、CH6-TS和CH6-TSY，这些突变体活性高，大体上来说：第一、突变体CH6-CotA的体积活性、比活力分别是野生型漆酶NH6-CotA的7倍和2.4倍；第二，与野生型漆酶NH6-CotA相比，各突变体的Kcat/Km（即催化效率）均有显著提升，其中突变体CH6-P455S、CH6-TS的Kcat/Km是NH6-CotA的3倍。
具体的方案为，高活性漆酶突变体蛋白的纯化步骤如下：
（1）往OD600到达0.4-0.6的培养物中分别加入终浓度为0.2 mmol/L的IPTG和终浓度为0.25 mmol/L的CuSO4，在25℃、180 rpm的条件下培养4-5 h后，于25℃摇床中静置过夜，离心收集菌体后重悬浮于20 mM Na2HPO4-NaH2PO4，pH 7.6，500 mM NaCl，20 mM咪唑的Buffer溶液中，超声破碎；
（2）将破碎后的菌体于10000-12000 rpm条件下离心30-35min，将上清液于68-72℃条件下加热13-17 min；
（3）将加热后的样品于10000-12000 rpm条件下离心30-35min，将上清液进行镍株亲和层析，用咪唑浓度为50–300mM的缓冲液洗脱蛋白质。
优选的，突变体蛋白在纯化前经过如下处理：
1）将保存在-80℃条件下的突变体菌株划线到LB固体平板中，在37-40℃条件下过夜活化；
2）挑起活化的单菌落于10 ml有氨苄抗性的液体LB培养基中，在37-40℃、150-300rpm的条件下过夜活化做种子液；
3）按0.4-0.6％的接种量将种子液接种于1L有氨苄抗性的液体LB培养基中，在37-40℃、150-300rpm的条件下培养；
4）当OD600为0.6时，向培养液中加入IPTG至终浓度为0.2 mM，加入硫酸铜至终浓度为0.25 mM，然后置于25℃、150-180 rpm的条件下诱导4h，之后在25℃的恒温摇床里静置过夜培养。
静置过夜培养实际上是使得培养物在微厌氧环境中培养，也就是说，通过引入静置过夜培养，这样在微厌氧环境下有助于铜离子加载到蛋白上，申请人经试验证明，静置过夜培养的野生型漆酶的酶活是非静置过夜培养的野生型漆酶酶活的20倍，而静置过夜培养的突变体蛋白酶活是非静置过夜培养的突变体蛋白酶活的40倍以上，这些高活性漆酶突变体蛋白的获得为漆酶更加广泛地应用于工业生产中奠定了基础。
附图说明
图1是野生型漆酶NH6-CotA及其突变体纯化后的SDS-PAGE电泳示意图；
图2是野生型漆酶NH6-CotA及其突变体在不同培养条件下的酶活柱状图。
具体实施方式
为了进一步说明本发明公开的技术方案，以下通过3个实施例来说明。
实施例1：突变体的构建
a）引物设计：CH6-CotA的正、反向引物的核苷酸序列分别如SEQ ID NO.7、SEQ IDNO.8所示；CH6-F207Y的正、反向引物的核苷酸序列分别如SEQ ID NO.9、SEQ ID NO.10所示；CH6-V403T的正、反向引物的核苷酸序列分别如SEQ ID NO.11、SEQ ID NO.12所示；CH6-P455S的正、反向引物的核苷酸序列分别如SEQ ID NO.13、SEQ ID NO.14所示；
b）DpnⅠ酶切消化：分别用各对正、反向引物对pET22b/CotA质粒进行扩增，将扩增产物用DpnⅠ酶在37℃下酶切5h，扩增条件：94℃，5min；94℃，1min；68℃，30s；72℃, 1min，循环30次；72℃，5min；4℃保温；
c）退火、纯化回收：将酶切产物混合，72℃反应10min后室温退火，将退火后的产物用试剂盒纯化回收；
d）转化、测序：将回收产物转化到感受态DH5α大肠杆菌中，平板培养，挑取单菌落测序，得测序正确的高活性漆酶衍生物的突变体CH6-CotA菌株、CH6-F207Y菌株、CH6-V403T菌株、CH6-P455S菌株；
e）以CH6-P455S的质粒为模板，SEQ ID NO.11、SEQ ID NO.12所示的核苷酸序列为正、反向引物对模板进行扩增，扩增产物按照步骤b）的条件DpnⅠ酶切消化，之后重复步骤c）、d），得双突变的突变体CH6-TS 菌株；
f）以CH6-TS的质粒为模板，SEQ ID NO.9、SEQ ID NO.10所示的核苷酸序列为正、反向引物对模板进行扩增，扩增产物按照步骤b）的条件DpnⅠ酶切消化，之后重复步骤c）、d），得三突变的突变体CH6-TSY菌株。
实施例2：野生型漆酶NH6-CotA及其突变体蛋白的纯化
1、蛋白的培养
1）将保存在-80℃条件下的野生型漆酶NH6-CotA/突变体菌株划线到LB固体平板中，在37℃条件下过夜活化；
2）挑起活化的单菌落于10ml有氨苄抗性的液体LB培养基中，在37℃、200rpm的条件下过夜活化做种子液；
3）按0.5％的接种量将种子液接种于1L有氨苄抗性的液体LB培养基中，在37℃、200rpm的条件下培养；
4）当OD600为0.6时，向培养液中加入IPTG至终浓度为0.2 mM，加入硫酸铜至终浓度为0.25 mM，然后置于25℃、180 rpm的条件下诱导4h，之后在25℃的恒温摇床里静置过夜（微厌氧环境）培养。
2、蛋白的纯化
（1）往OD600到达0.4-0.6的培养物中分别加入终浓度为0.2 mmol/L的IPTG和终浓度为0.25 mmol/L的CuSO4，25℃、180 rpm培养4-5 h后，于25℃摇床中静静过夜，离心收集菌体后重悬于20mM Na2HPO4-NaH2PO4，pH7.6，500mM NaCl，20 mM咪唑的Buffer溶液中，超声破碎；
（2）将破碎后的菌体于12000 rpm条件下离心30 min，将上清液于70 ℃条件下加热15min；
（3）将加热后的样品于12000 rpm条件下离心30 min，将上清液进行镍株亲和层析，用咪唑浓度为200mM的缓冲液洗脱蛋白质。
3、蛋白纯化结果
如图1所示为野生型漆酶NH6-CotA及其突变体纯化后的SDS-PAGE电泳图，其中蛋白泳道1-7分别代表野生型漆酶NH6-CotA及突变体CH6-CotA、CH6-F207Y、CH6-V403T、CH6-P455S、CH6-TS、CH6-TSY的电泳图。经SDS-PAGE电泳检测，野生型漆酶NH6-CotA及其突变体CH6-CotA、CH6-F207Y、CH6-V403T、CH6-P455S、CH6-TS、CH6-TSY的纯度均达到95%以上，可以用于活性测试实验。
实施例3：野生型漆酶NH6-CotA及其突变体蛋白的酶活检测
在酶标板中加入10 ul浓度为20 mM的ABTS底物和1-2 ul的酶，最后加入0.1M磷酸氢二钠-柠檬酸缓冲液（pH 4.0）构成200 ul的反应体系，用酶标仪在40℃条件下每5 s读取OD420的吸光值，然后经过线性拟合算出最大反应速率Vmax，单位为umol /min。
如表1所示为NH6-CotA和CH6-CotA的蛋白产量和活性检测结果，从表中可以看出，突变体CH6-CotA的蛋白产量是野生型漆酶NH6-CotA的4倍多，突变体CH6-CotA的体积活性、比活力分别是野生型漆酶NH6-CotA的7倍和2.4倍。
表1 NH6-CotA和CH6-CotA的蛋白产量和活性比较
如表2所示为NH6-CotA及其突变体的酶活测定结果，从表中可以看出，与野生型漆酶NH6-CotA相比，各突变体的Kcat/Km均有显著提升，其中突变体CH6-P455S、CH6-TS的Kcat/Km是NH6-CotA的3倍，说明与野生型漆酶相比，本发明公开的突变体蛋白的酶活具有显著的提升。
表2 NH6-CotA及其突变体的动力学参数分析
如图2所示为NH6-CotA及其突变体蛋白在不同培养条件下的酶活测定，其中静止培养是将蛋白置于25℃、180 rpm的条件下诱导4h后在静置过夜（微厌氧环境）培养，而非静止培养是诱导4h后未静置过夜。通过静置培养，这样在微厌氧环境下有助于铜离子加载到蛋白上，具体来说，结合图2及实验数据可知，静置过夜培养的野生型漆酶的酶活是非静置过夜培养的野生型漆酶酶活的20倍，而静置过夜培养的突变体蛋白酶活是非静置过夜培养的突变体蛋白酶活的40倍以上。
核苷酸或氨基酸序列表
SEQUENCE LISTING
<110> 中国科学院成都生物研究所
<120> 一种高活性漆酶突变体蛋白及其制备方法
<130>
<160> 20
<170> PatentIn version 3.3
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<213> 人工序列
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atgacacttg aaaaatttgt ggatgctctc ccaatcccag atacactaaa gccagtacag 60
caatcaaaag aaaaaacata ctacgaagtc accatggagg aatgcactca tcagctccat 120
cgcgatctcc ctccaacccg cctgtggggc tacaacggct tatttccggg accgaccatt 180
gaggttaaaa gaaatgaaaa cgtatatgta aaatggatga ataaccttcc ttccacgcat 240
ttccttccga ttgatcacac cattcatcac agtgacagcc agcatgaaga gcccgaggta 300
aagactgttg ttcatttaca cggcggcgtc acgccagatg atagtgacgg gtatccggag 360
gcttggtttt ccaaagactt tgaacaaaca ggaccttatt tcaaaagaga ggtttatcat 420
tatccaaacc agcagcgcgg ggctatattg tggtatcacg atcacgccat ggcgctcacc 480
aggctaaatg tctatgccgg acttgtcggt gcatatatca ttcatgaccc aaaggaaaaa 540
cgcttaaaac tgccttcaga cgaatacgat gtgccgcttc ttatcacaga ccgcacgatc 600
aatgaggatg gttctttgtt ttatccgagc gcaccggaaa acccttctcc gtcactgcct 660
aatccttcaa tcgttccggc tttttgcgga gaaaccatac tcgtcaacgg gaaggtatgg 720
ccatacttgg aagtcgagcc aaggaaatac cgattccgtg tcatcaacgc ctccaataca 780
agaacctata acctgtcact cgataatggc ggagatttta ttcagattgg ttcagatgga 840
gggctcctgc cgcgatctgt taaactgaat tctttcagcc ttgcgcctgc tgaacgttac 900
gatatcatca ttgacttcac agcatatgaa ggagaatcga tcattttggc aaacagcgcg 960
ggctgcggcg gtgacgtcaa tcctgaaaca gatgcgaata tcatgcaatt cagagtcaca 1020
aaaccattgg cacaaaaaga cgaaagcaga aagccgaagt acctcgcctc atacccttcg 1080
gtacagcatg aaagaataca aaacatcaga acgttaaaac tggcaggcac ccaggacgaa 1140
tacggcagac ccgtccttct gcttaataac aaacgctggc acgatcccgt cacagaaaca 1200
ccaaaagtcg gcacaactga aatatggtcc attatcaacc cgacacgcgg aacacatccg 1260
atccacctgc atctagtctc cttccgtgta ttagaccggc ggccgtttga tatcgcccgt 1320
tatcaagaaa gcggggaatt gtcctatacc ggtccggctg tcccgccgcc gccaagtgaa 1380
aagggctgga aagacaccat tcaagcgcat gcaggtgaag tcctgagaat cgcggcgaca 1440
ttcggtccgt acagcggacg atacgtatgg cattgccata ttctagagca tgaagactat 1500
gacatgatga gaccgatgga tataactgat ccccataaat aa 1542
<210> 2
<211> 1542
<212> DNA
<213> 人工序列
<400> 2
atgacacttg aaaaatttgt ggatgctctc ccaatcccag atacactaaa gccagtacag 60
caatcaaaag aaaaaacata ctacgaagtc accatggagg aatgcactca tcagctccat 120
cgcgatctcc ctccaacccg cctgtggggc tacaacggct tatttccggg accgaccatt 180
gaggttaaaa gaaatgaaaa cgtatatgta aaatggatga ataaccttcc ttccacgcat 240
ttccttccga ttgatcacac cattcatcac agtgacagcc agcatgaaga gcccgaggta 300
aagactgttg ttcatttaca cggcggcgtc acgccagatg atagtgacgg gtatccggag 360
gcttggtttt ccaaagactt tgaacaaaca ggaccttatt tcaaaagaga ggtttatcat 420
tatccaaacc agcagcgcgg ggctatattg tggtatcacg atcacgccat ggcgctcacc 480
aggctaaatg tctatgccgg acttgtcggt gcatatatca ttcatgaccc aaaggaaaaa 540
cgcttaaaac tgccttcaga cgaatacgat gtgccgcttc ttatcacaga ccgcacgatc 600
aatgaggatg gttctttgta ttatccgagc gcaccggaaa acccttctcc gtcactgcct 660
aatccttcaa tcgttccggc tttttgcgga gaaaccatac tcgtcaacgg gaaggtatgg 720
ccatacttgg aagtcgagcc aaggaaatac cgattccgtg tcatcaacgc ctccaataca 780
agaacctata acctgtcact cgataatggc ggagatttta ttcagattgg ttcagatgga 840
gggctcctgc cgcgatctgt taaactgaat tctttcagcc ttgcgcctgc tgaacgttac 900
gatatcatca ttgacttcac agcatatgaa ggagaatcga tcattttggc aaacagcgcg 960
ggctgcggcg gtgacgtcaa tcctgaaaca gatgcgaata tcatgcaatt cagagtcaca 1020
aaaccattgg cacaaaaaga cgaaagcaga aagccgaagt acctcgcctc atacccttcg 1080
gtacagcatg aaagaataca aaacatcaga acgttaaaac tggcaggcac ccaggacgaa 1140
tacggcagac ccgtccttct gcttaataac aaacgctggc acgatcccgt cacagaaaca 1200
ccaaaagtcg gcacaactga aatatggtcc attatcaacc cgacacgcgg aacacatccg 1260
atccacctgc atctagtctc cttccgtgta ttagaccggc ggccgtttga tatcgcccgt 1320
tatcaagaaa gcggggaatt gtcctatacc ggtccggctg tcccgccgcc gccaagtgaa 1380
aagggctgga aagacaccat tcaagcgcat gcaggtgaag tcctgagaat cgcggcgaca 1440
ttcggtccgt acagcggacg atacgtatgg cattgccata ttctagagca tgaagactat 1500
gacatgatga gaccgatgga tataactgat ccccataaat aa 1542
<210> 3
<211> 1542
<212> DNA
<213> 人工序列
<400> 3
atgacacttg aaaaatttgt ggatgctctc ccaatcccag atacactaaa gccagtacag 60
caatcaaaag aaaaaacata ctacgaagtc accatggagg aatgcactca tcagctccat 120
cgcgatctcc ctccaacccg cctgtggggc tacaacggct tatttccggg accgaccatt 180
gaggttaaaa gaaatgaaaa cgtatatgta aaatggatga ataaccttcc ttccacgcat 240
ttccttccga ttgatcacac cattcatcac agtgacagcc agcatgaaga gcccgaggta 300
aagactgttg ttcatttaca cggcggcgtc acgccagatg atagtgacgg gtatccggag 360
gcttggtttt ccaaagactt tgaacaaaca ggaccttatt tcaaaagaga ggtttatcat 420
tatccaaacc agcagcgcgg ggctatattg tggtatcacg atcacgccat ggcgctcacc 480
aggctaaatg tctatgccgg acttgtcggt gcatatatca ttcatgaccc aaaggaaaaa 540
cgcttaaaac tgccttcaga cgaatacgat gtgccgcttc ttatcacaga ccgcacgatc 600
aatgaggatg gttctttgtt ttatccgagc gcaccggaaa acccttctcc gtcactgcct 660
aatccttcaa tcgttccggc tttttgcgga gaaaccatac tcgtcaacgg gaaggtatgg 720
ccatacttgg aagtcgagcc aaggaaatac cgattccgtg tcatcaacgc ctccaataca 780
agaacctata acctgtcact cgataatggc ggagatttta ttcagattgg ttcagatgga 840
gggctcctgc cgcgatctgt taaactgaat tctttcagcc ttgcgcctgc tgaacgttac 900
gatatcatca ttgacttcac agcatatgaa ggagaatcga tcattttggc aaacagcgcg 960
ggctgcggcg gtgacgtcaa tcctgaaaca gatgcgaata tcatgcaatt cagagtcaca 1020
aaaccattgg cacaaaaaga cgaaagcaga aagccgaagt acctcgcctc atacccttcg 1080
gtacagcatg aaagaataca aaacatcaga acgttaaaac tggcaggcac ccaggacgaa 1140
tacggcagac ccgtccttct gcttaataac aaacgctggc acgatcccgt cacagaaaca 1200
ccaaaaacgg gcacaactga aatatggtcc attatcaacc cgacacgcgg aacacatccg 1260
atccacctgc atctagtctc cttccgtgta ttagaccggc ggccgtttga tatcgcccgt 1320
tatcaagaaa gcggggaatt gtcctatacc ggtccggctg tcccgccgcc gccaagtgaa 1380
aagggctgga aagacaccat tcaagcgcat gcaggtgaag tcctgagaat cgcggcgaca 1440
ttcggtccgt acagcggacg atacgtatgg cattgccata ttctagagca tgaagactat 1500
gacatgatga gaccgatgga tataactgat ccccataaat aa 1542
<210> 4
<211> 1542
<212> DNA
<213> 人工序列
<400> 4
atgacacttg aaaaatttgt ggatgctctc ccaatcccag atacactaaa gccagtacag 60
caatcaaaag aaaaaacata ctacgaagtc accatggagg aatgcactca tcagctccat 120
cgcgatctcc ctccaacccg cctgtggggc tacaacggct tatttccggg accgaccatt 180
gaggttaaaa gaaatgaaaa cgtatatgta aaatggatga ataaccttcc ttccacgcat 240
ttccttccga ttgatcacac cattcatcac agtgacagcc agcatgaaga gcccgaggta 300
aagactgttg ttcatttaca cggcggcgtc acgccagatg atagtgacgg gtatccggag 360
gcttggtttt ccaaagactt tgaacaaaca ggaccttatt tcaaaagaga ggtttatcat 420
tatccaaacc agcagcgcgg ggctatattg tggtatcacg atcacgccat ggcgctcacc 480
aggctaaatg tctatgccgg acttgtcggt gcatatatca ttcatgaccc aaaggaaaaa 540
cgcttaaaac tgccttcaga cgaatacgat gtgccgcttc ttatcacaga ccgcacgatc 600
aatgaggatg gttctttgtt ttatccgagc gcaccggaaa acccttctcc gtcactgcct 660
aatccttcaa tcgttccggc tttttgcgga gaaaccatac tcgtcaacgg gaaggtatgg 720
ccatacttgg aagtcgagcc aaggaaatac cgattccgtg tcatcaacgc ctccaataca 780
agaacctata acctgtcact cgataatggc ggagatttta ttcagattgg ttcagatgga 840
gggctcctgc cgcgatctgt taaactgaat tctttcagcc ttgcgcctgc tgaacgttac 900
gatatcatca ttgacttcac agcatatgaa ggagaatcga tcattttggc aaacagcgcg 960
ggctgcggcg gtgacgtcaa tcctgaaaca gatgcgaata tcatgcaatt cagagtcaca 1020
aaaccattgg cacaaaaaga cgaaagcaga aagccgaagt acctcgcctc atacccttcg 1080
gtacagcatg aaagaataca aaacatcaga acgttaaaac tggcaggcac ccaggacgaa 1140
tacggcagac ccgtccttct gcttaataac aaacgctggc acgatcccgt cacagaaaca 1200
ccaaaagtcg gcacaactga aatatggtcc attatcaacc cgacacgcgg aacacatccg 1260
atccacctgc atctagtctc cttccgtgta ttagaccggc ggccgtttga tatcgcccgt 1320
tatcaagaaa gcggggaatt gtcctatacc ggtccggctg tctctccgcc gccaagtgaa 1380
aagggctgga aagacaccat tcaagcgcat gcaggtgaag tcctgagaat cgcggcgaca 1440
ttcggtccgt acagcggacg atacgtatgg cattgccata ttctagagca tgaagactat 1500
gacatgatga gaccgatgga tataactgat ccccataaat aa 1542
<210> 5
<211> 1542
<212> DNA
<213> 人工序列
<400> 5
atgacacttg aaaaatttgt ggatgctctc ccaatcccag atacactaaa gccagtacag 60
caatcaaaag aaaaaacata ctacgaagtc accatggagg aatgcactca tcagctccat 120
cgcgatctcc ctccaacccg cctgtggggc tacaacggct tatttccggg accgaccatt 180
gaggttaaaa gaaatgaaaa cgtatatgta aaatggatga ataaccttcc ttccacgcat 240
ttccttccga ttgatcacac cattcatcac agtgacagcc agcatgaaga gcccgaggta 300
aagactgttg ttcatttaca cggcggcgtc acgccagatg atagtgacgg gtatccggag 360
gcttggtttt ccaaagactt tgaacaaaca ggaccttatt tcaaaagaga ggtttatcat 420
tatccaaacc agcagcgcgg ggctatattg tggtatcacg atcacgccat ggcgctcacc 480
aggctaaatg tctatgccgg acttgtcggt gcatatatca ttcatgaccc aaaggaaaaa 540
cgcttaaaac tgccttcaga cgaatacgat gtgccgcttc ttatcacaga ccgcacgatc 600
aatgaggatg gttctttgtt ttatccgagc gcaccggaaa acccttctcc gtcactgcct 660
aatccttcaa tcgttccggc tttttgcgga gaaaccatac tcgtcaacgg gaaggtatgg 720
ccatacttgg aagtcgagcc aaggaaatac cgattccgtg tcatcaacgc ctccaataca 780
agaacctata acctgtcact cgataatggc ggagatttta ttcagattgg ttcagatgga 840
gggctcctgc cgcgatctgt taaactgaat tctttcagcc ttgcgcctgc tgaacgttac 900
gatatcatca ttgacttcac agcatatgaa ggagaatcga tcattttggc aaacagcgcg 960
ggctgcggcg gtgacgtcaa tcctgaaaca gatgcgaata tcatgcaatt cagagtcaca 1020
aaaccattgg cacaaaaaga cgaaagcaga aagccgaagt acctcgcctc atacccttcg 1080
gtacagcatg aaagaataca aaacatcaga acgttaaaac tggcaggcac ccaggacgaa 1140
tacggcagac ccgtccttct gcttaataac aaacgctggc acgatcccgt cacagaaaca 1200
ccaaaaacgg gcacaactga aatatggtcc attatcaacc cgacacgcgg aacacatccg 1260
atccacctgc atctagtctc cttccgtgta ttagaccggc ggccgtttga tatcgcccgt 1320
tatcaagaaa gcggggaatt gtcctatacc ggtccggctg tctctccgcc gccaagtgaa 1380
aagggctgga aagacaccat tcaagcgcat gcaggtgaag tcctgagaat cgcggcgaca 1440
ttcggtccgt acagcggacg atacgtatgg cattgccata ttctagagca tgaagactat 1500
gacatgatga gaccgatgga tataactgat ccccataaat aa 1542
<210> 6
<211> 1542
<212> DNA
<213> 人工序列
<400> 6
atgacacttg aaaaatttgt ggatgctctc ccaatcccag atacactaaa gccagtacag 60
caatcaaaag aaaaaacata ctacgaagtc accatggagg aatgcactca tcagctccat 120
cgcgatctcc ctccaacccg cctgtggggc tacaacggct tatttccggg accgaccatt 180
gaggttaaaa gaaatgaaaa cgtatatgta aaatggatga ataaccttcc ttccacgcat 240
ttccttccga ttgatcacac cattcatcac agtgacagcc agcatgaaga gcccgaggta 300
aagactgttg ttcatttaca cggcggcgtc acgccagatg atagtgacgg gtatccggag 360
gcttggtttt ccaaagactt tgaacaaaca ggaccttatt tcaaaagaga ggtttatcat 420
tatccaaacc agcagcgcgg ggctatattg tggtatcacg atcacgccat ggcgctcacc 480
aggctaaatg tctatgccgg acttgtcggt gcatatatca ttcatgaccc aaaggaaaaa 540
cgcttaaaac tgccttcaga cgaatacgat gtgccgcttc ttatcacaga ccgcacgatc 600
aatgaggata tttctttgtt ttatccgagc gcaccggaaa acccttctcc gtcactgcct 660
aatccttcaa tcgttccggc tttttgcgga gaaaccatac tcgtcaacgg gaaggtatgg 720
ccatacttgg aagtcgagcc aaggaaatac cgattccgtg tcatcaacgc ctccaataca 780
agaacctata acctgtcact cgataatggc ggagatttta ttcagattgg ttcagatgga 840
gggctcctgc cgcgatctgt taaactgaat tctttcagcc ttgcgcctgc tgaacgttac 900
gatatcatca ttgacttcac agcatatgaa ggagaatcga tcattttggc aaacagcgcg 960
ggctgcggcg gtgacgtcaa tcctgaaaca gatgcgaata tcatgcaatt cagagtcaca 1020
aaaccattgg cacaaaaaga cgaaagcaga aagccgaagt acctcgcctc atacccttcg 1080
gtacagcatg aaagaataca aaacatcaga acgttaaaac tggcaggcac ccaggacgaa 1140
tacggcagac ccgtccttct gcttaataac aaacgctggc acgatcccgt cacagaaaca 1200
ccaaaaacgg gcacaactga aatatggtcc attatcaacc cgacacgcgg aacacatccg 1260
atccacctgc atctagtctc cttccgtgta ttagaccggc ggccgtttga tatcgcccgt 1320
tatcaagaaa gcggggaatt gtcctatacc ggtccggctg tctctccgcc gccaagtgaa 1380
aagggctgga aagacaccat tcaagcgcat gcaggtgaag tcctgagaat cgcggcgaca 1440
ttcggtccgt acagcggacg atacgtatgg cattgccata ttctagagca tgaagactat 1500
gacatgatga gaccgatgga tataactgat ccccataaat aa 1542
<210> 7
<211> 37
<212> DNA
<213> 人工序列
<400> 7
ggggagtcga tatgacactt gaaaaatttg tggatgc 37
<210> 8
<211> 38
<212> DNA
<213> 人工序列
<400> 8
gaatactcga gtttatgggg atcagttata tccatcgg 38
<210> 9
<211> 31
<212> DNA
<213> 人工序列
<400> 9
ggatggttct ttgtattatc cgagcgcacc g 31
<210> 10
<211> 31
<212> DNA
<213> 人工序列
<400> 10
cggtgcgctc ggataataca aagaaccatc c 31
<210> 11
<211> 36
<212> DNA
<213> 人工序列
<400> 11
cacagaaaca ccaaaaacgg gcacaactga aatatg 36
<210> 12
<211> 36
<212> DNA
<213> 人工序列
<400> 12
catatttcag ttgtgcccgt ttttggtgtt tctgtg 36
<210> 13
<211> 34
<212> DNA
<213> 人工序列
<400> 13
cggtccggct gtctctccgc cgccaagtga aaag 34
<210> 14
<211> 34
<212> DNA
<213> 人工序列
<400> 14
cttttcactt ggcggcggag agacagccgg accg 34
<210> 15
<211> 513
<212> PRT
<213> 人工序列
<400> 15
Met Thr Leu Glu Lys Phe Val Asp Ala Leu Pro Ile Pro Asp Thr Leu Lys
5 10 15
Pro Val Gln Gln Ser Lys Glu Lys Thr Tyr Tyr Glu Val Thr Met Glu Glu
20 25 30
Cys Thr His Gln Leu His Arg Asp Leu Pro Pro Thr Arg Leu Trp Gly Tyr
35 40 45 50
Asn Gly Leu Phe Pro Gly Pro Thr Ile Glu Val Lys Arg Asn Glu Asn Val
55 60 65
Tyr Val Lys Trp Met Asn Asn Leu Pro Ser Thr His Phe Leu Pro Ile Asp
70 75 80 85
His Thr Ile His His Ser Asp Ser Gln His Glu Glu Pro Glu Val Lys Thr
90 95 100
Val Val His Leu His Gly Gly Val Thr Pro Asp Asp Ser Asp Gly Tyr Pro
105 110 115
Glu Ala Trp Phe Ser Lys Asp Phe Glu Gln Thr Gly Pro Tyr Phe Lys Arg
120 125 130 135
Glu Val Tyr His Tyr Pro Asn Gln Gln Arg Gly Ala Ile Leu Trp Tyr His
140 145 150
Asp His Ala Met Ala Leu Thr Arg Leu Asn Val Tyr Ala Gly Leu Val Gly
155 160 165 170
Ala Tyr Ile Ile His Asp Pro Lys Glu Lys Arg Leu Lys Leu Pro Ser Asp
175 180 185
Glu Tyr Asp Val Pro Leu Leu Ile Thr Asp Arg Thr Ile Asn Glu Asp Gly
190 195 200
Ser Leu Phe Tyr Pro Ser Ala Pro Glu Asn Pro Ser Pro Ser Leu Pro Asn
205 210 215 220
Pro Ser Ile Val Pro Ala Phe Cys Gly Glu Thr Ile Leu Val Asn Gly Lys
225 230 235
Val Trp Pro Tyr Leu Glu Val Glu Pro Arg Lys Tyr Arg Phe Arg Val Ile
240 245 250 255
Asn Ala Ser Asn Thr Arg Thr Tyr Asn Leu Ser Leu Asp Asn Gly Gly
260 265 270
Asp Phe Ile Gln Ile Gly Ser Asp Gly Gly Leu Leu Pro Arg Ser Val Lys
275 280 285
Leu Asn Ser Phe Ser Leu Ala Pro Ala Glu Arg Tyr Asp Ile Ile Ile Asp
290 295 300 305
Phe Thr Ala Tyr Glu Gly Glu Ser Ile Ile Leu Ala Asn Ser Asn Gly Cys
310 315 320
Gly Gly Asp Val Asn Pro Glu Thr Asp Ala Asn Ile Met Gln Phe Arg
325 330 335
Val Thr Lys Pro Leu Ala Gln Lys Asp Glu Ser Arg Lys Pro Lys Tyr Leu
340 345 350 355
Ala Ser Tyr Pro Ser Val Gln His Glu Arg Ile Gln Asn Ile Arg Thr Leu
360 365 370
Lys Leu Ala Gly Thr Gln Asp Glu Tyr Gly Arg Pro Val Leu Leu Leu
375 380 385
Asn Asn Lys Arg Trp His Asp Pro Val Thr Glu Thr Pro Lys Val Gly Thr
390 395 400 405
Thr Glu Ile Trp Ser Ile Ile Asn Pro Thr Arg Gly Thr His Pro Ile His Leu
410 415 420
His Leu Val Ser Phe Arg Val Leu Asp Arg Arg Pro Phe Asp Ile Ala Arg
425 430 435 440
Tyr Gln Glu Ser Gly Glu Leu Ser Tyr Thr Gly Pro Ala Val Pro Pro Pro
445 450 455
Pro Ser Glu Lys Gly Trp Lys Asp Thr Ile Gln Ala His Ala Gly Glu Val
460 465 470
Leu Arg Ile Ala Ala Thr Phe Gly Pro Tyr Ser Gly Arg Tyr Val Trp His
475 480 485 490
Cys His Ile Leu Glu His Glu Asp Tyr Asp Met Met Arg Pro Met Asp Ile
495 500 505
Thr Asp Pro His Lys
510
<210> 16
<211> 513
<212> PRT
<213> 人工序列
<400> 16
Met Thr Leu Glu Lys Phe Val Asp Ala Leu Pro Ile Pro Asp Thr Leu Lys
5 10 15
Pro Val Gln Gln Ser Lys Glu Lys Thr Tyr Tyr Glu Val Thr Met Glu Glu
20 25 30
Cys Thr His Gln Leu His Arg Asp Leu Pro Pro Thr Arg Leu Trp Gly Tyr
35 40 45 50
Asn Gly Leu Phe Pro Gly Pro Thr Ile Glu Val Lys Arg Asn Glu Asn Val
55 60 65
Tyr Val Lys Trp Met Asn Asn Leu Pro Ser Thr His Phe Leu Pro Ile Asp
70 75 80 85
His Thr Ile His His Ser Asp Ser Gln His Glu Glu Pro Glu Val Lys Thr
90 95 100
Val Val His Leu His Gly Gly Val Thr Pro Asp Asp Ser Asp Gly Tyr Pro
105 110 115
Glu Ala Trp Phe Ser Lys Asp Phe Glu Gln Thr Gly Pro Tyr Phe Lys Arg
120 125 130 135
Glu Val Tyr His Tyr Pro Asn Gln Gln Arg Gly Ala Ile Leu Trp Tyr His
140 145 150
Asp His Ala Met Ala Leu Thr Arg Leu Asn Val Tyr Ala Gly Leu Val Gly
155 160 165 170
Ala Tyr Ile Ile His Asp Pro Lys Glu Lys Arg Leu Lys Leu Pro Ser Asp
175 180 185
Glu Tyr Asp Val Pro Leu Leu Ile Thr Asp Arg Thr Ile Asn Glu Asp Gly
190 195 200
Ser Leu Tyr Tyr Pro Ser Ala Pro Glu Asn Pro Ser Pro Ser Leu Pro Asn
205 210 215 220
Pro Ser Ile Val Pro Ala Phe Cys Gly Glu Thr Ile Leu Val Asn Gly Lys
225 230 235
Val Trp Pro Tyr Leu Glu Val Glu Pro Arg Lys Tyr Arg Phe Arg Val Ile
240 245 250 255
Asn Ala Ser Asn Thr Arg Thr Tyr Asn Leu Ser Leu Asp Asn Gly Gly
260 265 270
Asp Phe Ile Gln Ile Gly Ser Asp Gly Gly Leu Leu Pro Arg Ser Val Lys
275 280 285
Leu Asn Ser Phe Ser Leu Ala Pro Ala Glu Arg Tyr Asp Ile Ile Ile Asp
290 295 300 305
Phe Thr Ala Tyr Glu Gly Glu Ser Ile Ile Leu Ala Asn Ser Asn Gly Cys
310 315 320
Gly Gly Asp Val Asn Pro Glu Thr Asp Ala Asn Ile Met Gln Phe Arg
325 330 335
Val Thr Lys Pro Leu Ala Gln Lys Asp Glu Ser Arg Lys Pro Lys Tyr Leu
340 345 350 355
Ala Ser Tyr Pro Ser Val Gln His Glu Arg Ile Gln Asn Ile Arg Thr Leu
360 365 370
Lys Leu Ala Gly Thr Gln Asp Glu Tyr Gly Arg Pro Val Leu Leu Leu
375 380 385
Asn Asn Lys Arg Trp His Asp Pro Val Thr Glu Thr Pro Lys Val Gly Thr
390 395 400 405
Thr Glu Ile Trp Ser Ile Ile Asn Pro Thr Arg Gly Thr His Pro Ile His Leu
410 415 420
His Leu Val Ser Phe Arg Val Leu Asp Arg Arg Pro Phe Asp Ile Ala Arg
425 430 435 440
Tyr Gln Glu Ser Gly Glu Leu Ser Tyr Thr Gly Pro Ala Val Pro Pro Pro
445 450 455
Pro Ser Glu Lys Gly Trp Lys Asp Thr Ile Gln Ala His Ala Gly Glu Val
460 465 470
Leu Arg Ile Ala Ala Thr Phe Gly Pro Tyr Ser Gly Arg Tyr Val Trp His
475 480 485 490
Cys His Ile Leu Glu His Glu Asp Tyr Asp Met Met Arg Pro Met Asp Ile
495 500 505
Thr Asp Pro His Lys
510
<210> 17
<211> 513
<212> PRT
<213> 人工序列
<400> 17
Met Thr Leu Glu Lys Phe Val Asp Ala Leu Pro Ile Pro Asp Thr Leu Lys
5 10 15
Pro Val Gln Gln Ser Lys Glu Lys Thr Tyr Tyr Glu Val Thr Met Glu Glu
20 25 30
Cys Thr His Gln Leu His Arg Asp Leu Pro Pro Thr Arg Leu Trp Gly Tyr
35 40 45 50
Asn Gly Leu Phe Pro Gly Pro Thr Ile Glu Val Lys Arg Asn Glu Asn Val
55 60 65
Tyr Val Lys Trp Met Asn Asn Leu Pro Ser Thr His Phe Leu Pro Ile Asp
70 75 80 85
His Thr Ile His His Ser Asp Ser Gln His Glu Glu Pro Glu Val Lys Thr
90 95 100
Val Val His Leu His Gly Gly Val Thr Pro Asp Asp Ser Asp Gly Tyr Pro
105 110 115
Glu Ala Trp Phe Ser Lys Asp Phe Glu Gln Thr Gly Pro Tyr Phe Lys Arg
120 125 130 135
Glu Val Tyr His Tyr Pro Asn Gln Gln Arg Gly Ala Ile Leu Trp Tyr His
140 145 150
Asp His Ala Met Ala Leu Thr Arg Leu Asn Val Tyr Ala Gly Leu Val Gly
155 160 165 170
Ala Tyr Ile Ile His Asp Pro Lys Glu Lys Arg Leu Lys Leu Pro Ser Asp
175 180 185
Glu Tyr Asp Val Pro Leu Leu Ile Thr Asp Arg Thr Ile Asn Glu Asp Gly
190 195 200
Ser Leu Phe Tyr Pro Ser Ala Pro Glu Asn Pro Ser Pro Ser Leu Pro Asn
205 210 215 220
Pro Ser Ile Val Pro Ala Phe Cys Gly Glu Thr Ile Leu Val Asn Gly Lys
225 230 235
Val Trp Pro Tyr Leu Glu Val Glu Pro Arg Lys Tyr Arg Phe Arg Val Ile
240 245 250 255
Asn Ala Ser Asn Thr Arg Thr Tyr Asn Leu Ser Leu Asp Asn Gly Gly
260 265 270
Asp Phe Ile Gln Ile Gly Ser Asp Gly Gly Leu Leu Pro Arg Ser Val Lys
275 280 285
Leu Asn Ser Phe Ser Leu Ala Pro Ala Glu Arg Tyr Asp Ile Ile Ile Asp
290 295 300 305
Phe Thr Ala Tyr Glu Gly Glu Ser Ile Ile Leu Ala Asn Ser Asn Gly Cys
310 315 320
Gly Gly Asp Val Asn Pro Glu Thr Asp Ala Asn Ile Met Gln Phe Arg
325 330 335
Val Thr Lys Pro Leu Ala Gln Lys Asp Glu Ser Arg Lys Pro Lys Tyr Leu
340 345 350 355
Ala Ser Tyr Pro Ser Val Gln His Glu Arg Ile Gln Asn Ile Arg Thr Leu
360 365 370
Lys Leu Ala Gly Thr Gln Asp Glu Tyr Gly Arg Pro Val Leu Leu Leu
375 380 385
Asn Asn Lys Arg Trp His Asp Pro Val Thr Glu Thr Pro Lys Thr Gly Thr
390 395 400 405
Thr Glu Ile Trp Ser Ile Ile Asn Pro Thr Arg Gly Thr His Pro Ile His Leu
410 415 420
His Leu Val Ser Phe Arg Val Leu Asp Arg Arg Pro Phe Asp Ile Ala Arg
425 430 435 440
Tyr Gln Glu Ser Gly Glu Leu Ser Tyr Thr Gly Pro Ala Val Pro Pro Pro
445 450 455
Pro Ser Glu Lys Gly Trp Lys Asp Thr Ile Gln Ala His Ala Gly Glu Val
460 465 470
Leu Arg Ile Ala Ala Thr Phe Gly Pro Tyr Ser Gly Arg Tyr Val Trp His
475 480 485 490
Cys His Ile Leu Glu His Glu Asp Tyr Asp Met Met Arg Pro Met Asp Ile
495 500 505
Thr Asp Pro His Lys
510
<210> 18
<211> 513
<212> PRT
<213> 人工序列
<400> 18
Met Thr Leu Glu Lys Phe Val Asp Ala Leu Pro Ile Pro Asp Thr Leu Lys
5 10 15
Pro Val Gln Gln Ser Lys Glu Lys Thr Tyr Tyr Glu Val Thr Met Glu Glu
20 25 30
Cys Thr His Gln Leu His Arg Asp Leu Pro Pro Thr Arg Leu Trp Gly Tyr
35 40 45 50
Asn Gly Leu Phe Pro Gly Pro Thr Ile Glu Val Lys Arg Asn Glu Asn Val
55 60 65
Tyr Val Lys Trp Met Asn Asn Leu Pro Ser Thr His Phe Leu Pro Ile Asp
70 75 80 85
His Thr Ile His His Ser Asp Ser Gln His Glu Glu Pro Glu Val Lys Thr
90 95 100
Val Val His Leu His Gly Gly Val Thr Pro Asp Asp Ser Asp Gly Tyr Pro
105 110 115
Glu Ala Trp Phe Ser Lys Asp Phe Glu Gln Thr Gly Pro Tyr Phe Lys Arg
120 125 130 135
Glu Val Tyr His Tyr Pro Asn Gln Gln Arg Gly Ala Ile Leu Trp Tyr His
140 145 150
Asp His Ala Met Ala Leu Thr Arg Leu Asn Val Tyr Ala Gly Leu Val Gly
155 160 165 170
Ala Tyr Ile Ile His Asp Pro Lys Glu Lys Arg Leu Lys Leu Pro Ser Asp
175 180 185
Glu Tyr Asp Val Pro Leu Leu Ile Thr Asp Arg Thr Ile Asn Glu Asp Gly
190 195 200
Ser Leu Phe Tyr Pro Ser Ala Pro Glu Asn Pro Ser Pro Ser Leu Pro Asn
205 210 215 220
Pro Ser Ile Val Pro Ala Phe Cys Gly Glu Thr Ile Leu Val Asn Gly Lys
225 230 235
Val Trp Pro Tyr Leu Glu Val Glu Pro Arg Lys Tyr Arg Phe Arg Val Ile
240 245 250 255
Asn Ala Ser Asn Thr Arg Thr Tyr Asn Leu Ser Leu Asp Asn Gly Gly
260 265 270
Asp Phe Ile Gln Ile Gly Ser Asp Gly Gly Leu Leu Pro Arg Ser Val Lys
275 280 285
Leu Asn Ser Phe Ser Leu Ala Pro Ala Glu Arg Tyr Asp Ile Ile Ile Asp
290 295 300 305
Phe Thr Ala Tyr Glu Gly Glu Ser Ile Ile Leu Ala Asn Ser Asn Gly Cys
310 315 320
Gly Gly Asp Val Asn Pro Glu Thr Asp Ala Asn Ile Met Gln Phe Arg
325 330 335
Val Thr Lys Pro Leu Ala Gln Lys Asp Glu Ser Arg Lys Pro Lys Tyr Leu
340 345 350 355
Ala Ser Tyr Pro Ser Val Gln His Glu Arg Ile Gln Asn Ile Arg Thr Leu
360 365 370
Lys Leu Ala Gly Thr Gln Asp Glu Tyr Gly Arg Pro Val Leu Leu Leu
375 380 385
Asn Asn Lys Arg Trp His Asp Pro Val Thr Glu Thr Pro Lys Val Gly Thr
390 395 400 405
Thr Glu Ile Trp Ser Ile Ile Asn Pro Thr Arg Gly Thr His Pro Ile His Leu
410 415 420
His Leu Val Ser Phe Arg Val Leu Asp Arg Arg Pro Phe Asp Ile Ala Arg
425 430 435 440
Tyr Gln Glu Ser Gly Glu Leu Ser Tyr Thr Gly Pro Ala Val Ser Pro Pro
445 450 455
Pro Ser Glu Lys Gly Trp Lys Asp Thr Ile Gln Ala His Ala Gly Glu Val
460 465 470
Leu Arg Ile Ala Ala Thr Phe Gly Pro Tyr Ser Gly Arg Tyr Val Trp His
475 480 485 490
Cys His Ile Leu Glu His Glu Asp Tyr Asp Met Met Arg Pro Met Asp Ile
495 500 505
Thr Asp Pro His Lys
510
<210> 19
<211> 513
<212> PRT
<213> 人工序列
<400> 19
Met Thr Leu Glu Lys Phe Val Asp Ala Leu Pro Ile Pro Asp Thr Leu Lys
5 10 15
Pro Val Gln Gln Ser Lys Glu Lys Thr Tyr Tyr Glu Val Thr Met Glu Glu
20 25 30
Cys Thr His Gln Leu His Arg Asp Leu Pro Pro Thr Arg Leu Trp Gly Tyr
35 40 45 50
Asn Gly Leu Phe Pro Gly Pro Thr Ile Glu Val Lys Arg Asn Glu Asn Val
55 60 65
Tyr Val Lys Trp Met Asn Asn Leu Pro Ser Thr His Phe Leu Pro Ile Asp
70 75 80 85
His Thr Ile His His Ser Asp Ser Gln His Glu Glu Pro Glu Val Lys Thr
90 95 100
Val Val His Leu His Gly Gly Val Thr Pro Asp Asp Ser Asp Gly Tyr Pro
105 110 115
Glu Ala Trp Phe Ser Lys Asp Phe Glu Gln Thr Gly Pro Tyr Phe Lys Arg
120 125 130 135
Glu Val Tyr His Tyr Pro Asn Gln Gln Arg Gly Ala Ile Leu Trp Tyr His
140 145 150
Asp His Ala Met Ala Leu Thr Arg Leu Asn Val Tyr Ala Gly Leu Val Gly
155 160 165 170
Ala Tyr Ile Ile His Asp Pro Lys Glu Lys Arg Leu Lys Leu Pro Ser Asp
175 180 185
Glu Tyr Asp Val Pro Leu Leu Ile Thr Asp Arg Thr Ile Asn Glu Asp Gly
190 195 200
Ser Leu Phe Tyr Pro Ser Ala Pro Glu Asn Pro Ser Pro Ser Leu Pro Asn
205 210 215 220
Pro Ser Ile Val Pro Ala Phe Cys Gly Glu Thr Ile Leu Val Asn Gly Lys
225 230 235
Val Trp Pro Tyr Leu Glu Val Glu Pro Arg Lys Tyr Arg Phe Arg Val Ile
240 245 250 255
Asn Ala Ser Asn Thr Arg Thr Tyr Asn Leu Ser Leu Asp Asn Gly Gly
260 265 270
Asp Phe Ile Gln Ile Gly Ser Asp Gly Gly Leu Leu Pro Arg Ser Val Lys
275 280 285
Leu Asn Ser Phe Ser Leu Ala Pro Ala Glu Arg Tyr Asp Ile Ile Ile Asp
290 295 300 305
Phe Thr Ala Tyr Glu Gly Glu Ser Ile Ile Leu Ala Asn Ser Asn Gly Cys
310 315 320
Gly Gly Asp Val Asn Pro Glu Thr Asp Ala Asn Ile Met Gln Phe Arg
325 330 335
Val Thr Lys Pro Leu Ala Gln Lys Asp Glu Ser Arg Lys Pro Lys Tyr Leu
340 345 350 355
Ala Ser Tyr Pro Ser Val Gln His Glu Arg Ile Gln Asn Ile Arg Thr Leu
360 365 370
Lys Leu Ala Gly Thr Gln Asp Glu Tyr Gly Arg Pro Val Leu Leu Leu
375 380 385
Asn Asn Lys Arg Trp His Asp Pro Val Thr Glu Thr Pro Lys Thr Gly Thr
390 395 400 405
Thr Glu Ile Trp Ser Ile Ile Asn Pro Thr Arg Gly Thr His Pro Ile His Leu
410 415 420
His Leu Val Ser Phe Arg Val Leu Asp Arg Arg Pro Phe Asp Ile Ala Arg
425 430 435 440
Tyr Gln Glu Ser Gly Glu Leu Ser Tyr Thr Gly Pro Ala Val Ser Pro Pro
445 450 455
Pro Ser Glu Lys Gly Trp Lys Asp Thr Ile Gln Ala His Ala Gly Glu Val
460 465 470
Leu Arg Ile Ala Ala Thr Phe Gly Pro Tyr Ser Gly Arg Tyr Val Trp His
475 480 485 490
Cys His Ile Leu Glu His Glu Asp Tyr Asp Met Met Arg Pro Met Asp Ile
495 500 505
Thr Asp Pro His Lys
510
<210> 20
<211> 513
<212> PRT
<213> 人工序列
<400> 20
Met Thr Leu Glu Lys Phe Val Asp Ala Leu Pro Ile Pro Asp Thr Leu Lys
5 10 15
Pro Val Gln Gln Ser Lys Glu Lys Thr Tyr Tyr Glu Val Thr Met Glu Glu
20 25 30
Cys Thr His Gln Leu His Arg Asp Leu Pro Pro Thr Arg Leu Trp Gly Tyr
35 40 45 50
Asn Gly Leu Phe Pro Gly Pro Thr Ile Glu Val Lys Arg Asn Glu Asn Val
55 60 65
Tyr Val Lys Trp Met Asn Asn Leu Pro Ser Thr His Phe Leu Pro Ile Asp
70 75 80 85
His Thr Ile His His Ser Asp Ser Gln His Glu Glu Pro Glu Val Lys Thr
90 95 100
Val Val His Leu His Gly Gly Val Thr Pro Asp Asp Ser Asp Gly Tyr Pro
105 110 115
Glu Ala Trp Phe Ser Lys Asp Phe Glu Gln Thr Gly Pro Tyr Phe Lys Arg
120 125 130 135
Glu Val Tyr His Tyr Pro Asn Gln Gln Arg Gly Ala Ile Leu Trp Tyr His
140 145 150
Asp His Ala Met Ala Leu Thr Arg Leu Asn Val Tyr Ala Gly Leu Val Gly
155 160 165 170
Ala Tyr Ile Ile His Asp Pro Lys Glu Lys Arg Leu Lys Leu Pro Ser Asp
175 180 185
Glu Tyr Asp Val Pro Leu Leu Ile Thr Asp Arg Thr Ile Asn Glu Asp Gly
190 195 200
Ser Leu Tyr Tyr Pro Ser Ala Pro Glu Asn Pro Ser Pro Ser Leu Pro Asn
205 210 215 220
Pro Ser Ile Val Pro Ala Phe Cys Gly Glu Thr Ile Leu Val Asn Gly Lys
225 230 235
Val Trp Pro Tyr Leu Glu Val Glu Pro Arg Lys Tyr Arg Phe Arg Val Ile
240 245 250 255
Asn Ala Ser Asn Thr Arg Thr Tyr Asn Leu Ser Leu Asp Asn Gly Gly
260 265 270
Asp Phe Ile Gln Ile Gly Ser Asp Gly Gly Leu Leu Pro Arg Ser Val Lys
275 280 285
Leu Asn Ser Phe Ser Leu Ala Pro Ala Glu Arg Tyr Asp Ile Ile Ile Asp
290 295 300 305
Phe Thr Ala Tyr Glu Gly Glu Ser Ile Ile Leu Ala Asn Ser Asn Gly Cys
310 315 320
Gly Gly Asp Val Asn Pro Glu Thr Asp Ala Asn Ile Met Gln Phe Arg
325 330 335
Val Thr Lys Pro Leu Ala Gln Lys Asp Glu Ser Arg Lys Pro Lys Tyr Leu
340 345 350 355
Ala Ser Tyr Pro Ser Val Gln His Glu Arg Ile Gln Asn Ile Arg Thr Leu
360 365 370
Lys Leu Ala Gly Thr Gln Asp Glu Tyr Gly Arg Pro Val Leu Leu Leu
375 380 385
Asn Asn Lys Arg Trp His Asp Pro Val Thr Glu Thr Pro Lys Thr Gly Thr
390 395 400 405
Thr Glu Ile Trp Ser Ile Ile Asn Pro Thr Arg Gly Thr His Pro Ile His Leu
410 415 420
His Leu Val Ser Phe Arg Val Leu Asp Arg Arg Pro Phe Asp Ile Ala Arg
425 430 435 440
Tyr Gln Glu Ser Gly Glu Leu Ser Tyr Thr Gly Pro Ala Val Ser Pro Pro
445 450 455
Pro Ser Glu Lys Gly Trp Lys Asp Thr Ile Gln Ala His Ala Gly Glu Val
460 465 470
Leu Arg Ile Ala Ala Thr Phe Gly Pro Tyr Ser Gly Arg Tyr Val Trp His
475 480 485 490
Cys His Ile Leu Glu His Glu Asp Tyr Asp Met Met Arg Pro Met Asp Ile
495 500 505
Thr Asp Pro His Lys
510

Claims (2)

1.一种高活性漆酶突变体蛋白的制备方法，其特征在于：所述漆酶突变体蛋白的制备方法包括如下步骤：
a）引物设计：CH6-P455S的正、反向引物的核苷酸序列分别如SEQ ID NO.13、SEQ IDNO.14所示；
b）DpnⅠ酶切消化：分别用各对正、反向引物对pET22b/CotA质粒进行扩增，将扩增产物用DpnⅠ酶在37℃下酶切5h，扩增条件：94℃，5min；94℃，1min；68℃，30s；72℃, 1min，循环30次；72℃，5min；4℃保温；
c）退火、纯化回收：将酶切产物混合，72℃反应8-12min后室温退火，将退火后的产物用试剂盒纯化回收；
d）转化、测序：将回收产物转化到感受态DH5α大肠杆菌中，平板培养，挑取单菌落测序，得测序正确的高活性漆酶衍生物的突变体CH6-P455S菌株，所述CH6-P455S菌株表达的突变体漆酶的核苷酸序列如SEQ ID NO.4所示，所述CH6-P455S菌株表达的突变体漆酶的氨基酸序列如SEQ ID NO.18所示；
e）以CH6-P455S的质粒为模板，SEQ ID NO.11、SEQ ID NO.12所示的核苷酸序列为正、反向引物对模板进行扩增，扩增产物按照步骤b）的条件DpnⅠ酶切消化，之后重复步骤c）、d），得双突变的突变体CH6-TS 菌株，所述CH6-TS菌株表达的突变体漆酶的核苷酸序列如SEQ ID NO.5所示，所述CH6-TS菌株表达的突变体漆酶的氨基酸序列如SEQ ID NO.19所示；
f）以CH6-TS的质粒为模板，SEQ ID NO.9、SEQ ID NO.10所示的核苷酸序列为正、反向引物对模板进行扩增，扩增产物按照步骤b）的条件DpnⅠ酶切消化，之后重复步骤c）、d），得三突变的突变体CH6-TSY菌株，所述CH6-TSY菌株表达的突变体漆酶的核苷酸序列如SEQ IDNO.6所示，所述CH6-TSY菌株表达的突变体漆酶的氨基酸序列如SEQ ID NO.20所示；
将获得的各突变体菌株经过如下处理，进行蛋白表达，获得所需的漆酶突变体蛋白：
1）将保存在-80℃条件下的突变体菌株划线到LB固体平板中，在37℃条件下过夜活化；
2）挑起活化的单菌落于10 mL 有氨苄抗性的液体LB培养基中，在37℃、200rpm的条件下过夜活化做种子液；
3）按0.5％的接种量将种子液接种于1L有氨苄抗性的液体LB培养基中，在37℃、200rpm的条件下培养；
4）当OD600为0.6时，向培养液中加入IPTG至终浓度为0.2 mM，加入硫酸铜至终浓度为0.25 mM，然后置于25℃、180 rpm的条件下诱导4h，之后在25℃的恒温摇床里静置过夜培养。
2.根据权利要求1所述的高活性漆酶突变体蛋白的制备方法，其特征在于：所述漆酶突变体蛋白的纯化步骤如下：
（1）往OD600到达0.4-0.6的培养物分别加入终浓度为0.2 mmol/L的IPTG和终浓度为0.25 mmol/L的CuSO4，在25℃、180 rpm的条件下培养4-5 h后，于25℃摇床中静置过夜，离心收集菌体后重悬浮于20 mM Na2HPO4-NaH2PO4，pH 7.6，500mM NaCl，20 mM咪唑的Buffer溶液中，超声破碎；
（2）将破碎后的菌体于12000 rpm条件下离心30min，将上清液于70℃条件下加热15min；
（3）将加热后的样品于12000 rpm条件下离心30min，将上清液进行镍柱亲和层析，用咪唑浓度为200mM的缓冲液洗脱蛋白质。
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