CN113862237A - 提高植酸酶的热稳定性的方法及突变体、基因和应用 - Google Patents

提高植酸酶的热稳定性的方法及突变体、基因和应用 Download PDF

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CN113862237A
CN113862237A CN202111454117.3A CN202111454117A CN113862237A CN 113862237 A CN113862237 A CN 113862237A CN 202111454117 A CN202111454117 A CN 202111454117A CN 113862237 A CN113862237 A CN 113862237A
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涂涛
黄火清
王倩
姚斌
罗会颖
王苑
柏映国
苏小运
王亚茹
张�杰
秦星
王晓璐
张红莲
于会民
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Abstract

本发明涉及基因工程领域，具体涉及提高植酸酶的热稳定性的方法及突变体、基因和应用。野生型植酸酶经定点突变后得到热稳定性提高的突变体。本发明的植酸酶突变体具有良好的酶学性质，可以应用于能源、食品和饲料等行业。

Description

提高植酸酶的热稳定性的方法及突变体、基因和应用
技术领域
本发明涉及基因工程领域，具体涉及提高植酸酶的热稳定性的方法及突变体、基因和应用。
背景技术
植酸酶（Phytase）,即肌醇六磷酸磷酸水解酶（myo-inositol hexakisphosphatephosphohydrolases）是一类能够催化植酸盐水解为肌醇、肌醇磷酸酯和无机磷酸盐的磷酸酶。植酸酶在食品加工、环境保护、生物燃料生产等多种行业中存在广泛应用价值，目前最常见的则是作为饲料添加剂。在动物饲料中添加植酸酶来降解植酸而释放无机磷、矿物质等，去除了植酸的抗营养作用，显著增加磷的利用率和提高动物的生产性能，同时降低了磷对环境的污染。
由于饲料制粒过程需要短暂高温，因此饲用植酸酶需要具有良好的热稳定性才能发挥其功能，这是制约植酸酶工业化应用的一大难点。N-糖基化是真核生物中最普遍存在的翻译后修饰之一，对蛋白质的结构和功能有重要影响。这种修饰包括在特征序列NXT/S中将N-聚糖连接到天冬酰胺上，其中X表示除脯氨酸以外的任意氨基酸。然而，由于N-糖基化修饰的环境依赖性，N-糖基化并不总是改善酶的热稳定性。
发明内容
为了进一步优化来源于Yersinia intermedia的植酸酶Y4的酶学特性，提出并完成了本发明。
本发明的目的是提供热稳定性提高的植酸酶突变体。
本发明的再一目的是提供上述植酸酶突变体的编码基因。
本发明的再一目的是提供包含上述植酸酶突变体编码基因的重组载体。
本发明的再一目的是提供包含上述植酸酶突变体编码基因的重组菌株。
本发明的再一目的是提供一种制备热稳定性提高的植酸酶的方法。
本发明的再一目的是提供上述植酸酶突变体的应用。
本发明对野生型植酸酶Y4进行突变，得到热稳定性提高的植酸酶突变体，其中，野生型植酸酶Y4的氨基酸序列如SEQ ID NO:1所示。
根据本发明的具体实施方式，将野生型植酸酶Y4的第78-80位氨基酸由AAG突变为NQT得到突变体AAG78NQT。第111-113位氨基酸由GCG突变为NCT得到突变体GCG111NCT。第143-145位氨基酸由QTH突变为NVT得到突变体QTH143NVT。第171-173位氨基酸由GEI突变为NTT得到突变体GEI171NTT。第189-191位氨基酸由GKT突变为NES得到突变体GKT189NES。第210-212位氨基酸由KVS突变为NVS得到突变体KVS210NVS。第245-247位氨基酸由GED突变为NES得到突变体GED245NES。第324-326位氨基酸由NWQ突变为NWT得到突变体NWQ324NWT。
根据本发明的具体实施方式，在所述突变体AAG78NQT基础上进一步进行突变，第111-113位氨基酸由GCG突变为NCT得到突变体78/11；第143-145位氨基酸由QTH突变为NVT得到突变体78/143；第171-173位氨基酸由GEI突变为NTT得到突变体78/171；第189-191位氨基酸由GKT突变为NES得到突变体78/189；第210-212位氨基酸由KVS突变为NVS得到突变体78/210；第245-247位氨基酸由GED突变为NES，得到突变体78/245；第324-326位氨基酸由NWQ突变为NWT得到突变体78/324。
根据本发明的具体实施方式，进一步，在所述突变体78/245基础上进行突变，第111-113位氨基酸由GCG突变为NCT得到突变体78/111/245；第143-145位氨基酸由QTH突变为NVT得到突变体78/143/245；第171-173位氨基酸由GEI突变为NTT得到突变体78/171/245；第189-191位氨基酸由GKT突变为NES得到突变体78/189/245；第210-212位氨基酸由KVS突变为NVS得到突变体78/210/245；第324-326位氨基酸由NWQ突变为NWT得到突变体78/245/324。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体AAG78NQT的氨基酸序列SEQ ID NO:2所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体GCG111NCT的氨基酸序列SEQ ID NO:3所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体QTH143NVT的氨基酸序列SEQ ID NO:4所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体GEI171NTT的氨基酸序列SEQ ID NO:5所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体GKT189NES的氨基酸序列SEQ ID NO:6所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体KVS210NVS的氨基酸序列SEQ ID NO:7所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体GED245NES的氨基酸序列SEQ ID NO:8所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体NWQ324NW的氨基酸序列SEQ ID NO:9所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体78/111的氨基酸序列SEQ ID NO:10所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体78/143的氨基酸序列SEQ ID NO:11所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体78/171的氨基酸序列SEQ ID NO:12所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体78/189的氨基酸序列SEQ ID NO:13所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体78/210的氨基酸序列SEQ ID NO:14所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体78/245的氨基酸序列SEQ ID NO:15所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体78/324的氨基酸序列SEQ ID NO:16所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体78/111/245的氨基酸序列SEQ ID NO:17所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体78/143/245的氨基酸序列SEQ ID NO:18所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体78/171/245的氨基酸序列SEQ ID NO:19所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体78/189/245的氨基酸序列SEQ ID NO:20所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体78/210/245的氨基酸序列SEQ ID NO:21所示。
根据本发明的具体实施方式，所述野生型植酸酶Y4的突变体78/245/324的氨基酸序列SEQ ID NO:22所示。
本发明提供了编码上述野生型植酸酶Y4突变体的基因。
根据本发明的具体实施方式，植酸酶Y4的基因序列如SEQ ID NO：23所示。
根据本发明的提高植酸酶的热稳定性的方法包括以下步骤：
将野生型植酸酶Y4进行单点突变，将第78-80位氨基酸由AAG突变为NQT，或第111-113位氨基酸由GCG突变为NCT，或第143-145位氨基酸由QTH突变为NVT，或第171-173位氨基酸由GEI突变为NTT，或第189-191位氨基酸由GKT突变为NES，或第210-212位氨基酸由KVS突变为NVS，或第245-247位氨基酸由GED突变为NES，或第324-326位氨基酸由NWQ突变为NWT。
根据本发明的提高植酸酶的热稳定性的方法，包括将野生型植酸酶Y4进行双点突变的步骤，在将野生型植酸酶Y4的第78-80位氨基酸由AAG突变为NQT的基础上，
将第111-113位氨基酸由GCG突变为NCT，或第143-145位氨基酸由QTH突变为NVT，或第171-173位氨基酸由GEI突变为NTT，或第189-191位氨基酸由GKT突变为NES，或第210-212位氨基酸由KVS突变为NVS，或第245-247位氨基酸由GED突变为NES，或第324-326位氨基酸由NWQ突变为NWT。
根据本发明的提高植酸酶的热稳定性的方法，将野生型植酸酶Y4进行三点突变，在将野生型植酸酶Y4的第78-80位氨基酸由AAG突变为NQT、第245-247位氨基酸由GED突变为NES的基础上，将第111-113位氨基酸由GCG突变为NCT，或第143-145位氨基酸由QTH突变为NVT，或第171-173位氨基酸由GEI突变为NTT，或第189-191位氨基酸由GKT突变为NES，或第210-212位氨基酸由KVS突变为NVS，或第324-326位氨基酸由NWQ突变为NWT。
本发明提供了包含上述植酸酶突变体的编码基因的重组载体。
本发明还提供了包含上述植酸酶突变体的编码基因的重组菌株。
根据本发明的具体实施方式，制备热稳定性提高的植酸酶的方法如下所述：
(1)用含有植酸酶突变体的编码基因的重组载体转化宿主细胞，得到重组菌株；
(2)培养重组菌株，诱导植酸酶表达；
(3)回收并纯化所表达的植酸酶。
本发明的有益效果：
本发明主要是通过在植酸酶的结构中引入N-糖基化修饰来达到提高酶热稳定性的目的，研究发现引入N-糖基化后显著提高了酶的热稳定性。本发明对野生型植酸酶Y4进行突变，植酸酶突变体与野生型植酸酶相比，植酸酶热稳定性增强，其中最佳突变体78/111/245在100℃处理5 min后仍保留85%左右的活性，而野生型酶早已失活。本发明克服了现有技术的不足，提供了具有高热稳定性的适合于在能源、食品和饲料等领域中应用的植酸酶突变体。因此，本发明提供的植酸酶突变体可以很好的应用于能源、食品和饲料行业中，有广阔的应用前景。
附图说明
图1显示野生型植酸酶Y4与各单点突变体的比活对比；
图2显示野生型植酸酶Y4与各单点突变体的最适温度对比；
图3显示野生型植酸酶Y4与各单点突变体在65℃下处理的热稳定性对比；
图4显示野生型植酸酶Y4与各双点突变体在70℃下处理的热稳定性对比；
图5显示野生型植酸酶Y4与各三点突变体在100℃下处理的热稳定性对比。
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pPICZαA。
2、酶类及其它生化试剂：限制性内切酶购自TaKaRa公司和New England Biolabs(NEB) 公司。其它都为国产试剂（均可从普通生化试剂公司购买得到）。
3、培养基：
（1）大肠杆菌培养基低盐LB（LLB）（1 %蛋白胨、0.5 %酵母提取物、0.5 % NaCl，pH自然）。
（2）毕赤酵母培养基YPD（1%酵母提取物，2%蛋白胨，2%葡萄糖，pH自然）。
（3）BMGY 培养基(1%酵母提取物，2%蛋白胨，1%甘油，1.34% YNB，0.00004%生物素，pH自然)。
（4）BMMY培养基（1%酵母提取物，2%蛋白胨，0.5%甲醇，1.34% YNB，0.00004%生物素，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、植酸酶的定点突变
以来源于Yersinia intermedia的植酸酶Y4作为母本，将氨基酸序列如SEQ IDNO:1所示的野生型植酸酶Y4进行定点突变，获得所需突变体。具体地，首先，将野生型植酸酶Y4的第78-80位氨基酸由AAG突变为NQT得到突变体AAG78NQT。第111-113位氨基酸由GCG突变为NCT得到突变体GCG111NCT。第143-145位氨基酸由QTH突变为NVT得到突变体QTH143NVT。第171-173位氨基酸由GEI突变为NTT得到突变体GEI171NTT。第189-191位氨基酸由GKT突变为NES得到突变体GKT189NES。第210-212位氨基酸由KVS突变为NVS得到突变体KVS210NVS。第245-247位氨基酸由GED突变为NES得到突变体GED245NES。第324-326位氨基酸由NWQ突变为NWT得到突变体NWQ324NWT。
然后，在AAG78NQT基础上进行突变，第111-113位氨基酸由GCG突变为NCT得到突变体78/11；第143-145位氨基酸由QTH突变为NVT得到突变体78/143；第171-173位氨基酸由GEI突变为NTT得到突变体78/171；第189-191位氨基酸由GKT突变为NES得到突变体78/189；第210-212位氨基酸由KVS突变为NVS得到突变体78/210；第245-247位氨基酸由GED突变为NES，得到突变体78/245；第324-326位氨基酸由NWQ突变为NWT得到突变体78/324。
进一步，在78/245基础上进行突变，第111-113位氨基酸由GCG突变为NCT得到突变体78/111/245；第143-145位氨基酸由QTH突变为NVT得到突变体78/143/245；第171-173位氨基酸由GEI突变为NTT得到突变体78/171/245；第189-191位氨基酸由GKT突变为NES得到突变体78/189/245；第210-212位氨基酸由KVS突变为NVS得到突变体78/210/245；第324-326位氨基酸由NWQ突变为NWT得到突变体78/245/324。
定点突变参照Fast Mutagenesis System（北京全式金生物技术有限公司）说明书进行，引物如下表所示，通过PCR的方法进行相应突变体的构建。
表1 定点突变所需的引物
Figure 417843DEST_PATH_IMAGE001
实施例2 植酸酶工程菌株的构建
（1）表达载体的构建及在酵母的表达
以质粒pICZαA-y4为模板，使用含有相应突变位点的引物进行PCR扩增。之后对PCR扩增产物进行1%琼脂糖凝胶电泳分析，若条带大小与理论值一致，则表明PCR反应成功获得了目的产物。为了消除模板质粒对后续实验的干扰，根据模板质粒与PCR产物的甲基化差异，向PCR体系中加入1 µL限制性内切酶Dpn I，37℃酶切1~2 h。之后取10 µL产物转化大肠杆菌DMT感受态细胞。待测序正确后，提取重组质粒，利用限制性内切酶Pme I进行线性化，产物纯化回收并电击转化毕赤酵母GS115感受态细胞，获得毕赤酵母重组表达菌株。
实施例3 野生型及突变体植酸酶的制备
（1）蛋白的诱导表达
将得到的重组表达菌株接种至YPD培养基中进行种子培养，200 rpm，30℃培养48h后，以1%接种量转接至BMGY培养基中，200 rpm，30℃培养48 h。之后4500 rpm离心5 min，弃上清，收集菌体并加入含有0.5%甲醇的BMMY培养基进行诱导表达，每12 h补加0.5%甲醇，共诱导48 h。
（2）蛋白的纯化
将诱导表达后的菌液12000 rpm离心10 min，收集上清进行浓缩，再用20 mM pH8.0 Tris-HCl进行透析。然后将透析后的酶液进行阴离子交换层析，A液为20 mM pH 8.0Tris-HCl，B液为A液加1 M NaCl，纯化蛋白，收集洗脱液，进行SDS-PAGE分析。
实施例4 野生型及突变体植酸酶的性质测定
（1）植酸酶活性测定
将酶液用0.1 mol/L含有0.05% BSA和0.05% Triton X-100的pH 5.5 HAc-NaAc缓冲液进行稀释，将100 µL稀释后的酶液加入到900 µL植酸钠底物(用0.1 mol/L的pH 5.5的HAc-NaAc缓冲液配制)中，在37℃反应10 min，加入1 mL 10%(W/V)TCA终止反应，最后加入1 mL显色液[1%(W/V)四水合钼酸铵，3.2%(V/V)浓硫酸，7.32%(W/V)硫酸亚铁]进行显色。对照则是在加酶液之前先加入TCA混匀使酶变性，其他相同。显色后，在700 nm光吸收下测定OD值，计算酶活。
将纯化后的野生型和突变体在pH 5.5、37℃下进行酶促反应以测定其酶活性。如图1所示，野生型的酶比活为2077 U/mg，突变体KVS210NVS的比活为1757 U/mg，相对于野生型下降了18%，突变体AAG78NQT、GCG111NCT、QTH143NVT的比活与野生型相当，而突变体GEI171NTT、GKT189NES、GED245NES和NWQ324NWT的比活相对于野生型分别提高了12.5%、24.0%、14.0%和11.7%。
（2）最适温度测定
在0.1mol/L pH 5.5 HAc-NaAc缓冲液条件下，分别在不同温度（20、25、30、37、40、45、50、55、60、65和70℃）下对野生型和突变体的酶活性进行测定来确定最适温度，最适温度对应活性定义为100%，依次计算其余温度下的剩余酶活。如图2所示，除突变体GEI171NTT的最适温度为50℃以外，野生型和其余突变体的最适温度都为55℃，引入糖基化修饰基本未影响植酸酶Y4的最适温度。
（3）热稳定性测定
将纯化所得蛋白用0.1 mol/L含有0.05% BSA和0.05% Triton X-100的pH 5.5HAc-NaAc缓冲液稀释至合适倍数后，取100 μL于1.5 mL EP管中，分别在不同温度下（65、100℃）保温0、2、5、10、15和30 min，之后测定对应酶活，以保温0 min的活性为100%，计算不同保温时间下的剩余酶活。如图3所示，野生型在65℃下处理5 min后，剩余酶活为21.6%；而突变体AAG78NQT、GCG111NCT、QTH143NVT、GEI171NTT、GKT189NES、KVS210NVS、GED245NES和NWQ324NWT在65℃下处理5 min之后，剩余酶活分别为71.3%、68.1%、56.8%、52.5%、59.4%、58.3%、68.2%和59.0%。如图4所示，野生型在70℃下处理5 min后，剩余酶活为11.1%；而突变体78/111、78/143、78/171、78/189、78/210、78/245和78/324的剩余酶活分别为59.6%、52.1%、48.2%、54.1%、47.2%、69.9%和57.1%。如图5所示，野生型在100℃下处理2 min后，酶活基本消失；而突变体78/111/245、78/143/245、78/171/245、78/189/245、78/210/245和78/245/324在100℃下处理5 min后，剩余酶活分别为85.5%、71.5%、75.0%、82.2%、69.9%和68.0%。
以上实施例仅用于理解本申请的技术方案，不限制本申请的保护范围。
序列表
<110> 中国农业科学院北京畜牧兽医研究所
<120> 提高植酸酶的热稳定性的方法及突变体、基因和应用
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Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
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Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
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Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Ala Ala Gly
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Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
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Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
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Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
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Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
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Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
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His Ile
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<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
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Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
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Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 3
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Ala Ala Gly
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Asn Cys
100 105 110
Thr Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 4
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Ala Ala Gly
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Asn Val
130 135 140
Thr Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 5
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Ala Ala Gly
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Asn Thr Thr Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 6
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Ala Ala Gly
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Asn Glu Ser Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 7
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Ala Ala Gly
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Asn Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 8
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Ala Ala Gly
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Asn Glu Ser Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 9
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Ala Ala Gly
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Thr Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 10
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Asn Cys
100 105 110
Thr Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 11
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Asn Val
130 135 140
Thr Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 12
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Asn Thr Thr Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 13
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Asn Glu Ser Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 14
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Asn Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 15
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Asn Glu Ser Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 16
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Gly Glu Asp Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Thr Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 17
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Asn Cys
100 105 110
Thr Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Asn Glu Ser Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 18
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Asn Val
130 135 140
Thr Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Asn Glu Ser Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 19
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Asn Thr Thr Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Asn Glu Ser Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 20
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Asn Glu Ser Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Asn Glu Ser Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 21
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Asn Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Asn Glu Ser Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 22
<211> 418
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Ala Ala Pro Val Ala Ile Gln Pro Thr Gly Tyr Thr Leu Glu Arg Val
1 5 10 15
Val Ile Leu Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln
20 25 30
Leu Met Asn Asp Val Thr Pro Asp Thr Trp Pro Gln Trp Pro Val Ala
35 40 45
Ala Gly Tyr Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly
50 55 60
Gly Phe Tyr Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Asn Gln Thr
65 70 75 80
Cys Pro Thr Asp Ala Val Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg
85 90 95
Thr Arg Leu Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys
100 105 110
Gly Leu Lys Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu
115 120 125
Phe His Pro Val Asp Ala Gly Val Cys Lys Leu Asp Ser Thr Gln Thr
130 135 140
His Lys Ala Val Glu Glu Arg Leu Gly Gly Pro Leu Ser Glu Leu Ser
145 150 155 160
Lys Arg Tyr Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe
165 170 175
Ala Ala Ser Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Thr Cys
180 185 190
Asp Phe Ala Asn Phe Ala Ala Asn Lys Ile Thr Val Asn Lys Pro Gly
195 200 205
Thr Lys Val Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly
210 215 220
Glu Ile Phe Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp
225 230 235 240
His Arg Leu Thr Asn Glu Ser Asn Trp Ile Ser Leu Leu Ser Leu His
245 250 255
Asn Ala Gln Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His
260 265 270
Lys Gly Thr Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Val Leu Gln
275 280 285
Arg Asp Ala Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Ile
290 295 300
Leu Phe Leu Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met
305 310 315 320
Leu Gly Ala Asn Trp Thr Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro
325 330 335
Gly Gly Gly Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln
340 345 350
Arg Tyr Val Ala Val Lys Met Phe Tyr Gln Thr Met Gly Gln Leu Arg
355 360 365
Asn Ala Glu Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Arg Val Pro
370 375 380
Val Ala Ile Asp Gly Cys Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln
385 390 395 400
Leu Asp Thr Phe Gln Lys Lys Val Ala Gln Ala Ile Glu Pro Ala Cys
405 410 415
His Ile
<210> 23
<211> 1257
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gctgctccag tcgctatcca acctactggt tacactcttg agagagttgt catcttgtct 60
agacatggtg ttagatcccc aactaagcag acccaattga tgaacgatgt gacacctgac 120
acgtggcctc aatggccagt tgcagctggt tacttgacac caagaggtgc tcagttggtt 180
actttgatgg gtggattcta cggtgactat ttcagatccc aaggattgct tgctgccggc 240
tgtcctactg atgctgtcat ctacgcacaa gctgacgttg atcaaagaac tcgtttgacc 300
ggacaagcat tcttggatgg tatcgctcca ggatgtggct tgaaagttca ctaccaggct 360
gatttgaaga aggttgatcc actgttccac cctgttgatg caggtgtttg taagcttgac 420
tctactcaaa cccacaaagc tgttgaagag agattgggtg gtccattgag cgaactttcg 480
aagagatacg ccaaaccttt tgcacaaatg ggagagatcc tgaacttcgc agcgtcacct 540
tactgtaaga gtttgcaaca gcaaggtaag acttgcgact ttgccaactt cgctgccaac 600
aagatcactg tcaacaagcc tggaacgaaa gtatccttgt ctggtccatt ggctctgtct 660
tccactcttg gagaaatctt cttgctgcaa aactctcaag ctatgccaga tgttgcctgg 720
cacagattga ccggtgagga caactggatt tctttgctct ccttacacaa tgcccaattc 780
gatctgatgg caaagactcc ttacattgct agacacaaag gaactccctt gcttcagcaa 840
atcgaaactg ctttggtcct ccaaagggac gcccagggtc aaactttgcc attgtctcct 900
cagaccaaga tcctgttctt gggtggacac gatactaaca tcgcaaacat cgctgggatg 960
ttgggtgcta actggcaact tccacagcaa ccagacaaca ccccacctgg cggtggtcta 1020
gtcttcgagt tgtggcaaaa ccctgacaac caccagagat acgttgctgt aaagatgttc 1080
tatcagacta tgggacaatt gcgtaacgca gagaagttgg atttgaagaa caacccagcc 1140
ggtagggttc ctgtcgcaat tgacggttgt gagaactctg gagatgacaa gttgtgccag 1200
cttgatactt tccagaagaa ggttgctcag gccatagagc cagcttgtca catctaa 1257

Claims (9)

1.一种热稳定性提高的植酸酶突变体，其特征在于，将氨基酸序列如SEQ ID NO:1所示的野生型植酸酶Y4进行如下突变而得，
第78-80位氨基酸由AAG突变为NQT得到突变体AAG78NQT；
第111-113位氨基酸由GCG突变为NCT得到突变体GCG111NCT；
第143-145位氨基酸由QTH突变为NVT得到突变体QTH143NVT；
第171-173位氨基酸由GEI突变为NTT得到突变体GEI171NTT；
第189-191位氨基酸由GKT突变为NES得到突变体GKT189NES；
第210-212位氨基酸由KVS突变为NVS得到突变体KVS210NVS；
第245-247位氨基酸由GED突变为NES得到突变体GED245NES；或
第324-326位氨基酸由NWQ突变为NWT得到突变体NWQ324NWT。
2.一种热稳定性提高的植酸酶突变体，其特征在于，将权利要求1所得突变体AAG78NQT进行以下突变而得，
第111-113位氨基酸由GCG突变为NCT得到突变体78/11；
第143-145位氨基酸由QTH突变为NVT得到突变体78/143；
第171-173位氨基酸由GEI突变为NTT得到突变体78/171；
第189-191位氨基酸由GKT突变为NES得到突变体78/189；
第210-212位氨基酸由KVS突变为NVS得到突变体78/210；
第245-247位氨基酸由GED突变为NES，得到突变体78/245；或
第324-326位氨基酸由NWQ突变为NWT得到突变体78/324。
3.一种热稳定性提高的植酸酶突变体，其特征在于，将权利要求2所得突变体78/245进行以下突变而得，
第111-113位氨基酸由GCG突变为NCT得到突变体78/111/245；
第143-145位氨基酸由QTH突变为NVT得到突变体78/143/245；
第171-173位氨基酸由GEI突变为NTT得到突变体78/171/245；
第189-191位氨基酸由GKT突变为NES得到突变体78/189/245；
第210-212位氨基酸由KVS突变为NVS得到突变体78/210/245；或
第324-326位氨基酸由NWQ突变为NWT得到突变体78/245/324。
4.一种提高植酸酶的热稳定性的方法，其特征在于，所述方法包括以下步骤：
将野生型植酸酶Y4进行以下突变，
第78-80位氨基酸由AAG突变为NQT；
第111-113位氨基酸由GCG突变为NCT；
第143-145位氨基酸由QTH突变为NVT；
第171-173位氨基酸由GEI突变为NTT；
第189-191位氨基酸由GKT突变为NES；
第210-212位氨基酸由KVS突变为NVS；
第245-247位氨基酸由GED突变为NES；或
第324-326位氨基酸由NWQ突变为NWT；
其中，所述野生型植酸酶Y4的氨基酸序列如SEQ ID NO:1所示。
5.根据权利要求4所述的提高植酸酶的热稳定性的方法，其特征在于，当将野生型植酸酶Y4进的第78-80位氨基酸由AAG突变为NQT时，所述方法进包括以下步骤：
第111-113位氨基酸由GCG突变为NCT；
第143-145位氨基酸由QTH突变为NVT；
第171-173位氨基酸由GEI突变为NTT；
第189-191位氨基酸由GKT突变为NES；
第210-212位氨基酸由KVS突变为NVS；
第245-247位氨基酸由GED突变为NES；或
第324-326位氨基酸由NWQ突变为NWT。
6.根据权利要求5所述的提高植酸酶的热稳定性的方法，其特征在于，当将第245-247位氨基酸由GED突变为NES时，所述方法进包括以下步骤：
第111-113位氨基酸由GCG突变为NCT；
第143-145位氨基酸由QTH突变为NVT；
第171-173位氨基酸由GEI突变为NTT；
第189-191位氨基酸由GKT突变为NES；
第210-212位氨基酸由KVS突变为NVS；或
第324-326位氨基酸由NWQ突变为NWT。
7.植酸酶突变体基因，其特征在于，编码权利要求1-3中任意一项所述的热稳定性提高的植酸酶突变体。
8.制备热稳定性提高的植酸酶的方法，其特征在于，包括以下步骤：
(1)用包含编码权利要求1-3任意一项所述的热稳定性提高的植酸酶突变体的基因的重组载体转化宿主细胞，得重组菌株；
(2)培养重组菌株，诱导表达植酸酶；
(3)回收并纯化所表达的植酸酶。
9.权利要求1-3任意一项所述的热稳定性提高的植酸酶突变体用于水解植酸盐的应用。
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CN114807087A (zh) * 2022-06-28 2022-07-29 中国农业科学院北京畜牧兽医研究所 一种提高植酸酶热稳定性的方法及突变体和应用
CN114807088A (zh) * 2022-06-28 2022-07-29 中国农业科学院北京畜牧兽医研究所 一种提高植酸酶热稳定性的方法及突变体APPAmut6和应用
CN114807088B (zh) * 2022-06-28 2022-09-27 中国农业科学院北京畜牧兽医研究所 一种提高植酸酶热稳定性的方法及突变体APPAmut6和应用
CN114807089A (zh) * 2022-06-29 2022-07-29 中国农业科学院北京畜牧兽医研究所 一种提高植酸酶热稳定性的方法及突变体APPAmut7和应用
CN114807089B (zh) * 2022-06-29 2022-09-27 中国农业科学院北京畜牧兽医研究所 一种提高植酸酶热稳定性的方法及突变体APPAmut7和应用

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