CN106459945A - 具有增强活性的外切葡聚糖酶变体及其用途 - Google Patents

具有增强活性的外切葡聚糖酶变体及其用途 Download PDF

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CN106459945A
CN106459945A CN201580032311.9A CN201580032311A CN106459945A CN 106459945 A CN106459945 A CN 106459945A CN 201580032311 A CN201580032311 A CN 201580032311A CN 106459945 A CN106459945 A CN 106459945A
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C·佩尔西永
C·于尔曼
C·艾林哈克
O·邦宗
A·马尔若
H·马蒂斯
S·福尔
S·阿尔芒
S·普拉多
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Pu Lutaisi Co
Centre National de la Recherche Scientifique CNRS
IFP Energies Nouvelles IFPEN
Proteus SA
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Abstract

本发明涉及参与分解木质纤维素生物质的酶的表达和优化。本发明具体涉及里氏木霉的外切葡聚糖酶2的变体以及所述具有增强功效的变体在用于分解纤维素和用于生产生物燃料的方法中的用途。

Description

具有增强活性的外切葡聚糖酶变体及其用途
由于大量原料的可利用性和乙醇作为燃料的优势，用纤维素生产乙醇的可能性已经得到很大的关注。用于这类工艺的基于纤维素的天然原料被称为“生物质”。已经将许多类型的生物质，例如木材、农业残余物、草本作物和城市固体垃圾，考虑作为生产生物燃料的潜在原料。这些材料主要由纤维素、半纤维素和木质素组成。
纤维素是由β-1,4键连接的葡萄糖分子组成的聚合物，其对分解或解聚合非常有耐性。一旦纤维素转化为葡萄糖，后者容易用酵母发酵成生物燃料，如乙醇。
研究的用于将纤维素转化为葡萄糖的最古老的方法是基于酸水解。该工艺可以在浓酸或稀酸的存在下进行。然而，几个缺点(例如当使用浓酸时酸回收率很差，以及使用稀酸的情况下葡萄糖的产量低)对于酸水解工艺的经济性是不利的。
为了克服酸水解工艺的缺点，最近的纤维素转化工艺已经更多地与使用纤维素酶类型的酶的酶促水解相关。然而，该木质纤维素生物质(如纤维素)的酶促水解的缺点是其工业生产工艺较为昂贵。因此，有必要使用日益有效的分泌纤维素酶的微生物菌株。在这方面，很多微生物包含水解纤维素的酶，例如真菌-木霉属(Trichoderma)、曲霉属(Aspergilus)、腐质霉属(Humicola)、或镰刀菌属(Fusarium)，以及细菌例如高温单孢菌属(Thermomonospora)、芽孢杆菌属(Bacilus)、纤维单胞菌属(Celulomonas)和链霉菌属(Streptomyces)。由这些微生物分泌的酶具有用于将纤维素转化为葡萄糖的三种不同类型的活性，可以分为三组：随机攻击纤维素纤维内部的内切葡聚糖酶，攻击纤维末端释放纤维二糖的外切葡聚糖酶，和将该纤维二糖水解为葡萄糖的β-葡萄糖苷酶。其他类型的酶，例如半纤维素酶或最近发现的一类多糖单加氧酶也可以在水解功效中起作用。
工业上强烈需要降低酶促水解的成本，而这种将低涉及使用降低的酶剂量，从而使用更有效的酶鸡尾酒。因此，一些专利申请描述了活性高于里氏木霉(Trichodermareesei)的天然酶或通过基因工程改进的变体。可以提及的有涉及外切葡聚糖酶的专利申请US2010304464、WO 2010/066411和WO 2013/029176，涉及内切葡聚糖酶的申请WO 2007/109441、WO 2012/149192和WO 2010/076388，涉及β-葡萄糖苷酶的申请WO 2010/029259、WO2010/135836或WO 2010/022518，或涉及多糖单加氧酶的申请WO 12135659和WO 12149344。
主要基于结构标准，水解木质纤维素生物质的酶在CAZy系统中分类(Cantarel,B.L.,Coutinho,P.M.,Rancurel,C.,Bernard,T.,Lombard,V.,&Henrissat,B.(2009).TheCarbohydrate-Active EnZymes database(CAZy):an expert resource forGlycogenomics.Nucleic acids research,37,D233-8)。外切葡聚糖酶可属于GH 6、7、9、48和74家族。
为了使木质纤维素生物质水解有效且具有经济效益，酶混合物必须包含平衡比例的具有各种酶活性的酶，特别包括但不排除外切葡聚糖酶、内切葡聚糖酶、木聚糖酶和β-葡萄糖苷酶类型。举例而言，在里氏木霉的天然混合物中，一般注意到存在60-70％的外切葡聚糖酶、15-20％的内切葡聚糖酶、几个百分点的半纤维素酶和约的5-10％的β-葡萄糖苷酶。该混合物适于以可接受的收率水解大部分经预处理的物质(例如在酸条件下汽爆的小麦秸秆类型)。在混合物中已经存在相当比例的外切葡聚糖酶表明，增加这些酶的量而不损害其他活性将非常困难。里氏木霉的基因组包含两种外切葡聚糖酶，一种来自家族6(CBH2,cel6a)，另一种来自家族7(CBH1,Cel7a)。它们分别将纤维素的非还原(EC3.2.1.176)和还原(EC3.2.1.91)末端水解为纤维二糖。
水解和发酵可以根据各种方案进行。最常见的由独立的水解和发酵(SHF)组成。该方法能够通过维持最佳反应条件来优化各个步骤。该发酵在约28℃至约30℃的温度即时进行，而水解一般在至少45℃的温度进行。然而，在SHF中，在反应结束时释放的糖浓度非常高，导致对酶的抑制，降低了该方法的效率。为了避免这些缺点，可以设想另一种方法。在SSF中，两个步骤(己糖的水解和发酵)同时发生，防止糖积聚到抑制酶的浓度。由于使用单一反应器，投资成本也降低了。由于没有抑制，水解率更高，因为释放的糖立即被用于发酵成乙醇。在该方法中，反应器的温度必然在水解和发酵的最佳温度之间形成平衡，一般在约30℃至约35℃。然而，纤维素分解酶的活性在该温度降低约30％。
SSF也允许分解纤维素的酶在发酵糖的生物中表达，从而能够限制，或在极端情况下消除在单独步骤产生的酶的来源。
因此，获得在水解和发酵的最佳温度(即，30℃-50℃)下保留有效的外切葡聚糖酶活性的酶，同时保持混合物中所有酶的比例，对将木质纤维素生物质转化为生物燃料的工艺而言是显著的增益。
本发明人已经开发了具有增强的外切葡聚糖酶活性的多肽，尤其是与序列SEQ IDNO：2的CBH2参考蛋白的外切葡聚糖酶活性相比。CBH2对应于来自里氏木霉的外切葡聚糖酶2。
在这方面，值得赞扬的是，申请人在大量研究后已经分离或纯化与CBH2参考蛋白(SEQ ID NO：2)的外切葡聚糖酶活性相比，外切葡聚糖酶活性增强的多肽。
因此，本发明涉及选自以下的多肽：
i.选自SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26和SEQ ID NO:28的氨基酸序列；和
ii.与序列SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ IDNO:26和SEQ ID NO:28相比具有至少70％，优选至少75％、80％、85％、90％、95％、98％或99％百分比的相同残基(同一性百分比)的氨基酸序列。
优选的，上述多肽的特征在于，其在发酵生物中的表达至少与CBH2参考蛋白(SEQID NO：2)的表达相等。
根据本发明，给定序列相对于SEQ ID NO:4、6、8、10、12、14、16、18、20、22、24、26或28的同一性百分比对应于该给定序列与SEQ ID NO:4、6、8、10、12、14、16、18、20、22、24、26或28之间相同的残基数除以SEQ ID NO:4、6、8、10、12、14、16、18、20、22、24、26或28的残基数。
在一个优选的实施方案中，在约35℃和/或约50℃的温度下，与氨基酸序列SEQ IDNO：2的CBH2多肽的外切葡聚糖酶活性相比，本发明的多肽的外切葡聚糖酶活性增强至少10％，优选至少20％，优选至少30％，甚至更优选至少40％。
例如，本领域技术人员将能够使用底物例如纤维素纤维素PASC或使用显色底物(对硝基苯基糖苷)例如pNP乳糖苷来测定酶活性的增加，或换言之增强。将分别通过释放的还原糖或硝基苯酚的比色法分析来显示酶活性。
本领域技术人员能够用来测定根据本发明的多肽与CBH2参考蛋白(SEQ ID NO：2)相比，酶活性是否增强的实验方案的一个实例如下：
–制备表达根据本发明的重组酶的解脂耶罗维亚酵母(Y.lipolytica)的原种培养物，于28℃过夜；
–用一定体积的原种培养物接种表达培养基使其在培养开始时在600nm的光密度为0.2；
–于28℃培养所述细胞96小时；
–在8000rpm离心5分钟；
–于35℃和50℃用100μl含有1％还原纤维糊精(CD)的pH6的0.1M柠檬酸磷酸缓冲液孵育100μl上清液4小时；
–取出100μl反应液；
–加入100μl DNS试剂；
–于100℃孵育5分钟；
–在冰上孵育3分钟；
–在3000rpm离心10分钟；
–用150μl在540nm读取OD。
本发明的一个主题还是编码至少一种如上所述多肽的纯化或分离的核酸。下表1包含来自里氏木霉的参考基因CBH2、来自鞭毛藻丛赤壳菌(Nectria haematococca，NH)和来自小麦赤霉菌(Giberela zeae，GZ)的推定的外切葡聚糖酶的核酸和肽序列，以及本发明的多肽和核酸的鉴定。
表1
克隆 核酸 多肽
CBH2(野生型) SEQ ID NO:1 SEQ ID NO:2
35B7 SEQ ID NO:3 SEQ ID NO:4
95B7 SEQ ID NO:5 SEQ ID NO:6
100F11 SEQ ID NO:7 SEQ ID NO:8
139F12 SEQ ID NO:9 SEQ ID NO:10
157B11 SEQ ID NO:11 SEQ ID NO:12
161A1 SEQ ID NO:13 SEQ ID NO:14
161C12 SEQ ID NO:15 SEQ ID NO:16
189H8 SEQ ID NO:17 SEQ ID NO:18
196D9 SEQ ID NO:19 SEQ ID NO:20
198E11 SEQ ID NO:21 SEQ ID NO:22
251B4 SEQ ID NO:23 SEQ ID NO:24
251C4 SEQ ID NO:25 SEQ ID NO:26
382A2 SEQ ID NO:27 SEQ ID NO:28
GZ基因 SEQ ID NO:29 SEQ ID NO:30
NH-7基因 SEQ ID NO:31 SEQ ID NO:32
本发明的一个主题还是编码至少一种如上所述多肽的纯化或分离的核酸。
优选的，所述纯化或分离的核酸选自以下序列：SEQ ID NO:3、SEQ ID NO:5、SEQID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25和SEQ ID NO:27。
根据本发明，如上所述的核酸可以与启动子、终止子或其在宿主细胞中的表达所需的任何其他序列功能性连接。
本发明还涉及包含至少一个如上所述的核酸的载体。
根据本发明，术语“载体”意欲是指可能在其中插入外源核酸片段的任何DNA序列，载体能够将外源DNA引入宿主细胞。作为载体，可以提及(不排除其他)：质粒、黏粒、酵母人工染色体(YAC)、细菌人工染色体(BAC)、衍生自P1噬菌体的人工染色体(PAC)或来自病毒的载体。
根据本发明的载体还可以携带可选择标志物。术语“可选择标志物”意欲是指其表达赋予包含它的细胞使得能够选择它们这一特征的基因。例如抗生素抗性基因。
本发明的一个主题还是包含至少一种如上所述的多肽、或至少一种如上所述的核酸、或至少一种如上所述的载体的分离的宿主细胞。
本领域技术人员将能够通过熟知的常规方法将如上所述的多肽之一、核酸之一或载体之一引入宿主细胞。例如，可以提及氯化钙处理、电穿孔或使用基因枪。
根据一个实施方案，本领域技术人员将能够通过常规方法将编码根据本发明的具有增强的外切葡聚糖酶活性的多肽的核酸的几个拷贝引入宿主细胞。
根据一个实施方案，如上所述的分离的宿主细胞选自：木霉属(Trichoderma)、曲霉属(Aspergilus)、脉胞菌属(Neurospora)、腐质霉属(Humicola)、毁丝霉属(Myceliophthora)、金孢子菌属(Chrysosporium)、青霉属(Penicilium)、镰刀菌属(Fusarium)、高温单孢菌属(Thermomonospora)、芽孢杆菌属(Bacilus)、假单包菌属(Pesudomonas),埃希氏菌属(Escherichia)、梭状芽孢杆菌(Clostridium)、纤维单胞菌属(Celulomonas)、链霉菌属(Streptomyces)、耶罗维亚酵母属(Yarrowia)、毕赤酵母属(Pichia)和酵母属(Saccharomyces)。
根据一个优选的实施方案，如上所述的宿主细胞选自：里氏木霉、Trichodermaviridae、康氏木霉(Trichoderma koningi)、黑曲霉(Aspergilus niger)、构巢曲霉(Aspergilus nidulans)、文氏曲霉(Aspergilus wenti)、米曲霉(Aspergilus oryzae)、海枣曲霉(Aspergilus phoenicis)、嗜热毁丝霉(Myceliophthora thermopila)、Chrysosporium lucknowense、粗糙脉孢菌(Neurospora crassa)、灰腐质霉(Humicolagrisae)、嗜松青霉(Penicilium pinophilum)、草酸青霉(Penicilium oxalicum)、大肠杆菌(Escherichia coli)、丙酮丁醇梭菌(Clostridium acetobutylicum)、糖解梭菌(Clostridium saccharolyticum)、拜氏梭菌(Clostridium benjerincki)、丁酸梭菌(Clostridium butylicum)、毕赤酵母(Pichia pastoris)、解脂耶罗维亚酵母(Yarrowialipolityca)和酿酒酵母(Saccharomyces cerevisiae)。
根据一个优选的实施方案，如上所述的分离的宿主细胞选自里氏木霉和酿酒酵母。
本发明的一个主题还是如上所述的任何一种多肽用于纤维素水解的用途。
本发明的一个主题还是如上所述的任何一种多肽用于生产生物燃料的用途。
根据本发明，术语“生物燃料”可以定义为来自生物质转化且可以用于能量目的的任何产物。首先，不希望被限制于此，可以提及的实例有生物气、可引入燃料(任选地在随后转化之后)或其本身可以作为燃料的产物，例如醇(乙醇、丁醇和/或异丙醇，取决于所用发酵生物的类型)、溶剂(丙酮)、酸(丁酸)、脂质及其衍生物(短链或长链脂肪酸、脂肪酸酯)和氢。
优选的，根据本发明的生物燃料是醇，例如乙醇、丁醇和/或异丙醇。更优选的，根据本发明的生物燃料是乙醇。
在另一个实施方案中，生物燃料是生物气。
在另一个实施方案中，产物是在化学工业中具有优势的分子，例如另一种醇，诸如1,2-丙二醇、1,3-丙二醇、1,4-丙二醇或2,3-丁二醇，有机酸例如乙酸、丙酸、丙烯酸、丁酸、琥珀酸、苹果酸、延胡索酸、柠檬酸或衣康酸，或羟基酸例如乙醇酸，羟基丙酸和乳酸。
生产用于木质纤维素水解的酶鸡尾酒的一个实施方案描述如下。
在碳基底物例如乳糖或葡萄糖(选择用于微生物生长)存在下，在发酵器中培养能够表达至少一种根据本发明的多肽的丝状真菌(优选木霉属，更优选里氏木霉)的菌株。在一个实施方案中，取决于其性质，所述碳基底物在灭菌之前引入发酵器，或单独灭菌并在发酵器灭菌后引入发酵器，从而获得20-35g/l的初始浓度。
然后加入含有选择用于酶生产的底物的水性溶液。最后通过将培养基离心回收真菌产生的作用于木质纤维素生物质的酶组合物。该组合物特别包含根据本发明的β-葡萄糖苷酶、内切葡聚糖酶和外切葡聚糖酶。
在一个实施方案中，含有选择用于生产酶的底物的水性溶液以200-250g/l的浓度制备。该溶液优选还包含诱导底物，例如乳糖。在初始碳基底物耗尽后注入该水性溶液，以提供35-45mg/g细胞的最佳量(分批进料)。在该分批进料阶段期间，培养基中糖的残留浓度小于1g/l，通过真菌分泌作用于木质纤维素生物质的酶。所述酶可以通过离心培养基来回收。
本发明的一个主题是能够作用于木质纤维素生物质的酶组合物，所述酶组合物由丝状真菌产生，且包含至少一种与CBH2参考蛋白的外切葡聚糖酶活性相比，外切葡聚糖酶活性增强的多肽。术语“丝状真菌”特别意欲是指木霉属，更优选里氏木霉。
最后，本发明的一个主题是用于从生物质生产生物燃料的方法，包含以下连续步骤：
–将待水解的生物质悬浮于水相中；
–在如上所述的酶组合物存在下水解木质纤维素生物质，以产生含有葡萄糖的水解物；
–在发酵生物存在下发酵水解物的葡萄糖，以产生发酵液；
–将生物燃料与发酵液分离。
在一个实施方案中，待水解的生物质以6％-40％，优选20％-30％的固体量悬浮于水相。将pH调节至4-5.5，优选4.8-5.2，温度为40-60℃，优选45-50℃。通过加入作用于木质纤维素生物质的酶组合物启动水解反应，通常使用的量是10-30mg分泌蛋白/g预处理底物，或更少。反应通常持续15-48小时。反应之后分析释放的糖，尤其是葡萄糖。通过过滤或离心然后在发酵单元中处理将糖溶液与未水解的固体部分(主要由木质素组成)分离。
在发酵步骤后，例如通过蒸馏将生物燃料与发酵液分离。
本发明的另一个主题是用于从生物质生产生物燃料的方法，其特征在于，它包含以下连续步骤：
–将待水解的生物质悬浮于水相中；
–向悬浮液同时加入如上所述的酶组合物和发酵生物，发酵混合物以产生发酵液；
–将生物燃料与发酵液分离。
优选的，同时加入酶组合物和发酵生物，然后在30-50℃的温度下孵育以产生发酵液。
根据该实施方案，根据本领域技术人员已知的SSF(同时发生的糖化和发酵)方法，将存在于生物质中的纤维素转化为葡萄糖，同时，在同一反应器中，发酵生物(例如酵母)将葡萄糖转化为最终产物。取决于发酵生物的代谢和水解能力，可能需要加入或多或少显著量的外源细胞裂解混合物以使操作正确进行。
在另一个实施方案中，发酵生物通过分泌或在其细胞表面产生本发明主题的多肽，任选和作用于木质纤维素生物质的其他酶一起，从而限制或消除对由丝状真菌产生的酶的需要。优选的，发酵生物是如上所述的宿主细胞。
优选的，加入具有酶组合物的宿主细胞和/或发酵生物，然后在30-50℃的温度下孵育以产生发酵液。
因此，使用根据本发明的具有更好外切葡聚糖酶活性的多肽具有以下优势：获得更好的葡萄糖生产收率，同时使用比之前更少的酶，从而还具有经济上的优势。
本发明的其他方面、主题、优势和特征将通过实施例和附图的方式阅读以下描述本发明优选实施方案的非限制性描述展示。
图1是显示用于筛选的DP3-DP11纤维糊精的MALDI-TOF质谱图。
具体实施方式
实施例1：制备还原纤维糊精(DP3-11)
1-纤维素水解(改编自Y-H.Percival Zhang,L.R.Lynd AnalyticalBiochemistry 322(2003),225-232.)
向160ml冷却至0℃的盐酸溶液分批加入20g纤维素(Avicel,CAS Number 9004-34-6,Sigma-Aldrich Saint-Quentin Fallavier)，并剧烈搅拌。分几个步骤(4×10ml)向该溶液中加入预先冷却的硫酸。将该反应于24℃保持搅拌4小时，然后倒入冷却至-20℃的0.8l丙酮。搅拌2小时后，过滤出沉淀物，取400ml冷却的丙酮，然后再次过滤。然后将固体置于600ml水中，搅拌过夜以溶解纤维糊精(CD)。过滤出固体后，用300g Amberlite IRA400OH-树脂中和含有纤维糊精的可溶部分，然后冻干。然后在过滤前，在超声存在下，将冻干物重悬于500ml甲醇中30分钟以溶解低分子量的糖，然后再次冻干以获得6.8g DP3-11纤维糊精。
对于筛选，选择具有最好可能分子量的底物进行，以尽可能模拟纤维素的结构。然而，高分子量的纤维糊精是不可溶的，这使该测试不容易重复。
因此，选择DP5-7的纤维糊精，这表示所需的高分子量和仙湖微晶的溶解度之间的良好平衡。
图1表示根据如上所述的方法一般获得的MALDI-TOF质谱。
图1表明，分离的寡糖主要是DP5-7。
2-纤维糊精的还原
在稀释于120ml水的2g DP3-11纤维糊精中加入400mg硼氢化钠。在室温下搅拌3小时后，加入Amberlite H+ IR 120树脂中和溶液，过滤，然后冻干，以获得2g定量还原的纤维糊精(C.Schou,G.Rasmussen,M-B.Kaltoft,B.Henrissat,M.SchuleinEur.J.Biochem.217,947-953(1993))。
用BCA(二喹啉甲酸)分析分离的纤维糊精能够确认末端的完全还原(Y.-H.Percival Zhang,L.R.Lynd Biomacromolecules 2005,6,1510-1515)。
实施例2：通过L-洗牌(shuffling)的进化
将里氏木霉的纤维二糖水解酶2基因(SEQ ID NO：1)用与亲本基因CBH2(SEQ IDNO：1)分别具有63％和69％同源性的来自小麦赤霉菌PH-1的推测的外切葡聚糖酶基因(SEQID NO：29)和来自鞭毛藻丛赤壳菌mpVI的假想蛋白NECHADRAFT_73991基因(SEQ ID NO：31)根据专利EP1104457中所述的专利方法进行一轮L-洗牌。在克隆中删除编码信号肽的核酸序列(SEQ ID NO:33)，并用酵母的序列SEQ ID NO：34(对应的信号肽序列：SEQ ID NO:35)替换。
1-高通量筛选
开发高通量筛选试验以选择由洗牌产生的最佳克隆，即，与CBH2参考酶(SEQ IDNO：2)相比，显示纤维二糖水解酶活性增强至少20％的那些。
高通量筛选试验实验根据以下步骤进行：
–在琼脂上分离表达根据本发明的酶的L-洗牌变体的解脂耶罗维亚酵母的克隆，并于28℃在YNB casa培养基(酵母氮基1.7g/l、NH4Cl 10g/l、葡萄糖10g/l、酪蛋白氨基酸2g/l,pH 7)中预培养所述克隆36小时；
–用所述预培养接种补充有12.5μg/ml的四环素的5％YTD培养基(酵母提取物10g/l、胰蛋白胨20g/l、葡萄糖2.5g/l,pH 6.8)，然后于28℃孵育20小时；
–用之前的培养物接种含有20g/l量的诱导剂(油酸)的10％表达培养基，然后于28℃孵育96小时
-在1500rpm离心5分钟；
–取出100μl上清液；
–加入100μl在0.1M柠檬酸磷酸缓冲液(pH6)中的1g/l的还原CD，
–于35℃孵育24小时；
–在2500rpm离心5分钟；
–取出80μl上清液；
–加入80μl DNS试剂；
–于105℃孵育12分钟，然后在冰上孵育5分钟；
–用120μl在540nm读取光学密度(OD)。
在这些筛选条件下，发现一些克隆中的纤维二糖水解酶活性与CBH2参考酶(SEQID NO:2)相比增强(540nm的OD增加)。在这些克隆中，可以提及35B7、95B7、100F11、139F12、157B11、161A1、161C12、189H8、196D9、198E11、251B4、251C4和382A2，其分别编码酶SEQ IDNO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ IDNO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26和SEQID NO:28。
2-测定纤维二糖水解酶活性的增强
2-1、在还原纤维糊精底物上
为了评估在第一轮洗牌中选择的变体相对于CBH2参考酶(SEQ ID NO:2)的Kcat，进行以下过程：
–制备表达根据本发明的重组酶的解脂耶罗维亚酵母的原种培养物，于28℃过夜；
–用一定体积的原种培养物接种表达培养基使其在培养开始时在600nm的光密度为0.2；
–于28℃培养所述细胞96小时；
–在8000rpm离心5分钟；
–于35℃和50℃用100μl含有1％还原纤维糊精(CD)的pH6的0.1M柠檬酸磷酸缓冲液孵育100μl上清液4小时；
–取出100μl反应液；
–加入100μl DNS试剂；
–于100℃孵育5分钟；
–在冰上孵育3分钟；
–在3000rpm离心10分钟；
–用150μl在540nm读取光密度。
根据本发明，根据以下方法计算Kcat：
–将测试中在540nm的OD作为培养物上清液的体积的函数作曲线图；
–减去阴性对照的值；
–除以葡萄糖标准率系数(用DNS显示的葡萄糖的各种量)；
–除以反应时间(240分钟)。
表2给出了在这些实验条件下，与CBH2参考蛋白(SEQ ID NO：2)相比，分别编码酶SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26和SEQ ID NO:28的35B7、95B7、100F11、139F12、157B11、161A1、161C12、189H8、196D9、198E11、251B4、251C4和382A2克隆所得的Kcat值和增强倍数。
表2：在还原CD上纤维二糖水解酶活性的增强
结果表明，无论在35℃或在50℃，与CBH2参考蛋白(SEQ ID NO：2)相比，分别编码酶SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26和SEQ ID NO:28的35B7、95B7、100F11、139F12、157B11、161A1、161C12、189H8、196D9、198E11、251B4、251C4和382A2克隆的酶活性增强。
2-2、在Avicel底物上
然后在第二种底物：Avicel上测量分别编码酶SEQ ID NO:4、SEQ ID NO:6、SEQ IDNO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ IDNO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26和SEQ ID NO:28的35B7、95B7、100F11、139F12、157B11、161A1、161C12、189H8、196D9、198E11、251B4、251C4和382A2克隆的活性的增强。
根据以下实验方案通过端点测量进行在这种底物上活性增强的测定：
–制备表达根据本发明的重组酶的解脂耶罗维亚酵母的原种培养物，于28℃过夜；
–用一定体积的原种培养物接种表达培养基使其在培养开始时在600nm的光密度为0.2；
–于28℃培养所述细胞96小时；
–在8000rpm离心5分钟；
–于35℃和50℃用100μl含有1％Avicel的pH6的0.1M柠檬酸磷酸缓冲液孵育100μl上清液18小时；
–取出100μl反应液；
–加入100μl DNS试剂；
–于100℃孵育5分钟；
–在冰上孵育3分钟；
–在3000rpm离心10分钟；
–用150μl在540nm读取光密度。
表3示出了在这些实验条件下，35B7、95B7、100F11、139F12、157B11、161A1、161C12、189H8、196D9、198E11、251B4、251C4和382A2克隆在540nm的OD值(减去阴性对照值之后)以及增强倍数。
表3：在Avicel上纤维二糖水解酶活性的增强
表3的结果表明，198E11和251B4克隆(分别为SEQ ID No:22和24)在35℃的酶活性与CBH2参考酶(SEQ ID NO：2)相比增强，382A2克隆(SEQ ID NO:28)在35℃和50℃的酶活性与CBH2参考酶(SEQ ID NO：2)相比增强。
序列表
<110> IFP Energies Nouvelles et al
<120> 具有增强活性的外切葡聚糖酶变体及其用途
<130> BFF140338
<160> 35
<170> PatentIn version 3.5
<210> 1
<211> 1413
<212> DNA
<213> 里氏木霉
<400> 1
atgattgtcg gcattctcac cacgctggct acgctggcca cactcgcagc tagtgtgcct 60
ctagaggagc ggcaagcttg ctcaagcgtc tggggccaat gtggtggcca gaattggtcg 120
ggtccgactt gctgtgcttc cggaagcaca tgcgtctact ccaacgacta ttactcccag 180
tgtcttcccg gcgctgcaag ctcaagctcg tccacgcgcg ccgcgtcgac gacttctcga 240
gtatccccca caacatcccg gtcgagctcc gcgacgcctc cacctggttc tactactacc 300
agagtacctc cagtcggatc gggaaccgct acgtattcag gcaacccttt tgttggggtc 360
actccttggg ccaatgcata ttacgcctct gaagttagca gcctcgctat tcctagcttg 420
actggagcca tggccactgc tgcagcagct gtcgcaaagg ttccctcttt tatgtggcta 480
gatactcttg acaagacccc tctcatggag caaaccttgg ccgacatccg caccgccaac 540
aagaatggcg gtaactatgc cggacagttt gtggtgtatg acttgccgga tcgcgattgc 600
gctgcccttg cctcgaatgg cgaatactct attgccgatg gtggcgtcgc caaatataag 660
aactatatcg acaccattcg tcaaattgtc gtggaatatt ccgatatccg gaccctcctg 720
gttattgagc ctgactctct tgccaacctg gtgaccaacc tcggtactcc aaagtgtgcc 780
aatgctcagt cagcctacct tgagtgcatc aactacgccg tcacacagct gaaccttcca 840
aatgttgcga tgtatttgga cgctggccat gcaggatggc ttggctggcc ggcaaaccaa 900
gacccggccg ctcagctatt tgcaaatgtt tacaagaatg catcgtctcc gagagctctt 960
cgcggattgg caaccaatgt cgccaactac aacgggtgga acattaccag ccccccatcg 1020
tacacgcaag gcaacgctgt ctacaacgag aagctgtaca tccacgctat tggacctctt 1080
cttgccaatc acggctggtc caacgccttc ttcatcactg atcaaggtcg atcgggaaag 1140
cagcctaccg gacagcaaca gtggggagac tggtgcaatg tgatcggcac cggatttggt 1200
attcgcccat ccgcaaacac tggggactcg ttgctggatt cgtttgtctg ggtcaagcca 1260
ggcggcgagt gtgacggcac cagcgacagc agtgcgccac gatttgactc ccactgtgcg 1320
ctcccagatg ccttgcaacc ggcgcctcaa gctggtgctt ggttccaagc ctactttgtg 1380
cagcttctca caaacgcaaa cccatcgttc ctg 1413
<210> 2
<211> 471
<212> PRT
<213> 里氏木霉
<400> 2
Met Ile Val Gly Ile Leu Thr Thr Leu Ala Thr Leu Ala Thr Leu Ala
1 5 10 15
Ala Ser Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser Val Trp Gly
20 25 30
Gln Cys Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala Ser Gly
35 40 45
Ser Thr Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Gly
50 55 60
Ala Ala Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser Thr Thr Ser Arg
65 70 75 80
Val Ser Pro Thr Thr Ser Arg Ser Ser Ser Ala Thr Pro Pro Pro Gly
85 90 95
Ser Thr Thr Thr Arg Val Pro Pro Val Gly Ser Gly Thr Ala Thr Tyr
100 105 110
Ser Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr
115 120 125
Ala Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met
130 135 140
Ala Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu
145 150 155 160
Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile
165 170 175
Arg Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val
180 185 190
Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu
195 200 205
Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp
210 215 220
Thr Ile Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu
225 230 235 240
Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr
245 250 255
Pro Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr
260 265 270
Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala
275 280 285
Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala
290 295 300
Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu
305 310 315 320
Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp Asn Ile Thr
325 330 335
Ser Pro Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu Lys Leu
340 345 350
Tyr Ile His Ala Ile Gly Pro Leu Leu Ala Asn His Gly Trp Ser Asn
355 360 365
Ala Phe Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly
370 375 380
Gln Gln Gln Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly
385 390 395 400
Ile Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ser Phe Val
405 410 415
Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Ser Ser Ala
420 425 430
Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala
435 440 445
Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Thr
450 455 460
Asn Ala Asn Pro Ser Phe Leu
465 470
<210> 3
<211> 1353
<212> DNA
<213> 人工序列
<220>
<223> 克隆 35B7
<400> 3
gcccctcttg ttgaggagcg ccaggcttgc gctgcccaat gggcccagtg tggtggcttc 60
agctggaatg gtgctacctg ctgccagtct ggtagctact gcagcaagat caacgactat 120
tactctcagt gcattcctgg agaaggcccc gccacttcca agtcgagcac tcttcctgct 180
tccaccacaa caactcagcc aacttccact tcgactgctg gaacctcttc cactaccaag 240
cctcctccag ctggatcggg aaccgctacg tattcaggca acccttactc tggggtcaac 300
ctttgggcca atagctatta ccgctctgaa gttaccaacc tcgctattcc taagttgagc 360
ggagccatgg ccactgctgc agcaaaggtc gcagatgttc cctcttttca gtggatggat 420
tctttcgacc acatctccct catggaggac accttggtcg acatccgcaa ggccaacctg 480
gctggcggta actatgccgg acagtttgtg gtgtatgact tgccggatcg cgattgcgct 540
gccgccgcct cgaatggcga atactctctt gccgatggtg gcgtcgccaa atataaggcc 600
tatatcgcca agattaaggg tattctccag gactattccg atacccggat cattctggtt 660
attgagcctg actctcttgc caacctggtg accaacatga acgtcccaaa gtgtgccaat 720
gctgcttcag cctacaagga gctcaccatt cacgccctca aggagctgaa ccttccaaat 780
gtttccatgt atatcgacgc tggccatggt ggatggcttg gctggccggc aaaccaaggc 840
ccggccgcta aactatttgc aagcatctac aaggatgcag gcaagccggc cgctcttcgc 900
ggattggcaa ccaatgtcgc caactacaac gcctggagcc tcagcagcgc tccaccttac 960
acgcaaggcg cctctatcta cgacgagaag agcttcatcc acgctatggg acctcttctt 1020
gagcagaatg gctggcctgg tgcccacttc atcactgatc aaggtcgatc gggaaagcag 1080
cctaccggac agatccagtg gggagactgg tgcaattcca agggcaccgg atttggtatt 1140
cgcccatccg caaacactgg ggactcgttg ctggatgctt ttgtctgggt caagccaggc 1200
ggcgagtctg acggcaccag cgacaccagt gcggctcgat acgactacca ctgtggtatt 1260
gacggcgccg tcaagccggc gcctgaggct ggtacctggt tccaagccta ctttgagcag 1320
cttctcacaa acgcaaaccc atcgttcctg taa 1353
<210> 4
<211> 450
<212> PRT
<213> 人工序列
<220>
<223> 克隆 35B7
<400> 4
Ala Pro Leu Val Glu Glu Arg Gln Ala Cys Ala Ala Gln Trp Ala Gln
1 5 10 15
Cys Gly Gly Phe Ser Trp Asn Gly Ala Thr Cys Cys Gln Ser Gly Ser
20 25 30
Tyr Cys Ser Lys Ile Asn Asp Tyr Tyr Ser Gln Cys Ile Pro Gly Glu
35 40 45
Gly Pro Ala Thr Ser Lys Ser Ser Thr Leu Pro Ala Ser Thr Thr Thr
50 55 60
Thr Gln Pro Thr Ser Thr Ser Thr Ala Gly Thr Ser Ser Thr Thr Lys
65 70 75 80
Pro Pro Pro Ala Gly Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Tyr
85 90 95
Ser Gly Val Asn Leu Trp Ala Asn Ser Tyr Tyr Arg Ser Glu Val Thr
100 105 110
Asn Leu Ala Ile Pro Lys Leu Ser Gly Ala Met Ala Thr Ala Ala Ala
115 120 125
Lys Val Ala Asp Val Pro Ser Phe Gln Trp Met Asp Ser Phe Asp His
130 135 140
Ile Ser Leu Met Glu Asp Thr Leu Val Asp Ile Arg Lys Ala Asn Leu
145 150 155 160
Ala Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp Leu Pro Asp
165 170 175
Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu Tyr Ser Leu Ala Asp
180 185 190
Gly Gly Val Ala Lys Tyr Lys Ala Tyr Ile Ala Lys Ile Lys Gly Ile
195 200 205
Leu Gln Asp Tyr Ser Asp Thr Arg Ile Ile Leu Val Ile Glu Pro Asp
210 215 220
Ser Leu Ala Asn Leu Val Thr Asn Met Asn Val Pro Lys Cys Ala Asn
225 230 235 240
Ala Ala Ser Ala Tyr Lys Glu Leu Thr Ile His Ala Leu Lys Glu Leu
245 250 255
Asn Leu Pro Asn Val Ser Met Tyr Ile Asp Ala Gly His Gly Gly Trp
260 265 270
Leu Gly Trp Pro Ala Asn Gln Gly Pro Ala Ala Lys Leu Phe Ala Ser
275 280 285
Ile Tyr Lys Asp Ala Gly Lys Pro Ala Ala Leu Arg Gly Leu Ala Thr
290 295 300
Asn Val Ala Asn Tyr Asn Ala Trp Ser Leu Ser Ser Ala Pro Pro Tyr
305 310 315 320
Thr Gln Gly Ala Ser Ile Tyr Asp Glu Lys Ser Phe Ile His Ala Met
325 330 335
Gly Pro Leu Leu Glu Gln Asn Gly Trp Pro Gly Ala His Phe Ile Thr
340 345 350
Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln Ile Gln Trp Gly
355 360 365
Asp Trp Cys Asn Ser Lys Gly Thr Gly Phe Gly Ile Arg Pro Ser Ala
370 375 380
Asn Thr Gly Asp Ser Leu Leu Asp Ala Phe Val Trp Val Lys Pro Gly
385 390 395 400
Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser Ala Ala Arg Tyr Asp Tyr
405 410 415
His Cys Gly Ile Asp Gly Ala Val Lys Pro Ala Pro Glu Ala Gly Thr
420 425 430
Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr Asn Ala Asn Pro Ser
435 440 445
Phe Leu
450
<210> 5
<211> 1362
<212> DNA
<213> 人工序列
<220>
<223> 克隆 95B7
<400> 5
gtgcctctag aggagcggca agcttgctca agcgtctggg gccaatgtgg tggccagaat 60
tggtcgggtc cgacttgctg tgcttccgga agcacatgcg tctactccaa cgactattac 120
tcccagtgtc ttcccggcgc tgcaagctca agctcgtcca cgcgcgccgc gtcgacgact 180
tctcgagtat cccccacaac atcccggtcg agctccgcga cgcctccacc tggttctact 240
actaccagag tacctccagt cggatcggga accgctacgt attcaggcaa cccttttgtt 300
ggggtcactc cttgggccaa tgcatattac gcctctgaag ttagcagcct cgctattcct 360
agcttgactg gagccatggc cactgctgca gcagctgtcg caaaggttcc ctcttttatg 420
tggctagata ctcttgacaa gacccctctc atggagcaaa ccttggccga catccgcacc 480
gccaacaaga atggcggtaa ctatgccgga cagtttgtgg tgtatgactt gccggatcgc 540
gattgcgctg cccttgcctc gaatggcgaa tactctattg ccgatggtgg cgtcgccaaa 600
tataagaact atatcgacac cattcgtcaa attgtcgtgg aatattccga tatccggacc 660
ctcctggtta ttgagcctga ctctcttgcc aaccaggtga ccaacatgga tgtcgccaag 720
tgtgccaagg ctcatgatgc ctacatcagc ctgacgaact acgccgtcac agaactgaac 780
cttccaaatg ttgcgatgta tttggacgct ggcaatgcag gatggcttgg ctggccggca 840
aaccaaggcc cggccgctaa actatttgca agcatctaca aggatgcagg caagccggcc 900
gctcttcgcg gattggcaac caatgtcgcc aactacaacg cctggagcct cagcagcgct 960
ccaccttaca cgcaaggcgc ctctatctac gacgagaaga gcttcatcca cgctatggga 1020
cctcttcttg agcagaatgg ctggcctggt gcccacttca tcactgatca aggtcgatcg 1080
ggaaagcagc ctaccggaca gatccagtgg ggagactggt gcaattccaa gggcaccgga 1140
tttggtattc gcccatccgc aaacactggg gactcgttgc tggatgcttt tgtctgggtc 1200
aagccaggcg gcgagtctga cggcaccagc gacaccagtg cgacccgata cgactaccac 1260
tgtggtgctt ctgccgcctt gcaaccggcg cctgaggctg gtacctggtt ccaagcctac 1320
tttgagcagc ttctcaagaa cgcaaaccca tcgttcctgt aa 1362
<210> 6
<211> 453
<212> PRT
<213> 人工序列
<220>
<223> 克隆 95B7
<400> 6
Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser Val Trp Gly Gln Cys
1 5 10 15
Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr
20 25 30
Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala
35 40 45
Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser Thr Thr Ser Arg Val Ser
50 55 60
Pro Thr Thr Ser Arg Ser Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr
65 70 75 80
Thr Thr Arg Val Pro Pro Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly
85 90 95
Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser
100 105 110
Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala Thr
115 120 125
Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu Asp Thr
130 135 140
Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile Arg Thr
145 150 155 160
Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp
165 170 175
Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser
180 185 190
Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile
195 200 205
Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile
210 215 220
Glu Pro Asp Ser Leu Ala Asn Gln Val Thr Asn Met Asp Val Ala Lys
225 230 235 240
Cys Ala Lys Ala His Asp Ala Tyr Ile Ser Leu Thr Asn Tyr Ala Val
245 250 255
Thr Glu Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly Asn
260 265 270
Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Gly Pro Ala Ala Lys Leu
275 280 285
Phe Ala Ser Ile Tyr Lys Asp Ala Gly Lys Pro Ala Ala Leu Arg Gly
290 295 300
Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Leu Ser Ser Ala
305 310 315 320
Pro Pro Tyr Thr Gln Gly Ala Ser Ile Tyr Asp Glu Lys Ser Phe Ile
325 330 335
His Ala Met Gly Pro Leu Leu Glu Gln Asn Gly Trp Pro Gly Ala His
340 345 350
Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln Ile
355 360 365
Gln Trp Gly Asp Trp Cys Asn Ser Lys Gly Thr Gly Phe Gly Ile Arg
370 375 380
Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ala Phe Val Trp Val
385 390 395 400
Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser Ala Thr Arg
405 410 415
Tyr Asp Tyr His Cys Gly Ala Ser Ala Ala Leu Gln Pro Ala Pro Glu
420 425 430
Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Lys Asn Ala
435 440 445
Asn Pro Ser Phe Leu
450
<210> 7
<211> 1323
<212> DNA
<213> 人工序列
<220>
<223> 克隆 100F11
<400> 7
gctcctgttg aagagcgtca gtcttgcagc aacggagtct ggtctcaatg tggtggtcag 60
aactggtccg gtactccttg ctgcaccagt ggaaacaagt gtgtcaaggt caacgacttc 120
tactcccagt gccagcctgg atctgcagac ccttctccca catccaccat tgtctccgcg 180
acgaccacca aggctactac tactggtagt ggaggctctg tcacctcgcc tcctcctgtt 240
gccaccaaca accctttttc tggggtcgat ctgtgggcca ataactatta ccgctctgaa 300
gttagcactc tcgctattcc taagttgagc ggagccatgg ccactgctgc agcaaaggtc 360
gcagatgttc cctcttttca gtggatggat acttatgacc acatctcctt catggaggag 420
tctttggccg acatccgcaa ggccaacaag gctggcggta actatgccgg acagtttgtg 480
gtgtatgact tgccggatcg cgattgcgct gccgctgcct cgaatggcga atactctctt 540
gacaaggatg gcaagaacaa atataaggcc tatatcgcca agattaaggg tattctccag 600
gactattccg atacccggat cattctggtt attgagcctg actctcttgc caacatggtg 660
accaacatga acgtcccaaa gtgtgccaat gctgcttcag cctacaagga gctcaccatt 720
cacgccctca aggagctgaa ccttccaaat gtttccatgt atatcgacgc tggccatggt 780
ggatggcttg gctggccggc aaaccttcct ccggccgctc agctatacgg tcagctctac 840
aaggatgcag gcaagccgtc tcgccttcgc ggattggtca ccaatgtctc caactacaac 900
gcctggaagc tatccagcaa gccagactac acggagagca accccaacta cgacgagcag 960
aagtacatcc acgctctgtc tcctcttctt gagcaggagg gctggcccgg tgccaagttc 1020
atcgtcgatc aaggtcgatc gggaaagcag cctaccggac agaaggcttg gggagactgg 1080
tgcaatgctc ccggcaccgg atttggtatt cgcccatccg caaacactgg ggactcgttg 1140
ctggatgctt ttgtctgggt caagccaggc ggcgagtctg acggcaccag cgacaccagt 1200
gcgacccgat acgactacca ctgtggtgct tctgccgcct tgcaaccggc gcctgaggct 1260
ggtacctggt tccaagccta ctttgagcag cttctcacaa acgcaaaccc atcgttcctg 1320
taa 1323
<210> 8
<211> 440
<212> PRT
<213> 人工序列
<220>
<223> 克隆 100F11
<400> 8
Ala Pro Val Glu Glu Arg Gln Ser Cys Ser Asn Gly Val Trp Ser Gln
1 5 10 15
Cys Gly Gly Gln Asn Trp Ser Gly Thr Pro Cys Cys Thr Ser Gly Asn
20 25 30
Lys Cys Val Lys Val Asn Asp Phe Tyr Ser Gln Cys Gln Pro Gly Ser
35 40 45
Ala Asp Pro Ser Pro Thr Ser Thr Ile Val Ser Ala Thr Thr Thr Lys
50 55 60
Ala Thr Thr Thr Gly Ser Gly Gly Ser Val Thr Ser Pro Pro Pro Val
65 70 75 80
Ala Thr Asn Asn Pro Phe Ser Gly Val Asp Leu Trp Ala Asn Asn Tyr
85 90 95
Tyr Arg Ser Glu Val Ser Thr Leu Ala Ile Pro Lys Leu Ser Gly Ala
100 105 110
Met Ala Thr Ala Ala Ala Lys Val Ala Asp Val Pro Ser Phe Gln Trp
115 120 125
Met Asp Thr Tyr Asp His Ile Ser Phe Met Glu Glu Ser Leu Ala Asp
130 135 140
Ile Arg Lys Ala Asn Lys Ala Gly Gly Asn Tyr Ala Gly Gln Phe Val
145 150 155 160
Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly
165 170 175
Glu Tyr Ser Leu Asp Lys Asp Gly Lys Asn Lys Tyr Lys Ala Tyr Ile
180 185 190
Ala Lys Ile Lys Gly Ile Leu Gln Asp Tyr Ser Asp Thr Arg Ile Ile
195 200 205
Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn
210 215 220
Val Pro Lys Cys Ala Asn Ala Ala Ser Ala Tyr Lys Glu Leu Thr Ile
225 230 235 240
His Ala Leu Lys Glu Leu Asn Leu Pro Asn Val Ser Met Tyr Ile Asp
245 250 255
Ala Gly His Gly Gly Trp Leu Gly Trp Pro Ala Asn Leu Pro Pro Ala
260 265 270
Ala Gln Leu Tyr Gly Gln Leu Tyr Lys Asp Ala Gly Lys Pro Ser Arg
275 280 285
Leu Arg Gly Leu Val Thr Asn Val Ser Asn Tyr Asn Ala Trp Lys Leu
290 295 300
Ser Ser Lys Pro Asp Tyr Thr Glu Ser Asn Pro Asn Tyr Asp Glu Gln
305 310 315 320
Lys Tyr Ile His Ala Leu Ser Pro Leu Leu Glu Gln Glu Gly Trp Pro
325 330 335
Gly Ala Lys Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr
340 345 350
Gly Gln Lys Ala Trp Gly Asp Trp Cys Asn Ala Pro Gly Thr Gly Phe
355 360 365
Gly Ile Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ala Phe
370 375 380
Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser
385 390 395 400
Ala Thr Arg Tyr Asp Tyr His Cys Gly Ala Ser Ala Ala Leu Gln Pro
405 410 415
Ala Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu
420 425 430
Thr Asn Ala Asn Pro Ser Phe Leu
435 440
<210> 9
<211> 1323
<212> DNA
<213> 人工序列
<220>
<223> 克隆 139F12
<400> 9
gctcctgttg aagagcgtca gtcttgcagc aacggagtct ggtctcaatg tggtggtcag 60
aactggtccg gtactccttg ctgcaccagt ggaaacaagt gtgtcaaggt caacgacttc 120
tactcccagt gccagcctgg atctgcagac ccttctccca catccaccat tgtctccgcg 180
acgaccacca aggctactac tactggtagt ggaggctctg tcacctcgcc tcctcctgtt 240
gccaccaaca accctttttc tggggtcgat ctgtgggcca ataactatta ccgctctgaa 300
gttagcactc tcgctattcc taagttgagc ggagccatgg ccactgctgc agcaaaggtc 360
gcagatgttc cctcttttca gtggatggat acttatgacc acatctcctt catggaggag 420
tctttggccg acatccgcaa ggccaacaag gctggcggta actatgccgg acagtttgtg 480
gtgtatgact tgccggatcg cgattgcgct gccgctgcct cgaatggcga atactctctt 540
gacaaggatg gcaagaacaa atataaggcc tatatcgcca agattaaggg tattctccag 600
gactattccg atacccggat cattctggtt attgagcctg actctcttgc caacatggtg 660
accaacatga acgtcccaaa gtgtgccaat gctgcttcag cctacaagga gctcaccatt 720
cacgccctca aggagctgaa ccttccaaat gtttccatgt atatcgacgc tggccatggt 780
ggatggcttg gctggccggc aaaccttcct ccggccgctc agctatacgg tcagctctac 840
aaggatgcag gcaagccgtc tcgccttcgc ggattggtca ccaatgtctc caactacaac 900
gcctggaagc tatccagcaa gccagactac acggagagca accccaacta cgacgagcag 960
aagtacatcc acgctctgtc tcctcttctt gagcaggagg gctggcccgg tgccaagttc 1020
atcgtcgatc aaggtcgatc gggaaagcag cctaccggac agaaggcttg gggagactgg 1080
tgcaatgctc ccggcaccgg atttggtctc cgcccatccg caaacactgg ggacgccttg 1140
gtcgatgctt ttgtctgggt caagccaggc ggcgagtctg acggcaccag cgacaccagt 1200
gcggctcgat acgactacca ctgtggtgct tctgccgcct tgcaaccggc gcctgaggct 1260
ggtacctggt tccaagccta ctttgagcag cttctcaaga acgcaaaccc atcgttcctg 1320
taa 1323
<210> 10
<211> 440
<212> PRT
<213> 人工序列
<220>
<223> 克隆 139F12
<400> 10
Ala Pro Val Glu Glu Arg Gln Ser Cys Ser Asn Gly Val Trp Ser Gln
1 5 10 15
Cys Gly Gly Gln Asn Trp Ser Gly Thr Pro Cys Cys Thr Ser Gly Asn
20 25 30
Lys Cys Val Lys Val Asn Asp Phe Tyr Ser Gln Cys Gln Pro Gly Ser
35 40 45
Ala Asp Pro Ser Pro Thr Ser Thr Ile Val Ser Ala Thr Thr Thr Lys
50 55 60
Ala Thr Thr Thr Gly Ser Gly Gly Ser Val Thr Ser Pro Pro Pro Val
65 70 75 80
Ala Thr Asn Asn Pro Phe Ser Gly Val Asp Leu Trp Ala Asn Asn Tyr
85 90 95
Tyr Arg Ser Glu Val Ser Thr Leu Ala Ile Pro Lys Leu Ser Gly Ala
100 105 110
Met Ala Thr Ala Ala Ala Lys Val Ala Asp Val Pro Ser Phe Gln Trp
115 120 125
Met Asp Thr Tyr Asp His Ile Ser Phe Met Glu Glu Ser Leu Ala Asp
130 135 140
Ile Arg Lys Ala Asn Lys Ala Gly Gly Asn Tyr Ala Gly Gln Phe Val
145 150 155 160
Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly
165 170 175
Glu Tyr Ser Leu Asp Lys Asp Gly Lys Asn Lys Tyr Lys Ala Tyr Ile
180 185 190
Ala Lys Ile Lys Gly Ile Leu Gln Asp Tyr Ser Asp Thr Arg Ile Ile
195 200 205
Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn
210 215 220
Val Pro Lys Cys Ala Asn Ala Ala Ser Ala Tyr Lys Glu Leu Thr Ile
225 230 235 240
His Ala Leu Lys Glu Leu Asn Leu Pro Asn Val Ser Met Tyr Ile Asp
245 250 255
Ala Gly His Gly Gly Trp Leu Gly Trp Pro Ala Asn Leu Pro Pro Ala
260 265 270
Ala Gln Leu Tyr Gly Gln Leu Tyr Lys Asp Ala Gly Lys Pro Ser Arg
275 280 285
Leu Arg Gly Leu Val Thr Asn Val Ser Asn Tyr Asn Ala Trp Lys Leu
290 295 300
Ser Ser Lys Pro Asp Tyr Thr Glu Ser Asn Pro Asn Tyr Asp Glu Gln
305 310 315 320
Lys Tyr Ile His Ala Leu Ser Pro Leu Leu Glu Gln Glu Gly Trp Pro
325 330 335
Gly Ala Lys Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr
340 345 350
Gly Gln Lys Ala Trp Gly Asp Trp Cys Asn Ala Pro Gly Thr Gly Phe
355 360 365
Gly Leu Arg Pro Ser Ala Asn Thr Gly Asp Ala Leu Val Asp Ala Phe
370 375 380
Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser
385 390 395 400
Ala Ala Arg Tyr Asp Tyr His Cys Gly Ala Ser Ala Ala Leu Gln Pro
405 410 415
Ala Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu
420 425 430
Lys Asn Ala Asn Pro Ser Phe Leu
435 440
<210> 11
<211> 1323
<212> DNA
<213> 人工序列
<220>
<223> 克隆 157B11
<400> 11
gctcctgttg aagagcgtca gtcttgcagc aacggagtct ggtctcaatg tggtggtcag 60
aactggtccg gtactccttg ctgcaccagt ggaaacaagt gtgtcaaggt caacgacttc 120
tactcccagt gccagcctgg atctgcagac ccttctccca catccaccat tgtctccgcg 180
acgaccacca aggctactac tactggtagt ggaggctctg tcacctcgcc tcctcctgtt 240
gccaccaaca accctttttc tggggtcgat ctgtgggcca ataactatta ccgctctgaa 300
gttagcactc tcgctattcc taagttgagc ggagccatgg ccactgctgc agcaaaggtc 360
gcagatgttc cctcttttca gtggatggat acttatgacc acatctcctt catggaggag 420
tctttggccg acatccgcaa ggccaacaag gctggcggta actatgccgg acagtttgtg 480
gtgtatgact tgccggatcg cgattgcgct gccgccgcct cgaatggcga atactctctc 540
gacaatgatg gcgccaacaa atataaggcc tatatcgcca agattaaggg tattctccag 600
gactattccg atacccggat cattctggtt attgagcctg actctcttgc caacatggtg 660
accaacatga acgtcccaaa gtgtgccaat gctgcttcag cctacaagga gctcaccatt 720
cacgccctca aggagctgaa ccttccaaat gtttccatgt atatcgacgc tggccatggt 780
ggatggcttg gctggccggc aaaccttcct ccggccgctc agctatacgg tcagctctac 840
aaggatgcag gcaagccgtc tcgccttcgc ggattggtca ccaatgtctc caactacaac 900
gcctggaagc tatccagcaa gccagactac acggagagca accccaacta cgacgagcag 960
aagtacatcc acgctctgtc tcctcttctt gagcaggagg gctggcccgg tgccaagttc 1020
atcgtcgatc aaggtcgatc gggaaagcag cctaccggac agaaggcttg gggagactgg 1080
tgcaatgctc ccggcaccgg atttggtctc cgcccatccg caaacactgg ggactcgttg 1140
ctggatgctt ttgtctgggt caagccaggc ggcgagtctg acggcaccag cgacaccagt 1200
gcgacccgat acgactacca ctgtggtgct tctgccgcct tgcaaccggc gcctgaggct 1260
ggtacctggt tccaagccta ctttgagcag cttctcacaa acgcaaaccc atcgttcctg 1320
taa 1323
<210> 12
<211> 440
<212> PRT
<213> 人工序列
<220>
<223> 克隆 157B11
<400> 12
Ala Pro Val Glu Glu Arg Gln Ser Cys Ser Asn Gly Val Trp Ser Gln
1 5 10 15
Cys Gly Gly Gln Asn Trp Ser Gly Thr Pro Cys Cys Thr Ser Gly Asn
20 25 30
Lys Cys Val Lys Val Asn Asp Phe Tyr Ser Gln Cys Gln Pro Gly Ser
35 40 45
Ala Asp Pro Ser Pro Thr Ser Thr Ile Val Ser Ala Thr Thr Thr Lys
50 55 60
Ala Thr Thr Thr Gly Ser Gly Gly Ser Val Thr Ser Pro Pro Pro Val
65 70 75 80
Ala Thr Asn Asn Pro Phe Ser Gly Val Asp Leu Trp Ala Asn Asn Tyr
85 90 95
Tyr Arg Ser Glu Val Ser Thr Leu Ala Ile Pro Lys Leu Ser Gly Ala
100 105 110
Met Ala Thr Ala Ala Ala Lys Val Ala Asp Val Pro Ser Phe Gln Trp
115 120 125
Met Asp Thr Tyr Asp His Ile Ser Phe Met Glu Glu Ser Leu Ala Asp
130 135 140
Ile Arg Lys Ala Asn Lys Ala Gly Gly Asn Tyr Ala Gly Gln Phe Val
145 150 155 160
Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly
165 170 175
Glu Tyr Ser Leu Asp Asn Asp Gly Ala Asn Lys Tyr Lys Ala Tyr Ile
180 185 190
Ala Lys Ile Lys Gly Ile Leu Gln Asp Tyr Ser Asp Thr Arg Ile Ile
195 200 205
Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn
210 215 220
Val Pro Lys Cys Ala Asn Ala Ala Ser Ala Tyr Lys Glu Leu Thr Ile
225 230 235 240
His Ala Leu Lys Glu Leu Asn Leu Pro Asn Val Ser Met Tyr Ile Asp
245 250 255
Ala Gly His Gly Gly Trp Leu Gly Trp Pro Ala Asn Leu Pro Pro Ala
260 265 270
Ala Gln Leu Tyr Gly Gln Leu Tyr Lys Asp Ala Gly Lys Pro Ser Arg
275 280 285
Leu Arg Gly Leu Val Thr Asn Val Ser Asn Tyr Asn Ala Trp Lys Leu
290 295 300
Ser Ser Lys Pro Asp Tyr Thr Glu Ser Asn Pro Asn Tyr Asp Glu Gln
305 310 315 320
Lys Tyr Ile His Ala Leu Ser Pro Leu Leu Glu Gln Glu Gly Trp Pro
325 330 335
Gly Ala Lys Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr
340 345 350
Gly Gln Lys Ala Trp Gly Asp Trp Cys Asn Ala Pro Gly Thr Gly Phe
355 360 365
Gly Leu Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ala Phe
370 375 380
Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser
385 390 395 400
Ala Thr Arg Tyr Asp Tyr His Cys Gly Ala Ser Ala Ala Leu Gln Pro
405 410 415
Ala Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu
420 425 430
Thr Asn Ala Asn Pro Ser Phe Leu
435 440
<210> 13
<211> 1323
<212> DNA
<213> 人工序列
<220>
<223> 克隆 161A1
<400> 13
gctcctgttg aagagcgtca gtcttgcagc aacggagtct ggtctcaatg tggtggtcag 60
aactggtccg gtactccttg ctgcaccagt ggaaacaagt gtgtcaaggt caacgacttc 120
tactcccagt gccagcctgg atctgcagac ccttctccca catccaccat tgtctccgcg 180
acgaccacca aggctactac tactggtagt ggaggctctg tcacctcgcc tcctcctgtt 240
gccaccaaca accctttttc tggggtcgat ctgtgggcca ataactatta ccgctctgaa 300
gttagcactc tcgctattcc taagttgagc ggagccatgg ccactgctgc agcaaaggtc 360
gcagatgttc cctcttttca gtggatggat acttatgacc acatctcctt catggaggag 420
tctttggccg acatccgcaa ggccaacaag gctggcggta actatgccgg acagtttgtg 480
gtgtatgact tgccggatcg cgattgcgct gccgctgcct cgaatggcga atactctctt 540
gacaaggatg gcaagaacaa atataaggcc tatatcgcca agattaaggg tattctccag 600
gactattccg atacccggat cattctggtt attgagcctg actctcttgc caacatggtg 660
accaacatga acgtcccaaa gtgtgccaat gctgcttcag cctacaagga gctcaccatt 720
cacgccctca aggagctgaa ccttccaaat gtttccatgt atatcgacgc tggccatggt 780
ggatggcttg gctggccggc aaaccttcct ccggccgctc agctatacgg tcagctctac 840
aaggatgcag gcaagccgtc tcgccttcgc ggattggtca ccaatgtctc caactacaac 900
gcctggaagc tatccagcaa gccagactac acggagagca accccaacta cgacgagcag 960
aagtacatcc acgctctgtc tcctcttctt gagcaggagg gctggcccgg tgccaagttc 1020
atcgtcgatc aaggtcgatc gggaaagcag cctaccggac agaaggcttg gggagactgg 1080
tgcaatgctc ccggcaccgg atttggtatc cgcccatccg caaacactgg ggacgccttg 1140
gtcgatgctt ttgtctgggt caagccaggc ggcgagtctg acggcaccag cgacaccagt 1200
gcgacccgat acgactacca ctgtggtgct tctgccgcct tgcaaccggc gcctgaggct 1260
ggtacctggt tccaagccta ctttgagcag cttctcaaga acgcaaaccc atcgttcctg 1320
taa 1323
<210> 14
<211> 440
<212> PRT
<213> 人工序列
<220>
<223> 克隆 161A1
<400> 14
Ala Pro Val Glu Glu Arg Gln Ser Cys Ser Asn Gly Val Trp Ser Gln
1 5 10 15
Cys Gly Gly Gln Asn Trp Ser Gly Thr Pro Cys Cys Thr Ser Gly Asn
20 25 30
Lys Cys Val Lys Val Asn Asp Phe Tyr Ser Gln Cys Gln Pro Gly Ser
35 40 45
Ala Asp Pro Ser Pro Thr Ser Thr Ile Val Ser Ala Thr Thr Thr Lys
50 55 60
Ala Thr Thr Thr Gly Ser Gly Gly Ser Val Thr Ser Pro Pro Pro Val
65 70 75 80
Ala Thr Asn Asn Pro Phe Ser Gly Val Asp Leu Trp Ala Asn Asn Tyr
85 90 95
Tyr Arg Ser Glu Val Ser Thr Leu Ala Ile Pro Lys Leu Ser Gly Ala
100 105 110
Met Ala Thr Ala Ala Ala Lys Val Ala Asp Val Pro Ser Phe Gln Trp
115 120 125
Met Asp Thr Tyr Asp His Ile Ser Phe Met Glu Glu Ser Leu Ala Asp
130 135 140
Ile Arg Lys Ala Asn Lys Ala Gly Gly Asn Tyr Ala Gly Gln Phe Val
145 150 155 160
Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly
165 170 175
Glu Tyr Ser Leu Asp Lys Asp Gly Lys Asn Lys Tyr Lys Ala Tyr Ile
180 185 190
Ala Lys Ile Lys Gly Ile Leu Gln Asp Tyr Ser Asp Thr Arg Ile Ile
195 200 205
Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn
210 215 220
Val Pro Lys Cys Ala Asn Ala Ala Ser Ala Tyr Lys Glu Leu Thr Ile
225 230 235 240
His Ala Leu Lys Glu Leu Asn Leu Pro Asn Val Ser Met Tyr Ile Asp
245 250 255
Ala Gly His Gly Gly Trp Leu Gly Trp Pro Ala Asn Leu Pro Pro Ala
260 265 270
Ala Gln Leu Tyr Gly Gln Leu Tyr Lys Asp Ala Gly Lys Pro Ser Arg
275 280 285
Leu Arg Gly Leu Val Thr Asn Val Ser Asn Tyr Asn Ala Trp Lys Leu
290 295 300
Ser Ser Lys Pro Asp Tyr Thr Glu Ser Asn Pro Asn Tyr Asp Glu Gln
305 310 315 320
Lys Tyr Ile His Ala Leu Ser Pro Leu Leu Glu Gln Glu Gly Trp Pro
325 330 335
Gly Ala Lys Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr
340 345 350
Gly Gln Lys Ala Trp Gly Asp Trp Cys Asn Ala Pro Gly Thr Gly Phe
355 360 365
Gly Ile Arg Pro Ser Ala Asn Thr Gly Asp Ala Leu Val Asp Ala Phe
370 375 380
Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser
385 390 395 400
Ala Thr Arg Tyr Asp Tyr His Cys Gly Ala Ser Ala Ala Leu Gln Pro
405 410 415
Ala Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu
420 425 430
Lys Asn Ala Asn Pro Ser Phe Leu
435 440
<210> 15
<211> 1362
<212> DNA
<213> 人工序列
<220>
<223> 克隆 161C12
<400> 15
gtgcctctag aggagcggca agcttgctca agcgtctggg gccaatgtgg tggccagaat 60
tggtcgggtc cgacttgctg tgcttccgga agcacatgcg tctactccaa cgactattac 120
tcccagtgtc ttcccggcgc tgcaagctca agctcgtcca cgcgcgccgc gtcgacgact 180
tctcgagtat cccccacaac atcccggtcg agctccgcga cgcctccacc tggttctact 240
actaccagag tacctccagt cggatcggga accgctacgt attcaggcaa cccttttgtt 300
ggggtcactc cttgggccaa tgcatattac gcctctgaag ttagcagcct cgctattcct 360
agcttgactg gagccatggc cactgctgca gcagctgtcg caaaggttcc ctcttttatg 420
tggctagata ctcttgacaa gacccctctc atggagcaaa ccttggccga catccgcacc 480
gccaacaaga atggcggtaa ctatgccgga cagtttgtgg tgtatgactt gccggatcgc 540
gattgcgctg cccttgcctc gaatggcgaa tactctctcg acaatgatgg cgccaacaaa 600
tataagaact atatccaaac cattaagaag attatccaga gctattccga tatccggata 660
ctcctggtta ttgagcctga ctctcttgcc aacctggtga ccaacatgga tgtcgccaag 720
tgtgccaagg ctcatgatgc ctacatcagc ctgacgaact acgccgtcac agaactgaac 780
cttccaaatg ttgcgatgta tttggacgct ggccatgcag gatggcttgg ctggccggca 840
aaccttcctc cggccgctca gctatacggt cagctctaca aggatgcagg caagccgtct 900
cgccttcgcg gattggtcac caatgtctcc aactacaacg cctggaagct atccagcaag 960
ccagactaca cggagagcaa ccccaactac gacgagcaga agtacatcca cgctctgtct 1020
cctcttcttg agcaggaggg ctggcccggt gccaagttca tcgtcgatca aggtcgatcg 1080
ggaaagcagc ctaccggaca gaaggcttgg ggagactggt gcaatgctcc cggcaccgga 1140
tttggtctcc gcccatccgc aaacactggg gacgccttgg tcgatgcttt tgtctgggtc 1200
aagccaggcg gcgagtctga cggcaccagc gacaccagtg cggctcgata cgactaccac 1260
tgtggtattg acggcgccgt caagccggcg cctgaggctg gtacctggtt ccaagcctac 1320
tttgagcagc ttctcaagaa cgcaaaccca tcgttcctgt aa 1362
<210> 16
<211> 453
<212> PRT
<213> 人工序列
<220>
<223> 克隆 161C12
<400> 16
Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser Val Trp Gly Gln Cys
1 5 10 15
Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr
20 25 30
Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala
35 40 45
Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser Thr Thr Ser Arg Val Ser
50 55 60
Pro Thr Thr Ser Arg Ser Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr
65 70 75 80
Thr Thr Arg Val Pro Pro Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly
85 90 95
Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser
100 105 110
Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala Thr
115 120 125
Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu Asp Thr
130 135 140
Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile Arg Thr
145 150 155 160
Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp
165 170 175
Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser
180 185 190
Leu Asp Asn Asp Gly Ala Asn Lys Tyr Lys Asn Tyr Ile Gln Thr Ile
195 200 205
Lys Lys Ile Ile Gln Ser Tyr Ser Asp Ile Arg Ile Leu Leu Val Ile
210 215 220
Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Met Asp Val Ala Lys
225 230 235 240
Cys Ala Lys Ala His Asp Ala Tyr Ile Ser Leu Thr Asn Tyr Ala Val
245 250 255
Thr Glu Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly His
260 265 270
Ala Gly Trp Leu Gly Trp Pro Ala Asn Leu Pro Pro Ala Ala Gln Leu
275 280 285
Tyr Gly Gln Leu Tyr Lys Asp Ala Gly Lys Pro Ser Arg Leu Arg Gly
290 295 300
Leu Val Thr Asn Val Ser Asn Tyr Asn Ala Trp Lys Leu Ser Ser Lys
305 310 315 320
Pro Asp Tyr Thr Glu Ser Asn Pro Asn Tyr Asp Glu Gln Lys Tyr Ile
325 330 335
His Ala Leu Ser Pro Leu Leu Glu Gln Glu Gly Trp Pro Gly Ala Lys
340 345 350
Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln Lys
355 360 365
Ala Trp Gly Asp Trp Cys Asn Ala Pro Gly Thr Gly Phe Gly Leu Arg
370 375 380
Pro Ser Ala Asn Thr Gly Asp Ala Leu Val Asp Ala Phe Val Trp Val
385 390 395 400
Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser Ala Ala Arg
405 410 415
Tyr Asp Tyr His Cys Gly Ile Asp Gly Ala Val Lys Pro Ala Pro Glu
420 425 430
Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Lys Asn Ala
435 440 445
Asn Pro Ser Phe Leu
450
<210> 17
<211> 1323
<212> DNA
<213> 人工序列
<220>
<223> 克隆 189H8
<400> 17
gctcctgttg aagagcgtca gtcttgcagc aacggagtct ggtctcaatg tggtggtcag 60
aactggtccg gtactccttg ctgcaccagt ggaaacaagt gtgtcaaggt caacgacttc 120
tactcccagt gccagcctgg atctgcagac ccttctccca catccaccat tgtctccgcg 180
acgaccacca aggctactac tactggtagt ggaggctctg tcacctcgcc tcctcctgtt 240
gccaccaaca accctttttc tggggtcgat ctgtgggcca ataactatta ccgctctgaa 300
gttagcactc tcgctattcc taagttgagc ggagccatgg ccactgctgc agcaaaggtc 360
gcagatgttc cctcttttca gtggatggat acttatgacc acatctccct catggaggac 420
accttggtcg acatccgcaa ggccaacctg gctggcggta actatgccgg acagtttgtg 480
gtgtatgact tgccggatcg cgattgcgct gccgctgcct cgaatggcga atactctctt 540
gacaaggatg gcaagaacaa atataaggcc tatatcgcca agattaaggg tattctccag 600
gactattccg atacccggat cattctggtt attgagcctg actctcttgc caacatggtg 660
accaacatga acgtcccaaa gtgtgccaat gctgcttcag cctacaagga gctcaccatt 720
cacgccctca aggagctgaa ccttccaaat gtttccatgt atatcgacgc tggccatggt 780
ggatggcttg gctggccggc aaaccaaggc ccggccgcta aactatttgc aagcatctac 840
aaggatgcag gcaagccggc cgctcttcgc ggattggcaa ccaatgtcgc caactacaac 900
gcctggagcc tcagcagcgc tccaccttac acgcaaggcg cctctatcta cgacgagaag 960
agcttcatcc acgctatggg acctcttctt gagcagaatg gctggcctgg tgccaagttc 1020
atcgtcgatc aaggtcgatc gggaaagcag cctaccggac agaaggcttg gggagactgg 1080
tgcaatgctc ccggcaccgg atttggtctc cgcccatccg caaacactgg ggactcgttg 1140
ctggatgctt ttgtctgggt caagccaggc ggcgagtctg acggcaccag cgacaccagt 1200
gcgacccgat acgactacca ctgtggtgct tctgccgcct tgcaaccggc gcctgaggct 1260
ggtacctggt tccaagccta ctttgagcag cttctcacaa acgcaaaccc atcgttcctg 1320
taa 1323
<210> 18
<211> 440
<212> PRT
<213> 人工序列
<220>
<223> 克隆 189H8
<400> 18
Ala Pro Val Glu Glu Arg Gln Ser Cys Ser Asn Gly Val Trp Ser Gln
1 5 10 15
Cys Gly Gly Gln Asn Trp Ser Gly Thr Pro Cys Cys Thr Ser Gly Asn
20 25 30
Lys Cys Val Lys Val Asn Asp Phe Tyr Ser Gln Cys Gln Pro Gly Ser
35 40 45
Ala Asp Pro Ser Pro Thr Ser Thr Ile Val Ser Ala Thr Thr Thr Lys
50 55 60
Ala Thr Thr Thr Gly Ser Gly Gly Ser Val Thr Ser Pro Pro Pro Val
65 70 75 80
Ala Thr Asn Asn Pro Phe Ser Gly Val Asp Leu Trp Ala Asn Asn Tyr
85 90 95
Tyr Arg Ser Glu Val Ser Thr Leu Ala Ile Pro Lys Leu Ser Gly Ala
100 105 110
Met Ala Thr Ala Ala Ala Lys Val Ala Asp Val Pro Ser Phe Gln Trp
115 120 125
Met Asp Thr Tyr Asp His Ile Ser Leu Met Glu Asp Thr Leu Val Asp
130 135 140
Ile Arg Lys Ala Asn Leu Ala Gly Gly Asn Tyr Ala Gly Gln Phe Val
145 150 155 160
Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly
165 170 175
Glu Tyr Ser Leu Asp Lys Asp Gly Lys Asn Lys Tyr Lys Ala Tyr Ile
180 185 190
Ala Lys Ile Lys Gly Ile Leu Gln Asp Tyr Ser Asp Thr Arg Ile Ile
195 200 205
Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn
210 215 220
Val Pro Lys Cys Ala Asn Ala Ala Ser Ala Tyr Lys Glu Leu Thr Ile
225 230 235 240
His Ala Leu Lys Glu Leu Asn Leu Pro Asn Val Ser Met Tyr Ile Asp
245 250 255
Ala Gly His Gly Gly Trp Leu Gly Trp Pro Ala Asn Gln Gly Pro Ala
260 265 270
Ala Lys Leu Phe Ala Ser Ile Tyr Lys Asp Ala Gly Lys Pro Ala Ala
275 280 285
Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Leu
290 295 300
Ser Ser Ala Pro Pro Tyr Thr Gln Gly Ala Ser Ile Tyr Asp Glu Lys
305 310 315 320
Ser Phe Ile His Ala Met Gly Pro Leu Leu Glu Gln Asn Gly Trp Pro
325 330 335
Gly Ala Lys Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr
340 345 350
Gly Gln Lys Ala Trp Gly Asp Trp Cys Asn Ala Pro Gly Thr Gly Phe
355 360 365
Gly Leu Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ala Phe
370 375 380
Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser
385 390 395 400
Ala Thr Arg Tyr Asp Tyr His Cys Gly Ala Ser Ala Ala Leu Gln Pro
405 410 415
Ala Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu
420 425 430
Thr Asn Ala Asn Pro Ser Phe Leu
435 440
<210> 19
<211> 1362
<212> DNA
<213> 人工序列
<220>
<223> 克隆 196D9
<400> 19
gtgcctctag aggagcggca agcttgctca agcgtctggg gccaatgtgg tggccagaat 60
tggtcgggtc cgacttgctg tgcttccgga agcacatgcg tctactccaa cgactattac 120
tcccagtgtc ttcccggcgc tgcaagctca agctcgtcca cgcgcgccgc gtcgacgact 180
tctcgagtat cccccacaac atcccggtcg agctccgcga cgcctccacc tggttctact 240
actaccagag tacctccagt cggatcggga accgctacgt attcaggcaa cccttttgtt 300
ggggtcactc cttgggccaa tgcatattac gcctctgaag ttagcagcct cgctattcct 360
agcttgactg gagccatggc cactgctgca gcagctgtcg caaaggttcc ctcttttatg 420
tggctagata ctcttgacaa gacccctctc atggagcaaa ccttggccga catccgcacc 480
gccaacaaga atggcggtaa ctatgccgga cagtttgtgg tgtatgactt gccggatcgc 540
gattgcgctg cccttgcctc gaatggcgaa tactctattg ccgatggtgg cgtcgccaaa 600
tataagaact atatcgacac cattcgtcaa attgtcgtgg aatattccga tatccggacc 660
ctcctggtta ttgagcctga ctctcttacc aacctggtga ccaacatgaa cgtcccaaag 720
tgtgccaatg ctcagtcagc ctaccttgag tgcatcaact acgccgtcac acagctgaac 780
cttccaaatg ttgcgatgta tttggacgct ggccatgcag gatggcttgg ctggccggca 840
aaccaagacc cggccgctca gctatttgca aatgtttaca agaatgcatc gtctccgaga 900
gctcttcgcg gattggcaac caatgtcgcc aactacaacg ggtggaacat taccagcccc 960
ccatcgtaca cgcaaggcaa cgctgtctac aacgagaagc tgtacatcca cgctattgga 1020
cctcttcttg ccaatcacgg ctggtccaac gccttcttca tcactgatca aggtcgatcg 1080
ggaaagcagc ctaccggaca gcaacagtgg ggagactggt gcaatgctcc cggcaccgga 1140
tttggtctcc gcccatccgc aaacactggg gacgccttgg tcgatgcttt tgtctgggtc 1200
aagccaggcg gcgagtctga cggcaccagc gacaccagtg cggctcgata cgactaccac 1260
tgtggtattg acggcgccgt caagccggcg cctgaggctg gtacctggtt ccaagcctac 1320
tttgagcagc ttctcaagaa cgcaaaccca tcgttcctgt aa 1362
<210> 20
<211> 453
<212> PRT
<213> 人工序列
<220>
<223> 克隆 196D9
<400> 20
Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser Val Trp Gly Gln Cys
1 5 10 15
Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr
20 25 30
Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala
35 40 45
Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser Thr Thr Ser Arg Val Ser
50 55 60
Pro Thr Thr Ser Arg Ser Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr
65 70 75 80
Thr Thr Arg Val Pro Pro Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly
85 90 95
Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser
100 105 110
Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala Thr
115 120 125
Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu Asp Thr
130 135 140
Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile Arg Thr
145 150 155 160
Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp
165 170 175
Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser
180 185 190
Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile
195 200 205
Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile
210 215 220
Glu Pro Asp Ser Leu Thr Asn Leu Val Thr Asn Met Asn Val Pro Lys
225 230 235 240
Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr Ala Val
245 250 255
Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly His
260 265 270
Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala Gln Leu
275 280 285
Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu Arg Gly
290 295 300
Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp Asn Ile Thr Ser Pro
305 310 315 320
Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu Lys Leu Tyr Ile
325 330 335
His Ala Ile Gly Pro Leu Leu Ala Asn His Gly Trp Ser Asn Ala Phe
340 345 350
Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln Gln
355 360 365
Gln Trp Gly Asp Trp Cys Asn Ala Pro Gly Thr Gly Phe Gly Leu Arg
370 375 380
Pro Ser Ala Asn Thr Gly Asp Ala Leu Val Asp Ala Phe Val Trp Val
385 390 395 400
Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser Ala Ala Arg
405 410 415
Tyr Asp Tyr His Cys Gly Ile Asp Gly Ala Val Lys Pro Ala Pro Glu
420 425 430
Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Lys Asn Ala
435 440 445
Asn Pro Ser Phe Leu
450
<210> 21
<211> 1362
<212> DNA
<213> 人工序列
<220>
<223> 克隆 198E11
<400> 21
gtgcctctag aggagcggca agcttgctca agcgtctggg gccaatgtgg tggccagaat 60
tggtcgggtc cgacttgctg tgcttccgga agcacatgcg tctactccaa cgactattac 120
tcccagtgtc ttcccggcgc tgcaagctca agctcgtcca cgcgcgccgc gtcgacgact 180
tctcgagtat cccccacaac atcccggtcg agctccgcga cgcctccacc tggttctact 240
actaccagag tacctccagt cggatcggga accgctacgt attcaggcaa cccttttgtt 300
ggggtcactc cttgggccaa tgcatattac gcctctgaag ttagcagcct cgctattcct 360
agcttgactg gagccatggc cactgctgca gcagctgtcg caaaggttcc ctcttttatg 420
tggctagata ctcttgacaa gacccctctc atggagcaaa ccttggccga catccgcacc 480
gccaacaaga atggcggtaa ctatgccgga cagtttgtgg tgtatgactt gccggatcgc 540
gattgcgctg ccgctgcctc gaatggcgaa tactctcttg acaaggatgg caagaacaaa 600
tataaggcct atatcgccaa gattaagggt attctccagg actattccga tacccggatc 660
attctggtta ttgagcctga ctctcttgcc aacatggtga ccaacatgaa cgtcccaaag 720
tgtgccaatg ctgcttcagc ctacaaggag ctcaccattc acgccctcaa ggagctgaac 780
cttccaaatg tttccatgta tatcgacgct ggccatggtg gatggcttgg ctggccggca 840
aaccttcctc cggccgctca gctatacggt cagctctaca aggatgcagg caagccgtct 900
cgccttcgcg gattggtcac caatgtctcc aactacaacg cctggaagct atccagcaag 960
ccagactaca cggagagcaa ccccaactac gacgagcaga agtacatcca cgctctgtct 1020
cctcttcttg agcaggaggg ctggcccggt gccaagttca tcgtcgatca aggtcgatcg 1080
ggaaagcagc ctaccggaca gaaggcttgg ggagactggt gcaatgtgat cggcaccgga 1140
tttggtattc gcccatccgc aaacactggg gactcgttgc tggattcgtt tgtctgggtc 1200
aagccaggcg gcgagtctga cggcaccagc gacaccagtg cgacccgata cgactaccac 1260
tgtggtgctt ctgccgcctt gcaaccggcg cctgaggctg gtacctggtt ccaagcctac 1320
tttgagcagc ttctcacaaa cgcaaaccca tcgttcctgt aa 1362
<210> 22
<211> 453
<212> PRT
<213> 人工序列
<220>
<223> 克隆 198E11
<400> 22
Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser Val Trp Gly Gln Cys
1 5 10 15
Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr
20 25 30
Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala
35 40 45
Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser Thr Thr Ser Arg Val Ser
50 55 60
Pro Thr Thr Ser Arg Ser Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr
65 70 75 80
Thr Thr Arg Val Pro Pro Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly
85 90 95
Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser
100 105 110
Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala Thr
115 120 125
Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu Asp Thr
130 135 140
Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile Arg Thr
145 150 155 160
Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp
165 170 175
Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu Tyr Ser
180 185 190
Leu Asp Lys Asp Gly Lys Asn Lys Tyr Lys Ala Tyr Ile Ala Lys Ile
195 200 205
Lys Gly Ile Leu Gln Asp Tyr Ser Asp Thr Arg Ile Ile Leu Val Ile
210 215 220
Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn Val Pro Lys
225 230 235 240
Cys Ala Asn Ala Ala Ser Ala Tyr Lys Glu Leu Thr Ile His Ala Leu
245 250 255
Lys Glu Leu Asn Leu Pro Asn Val Ser Met Tyr Ile Asp Ala Gly His
260 265 270
Gly Gly Trp Leu Gly Trp Pro Ala Asn Leu Pro Pro Ala Ala Gln Leu
275 280 285
Tyr Gly Gln Leu Tyr Lys Asp Ala Gly Lys Pro Ser Arg Leu Arg Gly
290 295 300
Leu Val Thr Asn Val Ser Asn Tyr Asn Ala Trp Lys Leu Ser Ser Lys
305 310 315 320
Pro Asp Tyr Thr Glu Ser Asn Pro Asn Tyr Asp Glu Gln Lys Tyr Ile
325 330 335
His Ala Leu Ser Pro Leu Leu Glu Gln Glu Gly Trp Pro Gly Ala Lys
340 345 350
Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln Lys
355 360 365
Ala Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly Ile Arg
370 375 380
Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ser Phe Val Trp Val
385 390 395 400
Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser Ala Thr Arg
405 410 415
Tyr Asp Tyr His Cys Gly Ala Ser Ala Ala Leu Gln Pro Ala Pro Glu
420 425 430
Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr Asn Ala
435 440 445
Asn Pro Ser Phe Leu
450
<210> 23
<211> 1323
<212> DNA
<213> 人工序列
<220>
<223> 克隆 251B4
<400> 23
gctcctgttg aagagcgtca gtcttgcagc aacggagtct ggtctcaatg tggtggtcag 60
aactggtccg gtactccttg ctgcaccagt ggaaacaagt gtgtcaaggt caacgacttc 120
tactcccagt gccagcctgg atctgcagac ccttctccca catccaccat tgtctccgcg 180
acgaccacca aggctactac tactggtagt ggaggctctg tcacctcgcc tcctcctgtt 240
gccaccaaca accctttttc tggggtcgat ctgtgggcca ataactatta ccgctctgaa 300
gttagcactc tcgctattcc taagttgagc ggagccatgg ccactgctgc agcaaaggtc 360
gcagatgttc cctcttttca gtggatggat acttatgacc acatctcctt catggaggag 420
tctttggccg acatccgcac cgccaacaag aatggcggta actatgccgg acagtttgtg 480
gtgtatgact tgccggatcg cgattgcgct gccgccgcct cgaatggcga atactctctc 540
gacaatgatg gcgccaacaa atataagaac tatatccaaa ccattaagaa gattatccag 600
agctattccg atatccggat actcctggtt attgagcctg actctcttgc caacctggtg 660
accaacatgg atgtcgccaa gtgtgccaag gctcatgatg cctacatcag cctgacgaac 720
tacgccgtca cagaactgaa ccttccaaat gttgcgatgt atttggacgc tggccatgca 780
ggatggcttg gctggccggc aaaccaaggc ccggccgcta aactatttgc aagcatctac 840
aaggatgcag gcaagccggc cgctcttcgc ggattggcaa ccaatgtcgc caactacaac 900
gcctggaagc tatccagcaa gccagactac acggagagca accccaacta cgacgagcag 960
aagtacatcc acgctctgtc tcctcttctt gagcaggagg gctggcccgg tgccaagttc 1020
atcgtcgatc aaggtcgatc gggaaagcag cctaccggac agaaggcttg gggagactgg 1080
tgcaatgctc ccggcaccgg atttggtctc cgcccatccg caaacactgg ggacgccttg 1140
gtcgatgctt ttgtctgggt caagccaggc ggcgagtgtg acggcaccag cgacaccagt 1200
gcggctcgat acgactacca ctgtggtatt gacggcgccg tcaagccggc gcctgaggct 1260
ggtacctggt tccaagccta ctttgagcag cttctcacaa acgcaaaccc atcgttcctg 1320
taa 1323
<210> 24
<211> 440
<212> PRT
<213> 人工序列
<220>
<223> 克隆 251B4
<400> 24
Ala Pro Val Glu Glu Arg Gln Ser Cys Ser Asn Gly Val Trp Ser Gln
1 5 10 15
Cys Gly Gly Gln Asn Trp Ser Gly Thr Pro Cys Cys Thr Ser Gly Asn
20 25 30
Lys Cys Val Lys Val Asn Asp Phe Tyr Ser Gln Cys Gln Pro Gly Ser
35 40 45
Ala Asp Pro Ser Pro Thr Ser Thr Ile Val Ser Ala Thr Thr Thr Lys
50 55 60
Ala Thr Thr Thr Gly Ser Gly Gly Ser Val Thr Ser Pro Pro Pro Val
65 70 75 80
Ala Thr Asn Asn Pro Phe Ser Gly Val Asp Leu Trp Ala Asn Asn Tyr
85 90 95
Tyr Arg Ser Glu Val Ser Thr Leu Ala Ile Pro Lys Leu Ser Gly Ala
100 105 110
Met Ala Thr Ala Ala Ala Lys Val Ala Asp Val Pro Ser Phe Gln Trp
115 120 125
Met Asp Thr Tyr Asp His Ile Ser Phe Met Glu Glu Ser Leu Ala Asp
130 135 140
Ile Arg Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val
145 150 155 160
Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly
165 170 175
Glu Tyr Ser Leu Asp Asn Asp Gly Ala Asn Lys Tyr Lys Asn Tyr Ile
180 185 190
Gln Thr Ile Lys Lys Ile Ile Gln Ser Tyr Ser Asp Ile Arg Ile Leu
195 200 205
Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Met Asp
210 215 220
Val Ala Lys Cys Ala Lys Ala His Asp Ala Tyr Ile Ser Leu Thr Asn
225 230 235 240
Tyr Ala Val Thr Glu Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp
245 250 255
Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Gly Pro Ala
260 265 270
Ala Lys Leu Phe Ala Ser Ile Tyr Lys Asp Ala Gly Lys Pro Ala Ala
275 280 285
Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Lys Leu
290 295 300
Ser Ser Lys Pro Asp Tyr Thr Glu Ser Asn Pro Asn Tyr Asp Glu Gln
305 310 315 320
Lys Tyr Ile His Ala Leu Ser Pro Leu Leu Glu Gln Glu Gly Trp Pro
325 330 335
Gly Ala Lys Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr
340 345 350
Gly Gln Lys Ala Trp Gly Asp Trp Cys Asn Ala Pro Gly Thr Gly Phe
355 360 365
Gly Leu Arg Pro Ser Ala Asn Thr Gly Asp Ala Leu Val Asp Ala Phe
370 375 380
Val Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr Ser
385 390 395 400
Ala Ala Arg Tyr Asp Tyr His Cys Gly Ile Asp Gly Ala Val Lys Pro
405 410 415
Ala Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu
420 425 430
Thr Asn Ala Asn Pro Ser Phe Leu
435 440
<210> 25
<211> 1362
<212> DNA
<213> 人工序列
<220>
<223> 克隆 251C4
<400> 25
gtgcctctag aggagcggca agcttgctca agcgtctggg gccaatgtgg tggccagaat 60
tggtcgggtc cgacttgctg tgcttccgga agcacatgcg tctactccaa cgactattac 120
tcccagtgtc ttcccggcgc tgcaagctca agctcgtcca cgcgcgccgc gtcgacgact 180
tctcgagtat cccccacaac atcccggtcg agctccgcga cgcctccacc tggttctact 240
actaccagag tacctccagt cggatcggga accgctacgt attcaggcaa cccttttgtt 300
ggggtcactc cttgggccaa tgcatattac gcctctgaag ttagcagcct cgctattcct 360
agcttgactg gagccatggc cactgctgca gcagctgtcg caaaggttcc ctcttttatg 420
tggctagata ctcttgacaa gacccctctc atggagcaaa ccttggccga catccgcacc 480
gccaacaaga atggcggtaa ctatgccgga cagtttgtgg tgtatgactt gccggatcgc 540
gattgcgctg ccgctgcctc gaatggcgaa tactctattg ccgatggtgg cgtcgccaaa 600
tataagaact atatcgacac cattcgtcaa attgtcgtgg aatattccga tatccggacc 660
ctcctggtta ttgagcctga ctctcttgcc aacctggtga ccaacctcgg tactccaaag 720
tgtgccaatg ctcagtcagc ctaccttgag tgcatcaact acgccgtcac acagctgaac 780
cttccaaatg ttgcgatgta tttggacgct ggccatgcag gatggcttgg ctggccggca 840
aaccaagacc cggccgctca gctatttgca aatgtttaca agaatgcatc gtctccgaga 900
gctcttcgcg gattggcaac caatgtcgcc aactacaacg ggtggaacat taccagcccc 960
ccatcgtaca cgcaaggcaa cgctgtctac aacgagaagc tgtacatcca cgctattgga 1020
cctcttcttg ccaatcacgg ctggtccaac gccttcttca tcactgatca aggtcgatcg 1080
ggaaagcagc ctaccggaca gcaacagtgg ggagactggt gcaatgtgat cggcaccgga 1140
tttggtattc gcccatccgc aaacactggg gactcgttgc tggattcgtt tgtctgggtc 1200
aagccaggcg gcgagtgtga cggcaccagc gacaccagtg cgccacgatt tgactcccac 1260
tgtgcgctcc cagatgcctt gcaaccggcg cctgaggctg gtacctggtt ccaagcctac 1320
tttgagcagc ttctcacaaa cgcaaaccca tcgttcctgt aa 1362
<210> 26
<211> 453
<212> PRT
<213> 人工序列
<220>
<223> 克隆 251C4
<400> 26
Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser Val Trp Gly Gln Cys
1 5 10 15
Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr
20 25 30
Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala
35 40 45
Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser Thr Thr Ser Arg Val Ser
50 55 60
Pro Thr Thr Ser Arg Ser Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr
65 70 75 80
Thr Thr Arg Val Pro Pro Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly
85 90 95
Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser
100 105 110
Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala Thr
115 120 125
Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu Asp Thr
130 135 140
Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile Arg Thr
145 150 155 160
Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp
165 170 175
Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu Tyr Ser
180 185 190
Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile
195 200 205
Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile
210 215 220
Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr Pro Lys
225 230 235 240
Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr Ala Val
245 250 255
Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly His
260 265 270
Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala Gln Leu
275 280 285
Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu Arg Gly
290 295 300
Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp Asn Ile Thr Ser Pro
305 310 315 320
Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu Lys Leu Tyr Ile
325 330 335
His Ala Ile Gly Pro Leu Leu Ala Asn His Gly Trp Ser Asn Ala Phe
340 345 350
Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln Gln
355 360 365
Gln Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly Ile Arg
370 375 380
Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ser Phe Val Trp Val
385 390 395 400
Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr Ser Ala Pro Arg
405 410 415
Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala Pro Glu
420 425 430
Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr Asn Ala
435 440 445
Asn Pro Ser Phe Leu
450
<210> 27
<211> 1353
<212> DNA
<213> 人工序列
<220>
<223> 克隆 382A2
<400> 27
gcccctcttg ttgaggagcg ccaggcttgc gctgcccaat gggcccagtg tggtggcttc 60
agctggaatg gtgctacctg ctgccagtct ggtagctact gcagcaagat caacgactat 120
tactctcagt gcattcctgg agaaggcccc gccacttcca agtcgagcac tcttcctgct 180
tccaccacaa caactcagcc aacttccact tcgactgctg gaacctcttc cactaccaag 240
cctcctccag ctggatcggg aaccgctacg tattcaggca acccttactc tggggtcaac 300
ctttgggcca atagctatta ccgctctgaa gttaccaacc tcgctattcc taagttgagc 360
ggagccatgg ccactgctgc agcaaaggtc gcagatgttc cctcttttca gtggatggat 420
acttatgacc acatctcctt catggaggag tctttggccg acatccgcaa ggccaacaag 480
gctggcggta actatgccgg acagtttgtg gtgtatgact tgccggatcg cgattgcgct 540
gccgctgcct cgaatggcga atactctctt gacaaggatg gcaagaacaa atataaggcc 600
tatatcgcca agattaaggg tattctccag gactattccg atacccggat cattctggtt 660
attgagcctg actctcttgc caacatggtg accaacatga acgtcccaaa gtgtgccaat 720
gctgcttcag cctacaagga gctcaccatt cacgccctca aggagctgaa ccttccaaat 780
gtttccatgt atatcgacgc tggccatggt ggatggcttg gctggccggc aaaccttcct 840
ccggccgctc agctatacgg tcagctctac aaggatgcag gcaagccgtc tcgccttcgc 900
ggattggtca ccaatgtctc caactacaac gcctggaagc tatccagcaa gccagactac 960
acggagagca accccaacta cgacgagcag aagtacatcc acgctctgtc tcctcttctt 1020
gagcaggagg gctggcccgg tgccaagttc atcgtcgatc aaggtcgatc gggaaagcag 1080
cctaccggac agaaggcttg gggagactgg tgcaatgctc ccggcaccgg atttggtctc 1140
cgcccatccg caaacactgg ggacgccttg gtcgatgctt ttgtctgggt caagccaggc 1200
ggcgagtctg acggcaccag cgacaccagt gcggctcgat acgactacca ctgtggtatt 1260
gacggcgccg tcaagccggc gcctgaggct ggtacctggt tccaagccta ctttgagcag 1320
cttctcaaga acgcaaaccc atcgttcctg taa 1353
<210> 28
<211> 450
<212> PRT
<213> 人工序列
<220>
<223> 克隆 382A2
<400> 28
Ala Pro Leu Val Glu Glu Arg Gln Ala Cys Ala Ala Gln Trp Ala Gln
1 5 10 15
Cys Gly Gly Phe Ser Trp Asn Gly Ala Thr Cys Cys Gln Ser Gly Ser
20 25 30
Tyr Cys Ser Lys Ile Asn Asp Tyr Tyr Ser Gln Cys Ile Pro Gly Glu
35 40 45
Gly Pro Ala Thr Ser Lys Ser Ser Thr Leu Pro Ala Ser Thr Thr Thr
50 55 60
Thr Gln Pro Thr Ser Thr Ser Thr Ala Gly Thr Ser Ser Thr Thr Lys
65 70 75 80
Pro Pro Pro Ala Gly Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Tyr
85 90 95
Ser Gly Val Asn Leu Trp Ala Asn Ser Tyr Tyr Arg Ser Glu Val Thr
100 105 110
Asn Leu Ala Ile Pro Lys Leu Ser Gly Ala Met Ala Thr Ala Ala Ala
115 120 125
Lys Val Ala Asp Val Pro Ser Phe Gln Trp Met Asp Thr Tyr Asp His
130 135 140
Ile Ser Phe Met Glu Glu Ser Leu Ala Asp Ile Arg Lys Ala Asn Lys
145 150 155 160
Ala Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp Leu Pro Asp
165 170 175
Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu Tyr Ser Leu Asp Lys
180 185 190
Asp Gly Lys Asn Lys Tyr Lys Ala Tyr Ile Ala Lys Ile Lys Gly Ile
195 200 205
Leu Gln Asp Tyr Ser Asp Thr Arg Ile Ile Leu Val Ile Glu Pro Asp
210 215 220
Ser Leu Ala Asn Met Val Thr Asn Met Asn Val Pro Lys Cys Ala Asn
225 230 235 240
Ala Ala Ser Ala Tyr Lys Glu Leu Thr Ile His Ala Leu Lys Glu Leu
245 250 255
Asn Leu Pro Asn Val Ser Met Tyr Ile Asp Ala Gly His Gly Gly Trp
260 265 270
Leu Gly Trp Pro Ala Asn Leu Pro Pro Ala Ala Gln Leu Tyr Gly Gln
275 280 285
Leu Tyr Lys Asp Ala Gly Lys Pro Ser Arg Leu Arg Gly Leu Val Thr
290 295 300
Asn Val Ser Asn Tyr Asn Ala Trp Lys Leu Ser Ser Lys Pro Asp Tyr
305 310 315 320
Thr Glu Ser Asn Pro Asn Tyr Asp Glu Gln Lys Tyr Ile His Ala Leu
325 330 335
Ser Pro Leu Leu Glu Gln Glu Gly Trp Pro Gly Ala Lys Phe Ile Val
340 345 350
Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln Lys Ala Trp Gly
355 360 365
Asp Trp Cys Asn Ala Pro Gly Thr Gly Phe Gly Leu Arg Pro Ser Ala
370 375 380
Asn Thr Gly Asp Ala Leu Val Asp Ala Phe Val Trp Val Lys Pro Gly
385 390 395 400
Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser Ala Ala Arg Tyr Asp Tyr
405 410 415
His Cys Gly Ile Asp Gly Ala Val Lys Pro Ala Pro Glu Ala Gly Thr
420 425 430
Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Lys Asn Ala Asn Pro Ser
435 440 445
Phe Leu
450
<210> 29
<211> 1323
<212> DNA
<213> 小麦赤霉菌
<400> 29
gctcctgttg aagagcgtca gtcttgcagc aacggagtct ggtctcaatg tggtggtcag 60
aactggtccg gtactccttg ctgcaccagt ggaaacaagt gtgtcaaggt caacgacttc 120
tactcccagt gccagcctgg atctgcagac ccttctccca catccaccat tgtctccgcg 180
acgaccacca aggctactac tactggtagt ggaggctctg tcacctcgcc tcctcctgtt 240
gccaccaaca accctttttc tggggtcgat ctgtgggcca ataactatta ccgctctgaa 300
gttagcactc tcgctattcc taagttgagc ggagccatgg ccactgctgc agcaaaggtc 360
gcagatgttc cctcttttca gtggatggat acttatgacc acatctcctt catggaggag 420
tctttggccg acatccgcaa ggccaacaag gctggcggta actatgccgg acagtttgtg 480
gtgtatgact tgccggatcg cgattgcgct gccgctgcct cgaatggcga atactctctt 540
gacaaggatg gcaagaacaa atataaggcc tatatcgcca agattaaggg tattctccag 600
gactattccg atacccggat cattctggtt attgagcctg actctcttgc caacatggtg 660
accaacatga acgtcccaaa gtgtgccaat gctgcttcag cctacaagga gctcaccatt 720
cacgccctca aggagctgaa ccttccaaat gtttccatgt atatcgacgc tggccatggt 780
ggatggcttg gctggccggc aaaccttcct ccggccgctc agctatacgg tcagctctac 840
aaggatgcag gcaagccgtc tcgccttcgc ggattggtca ccaatgtctc caactacaac 900
gcctggaagc tatccagcaa gccagactac acggagagca accccaacta cgacgagcag 960
aagtacatcc acgctctgtc tcctcttctt gagcaggagg gctggcccgg tgccaagttc 1020
atcgtcgatc aaggtcgatc gggaaagcag cctaccggac agaaggcttg gggagactgg 1080
tgcaatgctc ccggcaccgg atttggtctc cgcccatccg caaacactgg ggacgccttg 1140
gtcgatgctt ttgtctgggt caagccaggc ggcgagtctg acggcaccag cgacaccagt 1200
gcggctcgat acgactacca ctgtggtatt gacggcgccg tcaagccggc gcctgaggct 1260
ggtacctggt tccaagccta ctttgagcag cttctcaaga acgcaaaccc atcgttcctg 1320
taa 1323
<210> 30
<211> 440
<212> PRT
<213> 小麦赤霉菌
<400> 30
Ala Pro Val Glu Glu Arg Gln Ser Cys Ser Asn Gly Val Trp Ser Gln
1 5 10 15
Cys Gly Gly Gln Asn Trp Ser Gly Thr Pro Cys Cys Thr Ser Gly Asn
20 25 30
Lys Cys Val Lys Val Asn Asp Phe Tyr Ser Gln Cys Gln Pro Gly Ser
35 40 45
Ala Asp Pro Ser Pro Thr Ser Thr Ile Val Ser Ala Thr Thr Thr Lys
50 55 60
Ala Thr Thr Thr Gly Ser Gly Gly Ser Val Thr Ser Pro Pro Pro Val
65 70 75 80
Ala Thr Asn Asn Pro Phe Ser Gly Val Asp Leu Trp Ala Asn Asn Tyr
85 90 95
Tyr Arg Ser Glu Val Ser Thr Leu Ala Ile Pro Lys Leu Ser Gly Ala
100 105 110
Met Ala Thr Ala Ala Ala Lys Val Ala Asp Val Pro Ser Phe Gln Trp
115 120 125
Met Asp Thr Tyr Asp His Ile Ser Phe Met Glu Glu Ser Leu Ala Asp
130 135 140
Ile Arg Lys Ala Asn Lys Ala Gly Gly Asn Tyr Ala Gly Gln Phe Val
145 150 155 160
Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly
165 170 175
Glu Tyr Ser Leu Asp Lys Asp Gly Lys Asn Lys Tyr Lys Ala Tyr Ile
180 185 190
Ala Lys Ile Lys Gly Ile Leu Gln Asp Tyr Ser Asp Thr Arg Ile Ile
195 200 205
Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn
210 215 220
Val Pro Lys Cys Ala Asn Ala Ala Ser Ala Tyr Lys Glu Leu Thr Ile
225 230 235 240
His Ala Leu Lys Glu Leu Asn Leu Pro Asn Val Ser Met Tyr Ile Asp
245 250 255
Ala Gly His Gly Gly Trp Leu Gly Trp Pro Ala Asn Leu Pro Pro Ala
260 265 270
Ala Gln Leu Tyr Gly Gln Leu Tyr Lys Asp Ala Gly Lys Pro Ser Arg
275 280 285
Leu Arg Gly Leu Val Thr Asn Val Ser Asn Tyr Asn Ala Trp Lys Leu
290 295 300
Ser Ser Lys Pro Asp Tyr Thr Glu Ser Asn Pro Asn Tyr Asp Glu Gln
305 310 315 320
Lys Tyr Ile His Ala Leu Ser Pro Leu Leu Glu Gln Glu Gly Trp Pro
325 330 335
Gly Ala Lys Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr
340 345 350
Gly Gln Lys Ala Trp Gly Asp Trp Cys Asn Ala Pro Gly Thr Gly Phe
355 360 365
Gly Leu Arg Pro Ser Ala Asn Thr Gly Asp Ala Leu Val Asp Ala Phe
370 375 380
Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser
385 390 395 400
Ala Ala Arg Tyr Asp Tyr His Cys Gly Ile Asp Gly Ala Val Lys Pro
405 410 415
Ala Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu
420 425 430
Lys Asn Ala Asn Pro Ser Phe Leu
435 440
<210> 31
<211> 1353
<212> DNA
<213> 鞭毛藻丛赤壳菌
<400> 31
gcccctcttg ttgaggagcg ccaggcttgc gctgcccaat gggcccagtg tggtggcttc 60
agctggaatg gtgctacctg ctgccagtct ggtagctact gcagcaagat caacgactat 120
tactctcagt gcattcctgg agaaggcccc gccacttcca agtcgagcac tcttcctgct 180
tccaccacaa caactcagcc aacttccact tcgactgctg gaacctcttc cactaccaag 240
cctcctccag ctggatcggg aaccgctacg tattcaggca acccttactc tggggtcaac 300
ctttgggcca atagctatta ccgctctgaa gttaccaacc tcgctattcc taagttgagc 360
ggagccatgg ccactgctgc agcaaaggtc gcagatgttc cctcttatca gtggatggat 420
tctttcgacc acatctccct catggaggac accttggtcg acatccgcaa ggccaacctg 480
gctggcggta actatgccgg acagtttgtg gtgtatgact tgccggatcg cgattgcgct 540
gccgccgcct cgaatggcga atactctctc gacaatgatg gcgccaacaa atataagaac 600
tatatccaaa ccattaagaa gattatccag agctattccg atatccggat actcctggtt 660
attgagcctg actctcttgc caacctggtg accaacatgg atgtcgccaa gtgtgccaag 720
gctcatgatg cctacatcag cctgacgaac tacgccgtca cagaactgaa ccttccaaat 780
gttgcgatgt atttggacgc tggccatgca ggatggcttg gctggccggc aaaccaaggc 840
ccggccgcta aactatttgc aagcatctac aaggatgcag gcaagccggc cgctcttcgc 900
ggattggcaa ccaatgtcgc caactacaac gcctggagcc tcagcagcgc tccaccttac 960
acgcaaggcg cctctatcta cgacgagaag agcttcatcc acgctatggg acctcttctt 1020
gagcagaatg gctggcctgg tgcccacttc atcactgatc aaggtcgatc gggaaagcag 1080
cctaccggac agatccagtg gggagactgg tgcaattcca agggcaccgg atttggtatt 1140
cgcccatccg caaacactgg ggactcgttg ctggatgctt ttgtctgggt caagccaggc 1200
ggcgagtctg acggcaccag cgacaccagt gcgacccgat acgactacca ctgtggtgct 1260
tctgccgcct tgcaaccggc gcctgaggct ggtacctggt tccaagccta ctttgagcag 1320
cttctcacaa acgcaaaccc atcgttcctg taa 1353
<210> 32
<211> 450
<212> PRT
<213> 鞭毛藻丛赤壳菌
<400> 32
Ala Pro Leu Val Glu Glu Arg Gln Ala Cys Ala Ala Gln Trp Ala Gln
1 5 10 15
Cys Gly Gly Phe Ser Trp Asn Gly Ala Thr Cys Cys Gln Ser Gly Ser
20 25 30
Tyr Cys Ser Lys Ile Asn Asp Tyr Tyr Ser Gln Cys Ile Pro Gly Glu
35 40 45
Gly Pro Ala Thr Ser Lys Ser Ser Thr Leu Pro Ala Ser Thr Thr Thr
50 55 60
Thr Gln Pro Thr Ser Thr Ser Thr Ala Gly Thr Ser Ser Thr Thr Lys
65 70 75 80
Pro Pro Pro Ala Gly Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Tyr
85 90 95
Ser Gly Val Asn Leu Trp Ala Asn Ser Tyr Tyr Arg Ser Glu Val Thr
100 105 110
Asn Leu Ala Ile Pro Lys Leu Ser Gly Ala Met Ala Thr Ala Ala Ala
115 120 125
Lys Val Ala Asp Val Pro Ser Tyr Gln Trp Met Asp Ser Phe Asp His
130 135 140
Ile Ser Leu Met Glu Asp Thr Leu Val Asp Ile Arg Lys Ala Asn Leu
145 150 155 160
Ala Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp Leu Pro Asp
165 170 175
Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu Tyr Ser Leu Asp Asn
180 185 190
Asp Gly Ala Asn Lys Tyr Lys Asn Tyr Ile Gln Thr Ile Lys Lys Ile
195 200 205
Ile Gln Ser Tyr Ser Asp Ile Arg Ile Leu Leu Val Ile Glu Pro Asp
210 215 220
Ser Leu Ala Asn Leu Val Thr Asn Met Asp Val Ala Lys Cys Ala Lys
225 230 235 240
Ala His Asp Ala Tyr Ile Ser Leu Thr Asn Tyr Ala Val Thr Glu Leu
245 250 255
Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly Trp
260 265 270
Leu Gly Trp Pro Ala Asn Gln Gly Pro Ala Ala Lys Leu Phe Ala Ser
275 280 285
Ile Tyr Lys Asp Ala Gly Lys Pro Ala Ala Leu Arg Gly Leu Ala Thr
290 295 300
Asn Val Ala Asn Tyr Asn Ala Trp Ser Leu Ser Ser Ala Pro Pro Tyr
305 310 315 320
Thr Gln Gly Ala Ser Ile Tyr Asp Glu Lys Ser Phe Ile His Ala Met
325 330 335
Gly Pro Leu Leu Glu Gln Asn Gly Trp Pro Gly Ala His Phe Ile Thr
340 345 350
Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln Ile Gln Trp Gly
355 360 365
Asp Trp Cys Asn Ser Lys Gly Thr Gly Phe Gly Ile Arg Pro Ser Ala
370 375 380
Asn Thr Gly Asp Ser Leu Leu Asp Ala Phe Val Trp Val Lys Pro Gly
385 390 395 400
Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser Ala Thr Arg Tyr Asp Tyr
405 410 415
His Cys Gly Ala Ser Ala Ala Leu Gln Pro Ala Pro Glu Ala Gly Thr
420 425 430
Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr Asn Ala Asn Pro Ser
435 440 445
Phe Leu
450
<210> 33
<211> 54
<212> DNA
<213> 人工序列
<220>
<223> CBH2的肽信号
<400> 33
atgattgtcg gcattctcac cacgctggct acgctggcca cactcgcagc tagt 54
<210> 34
<211> 99
<212> DNA
<213> 酿酒酵母
<400> 34
atgaagcttt ccaccatcct tttcacagcc tgcgctaccc tggctgccgc cctcccttcc 60
cccatcactc cttctgaggc cgcagttctg cagaagagg 99
<210> 35
<211> 33
<212> PRT
<213> 酿酒酵母
<400> 35
Met Lys Leu Ser Thr Ile Leu Phe Thr Ala Cys Ala Thr Leu Ala Ala
1 5 10 15
Ala Leu Pro Ser Pro Ile Thr Pro Ser Glu Ala Ala Val Leu Gln Lys
20 25 30
Arg

Claims (13)

1.一种分离或纯化的多肽，其特征在于，与SEQ ID NO：2的CBH2参考蛋白的外切葡聚糖酶活性相比，它具有增强的外切葡聚糖酶活性，所述多肽选自：
i.选自SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQID NO:26和SEQ ID NO:28的氨基酸序列；和
ii.与序列SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26和SEQ ID NO:28相比，具有至少70％，优选至少75％、80％、85％、90％、95％、98％或99％同一性百分比的氨基酸序列。
2.一种纯化或分离的核酸，其特征在于，它编码至少一种如权利要求1所述的多肽。
3.如权利要求2所述的纯化或分离的核酸，选自以下序列：SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25和SEQ ID NO:27。
4.一种载体，其特征在于，它包含至少一种如权利要求2或3所述的核酸。
5.一种分离的宿主细胞，其特征在于，它包含至少一种如权利要求1所述的多肽，或至少一种如权利要求2或3所述的核酸，或至少一种如权利要求4所述的载体。
6.如权利要求5所述的分离的宿主细胞，其特征在于，它选自：木霉属、曲霉属、脉胞菌属、腐质霉属、毁丝霉属、金孢子菌属、青霉属、镰刀菌属、高温单孢菌属、芽孢杆菌属、假单包菌属,埃希氏菌属、梭状芽孢杆菌、纤维单胞菌属、链霉菌属、耶罗维亚酵母属、毕赤酵母属和酵母属。
7.如权利要求5或6所述的分离的宿主细胞，其特征在于，它选自：里氏木霉、Trichoderma viridae、康氏木霉、黑曲霉、构巢曲霉、文氏曲霉、米曲霉、海枣曲霉、嗜热毁丝霉、Chrysosporium lucknowense、粗糙脉孢菌、灰腐质霉、嗜松青霉、草酸青霉、大肠杆菌、丙酮丁醇梭菌、糖解梭菌、拜氏梭菌、丁酸梭菌、毕赤酵母、解脂耶罗维亚酵母和酿酒酵母。
8.如权利要求1所述的多肽用于纤维素水解的用途。
9.如权利要求1所述的多肽用于生物燃料生产的用途。
10.一种能够作用于木质纤维素生物质的酶组合物，所述酶组合物由丝状真菌产生且包含至少一种如权利要求1所述的多肽。
11.一种用于从木质纤维素生物质生产生物燃料的方法，其特征在于，它包含以下连续步骤：
–将待水解的生物质悬浮于水相中；
–在如权利要求10所述的酶组合物存在下水解所述木质纤维素生物质，以产生含有葡萄糖的水解物；
–在发酵生物存在下发酵水解物的葡萄糖，以产生发酵液；
–将生物燃料与发酵液分离。
12.一种用于从生物质生产生物燃料的方法，其特征在于，它包含以下连续步骤：
–将待水解的生物质悬浮于水相中；
–向所述悬浮液同时加入如权利要求10所述的酶组合物和发酵生物，并发酵混合物以产生发酵液；
–将生物燃料与发酵液中分离。
13.如权利要求11或12所述的方法，其中所述发酵生物是如权利要求5-7任一项所述的宿主细胞。
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