CN110846297B

CN110846297B - 一种多功能融合酶和多功能融合酶真核表达载体及其构建方法

Info

Publication number: CN110846297B
Application number: CN201911170441.5A
Authority: CN
Inventors: 张献伟; 莫健新; 王豪强; 李国玲; 阮晓芳; 钟翠丽; 黄广燕; 蔡更元; 吴珍芳
Original assignee: South China Agricultural University; Wens Foodstuff Group Co Ltd
Current assignee: South China Agricultural University; Wens Foodstuff Group Co Ltd
Priority date: 2019-11-26
Filing date: 2019-11-26
Publication date: 2023-04-07
Anticipated expiration: 2039-11-26
Also published as: CN110846297A

Abstract

本发明公开了一种多功能融合酶和多功能融合酶真核表达载体及其构建方法，该多功能融合酶可以表达木聚糖酶、植酸酶、果胶酶，葡聚糖酶和纤维素酶，将其用于后期制备相应的转基因动物，该动物将自身分泌这些酶，起到消化饲料中木聚糖、植酸、果胶、葡聚糖酶和纤维素等抗营养因子，达到提高饲料利用率，减少污染排放的功效。同时，解决刚性肽介导两个以上融合蛋白共表达时相互干扰问题；解决2A连接肽多基因共表达效率低的问题，及多基因共表达中2A多肽残基对上游酶蛋白干扰的问题，达到多基因高效共表达的目的。

Description

一种多功能融合酶和多功能融合酶真核表达载体及其构建方法

技术领域

本发明涉及生物技术领域，特别涉及一种多功能融合酶和多功能融合酶真核表达载体及其构建方法。

背景技术

重组融合蛋白的构建需要两个因素：组成蛋白和连接肽。组成蛋白是根据所需融合蛋白产物的功能来进行选择的，在大多数情况下是相对简单的。而选择合适的连接肽却较困难，且在融合蛋白的设计过程中容易被忽略。若功能域不用连接肽直接进行融合会导致一些不良结果，如融合蛋白的错误折叠，产量低或者活性受损等。因此，连接肽的理性设计和选择对于融合蛋白的构建十分重要。

柔性连接肽因其灵活的伸展与弯曲性更适用于靶点不同、自身无相互作用的两种蛋白的融合。柔性连接肽一般由分子量小的非极性氨基酸(如甘氨酸)或者极性氨基酸(如丝氨酸或苏氨酸)组成。最典型的一个例子就是由Huston等人提出的(GGGGS)n(一般n≤6)序列，通过调整重复数n，优化GS连接肽的长度使得功能域适当分开或者保持域间的作用。随着柔性连接肽的广泛应用，一些问题随之而来。柔软的连接肽使得两端的功能蛋白可以随意游动，当形成并肩靠拢的构象时，这种“肩并肩”的结构使得融合蛋白整体紧凑缠绕容易形成二聚体，靠拢挤压的构象也使得活性位点可能被缠绕或遮蔽，导致活性的下降。

2A自剪切肽，其上游基因带着一个2A尾巴残基，下游基因带一个p氨酸残基，对蛋白活性存在潜在不良影响，部分蛋白结构也会影响2A自剪切。因此，2A介导的多基因融合表达存在共表达效率不高，多基因融合酶的首尾基因表达不一致的问题。

刚性连接肽使用一种成α-螺旋结构的连接肽，其刚性结构有效的控制了两端功能蛋白的相对位置和距离，充分的分隔开不同的结构域，使其相互影响达到最小。最常用的一种刚性连接肽氨基酸序列为EAAAK，其中的Glu(E)-…Lys+(K)形成的盐桥使这种螺旋具有稳定的二级结构，可以通过控制氨基酸排列来有效控制两端蛋白的距离。

连接肽最重要的指标是氨基酸链的长度，刘磊等在对白蛋白与干扰素融合蛋白的研究中发现，不同长度的GGGGS连接肽对融合蛋白的表达量和活性有着不同的影响，并不是连接肽越长表达量就越高。

刚性肽和柔性肽的缺点，仅用于两个功能蛋白的组合，不能用于2个以上蛋白的组合，在多功能酶构建时单独使用刚性态或柔性肽或2A都会影响其中一个或多个的基因表达或其生物活性。

由于饲料中含有大量的抗营养因子，而在饲料中添加这些抗营养因子的酶，不仅成本高，而且饲料加工、储存过程中酶活不稳定，容易失活，也增加了饲料的生产成本和储存成本。若是动物自身可以分泌这些酶，将饲料中的抗营养因子经自身分泌的酶进行消化吸收，不仅可以降低饲料的生产成本，还可以提高饲料的利用率，更可以减少饲料中未消化的氮、磷及其组成的有机化合物的污染排放。

发明内容

本公开提供一种多功能融合酶和多功能融合酶真核表达载体及其构建方法，该多功能融合酶可以表达木聚糖酶、植酸酶、果胶酶，葡聚糖酶和纤维素酶，将其用于后期制备相应的转基因动物，该动物将自身分泌这些酶，起到消化饲料中木聚糖、植酸、果胶、葡聚糖、纤维素等抗营养因子，达到提高饲料利用率，减少污染排放的功效。同时，解决刚性肽介导两个以上融合蛋白共表达时相互干扰问题；解决2A连接肽多基因共表达效率低的问题，及多基因共表达中2A多肽残基对上游酶蛋白干扰的问题，达到多基因高效共表达的目的。

根据本公开的一个方面，提供了多功能融合酶，该多功能酶可以同时表达木聚糖酶、植酸酶、果胶酶、葡聚糖酶和纤维素酶活性。

在某些实施方式中，该多功能融合酶基因由木聚糖酶基因-A3-植酸酶基因-furin-P2A-果胶酶基因-A3'-纤维素酶基因组成。由于在2个以上蛋白共表达时，单纯使用2A或刚性肽都很难实现所有基因共表达，本发明通过组合应用刚性肽A3和2A，解决刚性肽介导两个以上融合蛋白共表达时相互干扰问题；解决2A连接肽多基因共表达效率低的问题，及多基因共表达中2A多肽残基对上游酶蛋白干扰的问题，达到多基因高效共表达的效果。同时，与单纯2A连接肽相比，本发明利用A3和furin P2A构建XAPT显著提高四种酶不同PH条件下的活性，消除2A多肽对上游基因的影响，也避免部分蛋白空间结构抑制2A反应活性的问题，同时还避开对不同蛋白顺序优化繁琐试验过程，酶表达量比2A更高。刚性肽只能用于两种融合酶的构建，本发明将2A和A3巧妙结合，即保留A3优势，又增强融合酶的多基因共表达能力。在融合酶第二和第三号基因连接处采用自剪切效率最高的P2A，同时在其N端添加furin酶识别基序RVKR，RVKR可在细胞器高尔基体高效剪切，仅残留4个氨基酸残基，该设计与一般2A序列，具有更高的剪切效率，对表达产物影响最小。由此，通过本公开设计获得的XAPT基因序列，可以高效表达木聚糖酶、植酸酶、果胶酶、葡聚糖酶和纤维素酶活性，解决了四种基因共表达的难题，更为以后将其用于转基因动物和酶发酵工业生产来提高基因转移和酶的生产效率提供了基础。同时，在公开号为CN106086068A专利中公开的，采用2A连接两个基因序列时，两个基因的连接顺序都会影响基因的表达及功能的发挥，而且两个基因的酶活都不及单基因的酶活高；在多基因连接时，若只采用2A连接态，这种位置效应会更加明显，对于每个基因酶活的影响会更加突出。但是本公开采用A3和2A连接态组合使用构建的上述多功能酶基因，可以克服位置效应，每个基因表达的酶活与单基因相当。

在某些实施方式中，该多功能融合酶基因中A3的基因序列如SEQ ID No:5所示。

在某些实施方式中，该多功能融合酶基因中A3'的基因序列如SEQ ID No:6所示。

在某些实施方式中，该多功能融合酶基因序列如SEQ ID No:11所示。由于2A多肽高效的自剪切功能，可较好实现前后两个蛋白的共表达，被誉为蛋白融合表达最可靠的linker。但是，部分酶蛋白融合2A的C端多肽后功能显著受损，后一个蛋白的表达量降低。多基因共表达时，对蛋白组合顺序要求苛刻，需要繁琐验证排列组合，才能实现低水平的共表达(公开号为CN106086068A专利中已表明此现象)，不利于多功能融合酶的构建。2A序列剪切效率与上游多肽基序有关，部分蛋白氨基酸基序会严重影响2A切割活性，导致2A介导前后两个酶因无法完全切割，导致二级结构折叠异常，还有可能被上游基因信号肽迁移到靶向错误的细胞器，无法正确的加工和分泌，导致功能尚失，表达失败。本公开中，采用A3与2A连接态组合使用的方式来构建4种基因融合表达的多功能融合酶，可以克服2A及A3连接态各自的缺点，使该多功能融合酶可以高效的表达四种酶活，可为获得表达该四种酶的转基因动物做好基础。

在某些实施方式中，该多功能融合酶具有如SEQ ID No:12所示的氨基酸序列。连接肽最重要的指标是氨基酸链的长度，不同长度的GGGGS连接肽对融合蛋白的表达量和活性有着不同的影响，并不是连接肽越长表达量就越高。融合酶对柔性肽要求比较苛刻，过长或过短的柔性肽都不利于融合酶活性。分子质量大、结构复杂的蛋白需要的折叠空间也就越大，连接肽也应加长，但过长的肽链可能会增加抗原性，同时也易被酶水解断裂。连接肽过短形成空间位阻效应会影响蛋白的正确折叠，也提高了聚体形成的几率。刚性连接肽由于二级结构的稳固，不可伸展弯曲的特性，常用来固定两端功能蛋白间距，保证功能域的完整。由此，设计的刚性连接态A3/A3'与P2A共同作用连接4个基因，实现4个基因的共表达，A3/A3'与P2A连接态序列经过优化设计，使得木聚糖酶、植酸酶、果胶酶、葡聚糖酶和纤维素酶均可高效表达。

根据本公开的另一个方面，提供了一种多功能融合酶真核表达载体，该真核表达载体包括如权利要求5或6所述的多功能融合酶。

在某些实施方式中，多功能融合酶真核表达载体的基因序列如SEQ ID No:13所示。

根据本公开的再一个方面，提供了一种多功能融合酶真核表达载体的构建方法，其包括如下步骤：

候选目的基因的筛选和优化；

连接态的设计；

目的基因与连接态连接；

多功能融合酶基因XAPT的合成；

构建CEP112位点定点转XAPT基因表达载体。

在某些实施方式中，构建CEP112位点定点转XAPT基因表达载体包括如下步骤：

将XAPT多顺反子替换CEP112-LA340RA3219载体中BEXA顺反子，构建新载体Cep112-mPSP-XAPT；

用PacI和sexAI线性化Cep112-mPSP-XAPT，然后用inf-npsp引物扩增npsp上游调控区，并替换现有mpsp序列；

构建CEP112位点定点转XAPT基因表达载体Cep112-npsp-XAPT。

本公开的有益效果：

1)本公开设计构建xynB-A3-APPA-furin-P2A-pg7fnss-A3'-TeEG(XAPT)融合酶，具有共表达酸性木聚糖酶，植酸酶，果胶酶和葡聚糖酶和纤维素酶五种酶活性，囊括饲料中消除主要抗营养因子所需要水解酶，对提高饲料转化率具有重要价值。该设计提高了基因表达效率，若将其用于转基因动物和酶发酵工业生产，能显著提高基因转移和酶的生产效率，具有重要的经济价值；

2)与单纯2A连接肽相比，本发明获得XAPT显著提高四种酶不同PH条件下的活性，消除2A多肽对上游基因的影响，也避免部分蛋白空间结构抑制2A反应活性的问题，同时还避开对不同蛋白顺序优化繁琐试验过程；

3)刚性肽只能用于两种融合酶的构建，本发明将2A和A3巧妙结合，即保留A3优势，又增强融合酶的多基因共表达能力；

4)本发明在融合酶第二和第三号基因连接处采用自剪切效率最高的P2A，同时在其N端添加furin酶识别基序RVKR，RVKR可在细胞器高尔基体高效剪切，仅残留4个氨基酸残基，该设计与一般2A序列，具有更高的剪切效率，对表达产物影响最小。

5)本公开携带XAPT由猪CEP112位点高效定点整合载体运载，可高效制备转基因猪，快速获得整合位置一致的转基因家系，培育转基因猪新品种。

附图说明

图1为pxynB-A3-APPA-furin-P2A-ppg7fns-A3'-TeEG(XAPT)结构示意图；

图2为木聚糖酶(xynB)-植酸酶(appA)双顺反子优化组合与表达结果：A.木聚糖酶-植酸酶双顺反子优化组合设计示意图；B.xynB的pH范围；C.appA的pH范围；D.xynB的pH稳定性(39.℃，2h)；appA的pH稳定性(39.℃，2h)；

图3为果胶酶(pg7fns)-纤维素酶(TeEGⅠ)双顺反子优化组合与表达结果：A.pg7fns-TeEGⅠ双顺反子优化组合设计示意图；B.果胶酶最适pH酶活比较；C.纤维素酶最适pH酶活比较；D.葡聚糖酶的最适pH酶活比较；

图4为多顺反子XAPT在pK15细胞中表达检测结果；

图5为Cep112-npsp-XAPT质粒图。

具体实施方式

一、目的基因的筛选

分别将来源于黑曲霉的木聚糖酶基因xynB(Guo et al.,2013)，来源于大肠杆菌的植酸酶基因appA，来源于蟋蟀的纤维素酶基因TeEGⅠ(Kim et al.,2008)，来源于耐热真菌沙栖梭孢壳XZ7的果胶酶基因pg7fns(Tu et al.,2014)。经SignalP 4.1Server预测信号肽后，分别去掉其自身的信号肽，然后根据猪密码子偏好进行优化，并将猪或牛来源的腮腺分泌蛋白(parotid secretory protein,PSP)信号肽(signal peptide，sp)序列分别添加到密码子优化后的候选基因的氨基酸序列的N端，如pigPSP-SP-xynB,pigPSP-SP-appA,pigPSP-SP-teEGⅠ,bosPSP-sp-pg7fns,分别简写为pSPxyn,pSPappA，pSPTeEG,bSPpg7，密码优化后的成熟肽基因分别命名为pxyn(基因序列如SEQ ID No:1所示),pappA(基因序列如SEQ ID No:2所示),pTEGI(基因序列如SEQ ID No:3所示)，ppg7fnss(基因序列如SEQ IDNo:4所示)。

二、构建木聚糖酶-植酸酶-果胶酶-纤维素酶多顺反子基因序列

利用A3刚性肽，分别将木聚糖酶(xynB)-植酸酶(appA)，果胶酶(pg7fns)-纤维素酶(TeEGⅠ)连接构建双功能融合酶，去掉A3上游基因终止密码子，同时去掉A3C端下游基因信号肽。经优化突变后的A3序列如下：

A3(SEQ ID No:5)：GAGGCTGCCGCCAAAGAAGCTGCCGCCAAGGAGGCTGCCGCCAAG

A3'(SEQ ID No:6)：GGCCGCCGCCAAGGAGGCCGCCGCCAAGGAGGCCGCCGCCAAGGG

经优化后A3连接的双功能酶基因序列为xynB-A3-APPA与pg7fns-A3'-TeEGI。将上述融合设计多功能酶顺反子经猪密码子优化，去除稀有密码子，选择猪细胞使用频率较高的密码子。

并分别将多顺反子内A3重复序列和猪腮腺蛋白信号肽再次优化，以减少重复序列对多顺反子结构稳定性的影响，优化后的序列进行人工合成。优化后的pxynB-A3-APPA基因序列如SEQ ID No:7所示，氨基酸序列如SEQ ID No:8所示；优化后的ppg7fns-A3'-TeEGI基因序列如SEQ ID No:9所示，氨基酸序列如SEQ ID No:10所示。

利用Furin酶识别基因序列和高效自剪切P2A序列，将pxynB-A3-APPA，ppg7fns-A3'-TeEGI，连接构建多功能顺反子pxynB-A3-APPA-furin-P2A-ppg7fns-A3'-TeEGI(XAPT)(基因序列如SEQ ID No:11所示，基因结构图谱如图1所示；氨基酸序列如SEQ ID No:12所示)克隆到pcDNA3.1(+)真核表达载体多克隆位点BamHI/EcoRI上。

利用P2A分别将木聚糖酶(xynB)-植酸酶(appA)，果胶酶(pg7fns)-纤维素酶(TeEGⅠ)，连接构建双功能酶pxynB-p2A-pAPPA、ppg7fns-p2A-pTeEG，克隆到pcDNA3.1(+)真核表达载体上多克隆位点,作为对照组。

三、木聚糖酶-植酸酶-果胶酶-纤维素酶真核表达载体构建

将优化后的多功能酶顺反子XAPT插入真核表达载体pcDNA3.1多克隆位点BamHI/EcoRI上，经酶切和测序鉴定，多功能酶顺反子XAPT真核表达载体pCD-XAPT构建成功。

四、木聚糖酶-植酸酶-果胶酶-纤维素酶多功能酶的体外表达与功能验证

将pCD-XAPT真核表达载体按照转染试剂盒LipofectamineTM LTX+PLUSTMReagent(invitrogen)说明书瞬时转染猪肾pK15细胞系，48-72h，收集细胞上清液作为粗酶液测定酶活，检测其酶活力及其pH耐受力。酶活测定方法和定义参考纤维素酶《NYT/912-2004》、木聚糖酶《GBT/23874-2009》、植酸酶《GBT/18634-2009》、果胶酶测定方法参考张飞等(2004)《果胶酶果胶酶活力的测定方法研究》。

1、木聚糖酶(xynB)和植酸酶(appA)融合酶表达分析

将木聚糖酶(xynB)和植酸酶(appA)分别用furin-P2A和A3融合，并在猪pK15细胞表达，结果显示，融合后A3连接的融合酶成功表达木聚糖酶和植酸酶双功能酶，且其表达木聚糖酶和植酸酶在pH2.0～pH6.5均具有较高的生物学活，其中pxynB-A3-appA的木聚糖酶活性，在pH2.0～5.0稍高于pxynB-p2A-pappA，在pH5.0后略低于后者，但pxynB-A3-appA表达的木聚糖酶对pH2.0～pH7.0耐受能力明显高于pxynB-p2A-pappA。pxynB-A3-appA表达的植酸酶在不同pH缓冲液中活性和耐受性均优于pxynB-p2A-pappA，结果如图2所示。

2、果胶酶(pg7fns)-纤维素酶(TeEGⅠ)融合酶表达分析

将果胶酶(pg7fns)和纤维素酶(TeEGⅠ)通过不同linker融合，结果显示，ppg7fns-A3'-TeEGⅠ能同时表达果胶酶，纤维素酶和葡聚糖酶功能，且其最适pH条件下，相应功能酶的活性均高于ppg7fns-p2A-pTeEGⅠ。ppg7fns-A3'-TeEGⅠ表达的果胶酶和葡聚糖酶在不同pH缓冲液中，酶活走势与相应单体酶酶活变化基本一致，结果如图3所示。

3、多顺反子XAPT在pK15细胞中表达检测

分别用电转和脂质体化学转染方法，将多顺反子XAPT的真核表达载体导入PK15细胞，于48h后收集细胞上清培养液，测定其表达情况，结果显示，多顺反子XAPT成功表达出木聚糖酶，植酸酶，葡聚糖酶和纤维素酶及果胶酶活性五种酶功能活性，四种酶活与单体酶活相当，但是具有更好的PH耐受能力，且有更好的PH适应性，结果如图4所示。

五、定点整合到CEP112位点转木聚糖酶-植酸酶-果胶酶-纤维素酶(XAPT)基因表达载体构建

首先将XAPT多顺反子替换前期研究载体CEP112-LA340RA3219(来源于第“201711477805.5”号“一种定点整合外源DNA转基因猪的构建方法”，公开号“108285906A”专利)中的BEXA顺反子，构建了新载体Cep112-mPSP-XAPT，在其基础上，用PacI和sexAI线性化Cep112-mPSP-XAPT，然后用inf-npsp引物扩增npsp上游调控区，并替换现有序列，npsp(-11.5kb～-5.7kb)在原mpsp(-11.1kb～-5.7kb)基础上延长了调控区序列395bp，构建Cep112-npsp-XAPT载体(序列如SEQ ID No:13所示)，经酶切验证，切割条带大小与预期相符，经过测序验证，确定成功获得一个能在猪唾液腺中特异表达XAPT四种功能酶的转基因载体，质粒图谱如图5所示。六、转木聚糖酶-植酸酶-果胶酶-纤维素酶(XAPT)基因猪的获得

将构建成功的Cep112-npsp-XAPT载体转染猪成纤维细胞系，获得表达XAPT多顺反子的阳性细胞系，将阳性细胞系作为供核细胞进行核移植，通过体细胞克隆的方法获得转XAPT基因猪。

对获得的转XAPT基因猪进行基因及测序水平的鉴定，采集阳性猪的唾液，进行检测，发现转XAPT基因猪均可高效的表达植酸酶，木聚糖酶，果胶酶和纤维素酶，且酶的活性与转单基因猪酶的活性相当。

以上所述的仅是本发明的一些实施方式。对于本领域的普通技术人员来说，在不脱离本发明创造构思的前提下，还可以做出若干变形和改进，这些都属于发明的保护范围。

序列表

<110> 温氏食品集团股份有限公司；华南农业大学

<120> 一种多功能融合酶和多功能融合酶真核表达载体及其构建方法

<130> 2019.11.21

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Met Phe Gln Leu Trp Lys Leu Val Phe Leu Cys Gly Leu Leu Ile Gly

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Thr Ser Ala Ser Ser Thr Pro Ser Ser Thr Gly Glu Asn Asn Gly Phe

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Tyr Tyr Ser Phe Trp Thr Asp Gly Gly Gly Asp Val Thr Tyr Thr Asn

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

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

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Ser Gly Thr Phe Thr Pro Ser Gly Asn Gly Tyr Leu Ser Val Tyr Gly

85 90 95

Trp Thr Thr Asp Pro Leu Ile Glu Tyr Tyr Ile Val Glu Ser Tyr Gly

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

115 120 125

Asp Gly Ser Val Tyr Asp Ile Tyr Thr Ala Thr Arg Thr Asn Ala Ala

130 135 140

Ser Ile Gln Gly Thr Ala Thr Phe Thr Gln Tyr Trp Ser Val Arg Gln

145 150 155 160

Asn Lys Arg Val Gly Gly Thr Val Thr Thr Ser Asn His Phe Asn Ala

165 170 175

Trp Ala Lys Leu Gly Met Asn Leu Gly Thr His Asn Tyr Gln Ile Val

180 185 190

Ala Thr Glu Gly Tyr Gln Ser Ser Gly Ser Ser Ser Ile Thr Val Gln

195 200 205

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gln

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Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg His

225 230 235 240

Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val Thr

245 250 255

Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Trp Leu Thr Pro

260 265 270

Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gln Arg Gln Arg

275 280 285

Leu Val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gln Ser Gly

290 295 300

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

305 310 315 320

Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val His

325 330 335

Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu Lys

340 345 350

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

355 360 365

Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg Gln Thr

370 375 380

Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn Leu

385 390 395 400

Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln Ala

405 410 415

Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr Gly

420 425 430

Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln Gln

435 440 445

Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser His

450 455 460

Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Tyr Leu Leu

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Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp

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Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala Tyr

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Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp Thr

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Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu Pro

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Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Arg

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Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu Val

565 570 575

Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu Asn

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Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu Arg

595 600 605

Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val Asn

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Glu Ala Arg Ile Pro Ala Cys Ser Leu Arg Val Lys Arg Gly Ser Gly

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Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

645 650 655

Pro Gly Pro

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atggtgcagc tctggaaact cgtcctcctc tgtgggctcc tcgccggaac atctgcctca 60

aaccccgtcc ccgctaactc taatctgagc aagagagcca gctgcacctt caccgacgcc 120

accagcgcca tcagcggcaa gaagagctgc tccaccatca ccctgaagga catcaccgtg 180

ccagccggca ccaccctgga cctgaccaag ctgaacgacg gcaccaaggt aatcttctcc 240

ggcaccacca ccttcggcta caaggagtgg gagggcccac tgatcagcgt gtccggcaac 300

aacatcctgg tggagggcgc caccggccac gtgatcgacg gcaacggcgc caagtggtgg 360

gacggcaagg gcagcaacgg cggcaagacc aagccaaagt tcttctacgc ccactccatg 420

aagaacagca acatcaaggg cctgcacgtg aagaacaccc cagtgcaggc cttcagcatc 480

aacggcgcca ccaacctggg cgtgtacgac gtgagcctgg acaacagcgc cggcgacagc 540

gccggcggcc acaacaccga cgccttcgac gtgggctcca gcaacggcgt gtacatcagc 600

ggcgccgtgg tgaagaacca ggacgactgc ctggccatca acagcggcac caacatcacc 660

ttcaccggcg gcaagtgctc cggcggccac ggcctgtcca tcggcagcgt gggcggcaga 720

tccgacaaca ccgtgaagac cgtgagaatc ctgaacagct ccatcagcaa ctcccagaac 780

ggcgtgagaa tcaagaccgt gtacggcgcc accggctccg tgagcgacgt gaagtacgag 840

ggcatcaccc tgagcggcat caccaagtac ggcgtggtaa tcgagcagga ctacgagaac 900

ggctccccaa ccggcacccc caccgccggc gtgccaatca ccgacctgac cctgaacggc 960

gtgaccggca gcgtgtccag cggcgccacc gaggtgtaca tcctgtgcgc caagggcgcc 1020

tgcaagaact ggacctggaa caaggtgtcc gtgaccggcg gcaagaagtc cgccaagtgc 1080

gagaacgtgc ccagcccagc ctcctgcgag gccgccgcca aggaggccgc cgccaaggag 1140

gccgccgcca agggctccta cgactacgcc gacgtgatca agaagagcct gctgttctac 1200

caggcccaga ggagcggcag actgtccggc atggacccac tggtgtcctg gagaaaggac 1260

tccgccctga acgacagagg caacaacggc gaggacctga ccggcggcta ctacgacgcc 1320

ggcgacttcg tgaagttcgg cttcccaatg gcctacacca tcaccctgct gagctggggc 1380

gtgatcgact acgagaacac ctactccagc atcggcgccc tgtccgccgc cagagccgcc 1440

atcaagtggg gcaccgacta cttcatcaag gcccacgtga gcgccaacga gctgtacggc 1500

caggtgggca acggcggcgc cgaccactcc tggtggggca gaccagagga catgaacatg 1560

gacaggccag cctacaagat cgacaccagc agaccaggca gcgacctggc cgccgagacc 1620

gccgccgcca tggccgccgc cagcatcgtg ttcaagaacg ccgacagcaa ctacgccaac 1680

accctgctga gacacgccaa ggagctgtac aacttcgccg acaactaccg gggcaagtac 1740

tccgacagca tctccgacgc cgccgccttc tacaactcct acagctacga ggacgagctg 1800

gtgtggggcg ccatctggct gtggagagcc accaacgacc agaactacct gaacaaggcc 1860

acccagtact acaaccagta cagcatccag tacaagaaca gcccactgtc ctgggacgac 1920

aagagcaccg gcgccagcgc cctgctggcc aagctgaccg gcggcgacca gtacaagagc 1980

gccgtgcagt ccttctgcga cggcttctac tacaaccagc agaagacccc caagggcctg 2040

atctggtaca gcgactgggg ctccctgaga cagagcatga acgccgtgtg ggtgtgcctg 2100

caagccgccg acgccggcgt gaagaccggc gagtacagaa gcctggccaa gaagcagctg 2160

gactacgccc tgggcgacgc cggcagatcc ttcgtggtgg gcttcggcaa caacccccca 2220

agccacgagc agcacagagc cgcctcctgc ccagacgccc ccgccgcctg cgactggaac 2280

acctacaacg gcggccagtc caactaccac gtgctgtacg gcgccctggt gggcggccca 2340

gacgccaacg actactacaa cgacgtgaga tccgactacg tgcacaacga ggtggcctgt 2400

gactacaatg ccgggttcca gaatgtcctc gtgtccctca aagccaacgg atactga 2457

<210> 10

<211> 818

<212> PRT

<213> 人工合成()

<400> 10

Met Val Gln Leu Trp Lys Leu Val Leu Leu Cys Gly Leu Leu Ala Gly

1 5 10 15

Thr Ser Ala Ser Asn Pro Val Pro Ala Asn Ser Asn Leu Ser Lys Arg

20 25 30

Ala Ser Cys Thr Phe Thr Asp Ala Thr Ser Ala Ile Ser Gly Lys Lys

35 40 45

Ser Cys Ser Thr Ile Thr Leu Lys Asp Ile Thr Val Pro Ala Gly Thr

50 55 60

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

65 70 75 80

Gly Thr Thr Thr Phe Gly Tyr Lys Glu Trp Glu Gly Pro Leu Ile Ser

85 90 95

Val Ser Gly Asn Asn Ile Leu Val Glu Gly Ala Thr Gly His Val Ile

100 105 110

Asp Gly Asn Gly Ala Lys Trp Trp Asp Gly Lys Gly Ser Asn Gly Gly

115 120 125

Lys Thr Lys Pro Lys Phe Phe Tyr Ala His Ser Met Lys Asn Ser Asn

130 135 140

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

145 150 155 160

Asn Gly Ala Thr Asn Leu Gly Val Tyr Asp Val Ser Leu Asp Asn Ser

165 170 175

Ala Gly Asp Ser Ala Gly Gly His Asn Thr Asp Ala Phe Asp Val Gly

180 185 190

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

195 200 205

Asp Cys Leu Ala Ile Asn Ser Gly Thr Asn Ile Thr Phe Thr Gly Gly

210 215 220

Lys Cys Ser Gly Gly His Gly Leu Ser Ile Gly Ser Val Gly Gly Arg

225 230 235 240

Ser Asp Asn Thr Val Lys Thr Val Arg Ile Leu Asn Ser Ser Ile Ser

245 250 255

Asn Ser Gln Asn Gly Val Arg Ile Lys Thr Val Tyr Gly Ala Thr Gly

260 265 270

Ser Val Ser Asp Val Lys Tyr Glu Gly Ile Thr Leu Ser Gly Ile Thr

275 280 285

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

290 295 300

Gly Thr Pro Thr Ala Gly Val Pro Ile Thr Asp Leu Thr Leu Asn Gly

305 310 315 320

Val Thr Gly Ser Val Ser Ser Gly Ala Thr Glu Val Tyr Ile Leu Cys

325 330 335

Ala Lys Gly Ala Cys Lys Asn Trp Thr Trp Asn Lys Val Ser Val Thr

340 345 350

Gly Gly Lys Lys Ser Ala Lys Cys Glu Asn Val Pro Ser Pro Ala Ser

355 360 365

Cys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

370 375 380

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

385 390 395 400

Gln Ala Gln Arg Ser Gly Arg Leu Ser Gly Met Asp Pro Leu Val Ser

405 410 415

Trp Arg Lys Asp Ser Ala Leu Asn Asp Arg Gly Asn Asn Gly Glu Asp

420 425 430

Leu Thr Gly Gly Tyr Tyr Asp Ala Gly Asp Phe Val Lys Phe Gly Phe

435 440 445

Pro Met Ala Tyr Thr Ile Thr Leu Leu Ser Trp Gly Val Ile Asp Tyr

450 455 460

Glu Asn Thr Tyr Ser Ser Ile Gly Ala Leu Ser Ala Ala Arg Ala Ala

465 470 475 480

Ile Lys Trp Gly Thr Asp Tyr Phe Ile Lys Ala His Val Ser Ala Asn

485 490 495

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

500 505 510

Gly Arg Pro Glu Asp Met Asn Met Asp Arg Pro Ala Tyr Lys Ile Asp

515 520 525

Thr Ser Arg Pro Gly Ser Asp Leu Ala Ala Glu Thr Ala Ala Ala Met

530 535 540

Ala Ala Ala Ser Ile Val Phe Lys Asn Ala Asp Ser Asn Tyr Ala Asn

545 550 555 560

Thr Leu Leu Arg His Ala Lys Glu Leu Tyr Asn Phe Ala Asp Asn Tyr

565 570 575

Arg Gly Lys Tyr Ser Asp Ser Ile Ser Asp Ala Ala Ala Phe Tyr Asn

580 585 590

Ser Tyr Ser Tyr Glu Asp Glu Leu Val Trp Gly Ala Ile Trp Leu Trp

595 600 605

Arg Ala Thr Asn Asp Gln Asn Tyr Leu Asn Lys Ala Thr Gln Tyr Tyr

610 615 620

Asn Gln Tyr Ser Ile Gln Tyr Lys Asn Ser Pro Leu Ser Trp Asp Asp

625 630 635 640

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

645 650 655

Gln Tyr Lys Ser Ala Val Gln Ser Phe Cys Asp Gly Phe Tyr Tyr Asn

660 665 670

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

675 680 685

Leu Arg Gln Ser Met Asn Ala Val Trp Val Cys Leu Gln Ala Ala Asp

690 695 700

Ala Gly Val Lys Thr Gly Glu Tyr Arg Ser Leu Ala Lys Lys Gln Leu

705 710 715 720

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

725 730 735

Asn Asn Pro Pro Ser His Glu Gln His Arg Ala Ala Ser Cys Pro Asp

740 745 750

Ala Pro Ala Ala Cys Asp Trp Asn Thr Tyr Asn Gly Gly Gln Ser Asn

755 760 765

Tyr His Val Leu Tyr Gly Ala Leu Val Gly Gly Pro Asp Ala Asn Asp

770 775 780

Tyr Tyr Asn Asp Val Arg Ser Asp Tyr Val His Asn Glu Val Ala Cys

785 790 795 800

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

805 810 815

Gly Tyr

<210> 11

<211> 4434

<212> DNA

<213> 人工合成()

<400> 11

atgttccagc tgtggaagct ggtgttcctg tgcggactgc tgatcggcac cagcgcctcc 60

agcaccccct ccagcaccgg agagaacaac ggcttctact actccttctg gaccgacgga 120

ggaggcgacg tgacctacac caacggcgac gccggagctt acaccgtgga gtggagcaac 180

gtgggcaact tcgtgggagg caagggatgg aacccaggct ccgcccagga catcacctac 240

tccggcacct tcaccccaag cggcaacggc tacctgtccg tgtacggctg gaccaccgac 300

cccctgatcg agtactacat cgtggagagc tacggcgact acaacccagg ctccggaggc 360

acctacaagg gcaccgtgac cagcgacggc tccgtgtacg acatctacac cgctaccagg 420

accaacgctg ccagcatcca gggcaccgcc accttcaccc agtactggtc cgtgaggcag 480

aacaagagag tgggcggcac cgtgaccacc agcaaccact tcaacgcctg ggccaagctg 540

ggcatgaacc tgggcaccca caactaccag atcgtggcta ccgagggcta ccagtccagc 600

ggctccagct ccatcaccgt gcaggaggct gccgccaaag aagctgccgc caaggaggct 660

gccgccaagc agtccgagcc agagctgaag ctggagagcg tggtcatcgt gtcccgccac 720

ggcgtgcgcg ctccaaccaa ggccacccag ctgatgcagg acgtgacccc agacgcttgg 780

ccaacctggc cagtgaagct gggatggctg acccccaggg gcggagagct gatcgcctac 840

ctgggccact accagaggca gagactggtg gctgacggac tgctggccaa gaagggatgc 900

ccacagagcg gacaggtggc tatcatcgct gacgtggacg agcgcacccg gaagaccgga 960

gaggccttcg ccgccggcct ggccccagac tgcgctatca ccgtgcacac ccaggctgac 1020

accagctccc ccgacccact gttcaaccca ctgaagaccg gcgtgtgcca gctggacaac 1080

gccaacgtga ccgacgctat cctgagccgc gccggaggct ccatcgctga cttcaccgga 1140

cacaggcaga ccgccttcag ggagctggag agagtgctga acttccccca gtccaacctg 1200

tgcctgaagc gggagaagca ggacgagagc tgctccctga cccaggccct gccaagcgag 1260

ctgaaggtgt ccgccgacaa cgtgagcctg accggagccg tgagcctggc ctccatgctg 1320

accgagatct tcctgctcca gcaggctcag ggaatgccag agccaggatg gggaaggatc 1380

accgacagcc accagtggaa caccctgctg tccctgcaca acgcccagtt ctacctgctc 1440

cagcggaccc cagaggtggc taggagcaga gccaccccac tgctggacct gatcaagacc 1500

gccctgaccc cacacccacc acagaagcag gcctacggcg tgaccctgcc aacctccgtg 1560

ctgttcatcg ccggccacga caccaacctg gctaacctgg gaggcgccct ggagctgaac 1620

tggaccctgc caggacagcc agacaacacc ccaccaggag gagagctggt gttcgagagg 1680

tggcgccggc tgagcgacaa ctcccagtgg attcaggtgt ccctggtgtt ccagaccctc 1740

cagcagatga gagacaagac cccactgtcc ctgaacaccc caccaggaga ggtgaagctg 1800

accctggccg gatgcgagga gaggaacgct cagggaatgt gcagcctggc cggcttcacc 1860

cagatcgtga acgaggctag aatccccgcc tgctccctga gggtgaagag gggcagcgga 1920

gctaccaact tctccctgct gaagcaggct ggcgacgtgg aggagaaccc aggaccaatg 1980

gtgcagctct ggaaactcgt cctcctctgt gggctcctcg ccggaacatc tgcctcaaac 2040

cccgtccccg ctaactctaa tctgagcaag agagccagct gcaccttcac cgacgccacc 2100

agcgccatca gcggcaagaa gagctgctcc accatcaccc tgaaggacat caccgtgcca 2160

gccggcacca ccctggacct gaccaagctg aacgacggca ccaaggtaat cttctccggc 2220

accaccacct tcggctacaa ggagtgggag ggcccactga tcagcgtgtc cggcaacaac 2280

atcctggtgg agggcgccac cggccacgtg atcgacggca acggcgccaa gtggtgggac 2340

ggcaagggca gcaacggcgg caagaccaag ccaaagttct tctacgccca ctccatgaag 2400

aacagcaaca tcaagggcct gcacgtgaag aacaccccag tgcaggcctt cagcatcaac 2460

ggcgccacca acctgggcgt gtacgacgtg agcctggaca acagcgccgg cgacagcgcc 2520

ggcggccaca acaccgacgc cttcgacgtg ggctccagca acggcgtgta catcagcggc 2580

gccgtggtga agaaccagga cgactgcctg gccatcaaca gcggcaccaa catcaccttc 2640

accggcggca agtgctccgg cggccacggc ctgtccatcg gcagcgtggg cggcagatcc 2700

gacaacaccg tgaagaccgt gagaatcctg aacagctcca tcagcaactc ccagaacggc 2760

gtgagaatca agaccgtgta cggcgccacc ggctccgtga gcgacgtgaa gtacgagggc 2820

atcaccctga gcggcatcac caagtacggc gtggtaatcg agcaggacta cgagaacggc 2880

tccccaaccg gcacccccac cgccggcgtg ccaatcaccg acctgaccct gaacggcgtg 2940

accggcagcg tgtccagcgg cgccaccgag gtgtacatcc tgtgcgccaa gggcgcctgc 3000

aagaactgga cctggaacaa ggtgtccgtg accggcggca agaagtccgc caagtgcgag 3060

aacgtgccca gcccagcctc ctgcgaggcc gccgccaagg aggccgccgc caaggaggcc 3120

gccgccaagg gctcctacga ctacgccgac gtgatcaaga agagcctgct gttctaccag 3180

gcccagagga gcggcagact gtccggcatg gacccactgg tgtcctggag aaaggactcc 3240

gccctgaacg acagaggcaa caacggcgag gacctgaccg gcggctacta cgacgccggc 3300

gacttcgtga agttcggctt cccaatggcc tacaccatca ccctgctgag ctggggcgtg 3360

atcgactacg agaacaccta ctccagcatc ggcgccctgt ccgccgccag agccgccatc 3420

aagtggggca ccgactactt catcaaggcc cacgtgagcg ccaacgagct gtacggccag 3480

gtgggcaacg gcggcgccga ccactcctgg tggggcagac cagaggacat gaacatggac 3540

aggccagcct acaagatcga caccagcaga ccaggcagcg acctggccgc cgagaccgcc 3600

gccgccatgg ccgccgccag catcgtgttc aagaacgccg acagcaacta cgccaacacc 3660

ctgctgagac acgccaagga gctgtacaac ttcgccgaca actaccgggg caagtactcc 3720

gacagcatct ccgacgccgc cgccttctac aactcctaca gctacgagga cgagctggtg 3780

tggggcgcca tctggctgtg gagagccacc aacgaccaga actacctgaa caaggccacc 3840

cagtactaca accagtacag catccagtac aagaacagcc cactgtcctg ggacgacaag 3900

agcaccggcg ccagcgccct gctggccaag ctgaccggcg gcgaccagta caagagcgcc 3960

gtgcagtcct tctgcgacgg cttctactac aaccagcaga agacccccaa gggcctgatc 4020

tggtacagcg actggggctc cctgagacag agcatgaacg ccgtgtgggt gtgcctgcaa 4080

gccgccgacg ccggcgtgaa gaccggcgag tacagaagcc tggccaagaa gcagctggac 4140

tacgccctgg gcgacgccgg cagatccttc gtggtgggct tcggcaacaa ccccccaagc 4200

cacgagcagc acagagccgc ctcctgccca gacgcccccg ccgcctgcga ctggaacacc 4260

tacaacggcg gccagtccaa ctaccacgtg ctgtacggcg ccctggtggg cggcccagac 4320

gccaacgact actacaacga cgtgagatcc gactacgtgc acaacgaggt ggcctgtgac 4380

tacaatgccg ggttccagaa tgtcctcgtg tccctcaaag ccaacggata ctga 4434

<210> 12

<211> 1477

<212> PRT

<213> 人工合成()

<400> 12

Met Phe Gln Leu Trp Lys Leu Val Phe Leu Cys Gly Leu Leu Ile Gly

1 5 10 15

Thr Ser Ala Ser Ser Thr Pro Ser Ser Thr Gly Glu Asn Asn Gly Phe

20 25 30

Tyr Tyr Ser Phe Trp Thr Asp Gly Gly Gly Asp Val Thr Tyr Thr Asn

35 40 45

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

50 55 60

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

65 70 75 80

Ser Gly Thr Phe Thr Pro Ser Gly Asn Gly Tyr Leu Ser Val Tyr Gly

85 90 95

Trp Thr Thr Asp Pro Leu Ile Glu Tyr Tyr Ile Val Glu Ser Tyr Gly

100 105 110

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

115 120 125

Asp Gly Ser Val Tyr Asp Ile Tyr Thr Ala Thr Arg Thr Asn Ala Ala

130 135 140

Ser Ile Gln Gly Thr Ala Thr Phe Thr Gln Tyr Trp Ser Val Arg Gln

145 150 155 160

Asn Lys Arg Val Gly Gly Thr Val Thr Thr Ser Asn His Phe Asn Ala

165 170 175

Trp Ala Lys Leu Gly Met Asn Leu Gly Thr His Asn Tyr Gln Ile Val

180 185 190

Ala Thr Glu Gly Tyr Gln Ser Ser Gly Ser Ser Ser Ile Thr Val Gln

195 200 205

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gln

210 215 220

Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg His

225 230 235 240

Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val Thr

245 250 255

Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Trp Leu Thr Pro

260 265 270

Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gln Arg Gln Arg

275 280 285

Leu Val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gln Ser Gly

290 295 300

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

305 310 315 320

Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val His

325 330 335

Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu Lys

340 345 350

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

355 360 365

Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg Gln Thr

370 375 380

Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn Leu

385 390 395 400

Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln Ala

405 410 415

Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr Gly

420 425 430

Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln Gln

435 440 445

Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser His

450 455 460

Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Tyr Leu Leu

465 470 475 480

Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp

485 490 495

Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala Tyr

500 505 510

Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp Thr

515 520 525

Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu Pro

530 535 540

Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Arg

545 550 555 560

Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu Val

565 570 575

Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu Asn

580 585 590

Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu Arg

595 600 605

Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val Asn

610 615 620

Glu Ala Arg Ile Pro Ala Cys Ser Leu Arg Val Lys Arg Gly Ser Gly

625 630 635 640

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

645 650 655

Pro Gly Pro Met Val Gln Leu Trp Lys Leu Val Leu Leu Cys Gly Leu

660 665 670

Leu Ala Gly Thr Ser Ala Ser Asn Pro Val Pro Ala Asn Ser Asn Leu

675 680 685

Ser Lys Arg Ala Ser Cys Thr Phe Thr Asp Ala Thr Ser Ala Ile Ser

690 695 700

Gly Lys Lys Ser Cys Ser Thr Ile Thr Leu Lys Asp Ile Thr Val Pro

705 710 715 720

Ala Gly Thr Thr Leu Asp Leu Thr Lys Leu Asn Asp Gly Thr Lys Val

725 730 735

Ile Phe Ser Gly Thr Thr Thr Phe Gly Tyr Lys Glu Trp Glu Gly Pro

740 745 750

Leu Ile Ser Val Ser Gly Asn Asn Ile Leu Val Glu Gly Ala Thr Gly

755 760 765

His Val Ile Asp Gly Asn Gly Ala Lys Trp Trp Asp Gly Lys Gly Ser

770 775 780

Asn Gly Gly Lys Thr Lys Pro Lys Phe Phe Tyr Ala His Ser Met Lys

785 790 795 800

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

805 810 815

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

820 825 830

Asp Asn Ser Ala Gly Asp Ser Ala Gly Gly His Asn Thr Asp Ala Phe

835 840 845

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

850 855 860

Asn Gln Asp Asp Cys Leu Ala Ile Asn Ser Gly Thr Asn Ile Thr Phe

865 870 875 880

Thr Gly Gly Lys Cys Ser Gly Gly His Gly Leu Ser Ile Gly Ser Val

885 890 895

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

900 905 910

Ser Ile Ser Asn Ser Gln Asn Gly Val Arg Ile Lys Thr Val Tyr Gly

915 920 925

Ala Thr Gly Ser Val Ser Asp Val Lys Tyr Glu Gly Ile Thr Leu Ser

930 935 940

Gly Ile Thr Lys Tyr Gly Val Val Ile Glu Gln Asp Tyr Glu Asn Gly

945 950 955 960

Ser Pro Thr Gly Thr Pro Thr Ala Gly Val Pro Ile Thr Asp Leu Thr

965 970 975

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

980 985 990

Ile Leu Cys Ala Lys Gly Ala Cys Lys Asn Trp Thr Trp Asn Lys Val

995 1000 1005

Ser Val Thr Gly Gly Lys Lys Ser Ala Lys Cys Glu Asn Val Pro Ser

1010 1015 1020

Pro Ala Ser Cys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala

1025 1030 1035 1040

Ala Ala Lys Gly Ser Tyr Asp Tyr Ala Asp Val Ile Lys Lys Ser Leu

1045 1050 1055

Leu Phe Tyr Gln Ala Gln Arg Ser Gly Arg Leu Ser Gly Met Asp Pro

1060 1065 1070

Leu Val Ser Trp Arg Lys Asp Ser Ala Leu Asn Asp Arg Gly Asn Asn

1075 1080 1085

Gly Glu Asp Leu Thr Gly Gly Tyr Tyr Asp Ala Gly Asp Phe Val Lys

1090 1095 1100

Phe Gly Phe Pro Met Ala Tyr Thr Ile Thr Leu Leu Ser Trp Gly Val

1105 1110 1115 1120

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

1125 1130 1135

Arg Ala Ala Ile Lys Trp Gly Thr Asp Tyr Phe Ile Lys Ala His Val

1140 1145 1150

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

1155 1160 1165

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

1170 1175 1180

Lys Ile Asp Thr Ser Arg Pro Gly Ser Asp Leu Ala Ala Glu Thr Ala

1185 1190 1195 1200

Ala Ala Met Ala Ala Ala Ser Ile Val Phe Lys Asn Ala Asp Ser Asn

1205 1210 1215

Tyr Ala Asn Thr Leu Leu Arg His Ala Lys Glu Leu Tyr Asn Phe Ala

1220 1225 1230

Asp Asn Tyr Arg Gly Lys Tyr Ser Asp Ser Ile Ser Asp Ala Ala Ala

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275 1280

Gln Tyr Tyr Asn Gln Tyr Ser Ile Gln Tyr Lys Asn Ser Pro Leu Ser

1285 1290 1295

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

1300 1305 1310

Gly Gly Asp Gln Tyr Lys Ser Ala Val Gln Ser Phe Cys Asp Gly Phe

1315 1320 1325

Tyr Tyr Asn Gln Gln Lys Thr Pro Lys Gly Leu Ile Trp Tyr Ser Asp

1330 1335 1340

Trp Gly Ser Leu Arg Gln Ser Met Asn Ala Val Trp Val Cys Leu Gln

1345 1350 1355 1360

Ala Ala Asp Ala Gly Val Lys Thr Gly Glu Tyr Arg Ser Leu Ala Lys

1365 1370 1375

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

1380 1385 1390

Gly Phe Gly Asn Asn Pro Pro Ser His Glu Gln His Arg Ala Ala Ser

1395 1400 1405

Cys Pro Asp Ala Pro Ala Ala Cys Asp Trp Asn Thr Tyr Asn Gly Gly

1410 1415 1420

Gln Ser Asn Tyr His Val Leu Tyr Gly Ala Leu Val Gly Gly Pro Asp

1425 1430 1435 1440

Ala Asn Asp Tyr Tyr Asn Asp Val Arg Ser Asp Tyr Val His Asn Glu

1445 1450 1455

Val Ala Cys Asp Tyr Asn Ala Gly Phe Gln Asn Val Leu Val Ser Leu

1460 1465 1470

Lys Ala Asn Gly Tyr

1475

<210> 13

<211> 26707

<212> DNA

<213> 人工合成()

<400> 13

gcggccgccc atccatagtg tgtccttcac cctctgaagt tcatgtgcga agttggctgc 60

gtctcttctc ataaaaatga cacaaaggaa aaagtacatc agttgtaatg aagtagcatt 120

gttttatgct ccagagggcc tttgacttcc tagacctatt ttttgttttt accataatca 180

taaactttct catctgaggt gaagagtgtg gaattaacac attttgttcc tttgttaggc 240

aaagactctg aggctgaaca atcgggaggt tctatcgctt aaataagaaa agttaagata 300

attaactggc attgagcact tgtccacatt cttgtgctgt ggtcagaggt aggacacagt 360

ctcccatccc cgggttaatt aagtgcctcc aacaaagggg tactgttgcc cacatagaaa 420

gatctaaact aattaattaa tccctcaccc gcaaatcttt cagtcactaa gttagcacga 480

ttgttgaaca agttctccaa aggagagata cagatgagtg cgtatagggt ggacctggct 540

gctgaggaga cacctgcatc tgactaagaa gagccacggt gttagttgaa tggtgtggag 600

tagggtggtt ctgtgggaca gtagaaaatc gagaggcatg tgccgtttag tgaactgatg 660

gaagctaccc caaacgacag agattgtcag tcaggccaat ccgtttcgag tttgatgggc 720

agccggacag tgagacagac acacctactc agttggagga aggatgagaa caatggccag 780

cagggattga gagaccctga caggcgcaag gccctaacac acacacctac cacctcactt 840

gacaaagctg ccaaagacca aagacttgtt ctccattaga aatgacagct ggcttgaccc 900

gacagcataa taagcagagt gtactctgat tggagaactt taatgtgttt cattcagtat 960

tataaaagga cagtattaca gattttgttg tacactgctg ttacatgtgg ggcagtgtgt 1020

ctttaagtag ggtaaagtac tctttaaaaa tgggtcctag atattttttc ctttaactca 1080

agtctcttac tgtttaaatg atttttattt tgtttaatat ggaggaaaaa gaagcgtaaa 1140

tggacaatat atatttagag aaagatggtt agctgtcaga aaaatatgca aatcaaaatc 1200

acaccaagac tgcagcacac ccctgtcaga tggctgtgat caagaaaata aatgacaatg 1260

agtggtggtg aagatgtact aaagggaaac acacacacac acacacacac acacacacac 1320

acacactgga gcaaccactg tggaaatcag tatgaatggt cctcaaaaac ctgaagatag 1380

agcggggcgt ggtggcatac acttttattc ccagcactgg ggaggcagag gcaggtggat 1440

ctctgagttc caggccagcc tggtctatag cacaggttct aggacagcca gggctacaca 1500

gaaaaaccct gccttgatta aaccaaacca aaccaaacca aaccaaacca aaccaaacca 1560

aaccaaacca aaccaaacca gaccaaacca aaacactgaa gatagaactt cagtattcca 1620

ttcctagata tatacccaat ggagactaag tcagcaagac acctgcacag ccatgttcac 1680

tactacactg ttcaccacag ccaggctgtg gaaccagcct gagtgtccat gataaatgaa 1740

tggataggta actttcaagg taaatggact ctgctgtgta catgcctcac attctgttta 1800

ttcatttttc tttatgaggt gtccattcag gagtcacatg gtagttctat tttcagtctt 1860

ctgaagatac tacactggtc cccacagttt acacttttat cagcagtgaa taagggttcc 1920

tctatcctta ccatcatttg ttgtaatttt tcttgatgac cctctttctg acagggatag 1980

gatgtaatat cagtgtgagg aagtacaact tgttttctaa gtatttattg gccccttgca 2040

tttcttcttt tgaaaactgt cggttcctga catctgctca ggtattcatt ggatgttgtt 2100

tctttggtgt ttgagttctt atgaattcta gatgttaaat ccctgcctgt ggttctctcc 2160

cattctgtag gctgcctcct caccctggca attgttgtcc ttgttttgca gaaacttttg 2220

acttcatgga atctcatttg tcagttttcc ctcctctgct atagcctgag ctaatgcact 2280

ggtttttaca gagccctggt ctatgccttt atcctcctct ggcagcttcg gagtttcatt 2340

tcttacattt agatctttga tccactttga acaagttttg gagcagggtg agagatacga 2400

atctagttcc attcttccat atgtgatcct agtttacata gcatcgttgg ttgaagaggt 2460

tttattttat ttttaaataa tgtgtcataa aaaacgaggt ggttgtagca gtgtggattt 2520

gtttctttgt cctttgatct acaggtcttg ttttgtgtca gtctcatgat gttttattgc 2580

tatggctctg tcatacagtc tgaggtcagg tattgtgata taccttcagt attgctccct 2640

cagactcagg tttgctttgg ccaggagtca tcttactcag tgctcttaga gctcccccag 2700

catgtagctg ctactattct tagttgataa atcaggaaac tggggctcag agagattaac 2760

tgtcttgaac tacttctggg gaggtgaaac gtggagacac taaactgtgt ttaccctgta 2820

ctgctccagt agctgtcggg tgctgggcta cagcaaagca cctatactat atattactca 2880

ggaggtggaa aaactcagcc tcccttgggg ttcccaagct cccaggtgtc cagtcactgc 2940

tggaaacctc atggagtctg aaaggaaggg ttgagggtac atggggcagc gatgaggagc 3000

ctggggctgg gatctcccaa acacctggat atccagatgc cactgggtca gggggagttg 3060

ggaacagagt tgggatgtcc atggacctgt gacaaggcca gggccagggg gaggataact 3120

ctggctttac taatttgcga aagtccttag cttagcagca gttgtctggg agcacagagg 3180

ggccttctgt aagaggctca ggcagtgccg ctctgtaggc gaaggtcttc tccatgttcc 3240

ccatggtggt tcttgatgaa agagacagtc cttggctcca aactggttta ttgattgttc 3300

attgtggaaa atgggtgcac accaccttct cagggtggac cagagatcaa ataccttttg 3360

cagggaggaa tatctgggaa gggacgctta ctggctaaac cctcagggcc tctagataca 3420

tcattagcat ggagaactct gttctgggct acatgaccac aggccacatt tccacaagcc 3480

acatgtggga agtgtggcac atgttctagg ccaggaatct ggtagggagc gtggagccac 3540

ctaccatccc aggtgggtgc ctgggtgcca gggaccctga acccgctcaa ccttaccaag 3600

tttcctggca gggtccactg tcctacacag aagctggagg aggtgtgagg gttgtgtctt 3660

tgtggaatgt cccatgctgc ttggggctca gtttctccac ctgtacctca ttggtttggg 3720

tataaaaagt ggggatactt tattattctc tgactcggtc ctgaggaaaa agcatcgtgg 3780

cagtccagga accacaccct gaggttcctg cactgaaggg actccctaag tctctggagt 3840

ctctcccctt cacagagctg ccaaagtcta ggttcttttg aggataacag agccatgctt 3900

ggtaagcaga caacagcatt tgtttactca accttctttt gtcagctccc tcttcataaa 3960

caagttgaga caccatgctg gcttgaggaa gacttctaaa gccagacaac tgtgcaagga 4020

agaagaagaa ggggcaagtg gagttagcct ggatgtagcc ctcaaagtct ccagagacca 4080

gccatgaagg ctcaagtgga gggcaagacc tgcagcagcc aagcatctgg caggagagga 4140

tcctgggaac ccctctacca tgacacacat tcttcctgca ggtcacactt aataggccat 4200

ttcttatttg gatctatcat ggtgttctgt gcgagattaa tgaggtgtta tgctgcgaac 4260

agaaagttat ataaaaacaa gtcccccccc cttgtcactg ctgctaagaa tgtagcagaa 4320

attgtctcaa gtgtctctct aatcagaaac aataaaggtc tccttggatt caagccctcc 4380

agtttcctcc ttccttgctg agccttggac acccatacaa acctcctgga tgctacagct 4440

ctgggcagag actccaaggt ggggagagac tgatggtaca aaagcaaaat acttgtttgg 4500

gggtacaccc actcctctgc ctgtgtggtt cctgcagtca gtcctgcaga caggccctca 4560

gtgggtcttc catgggcaac acgcagaggg aggcaatgga tgggaatacc cacaccctgg 4620

ttagtttacc ccggccatgc tctctgctct tcatccctcc tctgccctct gccacggctt 4680

tctctgcagg aatcatatct tcatattggc ccacaggtgt tctcctcacc ctagctatga 4740

tgtttacttt agagtgacct tagcagggct ggtgggaatg agttctagaa ggctcacgga 4800

gatgctaggg aagaaacgtc ttctaactac tgaggttact aagttcctgg tggttgtctc 4860

tgcctttccc ttgttaaagt caccttgaag ttagtgcaga agaaatcaga gcccagtcac 4920

agagtaaata tggtcctgaa gatttccttt gagtgcccag aatccatgac atttcaagag 4980

ccctctttgt accttaagtc atttggggtt gtatcttctg cttgatgtat gtgtgtgtgt 5040

ttatcaaaga gtgagatggt tacataagag gtgctctaaa ggacagagag gatttgcaat 5100

tgtggcatgt gacatcctca ggccttgctc tggtgccagg aggaactgat gcagaaaaga 5160

gtaagaggtc atttcctgga ggctgtcact atagaggaga tcttacagtg cattccctcc 5220

tccaggccct gcctgaggat agacatgtgc tgactgcaac tgaaacagag gcttgggatg 5280

gagagttagg ttcacagaag ggagggtggg agatggatgc ttgctgggtt ctgggtctca 5340

tcaccagctc ctgaccaccc ggtcagccca tgtgcttatt ccatagcttt cttttgctat 5400

gtttactcag tgtggtgttt gttgggaccc agcagaagcc agtcccaggc tgacagctgt 5460

ggatacacag ggcagcatga gggtcctcag cctgaagcag tcaggctggc agaagagaaa 5520

gaccagcaca cattccttca accaactatg tcttgaaaaa caaacatatt atatcacata 5580

tattgcattt atgagacagc taaaatgtac tcgggtagca tgactccagg tggggatatc 5640

tgcaagtgcc atgagtggca gagggacagc caatgtgagg caagaaggaa ttctggctca 5700

acacagctta gctccctggt gttggttcaa actttgagag tttgaccaca agcactttat 5760

ttttgacata tttaaacaga gcacaacttt gggaaaaagt tttcttatga aaattatcac 5820

aataaagctt aaggcatgac tacattaaaa tgcctttgca aagtatatgt gccctcttcc 5880

acaagaatgg ttctattgac tgagaaataa tgttcaggat aaagatccag gaagaaaaga 5940

tcagggataa gtaaaatact aaactctttt gcaaagtaca tagaccctct ttcataacaa 6000

tgggttctat tgactgacaa gcactgctca ggagttggga aagagtctag cataagcacg 6060

atagcctgga gactctagtg aggtctagtc ttacagacag caaaaatcac caggttacaa 6120

actacattca tttccagttt tctgatcagg cacaggtatg aatcccttct gttgaagaga 6180

aaagtccatg tgtttaaaat atctggtttc tccagtgcta ttagcgagaa gacttgagcc 6240

ctatacaact cccacctgga gtgacatcct gtcttcatgg tatattacat acctagacac 6300

gctcatctca cagacttagg actttgtctt ctgatctcca tttctgatcc cacttccacc 6360

tttgccttga tagtgtcatt ttcttcactg ccttggtgac aaccatgtta tcctctgtgt 6420

atttgagtgt taccattttc agattttacc tgtatgcaag atcacacagt ctttgtcttt 6480

ctgtctggat gcatgctaat ctctacacaa caacccttcc ccgtcactca gatcttcctc 6540

cattaacaca tacatggtgc tgaagaggct agggagcttc ccttcagtgg ggagctagct 6600

ggctattggg cctttttgac tgtccaggaa ggcccccaat tgctgagaca agaacttaga 6660

ttcttcatta ttgactctaa ctcatgtatc aagcagaagc taatgaatag ttatcaacag 6720

gatcagaggt tccagtgtaa gacactttga catgaaagaa cggaggaagg acagatggat 6780

gcataaaagc aggaccactg ccccaggaag gtcctggaaa ctgatgcagg gcaaaggaca 6840

ggttataaac caaatcttag ggagtcagga agagcacaga ggagctcaac caactgacca 6900

ctgcttaggg gctaccaacc caatcctccc tgtgggaaca gctaagctat cagccaaggg 6960

taataaacag gcaggacctg tggatgacat ggagagcata gggaccctgg gtccagcctt 7020

tagcacctgc actctcagga tactccacca ttgtgtctta gagagcctag ggatactggg 7080

tccagccttt ggtaccttca ctctcagggt accccatcac tgtgtcttgg agagcctagg 7140

caccctgggt ccagccttca gtacctgcgc tctcaggaca ccccaccatt gtctcttgcc 7200

ccgtctcttc ttcctcttcc tccctttcat tgtctcttct ctgtttcttt cttgactctc 7260

ctttcccctc acaccctcac tctagttctc cccttccctc tctgcatcac cctattctct 7320

ctgtggtccc tccactttcc tttatctctc atgcttctct cctccctcaa atacttgtca 7380

cccactatac ttcaggggcc agctctagtg acaaagctgt taatagcaag actctcagat 7440

ctccaacggc tcagaggagc cagacccacc aagaactctc tccaggtcca atttcaggtt 7500

ccttcgaaag ctttcagcaa atgctcaggg aacatgccac taacaagaag atgcaaattc 7560

cagttgagag tgggaaaggc ccttgcgtag gtcccatctt ccaggccaag gtcagagggg 7620

ctctgtgtaa tccggattga cagggctcag aacaatgttt tgtttttaag gtttatttat 7680

tttaggtgtt agtgtctttg cttgcatgac cttatgtgca tcatgtgtgt gcaggttcct 7740

gatgacagta gaggagggct ttgaatccct ggggatagga agttacagga aattataagc 7800

tgctttgtgg gtcttctagc tttcccaaca gaagtgaatg ctcttcacca ctgagccatc 7860

tctctaggcc caagagacat tgctttatgg atataattgt gtgtgtgtgt caacattgag 7920

gaaagggaaa taaaaaaaaa acttcagccg ctaaggttgt acagtttcac taattgctac 7980

ttttagttgt gataaaatgg caggtgcttc aacatttata tatacaaaaa cttccctgct 8040

ggtggttcaa ctgtgagaac tggggtaagt gggtgagttc tctttttctg tctctgtctc 8100

tgtctctctc cttccattct ttcttaaagg aaataaacat tgcagctggg ttatagctca 8160

tcaatatgga agttacagaa gtgaaaaaag gcattgcctt ggtgggtggt gttaccagct 8220

gatttttggt tgtcctgcaa ggaggtctgg ggactggctg ctctgtctct gtctgtatga 8280

gtgagggaag tctggggagc agattcccta accttcagcc tggcctggtt cctgagtgaa 8340

cccagcctct ctggtcctag tagctttttc caaacaggaa tctgagtggt gacagggaac 8400

aagtaccagc ccattgctta agtgccaggg ttagtgaggg caggaagctg ccatagctgg 8460

gattagtagt tgtattggat gtaggaagtc ctatcctggg acagctaatc cttaatgctt 8520

cactggagat tttcaatgag aaatttatcc cacggcccat atggccccat ccttttgtct 8580

ccaacagcca agtattttcc attagaggag acttcctgta cacttgatgg atgctcattt 8640

caaggtgact tggggcagtc agtacagact tgggatgacc tctgacagcc taacctctcc 8700

ccaacaaggg ccctctatgt ttgctatgta atgtaatgtc agacattgtc aggagtgtcc 8760

gcagcacagc ctgcccagtg tgagggctct cataggtttc ccactgtctt atctacacag 8820

ggataacgag gaggtaagct gcagttccca gtctcacttc acagaggaag agataacccc 8880

atcccaggtc atgtagccag cagtggaaag aatgaggatt tgaactcagg tcttccaagt 8940

cccattgata gcatctcctc acaagtccct tgccaccctc acgatgcctt agacacttgc 9000

ctgcccttta tactaaggag atgcaggtac aaggggttta cccatgtagc agctgaggca 9060

gctggggata gataccagca gcaggcctga tgtcaccact ctaactccag catccccagt 9120

ctgtgttcct ggagtgtgaa aatccctact taacaagatt gtgcaacagt ccttggctct 9180

gtgacccata gctggaaaca ggattctcat tgatttgtgg aacatggtgg cagccagcca 9240

aaaagagggt ctgcatacag aagacagctg tggcaaggcc acagcagact ctgactacct 9300

tagcttacag aattacaagg tcataatgtc ctctgctttg gtcacctcat gttaaggaca 9360

ggccctaatg aagatggggc agaagactga aggaatggcc aaccaataac tggcccaact 9420

tgagacccat cctacaggca agcatcaatt cctgacacta ctaatgatac tctgttatgc 9480

ttgcagacag aagcctagca taactatcct ccgagaggtc cacccagcaa ctgactgaaa 9540

cagaaaaaga tatccacagg caaacagtgg atggaggtca gggactatta tgggagagct 9600

gtgggaagga ttaaaaaccc tgaaggggat aggaacccca caggaagacc aacagagtca 9660

actaagagac ctgtgggagc tctcagagac tgagccacca accaaagagc atacacaggc 9720

cggtccgagg cacctggcac gtgtgaagca gacatgcagc tcagtctcca tgtaggtcct 9780

ccaataagcg gtagcctgac tgcagtatcc aatccctaac agggctgcac agtctggcct 9840

cagtggggga gggtgcccct aatcctgcag agacttgatg agtggagagc tatccagggg 9900

gaacccaccc tctctgagaa gggaatgggg atgggggagg gactctgtga agaggggaca 9960

aggacaaaca agaacctcaa ataggtcagg ccctaaaggc ttgctaagta gcagtggccc 10020

agctctgtcc tgttcctcag cccaaggctc agctcccacc tgtttctgtg tttttctggc 10080

ttttcatggg cctaggactt ggtggccagt tcaaacaatg gggcctgtgg aagacacaat 10140

atacaagact agggacattc ctgttctgct gactatccac agcctgatgt aggtggaagg 10200

acccaatcac tggatttcta cccttgcgca accttgacag ctgagggcct ctcagaaacc 10260

tatttcttcc actgaaaaat gagactctca aatgaacgtc ctgacaatca tcaggcttat 10320

taaagaggtg tatctaacct gaatggcaag cagacagcag gcaaatgtct gtatcaacct 10380

ctaggaagga caagaactgc tcactgctgc cccccaggag gccatttgct gaaacagctg 10440

ctctcctgct ggtgcacagg ccctgccttc tcattgcagc tacagcccct tcctgtctga 10500

acctcctgtc aggtcactgg gaaacagatc aagatggaac aggacagctc ctgatggtaa 10560

ataaaaaaca gtggtcatgg ctattcatag gggtttatgc ttcttcagtc cacactgtga 10620

agagctgtgg gcatgaacca cagtgttcga ggtagagttg gggttctgaa attcacagtg 10680

gggtgagctc agtaaatgtg agctggaggt cactcgtgag acacacagtc ctgctgcttc 10740

tgttcccaat atcctgagga gacgacacat ctactttgtt cagaggccac agtctagttg 10800

acctgagagt taccagtttc ttatttgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 10860

gtgttgttcg tgtgtgagtg caggtgcaca tatgatagcg tacacgttga ggtcagagga 10920

taactatcag gcgttgtccc ctcctacttt tcctcggact ctggagaaca aacatgggtc 10980

cttattccag gggagcaagt gcctgttggc tgacacatct tgctcacata cattttacct 11040

agacaatgga gcctccatca gagtattact ttagctcctc accgatggca atgcaccacc 11100

tctctaccca cataggagtt gggtctccac acacccccac acccccttca ccaaaacgtt 11160

ttcagttact ttatctggta aagttcatca gagaatgaag ccagtattaa gaacatggaa 11220

tcatttggga acctggatct agcaataccc caccctagat ggagttgctg agttttcacc 11280

tcagattata attcccccct agcttctatg gtttattctg aaaccagggg aactcgattc 11340

ctccctttgg accacagaca tcctggcttg tgaattcaca tgtcatctac tgctaatcca 11400

ttggtagtat gtggctcaca gagacacact acagtcatgg ccaatgtcaa ggtaggacag 11460

atgtgaatca ttcccccagt cctgctgttt tcatgactaa ccctcctcag cacagtgacc 11520

atgaacctac ttttcccctc cttttatttt tagaattgct ggaattttct attttgagaa 11580

ataatagcct tggggcagca ttaaacaaaa tcatctagaa agctggttta aaatacagat 11640

ggttgagtca gtgaaagagt gaggaatgtc attattggcc cctcacagag gctggctcac 11700

tccagcagag gtggttgaag ctcttggaca cgggtcaggt gcataggaag ggtggtctgg 11760

gacacctgag aaccacaatt gaacaaacag aagctgctgg cttttttttt tttaaatgag 11820

ttctcaaaaa atgactgggc tagcttaggc aaatacttcg agccaaccca acagaacatt 11880

cttccattga ttcattctgg atcttctttc tagacaatac tgaactgacc ccttgttggc 11940

agtctcaagt ttgacaacat agggctttga acttggcaca aggtccatca ctgtcaccca 12000

agcatcctgg gtgacctttg ggttggaata tcttggctaa ccttagatat tttctttgga 12060

gtatctttag aacatccagg aaatagggct tgattctcat cctgggacca caatataagt 12120

caccctagaa tcccaggaga tcgtgcagag aaacaaggat ctctctcgtg tgcatccttc 12180

ttcaaagcag tgagtagtga ctccactaaa ctgagttccc atctgagagt ccacaggagg 12240

ctttggggca agaagcagag ggaaggcact gtttgtgttg gtaaagtttt gactctaaca 12300

aatttgaaga catagatgac attgtgtcag actaacaaca acctagactc atgtgggttc 12360

tgtttaggga tcagatttta ttcatcaatg acttgtctta gtgtatagag aaaggcttcc 12420

tactggagtg taggctcaat aatgacagaa gagatagcta tttcccctag ggactgtgct 12480

gctccaagtt tggtggagaa aggcagtggg gaacctagat gtgctctctg gggagggggt 12540

ctgaagctgg cttcatagaa ggtgtgaagt tttgctgaaa catctaaaca gaattatagc 12600

ttaggaaagt gagcaggcaa ggcagggaat gtgttgcata tgtatatgta catgaatata 12660

ttatgttata gatacacaca catttgaacc tcatttgcag atgacagaaa ataggttatt 12720

ttgcctctct taactgctaa gcacaatgac ttccagttcc atccatttcc tgaaatgcca 12780

caatttcatt tttcattgtg gctgaataaa attccattgc agactgggcc ctacttcatc 12840

cactcctgag ggcaggcata tcccctggct ccatttctta cctattgtga agagaagtgc 12900

aactgtcttg ttgaaaggca agcgtgagag aggcaggcac taattgtggg tttttgtttc 12960

ttcttcctgc tatgactctc catttgtcag ggcgcgccgc caccatgttc cagctgtgga 13020

agctggtgtt cctgtgcgga ctgctgatcg gcaccagcgc ctccagcacc ccctccagca 13080

ccggagagaa caacggcttc tactactcct tctggaccga cggaggaggc gacgtgacct 13140

acaccaacgg cgacgccgga gcttacaccg tggagtggag caacgtgggc aacttcgtgg 13200

gaggcaaggg atggaaccca ggctccgccc aggacatcac ctactccggc accttcaccc 13260

caagcggcaa cggctacctg tccgtgtacg gctggaccac cgaccccctg atcgagtact 13320

acatcgtgga gagctacggc gactacaacc caggctccgg aggcacctac aagggcaccg 13380

tgaccagcga cggctccgtg tacgacatct acaccgctac caggaccaac gctgccagca 13440

tccagggcac cgccaccttc acccagtact ggtccgtgag gcagaacaag agagtgggcg 13500

gcaccgtgac caccagcaac cacttcaacg cctgggccaa gctgggcatg aacctgggca 13560

cccacaacta ccagatcgtg gctaccgagg gctaccagtc cagcggctcc agctccatca 13620

ccgtgcagga ggctgccgcc aaagaagctg ccgccaagga ggctgccgcc aagcagtccg 13680

agccagagct gaagctggag agcgtggtca tcgtgtcccg ccacggcgtg cgcgctccaa 13740

ccaaggccac ccagctgatg caggacgtga ccccagacgc ttggccaacc tggccagtga 13800

agctgggatg gctgaccccc aggggcggag agctgatcgc ctacctgggc cactaccaga 13860

ggcagagact ggtggctgac ggactgctgg ccaagaaggg atgcccacag agcggacagg 13920

tggctatcat cgctgacgtg gacgagcgca cccggaagac cggagaggcc ttcgccgccg 13980

gcctggcccc agactgcgct atcaccgtgc acacccaggc tgacaccagc tcccccgacc 14040

cactgttcaa cccactgaag accggcgtgt gccagctgga caacgccaac gtgaccgacg 14100

ctatcctgag ccgcgccgga ggctccatcg ctgacttcac cggacacagg cagaccgcct 14160

tcagggagct ggagagagtg ctgaacttcc cccagtccaa cctgtgcctg aagcgggaga 14220

agcaggacga gagctgctcc ctgacccagg ccctgccaag cgagctgaag gtgtccgccg 14280

acaacgtgag cctgaccgga gccgtgagcc tggcctccat gctgaccgag atcttcctgc 14340

tccagcaggc tcagggaatg ccagagccag gatggggaag gatcaccgac agccaccagt 14400

ggaacaccct gctgtccctg cacaacgccc agttctacct gctccagcgg accccagagg 14460

tggctaggag cagagccacc ccactgctgg acctgatcaa gaccgccctg accccacacc 14520

caccacagaa gcaggcctac ggcgtgaccc tgccaacctc cgtgctgttc atcgccggcc 14580

acgacaccaa cctggctaac ctgggaggcg ccctggagct gaactggacc ctgccaggac 14640

agccagacaa caccccacca ggaggagagc tggtgttcga gaggtggcgc cggctgagcg 14700

acaactccca gtggattcag gtgtccctgg tgttccagac cctccagcag atgagagaca 14760

agaccccact gtccctgaac accccaccag gagaggtgaa gctgaccctg gccggatgcg 14820

aggagaggaa cgctcaggga atgtgcagcc tggccggctt cacccagatc gtgaacgagg 14880

ctagaatccc cgcctgctcc ctgagggtga agaggggcag cggagctacc aacttctccc 14940

tgctgaagca ggctggcgac gtggaggaga acccaggacc aatggtgcag ctctggaaac 15000

tcgtcctcct ctgtgggctc ctcgccggaa catctgcctc aaaccccgtc cccgctaact 15060

ctaatctgag caagagagcc agctgcacct tcaccgacgc caccagcgcc atcagcggca 15120

agaagagctg ctccaccatc accctgaagg acatcaccgt gccagccggc accaccctgg 15180

acctgaccaa gctgaacgac ggcaccaagg taatcttctc cggcaccacc accttcggct 15240

acaaggagtg ggagggccca ctgatcagcg tgtccggcaa caacatcctg gtggagggcg 15300

ccaccggcca cgtgatcgac ggcaacggcg ccaagtggtg ggacggcaag ggcagcaacg 15360

gcggcaagac caagccaaag ttcttctacg cccactccat gaagaacagc aacatcaagg 15420

gcctgcacgt gaagaacacc ccagtgcagg ccttcagcat caacggcgcc accaacctgg 15480

gcgtgtacga cgtgagcctg gacaacagcg ccggcgacag cgccggcggc cacaacaccg 15540

acgccttcga cgtgggctcc agcaacggcg tgtacatcag cggcgccgtg gtgaagaacc 15600

aggacgactg cctggccatc aacagcggca ccaacatcac cttcaccggc ggcaagtgct 15660

ccggcggcca cggcctgtcc atcggcagcg tgggcggcag atccgacaac accgtgaaga 15720

ccgtgagaat cctgaacagc tccatcagca actcccagaa cggcgtgaga atcaagaccg 15780

tgtacggcgc caccggctcc gtgagcgacg tgaagtacga gggcatcacc ctgagcggca 15840

tcaccaagta cggcgtggta atcgagcagg actacgagaa cggctcccca accggcaccc 15900

ccaccgccgg cgtgccaatc accgacctga ccctgaacgg cgtgaccggc agcgtgtcca 15960

gcggcgccac cgaggtgtac atcctgtgcg ccaagggcgc ctgcaagaac tggacctgga 16020

acaaggtgtc cgtgaccggc ggcaagaagt ccgccaagtg cgagaacgtg cccagcccag 16080

cctcctgcga ggccgccgcc aaggaggccg ccgccaagga ggccgccgcc aagggctcct 16140

acgactacgc cgacgtgatc aagaagagcc tgctgttcta ccaggcccag aggagcggca 16200

gactgtccgg catggaccca ctggtgtcct ggagaaagga ctccgccctg aacgacagag 16260

gcaacaacgg cgaggacctg accggcggct actacgacgc cggcgacttc gtgaagttcg 16320

gcttcccaat ggcctacacc atcaccctgc tgagctgggg cgtgatcgac tacgagaaca 16380

cctactccag catcggcgcc ctgtccgccg ccagagccgc catcaagtgg ggcaccgact 16440

acttcatcaa ggcccacgtg agcgccaacg agctgtacgg ccaggtgggc aacggcggcg 16500

ccgaccactc ctggtggggc agaccagagg acatgaacat ggacaggcca gcctacaaga 16560

tcgacaccag cagaccaggc agcgacctgg ccgccgagac cgccgccgcc atggccgccg 16620

ccagcatcgt gttcaagaac gccgacagca actacgccaa caccctgctg agacacgcca 16680

aggagctgta caacttcgcc gacaactacc ggggcaagta ctccgacagc atctccgacg 16740

ccgccgcctt ctacaactcc tacagctacg aggacgagct ggtgtggggc gccatctggc 16800

tgtggagagc caccaacgac cagaactacc tgaacaaggc cacccagtac tacaaccagt 16860

acagcatcca gtacaagaac agcccactgt cctgggacga caagagcacc ggcgccagcg 16920

ccctgctggc caagctgacc ggcggcgacc agtacaagag cgccgtgcag tccttctgcg 16980

acggcttcta ctacaaccag cagaagaccc ccaagggcct gatctggtac agcgactggg 17040

gctccctgag acagagcatg aacgccgtgt gggtgtgcct gcaagccgcc gacgccggcg 17100

tgaagaccgg cgagtacaga agcctggcca agaagcagct ggactacgcc ctgggcgacg 17160

ccggcagatc cttcgtggtg ggcttcggca acaacccccc aagccacgag cagcacagag 17220

ccgcctcctg cccagacgcc cccgccgcct gcgactggaa cacctacaac ggcggccagt 17280

ccaactacca cgtgctgtac ggcgccctgg tgggcggccc agacgccaac gactactaca 17340

acgacgtgag atccgactac gtgcacaacg aggtggcctg tgactacaat gccgggttcc 17400

agaatgtcct cgtgtccctc aaagccaacg gatactgagg gcgcgccgat caattctcta 17460

gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 17520

cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 17580

aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 17640

aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 17700

ctatggcttc tgaggcggaa agaaccagct ggtttaaact cgattataac ttcgtatagc 17760

atacattata cgaagttatg atcgatatga agaatctgct tagggttagg cgttttgcgc 17820

tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat 17880

agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 17940

ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 18000

tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 18060

atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 18120

ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 18180

gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 18240

ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 18300

tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 18360

aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 18420

tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa 18480

ttaatacgac tcactatagg gagacccaag ctggctagcg tttaaactta agcttggtac 18540

cgagctcgga tccactagtc cagtgtggtg gaattcgcca ccatgggatc ggccattgaa 18600

caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac 18660

tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg 18720

cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag 18780

gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt 18840

gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg 18900

tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg 18960

catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga 19020

gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag 19080

gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgatgat 19140

ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt 19200

tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg 19260

gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt 19320

tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc 19380

ttcgagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg 19440

gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 19500

gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 19560

aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 19620

gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 19680

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 19740

aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 19800

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 19860

ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 19920

atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 19980

cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 20040

ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 20100

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaatct 20160

agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 20220

gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 20280

tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 20340

ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 20400

gatgcggtgg gctctatggc ttctgaagat ctataacttc gtatagcata cattatacga 20460

agttatggat ctgtcgacca tagtgtgtcc ttcacacatc acggttacaa ttaggcagtg 20520

ctgactctaa atcaagagac ctcattacat gttcctgact cttttttttc tcactttttt 20580

ttccattttt ttattactca aatgaattta tcacatctgt agttgtgcaa tgatcaaaac 20640

aatctgattt cacaggattt ccacattttt tattttgtct tttcactcaa gtgtctttgc 20700

aggcttccat ccctttatcc tcaagaatca ttttcgggct ctaagaaatc ttttttgtac 20760

tttttctatt tattttcaga gcctcttgtt ttccttttat agatgtaata aaacttcctc 20820

tcttcaagtt tatgaattag aattttttag tgccattccc caggttatct catttcttca 20880

ggtttttttc agttatcttt taatttctct tttatgtttc tcactcttgg cttttgtgct 20940

tgatgattct ttctctttat tcatattgac aaaagatgaa tattatatta gttgcctgtt 21000

gctattataa caaatcacaa caaattttgt ggcttaaaac aacagaaatt tattatctca 21060

gagttgtgaa ggtgaccatt ccaaaattag tcttagaata ctaaaatcaa taacatgact 21120

ctgtacgtca actatactcc aatacatatt tttttaattg agaaaaaaaa aaaagaaaat 21180

caaaaccaac ttgtcagcag ggatttcctt ccagaggctc cagggaagga tctgcttgct 21240

tgcctttccc agcttccaga agccacttaa cattcctgct catggccctg cacgcatcgc 21300

tcttcctcgt gctgctttca tcctgaccct ggcccttctc ctctcttaca aggacctttg 21360

aggttgcacc agccccacag gtgcacccag gatgctctcc catctcagga tccgttatca 21420

cacccgtgtt gccacgtaag gtaacatgtt caccggtgcc gagggttagg atgtgggcat 21480

ctttgggcag aggggcgttc ttcaacttac cgcagacagg gtttctagac agctgatttg 21540

ctgtttaaat agagtttctt tcctaatatg tctctccctg aggagaaagt caaaatgagt 21600

tctggggtgg gaatgacaca cggcctggag gcgatattag aatccaggcc ccttaaatat 21660

gacagtgagg agggctttat tctgggtttg tagaatccac acttgaagtc ttggtttccc 21720

ctggattgtt cattcacatt tatttttagg ggtgagcctt ctgtttttca tctcgtttgc 21780

ccacagtctg caggttggga tggggctcgc ctcctctata cacagacctt taaggagctt 21840

gtttttatct tcacttctca ccaggctctt tgccatcttt gcggtatgtg aggccagaac 21900

ttctctagag ggctggatag acagaggcgc cagctctgcg aacgcctccc tattagctag 21960

gctttcacag cttgttttat ccccaaacat ctcagtattc tcttacctcc tttccatcct 22020

gcagaagtgg actgagagct gttctcagtt ggtgaatgtc tcccaaaaat gactattcct 22080

gttctacacc tgactgtgtt atacagcaat ccgtttttat tttactcaag attgtttctt 22140

agctgttcag tattggttaa aggtcactaa aaagcagaat tcttaatgta ttgtaataat 22200

cacttaagat ataaaaatat ttgtgcatat aatgactaaa tgctgcattc aaggaatgaa 22260

tcttggttaa aactttttgc caatctgtat ctgataacaa aataatttga aacatattac 22320

attttaaacg aatggccctt aaaatttgaa tgaaggataa ctagacattt taatagaagt 22380

gcagcatgat actttctttg caatttcaca ttataaaata atgcaattac gaagcatatc 22440

attaggaact taattgtgct cagtgttgtt gtggctcagg ttattctgga aagagagcct 22500

gataacatat gagtacttat tggggaggga attccaggaa tttgaggtaa gtgaatggag 22560

ataaggaaac agagaaggga aaagccaata aggggagcct tattgatgga gttactgctg 22620

agagtgaagg gggtctccat cccactgagg accctgaatg atccttcagg acataatcat 22680

ggaatcgtcc catcagagaa tggtagcctg gagtatttag ccacacaagt ccagcccctt 22740

ttattgaggg tgctcctaaa ggacatctga ccctgctctt cctgctcctg cacttcctgt 22800

ctgcccctgc acttcctgcc tgctcctgca cttcctgtct tcccctgcac ttcctgcccc 22860

tgaacttcct gcctgctcct gcacttcctg tcttcccctg cacttcctgc ccctgcactt 22920

ccttgctcct acacttcccg ccttcttttg tacttcttgc tgctcctgca cttccttcct 22980

gactctgcac ttcctgcctg ctcctgtact tccctccttc gtttgcactt cctgtctgct 23040

cctgcacttc cttctcctac actccctgcc tgctcctgct cttcctgctc ctgcatttcc 23100

tgctttttcc ttttcctgct cctgcatctc ctacctgctc ctggagttcc ttcctgctcc 23160

tacacttcct gtctgctcct gcacttctgg cttcacctgc tcttggactg aatgaccttc 23220

cctagcttta aagaaagctt gaggtggaaa aactaagccg tcccacagcc cagttgaggg 23280

ggaatcaggt atgagttgcc tgtctcagct gggttgcaat cagatggatc aaaaagatgt 23340

ggcaggatgc cagaagcatc tagaattgaa tggaaacagt gaaagtggat cagaaataga 23400

gatgcatctt tctctacaca gtagtcttcc ctccataact gcattaaaac agcgttcaaa 23460

gatttgaatc atgtttatat aaaacatacc caaagaagcc ccccaaaatt agactacatg 23520

agtttacttt ttcatctctt acaaggcccc cttaaaatga caaaaatcaa tcccaaaggc 23580

gtaagtctgc cacaacaaag gaaacacaaa ggtggccgtc agcaggcaag acttggacat 23640

cttcctggaa gcaatgctga gatggccagc ttctcgctac acaggaagcc agttatagcc 23700

ttagaaagag ctcgaggatc tgcaatcccg cggccatggc ggccgggagc atgcgacgtc 23760

gggcccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt tttacaacgt 23820

cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc 23880

gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 23940

ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 24000

cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 24060

cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 24120

tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 24180

gctcacgtag tgggccatcg ccctgataga cggattttcg ccctttgacg ttggagtcca 24240

cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 24300

attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 24360

tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tcctgatgcg 24420

gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atcaggtggc acttttcggg 24480

gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 24540

tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 24600

ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 24660

ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 24720

gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 24780

gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 24840

acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 24900

actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 24960

ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 25020

cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 25080

gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 25140

caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 25200

aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 25260

ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 25320

tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 25380

ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 25440

ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 25500

ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 25560

tcccttaacg tgagttttcg ttccactgag catcagaccc cgtagaaaag atcaaaggat 25620

cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 25680

taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 25740

gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 25800

acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 25860

ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 25920

ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 25980

cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 26040

aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 26100

gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 26160

gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 26220

gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 26280

ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 26340

ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 26400

caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 26460

ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 26520

attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 26580

gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctatttaggt 26640

gacactatag aatactcaag ctatgcatcc aacgcgttgg gagctctccc atatggtcga 26700

cctgcag 26707

Claims

1.一种多功能融合酶XAPT，其特征在于，所述的多功能融合酶XAPT可以表达木聚糖酶、植酸酶、果胶酶、葡聚糖酶和纤维素酶活性，所述的多功能融合酶XAPT基因由木聚糖酶基因-A3-植酸酶基因-furin-P2A-果胶酶基因-A3'-纤维素酶基因组成，所述的A3的基因序列如SEQ ID No:5所示，所述的A3'基因序列如SEQ ID No:6所示，所述的多功能融合酶XAPT基因序列如SEQ ID No:11所示。

2.根据权利要求1所述的多功能融合酶XAPT，其特征在于，所述的多功能融合酶XAPT具有如SEQ ID No:12所示的氨基酸序列。

3.一种多功能融合酶XAPT真核表达载体，其特征在于，所述的真核表达载体可以表达如权利要求1或2所述的多功能融合酶XAPT。

4.根据权利要求3所述的多功能融合酶XAPT真核表达载体，其特征在于，所述的真核表达载体的基因序列如SEQ ID No:13所示。

5.根据权利要求4所述的多功能融合酶XAPT真核表达载体，其特征在于，所述的真核表达载体的构建方法，包括如下步骤：

候选目的基因的筛选和优化；

连接肽的设计；

目的基因与连接肽连接；

多功能融合酶基因XAPT的合成；

构建CEP112位点定点转XAPT基因表达载体：将XAPT多顺反子替换CEP112-LA340RA3219载体中BEXA顺反子，构建新载体Cep112-mPSP-XAPT；用PacI和sexAI线性化Cep112-mPSP-XAPT，然后用inf-npsp引物扩增npsp上游调控区，并替换现有mpsp序列；构建CEP112位点定点转XAPT基因表达载体Cep112-npsp-XAPT。