CN114736881B - 酸稳定性提高的葡萄糖氧化酶GoxM10突变体A4D及其衍生突变体和应用 - Google Patents
酸稳定性提高的葡萄糖氧化酶GoxM10突变体A4D及其衍生突变体和应用 Download PDFInfo
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Abstract
本发明属于饲料生物技术领域，具体涉及葡萄糖氧化酶GoxM10的酸稳定性提高的突变体A4D及其衍生突变体和应用。本发明以高热稳定性葡萄糖氧化酶GoxM10为研究材料，采用蛋白质工程改造技术对其进行酸稳定性的改良研究。最后获得酸稳定性提升的突变体，从而促进了Gox在饲料工业中的应用。
Description
技术领域
本发明属于饲料生物技术领域，具体涉及酸稳定性提高的葡萄糖氧化酶GoxM10突变体A4D及其衍生突变体和应用。
背景技术
葡萄糖氧化酶（Gox；EC1.1.3.4）是一种特性良好的需氧脱氢酶，在有氧条件下利用氧分子作为电子受体，催化β-d-葡萄糖生成d-葡萄糖酸内酯和过氧化氢，随后d-葡萄糖酸内酯水解成葡萄糖酸和水。近年来Gox作为一种新型抗生素替代添加剂受到饲料行业的普遍关注。Gox作为抗生素和生长促进剂的替代品，在不损害人类健康的前提下对保证动物健康和生产性能至关重要。大量应用研究表明，Gox作为一种绿色饲料添加剂，可用于预防牲畜胃肠道感染和腹泻，并有促进动物生长的作用。
当前大部分动物饲料均需要经过高温制粒，所以提高Gox的热稳定性对于其在饲料中的广泛应用至关重要。此外，能够在酸性环境中维持较高的活性，以便在动物胃肠道发挥最大作用也至关重要。所以为了满足Gox在饲料工业中的应用，Gox要具备良好的热稳定性和酸稳定性。对于黑曲霉（Aspergillus niger）来源Gox稳定性改进，现有技术首先通过改善氢键相互作用、π-π堆叠相互作用、疏水相互作用和酶表面电荷分布，获得了T 50增加7.5℃的突变体GoxM4，随后获得比GoxM4的T m提高了9 ℃的突变体GoxM10。
发明内容
本发明的目的是提供一种以来源于A. niger的高热稳定性葡萄糖氧化酶GoxM10作为母本经点突变获得的酸稳定性提高的突变体。
本发明的再一目的是提供编码上述突变体的基因。
本发明的再一目的是提供包含上述突变体基因的重组载体。
本发明的再一目的是提供包含上述突变体基因的重组菌株。
本发明的再一目的是提供上述突变体的应用。
本发明的再一目的是提供制备酸稳定性提高的葡萄糖氧化酶的方法。
本发明的再一目的是提供一种改善葡萄糖氧化酶的酸稳定性的方法。
根据本发明的具体实施方式，对氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶GoxM10进行定点突变。
根据本发明的具体实施方式，将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶进行4、133、471、581、4/133、4/471、4/581等氨基酸位点的突变，从而获得葡萄糖氧化酶酸稳定性提高的突变体。
根据本发明的具体实施方式，将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶进行A4D突变，得到氨基酸序列如SEQ ID NO:2所示的突变体。
根据本发明的具体实施方式，还提供了编码上述酸稳定性提高的葡萄糖氧化酶突变体A4D的基因，其核苷酸序列如SEQ ID NO:3所示，共1746 bp（包含终止子）。
根据本发明的具体实施方式，将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶进行S133E突变，得到氨基酸序列如SEQ ID NO:4所示的突变体。
根据本发明的具体实施方式，还提供了编码上述酸稳定性提高的葡萄糖氧化酶突变体S133E的基因，其核苷酸序列如SEQ IDNO:5所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶进行N471E突变，得到氨基酸序列如SEQ ID NO:6所示的突变。
根据本发明的具体实施方式，还提供了编码上述酸稳定性提高的葡萄糖氧化酶突变体N471E的基因，其核苷酸序列如SEQ ID NO:7所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶进行Q581E突变，得到氨基酸序列如SEQ ID NO:8所示的氨基酸序列。
根据本发明的具体实施方式，还提供了编码上述酸稳定性提高的葡萄糖氧化酶突变体Q581E的基因，其核苷酸序列如SEQ ID NO:9所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行A4D/S133E突变，得到氨基酸序列如SEQ ID NO:10所示。
根据本发明的具体实施方式，还提供了编码上述具有酸稳定性提高的葡萄糖氧化酶突变体A4D/S133E的基因，其核苷酸序列如SEQ ID NO:11所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶进行A4D/N471E突变，获得氨基酸序列如SEQ ID NO:12所示的突变体。
根据本发明的具体实施方式，还提供了编码上述酸稳定性提高的葡萄糖氧化酶突变体A4D/N471E的基因，其核苷酸序列如SEQ ID NO:13所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶进行A4D/Q581E突变，得到氨基酸序列如SEQ ID NO:14所示的突变体。
根据本发明的具体实施方式，还提供了编码上述酸稳定性提高的葡萄糖氧化酶突变体A4D/Q581E的基因，其核苷酸序列如SEQ ID NO:15所示。
根据本发明的具体实施方式，还提供了包含上述葡萄糖氧化酶突变体基因的重组载体。
根据本发明的具体实施例，所述重组表达载体的出发载体为pPIC9，所述重组表达载体具体为pPIC9-goxm10-A4D、pPIC9-goxm10-S133E、pPIC9-goxm10-N471E、pPIC9-goxm10-Q581E、pPIC9-goxm10-A4D/S133E、pPIC9-goxm10-A4D/N471E、pPIC9-goxm10-A4D/Q581E。
根据本发明的具体实施方式，还提供了包含上述葡萄糖氧化酶突变体基因的重组菌株，所述重组菌的出发菌株具体为GS115(pPIC9-goxm10)，所述重组菌株具体为GS115(pPIC9-goxm10-A4D、pPIC9-goxm10-S133E、pPIC9-goxm10-N471E，pPIC9-goxm10-Q581E、pPIC9-goxm10-A4D/S133E、pPIC9-goxm10-A4D/N471E，pPIC9-goxm10-A4D/Q581E。
根据本发明的制备具有酸稳定性提高的葡萄糖氧化酶的方法，包括以下步骤：
1）制备包含上述突变体基因的重组载体；
2）所述重组载体转化毕赤酵母GS115表达宿主；
3）发酵培养所述宿主，并分离葡萄糖氧化酶。
本发明的酸稳定性提高的葡萄糖氧化酶突变体与高热稳定性葡萄糖氧化酶GoxM10相比，酸稳定性均明显提高。突变体A4D、S133E、N471E、Q581E、A4D/S133E、A4D/N471E、A4D/Q581E纯化后的酶液在pH 2.75和37 ℃处理60 min后保留酶活力分别为37%、25%、27%、33%、32%、60%、26%左右，GoxM10的剩余酶活为24%。
根据本发明的改善葡萄糖氧化酶的酸稳定性的方法，所述方法包括将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶GoxM10进行A4D、S133E、Q581E或N471E突变的步骤。
根据本发明的改善葡萄糖氧化酶的酸稳定性的方法，所述方法包括：
将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶GoxM10进行A4D、S133E突变的步骤；或将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶GoxM10进行A4D、Q581E突变的步骤；或将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶GoxM10进行A4D、N471E突变的步骤。
本发明提供了上述具有酸稳定性的葡萄糖氧化酶突变体的应用，具体可以应用于食品和饲料领域中。
本发明提供了一种酸稳定性提高的、适合于在食品和饲料领域中应用的葡萄糖氧化酶突变体，能很好的满足食品和饲料等领域中的应用，有着非常广阔的应用前景。
附图说明
图1显示GoxM10和突变体在pH 2.5条件下处理30 min的酸稳定性对比；
图2显示GoxM10和突变体在pH 2.75条件下处理60 min的酸稳定性对比；
图3显示GoxM10和突变体的最适pH。
具体实施方式
试验材料和试剂
1、菌株及载体：表达宿主为Pichia pastoris GS115，表达质粒载体为pPIC9。
2、酶类及其它生化试剂：内切酶/高保真DNA聚合酶和重组酶。
3、大肠杆菌LB培养基（1%蛋白胨、0.5%酵母提取物、1% NaCl，pH自然）。毕赤酵母YPD培养基（1%酵母提取物，2%蛋白胨，2%葡萄糖，pH自然）；BMGY(1%酵母提取物，2%蛋白胨，10%无氨基酵母氮源，0.1%生物素，pH自然)；BMMY（1%酵母提取物，2%蛋白胨，10%无氨基酵母氮源，0.1%生物素，1%甲醇，pH自然）。
说明：以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》（第三版）J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行。
实施例1、重组菌株GS115(pPIC9-goxm10)的制备
1.扩增高热稳定性葡萄糖氧化酶GoxM10的核酸序列goxm10
采用PCR的方法扩增goxm10基因片段，采用双酶切方法获得载体pPIC9核酸片段，通过重组酶将两者连接，获得重组质粒pPIC9-goxm10，并转化毕赤酵母GS115，获得重组毕赤酵母菌株GS115(pPIC9-goxm10)。PCR所用引物如下：
GoxM10-F（SEQID NO: 16, 30bp）：GGTATTGAGGCTTCCTTGTTGACTGACCCA
GoxM10-R（SEQ ID NO: 17, 30bp）：TTGCATGGAGGCGTAGTCAGCCAAAACAGC，
其中，GoxM10-F及GoxM10-R用于扩增GoxM10的基因编码序列；载体pPIC9通过将保存的菌种接到液体LB培养基过夜培养后，质粒提取得到。
扩增结束后，将PCR产物以及提取质粒进行核酸电泳检测，goxm10与载体pPIC9条带大小分别为1746bp、8088bp，将载体用EcoRI和NotI进行酶切后，将PCR产物与酶切产物分别回收纯化。
2.构建重组菌株GS115(pPIC9-goxm10)
将回收的goxm10与pPIC9基因片段通过重组酶进行重组连接，然后将重组产物转化大肠杆菌JM109感受态，并涂布于LB（含100 μg/mL Amp）进行筛选。待测序正确后，利用BglⅡ限制性内切酶将重组质粒pPIC9-goxm10进行酶切，回收酶切产物后电击转化毕赤酵母GS115感受态细胞，得到重组表达菌株GS115(pPIC9-goxm10)。
实施例2、重组突变菌株GS115的制备
1.重组质粒的构建
根据突变体设计突变引物，以质粒pPIC9-goxm10为模板，通过点突变试剂盒引入突变氨基酸，含突变氨基酸的PCR产物用DpnⅠ消化处理以去除模板，消化后的PCR产物转化进入大肠杆菌JM109感受态细胞，并对其进行测序验证，获得含葡萄糖氧化酶突变质粒的大肠杆菌pPIC9-goxm10-A4D、pPIC9-goxm10-S133E，pPIC9-goxm10-N471E，pPIC9-goxm10-Q581E、，pPIC9-goxm10-A4D/S133E，pPIC9-goxm10-A4D/N471E，pPIC9-goxm10-A4D/Q581E，所用引物如下所示：
Gox-A4D-F（SEQ ID No：18），Gox-A4D-R（SEQ ID No：19）；Gox-S133E-F（SEQ IDNo：20），Gox-S133E-R（SEQ ID No：21）；Gox-N471E-F（SEQ ID No：22），
Gox-N471E-R（SEQ ID No：23）；Gox-Q581E-F（SEQ ID No：24），Gox-Q581E-R（SEQID No：25）。
2.重组菌株GS115构建
利用BglⅡ将测序正确的突变质粒进行酶切，回收酶切产物后电击转化毕赤酵母GS115感受态细胞，得到重组表达菌株GS115(ppPIC9-goxm10-A4D，GS115(pPIC9-goxm10-S133E)，GS115(pPIC9-goxm10-N471E)，GS115(pPIC9-goxm10-Q581E)，GS115(pPIC9-goxm10-A4D/S133E)，GS115(pPIC9-goxm10-A4D/N471E)，GS115(pPIC9-goxm10-A4D/Q581E)。
实施例3、葡萄糖氧化酶GoxM10及高酸稳定性和热稳定性突变体酶蛋白的获得
1.GoxM10及突变体的诱导表达
以1%的接种量将上述得到的重组酵母表达菌株接种至50 mL YPD培养基中进行种子液培养，200 rpm，30 ℃培养48 h后，以1%接种量转接至400 mL BMGY培养基中，200 rpm，30 ℃培养48 h，离心收集菌体转接到200 mL BMMY培养基中，每24 h补加1%的甲醇进行诱导表达，共培养72 h。
2.GoxM10及突变体的纯化
将诱导表达后的菌液12000 rpm, 10 min离心，收集上清后用10 kDa的膜包进行浓缩，再用10 mM磷酸氢二钠-柠檬酸缓冲液（pH 6.5）进行透析，然后将透析后的粗酶液用阴离子柱纯化，纯化用A液为10 mM磷酸氢二钠-柠檬酸缓冲液（pH 6.5），B液为A液加1 MNaCl，纯化蛋白，收集洗脱液。
3.GoxM10和突变体的酶学性质测定
（1）GoxM10和突变体的酸稳定性测定
采用两种测定方法评估GoxM10和突变体的pH稳定性。方法一：把相同浓度的GoxM10和突变体酶液置于准确配置的pH 2.5、2.75、3.0、4.0、5.0、6.0、7.0和8.0的0.1 M的磷酸氢二钠-柠檬酸缓冲液，然后于37 ℃保温处理1 h。保温结束后在标准条件下测定其剩余酶活力，以未处理样品的酶活力记为100%。方法二：把相同浓度的GoxM10和突变体酶液置于pH 2.5的0.1 M的磷酸氢二钠-柠檬酸缓冲液，然后于37 ℃保温处理10 min、20 min、30min、40 min、50 min和60 min。保温结束后在标准条件下测定GoxM10及其突变体酶液的相对剩余酶活。如图1所示，pH 2.5条件下处理30 min时，突变体A4D、S133E、N471E、Q581E、A4D/S133E、A4D/N471E、A4D/Q581E的相对剩余酶活力分别为30%、24%、24%、25%、30%、35%、26%，与GoxM10（21%）相比分别提高了约45%、14%、14%、19%、45%、67%、25%。如图2所示，在pH2.75和37 ℃条件下处理60 min，突变体A4D、S133E、N471E、Q581E、A4D/S133E、A4D/N471E、A4D/Q581E的相对剩余酶活力分别为37%、25%、27%、33%、32%、60%、26%，与GoxM10（24%）相比分别提高了约53%、6%、14%、40%、33%、150%、8%。
（2）GoxM10和突变体的最适pH测定
首先，分别配置不同pH梯度（3.0、4.0、5.0、6.0、7.0、8.0）的Gox酶活力测定的底物混合液，然后，在30 ℃下测定GoxM10及其突变体的酶活力，把最适反应pH条件下的酶活力设定为100%，然后计算其余pH条件下的相对酶活力，即可得到GoxM10及其突变体的最适反应pH。如图3示，突变体A4D、S133E、N471E、Q581E、A4D/S133E、A4D/N471E、A4D/Q581E的最适反应pH与GoxM10一致均为6.0。
以上实施例仅用于解释本申请的技术方案，不限定本申请的保护范围。
序列表
<110> 中国农业科学院北京畜牧兽医研究所
<120> 酸稳定性提高的葡萄糖氧化酶GoxM10突变体A4D及其衍生突变体和应用
<160> 25
<170> SIPOSequenceListing 1.0
<210> 1
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Lys Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala
405 410 415
Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 2
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Gly Ile Glu Asp Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Lys Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala
405 410 415
Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 3
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggtattgagg attccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60
atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120
gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180
gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240
gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300
tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360
gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420
gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480
tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540
tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600
gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660
caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720
caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780
agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840
cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900
atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960
ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020
gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080
gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140
agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200
attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260
ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320
ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380
ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440
tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctga cttgagagct 1500
tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560
atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620
ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680
gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740
caataa 1746
<210> 4
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Glu Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Lys Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala
405 410 415
Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 5
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ggtattgagg cttccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60
atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120
gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180
gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240
gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300
tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360
gagaccgtct tcggtaacga aggttggaat tgggatgaag tcgcagctta ctccttgcag 420
gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480
tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540
tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600
gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660
caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720
caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780
agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840
cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900
atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960
ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020
gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080
gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140
agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200
attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260
ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320
ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380
ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440
tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctga cttgagagct 1500
tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560
atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620
ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680
gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740
caataa 1746
<210> 6
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Lys Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala
405 410 415
Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Glu Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 7
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggtattgagg cttccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60
atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120
gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180
gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240
gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300
tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360
gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420
gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480
tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540
tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600
gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660
caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720
caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780
agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840
cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900
atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960
ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020
gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080
gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140
agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200
attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260
ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320
ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380
ggtcaggctg ccgctactca attggctaga gaaatctcta actccggtgc catgcaaact 1440
tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctga cttgagagct 1500
tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560
atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620
ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680
gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740
caataa 1746
<210> 8
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Lys Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala
405 410 415
Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp
565 570 575
Tyr Ala Ser Met Glu
580
<210> 9
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggtattgagg cttccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60
atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120
gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180
gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240
gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300
tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360
gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420
gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480
tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540
tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600
gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660
caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720
caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780
agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840
cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900
atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960
ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020
gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080
gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140
agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200
attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260
ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320
ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380
ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440
tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctga cttgagagct 1500
tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560
atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620
ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680
gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740
caagaa 1746
<210> 10
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Gly Ile Glu Asp Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Glu Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Lys Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala
405 410 415
Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 11
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ggtattgagg attccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60
atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120
gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180
gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240
gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300
tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360
gagaccgtct tcggtaacga aggttggaat tgggatgaag tcgcagctta ctccttgcag 420
gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480
tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540
tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600
gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660
caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720
caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780
agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840
cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900
atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960
ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020
gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080
gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140
agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200
attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260
ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320
ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380
ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440
tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctga cttgagagct 1500
tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560
atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620
ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680
gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740
caataa 1746
<210> 12
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Gly Ile Glu Asp Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Lys Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala
405 410 415
Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Glu Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 13
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggtattgagg attccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60
atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120
gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180
gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240
gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300
tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360
gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420
gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480
tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540
tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600
gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660
caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720
caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780
agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840
cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900
atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960
ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020
gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080
gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140
agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200
attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260
ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320
ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380
ggtcaggctg ccgctactca attggctaga gaaatctcta actccggtgc catgcaaact 1440
tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctga cttgagagct 1500
tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560
atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620
ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680
gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740
caataa 1746
<210> 14
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Gly Ile Glu Asp Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Lys Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala
405 410 415
Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp
565 570 575
Tyr Ala Ser Met Glu
580
<210> 15
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ggtattgagg attccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60
atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120
gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180
gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240
gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300
tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360
gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420
gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480
tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540
tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600
gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660
caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720
caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780
agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840
cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900
atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960
ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020
gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080
gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140
agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200
attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260
ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320
ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380
ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440
tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctga cttgagagct 1500
tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560
atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620
ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680
gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740
caagaa 1746
<210> 16
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ggtattgagg cttccttgtt gactgaccca 30
<210> 17
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ttgcatggag gcgtagtcag ccaaaacagc 30
<210> 18
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ttcggtattg aggattcctt gttgactgac cca 33
<210> 19
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
agtcaacaag gaatcctcaa taccgaattc ta 32
<210> 20
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tggaattggg atgaagtcgc agcttactcc tt 32
<210> 21
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gtaagctgcg acttcatccc aattccaacc ttc 33
<210> 22
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
caattggcta gagaaatctc taactccggt gc 32
<210> 23
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ggagttagag atttctctag ccaattgagt ag 32
<210> 24
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
tacgcctcca tggaataagc ggccgcgaat ta 32
<210> 25
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
cgcggccgct tattccatgg aggcgtagtc ag 32
Claims (10)
1.酸稳定性提高的葡萄糖氧化酶GoxM10突变体，其特征在于，具有将氨基酸序列如SEQID NO:1所示的葡萄糖氧化酶GoxM10进行A4D突变的氨基酸序列。
2.根据权利要求1所述的酸稳定性提高的葡萄糖氧化酶GoxM10突变体，其特征在于，具有将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶GoxM10进行A4D/S133E突变的氨基酸序列。
3.根据权利要求1所述的酸稳定性提高的葡萄糖氧化酶GoxM10突变体，其特征在于，具有将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶GoxM10进行A4D/Q581E突变的氨基酸序列。
4.根据权利要求1所述的酸稳定性提高的葡萄糖氧化酶GoxM10突变体，其特征在于，具有将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶GoxM10进行A4D/N471E突变的氨基酸序列。
5.一种葡萄糖氧化酶基因，其特征在于，所述基因编码权利要求1-4中任意一项所述酸稳定性提高的葡萄糖氧化酶GoxM10突变体。
6.包含权利要求5所述葡萄糖氧化酶基因的重组载体。
7.包含权利要求5所述葡萄糖氧化酶基因的重组菌株。
8.一种制备酸稳定性提高的葡萄糖氧化酶的方法，其特征在于，所述方法包括以下步骤：
构建包含编码权利要求1-4中任意一项所述酸稳定性提高的葡萄糖氧化酶GoxM10突变体的基因的重组载体；
将所述重组载体导入宿主细胞；
诱导表达、并分离葡萄糖氧化酶。
9.一种改善葡萄糖氧化酶的酸稳定性的方法，其特征在于，所述方法包括将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶GoxM10进行A4D突变的步骤。
10.根据权利要求9所述的改善葡萄糖氧化酶的酸稳定性的方法，其特征在于，所述方法包括：
将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶GoxM10进行A4D/S133E突变的步骤；或
将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶GoxM10进行A4D/Q581E突变的步骤；或
将氨基酸序列如SEQ IDNO:1所示的葡萄糖氧化酶GoxM10进行A4D/N471E突变的步骤。
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