CN111996182A

CN111996182A - β-半乳糖苷酶融合蛋白及其制备方法与用途

Info

Publication number: CN111996182A
Application number: CN202010910370.4A
Authority: CN
Inventors: 韩媛媛; 刘丽; 约瑟夫·弗戈迈尔
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2020-09-02
Filing date: 2020-09-02
Publication date: 2020-11-27

Abstract

本发明提供了一种β‑半乳糖苷酶融合蛋白及其制备方法与用途，通过使用柔性肽接头连接两个β‑半乳糖苷酶催化亚基刚性单元，使其在大肠杆菌原核系统中成功高效地异源表达出有活性的重组蛋白；本发明在二聚体间插入Linker的方法基于基因克隆、重组手段，具有操作便捷、经济成本低且重复性高等优点。

Description

β-半乳糖苷酶融合蛋白及其制备方法与用途

技术领域

本发明具体涉及一种β-半乳糖苷酶融合蛋白及其制备方法与用途。

技术背景

β-半乳糖苷酶，常简称为乳糖酶，广泛存在于各种动物、植物及微生物中。1889年，荷兰生物学家第一次报道了β-半乳糖苷酶可水解乳糖，β-半乳糖苷酶的最初应用也是利用其水解乳糖的性质来降低乳制品中的乳糖含量。利用各种技术手段研究β-半乳糖苷酶对乳中乳糖的水解作用，具有很强的现实意义。

鉴于Lactobacillus brevis源的β-半乳糖苷酶为由两个大小不一的亚基组成的异型二聚体，本申请以氨基酸序列组成的柔性肽接头以连接Lactobacillus brevis源β-半乳糖苷酶的两个蛋白亚基，基于克隆、基因重组等程序，对表达的重组蛋白进行β-半乳糖苷酶活性评估和应用。

发明内容

基于现有技术的状况，本发明的目的是提供一种β-半乳糖苷酶融合蛋白及其制备方法与用途；通过柔性氨基酸肽接头连接二聚体使其在大肠杆菌原核系统中异源表达，本发明建立了氨基酸柔性肽链(柔性肽接头)插入二聚体之间用以连接两个亚基的方法。

本发明解决其技术问题采用的技术方案是：

一种β-半乳糖苷酶融合蛋白，所述融合蛋白通过使用柔性肽接头连接两个β-半乳糖苷酶催化亚基刚性单元。

作为本申请的优选技术方案，所述β-半乳糖苷酶催化亚基刚性单元为同型亚基或异型亚基。

作为本申请的优选技术方案，所述β-半乳糖苷酶催化亚基来源于短乳酸杆菌Lactobacillus brevis。

作为本申请的优选技术方案，所述柔性肽接头为由几个氨基酸相互连接形成的含有多个肽键的一条柔性链状结构，用以提高β-半乳糖苷酶酶活力。

优选的，本发明提供了一种融合蛋白，该融合蛋白通过柔性肽接头连接两个β-半乳糖苷酶催化亚基刚性单元；所述两个亚基单元包含：

1.两个蛋白亚基为异型亚基，其编码的基因长度不一，蛋白结构不同。

2.两个亚基单元来源于短乳酸杆菌Lactobacillus brevis。

3.两个亚基单元具有β-半乳糖苷酶的活性，可催化β-1,4糖苷键相连的非还原端半乳糖基团水解。

作为本申请的优选技术方案，所述柔性肽接头选自甘氨酸、丝氨酸、甲硫氨酸、缬氨酸、赖氨酸、天冬氨酸、天冬酰胺或丙氨酸中的一种多种。

作为本申请的优选技术方案，所述柔性肽的分子式含有(GxSy)n的重复序列，其中，x、y、n为大于等于1的整数。

更优选的，所述柔性肽接头选自以下组：

Linker1：GSHM；

Linker2：GSGSGHM；

Linker3：GSGGSGGHM；

Linker4：GSGGGSGHM；

Linker5：GSGGGGSHM；

Linker6：GSGGGSGGGSHM。

更优选的，所述柔性肽接头选自：

Linker4：GSGGGSGHM；

Linker5：GSGGGGSHM；

Linker6：GSGGGSGGGSHM。

本发明还提供了上述柔性肽接头连接的异二聚体即融合蛋白的DNA序列，包括限制性酶切位点基因序列。

本发明还提供了上述柔性肽接头连接的异二聚体(即β-半乳糖苷酶融合蛋白)的氨基酸测序序列，如SEQ ID NO：10-15所示；

其中，柔性肽接头为Linker1时，所述融合蛋白的氨基酸序列如SEQ ID NO：10所示；

柔性肽接头为Linker2时，所述融合蛋白的氨基酸序列如SEQ ID NO：11所示；

柔性肽接头为Linker3时，所述融合蛋白的氨基酸序列如SEQ ID NO：12所示；

柔性肽接头为Linker4时，所述融合蛋白的氨基酸序列如SEQ ID NO：13所示；

柔性肽接头为Linker5时，所述融合蛋白的氨基酸序列如SEQ ID NO：14所示；

柔性肽接头为Linker6时，所述融合蛋白的氨基酸序列如SEQ ID NO：15所示。

一种β-半乳糖苷酶融合蛋白的制备方法，基于分子生物学通用方法，使用限制性内切酶将柔性肽接头插入在异二聚体之间构建表达载体进行原核表达；所述方法为包括：引物设计、基因克隆以及蛋白诱导表达等。

优选的，所述制备方法如下：

(1)设计含有酶切位点的DNA序列，两个亚基的N端、C端携带不同的酶切位点基因序列，对肽链部分的一端或两端设计相应的酶切位点；

(2)合成引物序列，以单亚基的DNA序列为模板，引物部分包括酶切位点、柔性肽接头的DNA序列、以及可与模板互补的DNA片段；

(3)使用引物进行PCR扩增，在原亚基模板上扩增出携带有柔性肽接头的DNA序列的长片段；

(4)对扩增产物进行酶切酶连，通过酶切位点分别连接两个亚基的N端和C端，在亚基之间插入柔性肽接头；所述柔性肽接头为通过相关酶切位点酶切连接、翻译后形成的氨基酸；(5)构建重组载体并在原核生物中表达。

更优选的，所述制备方法包括如下步骤：

(1)针对异二聚体(由大亚基LbGalA和小亚基LbGalB组成)中的小亚基LbGalB，设计PCR的引物，其中正向引物保持一致，均为LbGalB-FW：

5'-aaaccatggaccatcatcatcatcatcataacaccaccaaactgaacgt-3'，SEQ ID NO：1；

反向引物如下：

GSHM：5'-aaacatatgactaccaaccggggtcgg-3'，SEQ ID NO：2；

GSGSGHM：5'-aaacatatgaccactaccactaaccggggtcgg-3'，SEQ ID NO：3；

GSGGSGGHM：5'-aaacatatgaccaccactaccaccactaaccggggtcgg-3'，SEQ ID NO：4；

GSGGGSGHM：5'-aaacatatgaccactaccaccaccactaaccggggtcgg-3'，SEQ ID NO：5；

GSGGGGSHM：5'-aaacatatgactaccaccaccaccactaaccggggtcgg-3'，SEQ ID NO：6；

GSGGGSGGGSHM：5'-aaacatatgactaccaccaccactaccaccaccactaaccggggtcgg-3'，SEQ ID NO：7；

(2)据步骤(1)的设计，以BamH I和Nde I限制性核酸内切酶切开LbGalB和LbGalA之间的片段，将柔性肽接头的DNA序列加入LbGalB和LbGalA之间；

(3)再利用Nco I/Xho I同步酶切步骤(2)得到的目的基因、表达载体，并使用T4连接酶酶连，构建表达载体；

(3)使用大肠杆菌表达系统进行诱导表达，该大肠杆菌已敲除LacZ^-基因以消除菌株本身表达的β-半乳糖苷酶影响。

对表达的的重组蛋白(融合蛋白)进行β-半乳糖苷酶活性评估，方法如下：表达的蛋白稀释100倍，利用释放出pNP的量借助酶标仪在紫外分光光度计405nm处有吸收值，其值大小与酶活力呈正相关。

本发明还提供了一种β-半乳糖苷酶融合蛋白在催化水解β-1,4糖苷键相连的非还原端半乳糖基团中的应用。

本发明所述的基于氨基酸序列组成的柔性肽链，填补了其在大肠杆菌原核系统中异源高效表达的空白，操作简单，重复性好且成本低，同时表达的重组蛋白具有β-半乳糖苷酶活性，且较LbGalAB异二聚体更高，在研究乳糖酶应用方面具有一定潜在价值。

附图说明

图1表达载体示意图；

结合附图说明：肽链部分为箭头所指的区域，在BamH I/Nde I之间；

图2为基因扩增图；

图3为阳性菌落筛选图；

图4为β-半乳糖苷酶图释，其中A为原模板异二聚体未经连接的图释；

B为异二聚体经GS-Linker肽链连接的融合蛋白图释；

图5为β-半乳糖苷酶利用4-硝基苯基-β-D-半乳糖苷为底物活性检测原理图；

图6为β-半乳糖苷酶融合蛋白活性评估；

图7为乳样品的HPLC色谱图，分别对应人乳、牛和山羊奶；其中A为去除乳糖前，B为去除乳糖后。

具体实施方式

以下结合实施例对本发明做进一步详细说明。所用试剂或者仪器设备未注明生产厂商的，均视为可以通过市场购买的常规产品。

克隆菌株E.coli BMach T1以及已敲除β-半乳糖甘酶(LacZ^-)的表达菌株E.coliBL21(DE3)购自于天根生化科技(北京)有限公司，其感受态均由本实验中心(糖组学和糖生物研究中心)自主制备并冻存。克隆中间载体pGM-T由上海捷瑞生物工程有限公司购买，RSF-Duet载体均购自于Novagen公司。PCR所设计的引物由南京金斯瑞生物科技有限公司合成，基因测序由通用生物系统(安徽)有限公司完成；2×rTaq DNA聚合酶购买于Takara公司；10×Ligase Buffer和10×Fast Digest Buffer、T4连接酶、NdeΙ/BamH I/NcoRΙ/XhoΙ限制性核酸内切酶、去磷酸化Fast AP酶购于Thermo Scientific有限公司；DNA胶回收试剂盒、质粒提取试剂盒以及GoldviewΙ型核酸染色剂购自于北京索莱宝科技有限公司，琼脂粉、氨苄霉素、卡那霉素、氯霉素、5-溴-4-氯-3-吲哚-β-D-半乳糖苷(X-gal)、异丙基硫代半乳糖苷(IPTG)、氯化钠、酵母提取物、胰蛋白胨等分析纯试剂来自于南京杰汶达生物科技有限公司。

一、引物设计

以Lactobacillus brevis源的β-半乳糖苷酶为研究对象，其是由两个大小不一的亚基(大亚基LbGalA(SEQ ID NO：9)和小亚基LbGalB(SEQ ID NO：8))组成的异型二聚体。

使用计算机引物设计软件Primer Premier 5.0以小亚基LbGalB为模板设计引物，并在两端加入合适的酶切引物。在后引物上减去TAA终止密码子以及C端组氨酸标签序列，设计不同长度的后引物，以期添加不同长度的Linker，肽链接头的氨基酸序列和DNA序列如下表1所示。

表1引物设计

二、基因扩增与鉴定

基因扩增

分别以提取的亚基LbGalB质粒为扩增模板，在PCR rTaq缓冲液、前后引物、DNA聚合酶的参与下进行PCR扩增。试剂2×rTaq Mix中含有dNTP、Mg²⁺等所需要的碱基原料以及离子试剂等，PCR反应体系如下表2所示。首先95℃预变性5min，之后扩增程序为35个标准循环，每个循环包含三个单元，即变性：95℃，30s；复性退火：55℃，30s；延伸72℃，2min。退火温度取决于PCR反应中前后引物的T_m值，延伸时间根据扩增的DNA片段长度而定，每1000bp所需的时间为1min。

表2 PCR扩增反应体系

以LbGalB模板，设计含有不同Linker的后引物，利用前后引物扩增出与模板LbGalB末端不同的基因片段使末端带有不同长度的肽链，构建不同长度的Linker的表达载体。通过PCR扩增，在LbGalB基因上加入Linker基因序列及LbGalA基因，获得β半乳糖苷酶融合蛋白。PCR扩增结果如图2所示，因只相差12～36个碱基对，6个条带大小位置无太大变化，均在1000bp附近。

基因鉴定

以BamH I和Nde I限制性核酸内切酶切开LbGalB和LbGalA之间的片段，中间原约有188个碱基对，现使Linker在LbGalB和LbGalA之间，以替换掉原来的碱基对；再利用Nco I和Xho I双酶切扩增后的PCR产物以及RSF-Duet载体，连接后回收，并转化至E.coli BMachT1中培养，筛选阳性克隆，菌落鉴定如图3所示，框线所在的条带为成功构建融合载体的目的菌落。

三、表达载体的提取与转化

1.质粒的提取

使用高纯质粒提取试剂盒对筛选过的菌液进行质粒抽提，抽提出的质粒作为后续基因扩增的模板。抽提操作参考说明书，具体步骤如下：

(1)移液枪吸取2mL菌液加入EP管中，以8000rpm转速1min离心弃上清留沉淀；继续吸取2mL菌液加入该EP管中，重复一次；

(2)加250μL溶液S1于留有沉淀的EP管中，使用涡旋震荡器使沉淀充分裂解混匀悬浮在溶液中。(注意：需充分裂解混匀，避免有小的块状悬浮物，影响回收效率)；

(3)加250μL溶液S2于该EP管中，迅速温和地上下颠倒EP管4-8次，使之充分混匀至澄清透亮有粘稠感。(注意：此操作应迅速完成，尽量不超过5min，避免剧烈震荡操作，如未出现透亮粘稠感，则表明菌体量过多，可多加入溶液S2或少加入菌液减少菌体量)；

(4)继续加入350μL溶液S3于该EP管中，上下颠倒EP管4-8次迅速混匀，直至出现白色絮状沉淀，置于离心机中以12100rpm离心10min；

(5)移液枪吸取上一步离心管中的上清于2mL的纯化柱A收集管中，将其置于离心机中以8000rpm离心1min，弃滤液；

(6)加500μL溶液W1于收集管中，于离心机中12100rpm离心1min，弃滤液；

(7)继续加500μL溶液W1于收集管中，重复上述操作一次；

(8)将弃去滤液的收集管置于离心机中以12100rpm离心2min，充分去除收集管纯化柱膜上的漂洗液，离心结束后，将纯化柱放置于1.5mL干净无污染的EP管中，室温下开盖放置2-5min，使溶液W1中的乙醇挥发完全；

(9)加50μL洗脱液ES于上述纯化柱中的膜中央，避免膜被碰触损坏，盖好EP管盖，室温下静置2-5min后，置于离心机中以12100rpm离心1min，弃去纯化柱，收集1.5mL EP管中的液体即为抽提好的质粒模板；冻存于-20℃冰箱待备用。

2.感受态的制备

利用Ca²⁺化学法处理细胞使其成为易感的状态，加大细胞膜的通透性。其具体操作如下：

(1)接种敲除LacZ^-基因的BL21(DE3)细菌于5mL已灭菌的LB培养基中(不加抗生素)，置于37℃，200rpm的摇床中培养12-14小时，阴性对照不接菌。

(2)超净台中移取培养好的菌液1mL加入已灭菌冷却至室温的含有100mL液体培养基的锥形瓶中，将该瓶置于37℃，200rpm摇床中培养2小时，利用One-drop测量瓶中菌液的生长浓度，直至细胞生长浓度OD值在0.3-0.5之间。

(3)将达到细胞浓度要求的锥形瓶从摇床中取出置于冰水浴中冷却20min，使其停止生长。

(4)在超净台中将冷却的菌液转移至已灭菌的离心杯中，将离心杯配平置于台式冷冻离心机中，设置参数，温度为4℃，转速为4000rpm，时长10min，转子为50型号，离心结束后，取出离心杯，始终置于冰水中且避免剧烈震荡。

(5)在超净台将离心杯中的上清缓慢弃去，留沉淀置于冰水中，向沉淀中加入已灭菌预冷的100mM CaCl₂溶液20mL，轻晃使沉淀完全溶于CaCl₂溶液中，避免操作过程中用枪头剧烈吸打以及避免离心杯离开冰水混合物中。

(6)将离心杯拧紧，按照第4步操作重复一次，于超净台中弃去滤液。

(7)向沉淀中加入已灭菌预冷的100mM CaCl₂溶液6mL，轻晃使沉淀完全溶于CaCl₂溶液中，以及已灭菌冷却的甘油1mL，轻轻用枪头吸打悬浮菌液。

(8)将悬浮的菌液以每管100mL的体积量迅速分装至已灭菌冷却的1.5mL EP管中，液氮冻存已分装好的EP管，放置于-80℃冰箱留存备用。

3.质粒热击转化

将提取好的质粒通过热击的方法的转入E.coli BL21(DE3、LacZ^-)感受态细胞中，转化操作如下：

(1)取出按上述操作存于-80℃冰箱中的BL21(DE3、LacZ^-)感受态细胞，置于冰盒中解冻5min。

(2)移液枪吸取质粒1μL分别加入融化的感受态中，静置于冰盒中15-20min。同时开启水浴锅，温度参数设为42℃备用。

(3)将上述第2步的EP管迅速放入42℃水浴锅中热击1min，再迅速从水浴锅中拿出，置于冰盒中静置冰孵2min。

(4)在超净台移取200μL液体培养基加入该EP管中，置于37℃、850rpm的金属浴震荡培养45min进行复苏。

(5)复苏结束后，取出EP管，在超净台中开盖，用移液枪轻轻吸打混匀后，吸取200μL复苏液加入到含有50μg/mL卡那霉素(kana+)或34μg/mL氯霉素(chlo+)的固体培养基中进行筛选，利用涂布棒均匀涂布在固体培养基上。

(6)待涂布好的固体培养基吸收完全，将培养皿倒置于37℃隔水恒温培养箱中，过夜培养至形成肉眼可见的单个菌落。

四、柔性肽接头构建的异二聚体的表达载体的蛋白活性评估

重组蛋白的诱导

(1)在超净台中用移液枪从保藏管中挑取少量表达菌株E.coli BL21(DE3、LacZ-)接种于无菌且含50mg/mL卡那霉素LB培养基5mL的试管中，置于37℃、200rpm的摇床中培养12-14h。为防止摇菌活化过程中出现污染，应设置阴性对照。

(2)将培养结束的菌液在超净台中吸取4mL接种于含有400mL无菌LB培养基的大摇瓶中(体积比1:100)，置于37℃、200rpm的摇床中培养2h，由于接菌时，目的菌落为优势菌种，故不需要加抗生素。

(3)利用紫外分光光度计One-drop及时测定摇瓶中细胞浓度，测定600nm波长下的吸光值，直至在细胞生长对数阶段即OD600值在0.5-0.8期间。

(4)菌体培养至对数阶段时，取出大摇瓶，移取1mL菌液至EP管1.5mL中，留存备用，同时加入终浓度为1mM的IPTG，即400μL浓度为1M的IPTG用于诱导。

(5)提前设置摇床参数即温度为18℃，转速为200rpm。加入IPTG后，将大摇瓶放入已设置好参数的摇床中继续培养18-24h。(温度和转速过高或过快容易导致蛋白形成包涵体)

(6)取出表达完毕的大摇瓶，移取1mL菌液至EP管1.5mL中。对诱导前、后的各1mL菌液使用高速离心机12100rpm离心1min，弃上清，留菌体沉淀。以备后续SDS-page检测，放入-20℃冰箱中保存。

(7)将大摇瓶中剩下的培养基分两次倒入250mL离心杯中，使用高速冷冻离心机，需提前预冷，设置离心参数转子为50型号，温度为4℃，转速为4000rpm，时长20min。配平离心，弃离心杯中上清，留杯中沉淀置于冰水浴中。

(8)配制细胞裂解液：100mM的氯化钠、1％(V/V)的Triton X-100、50mM的Tris，调节pH至8.0。向留有沉淀的杯中加入裂解液，每400mL菌液得到的沉淀加入10mL裂解液，将充分吸打使沉淀悬浮在裂解中，至无块状菌体。操作过程应保持在冰水浴中，避免蛋白变性失活。

(9)将裂解好的细胞液转移至10mL离心管中(不可装的过满)放置-80℃冰箱中保存。

重组蛋白的粗酶液的制备

(1)解冻：从冰箱中取出裂解液，流水使其解冻融化。(注意解冻过程中将装有裂解液的试管始终放置在冰水浴中，保持低温)。

(2)加入蛋白酶抑制剂：将重悬体转移至50mL的离心管中，加入100μL浓度为100mM的苯甲基磺酰氟(PMSF)抑制粗酶液中蛋白酶的活性。

(3)超声：水清洗探头，防止探头上沾有杂质污染样品。将该离心管置于超声破碎仪中破碎，超声破碎仪参数设为：温度范围4-16℃，探头为Φ6型号，每次循环为超声时间1.5秒，间隔时间2.5秒，总运行时间为20分钟，功率为350W。超声过程中应将离心杯固定在含有冰水的烧杯中，防止超声工作释放大量热量，影响酶活。

(4)离心：设置高速冷冻离心机参数：转子为18型号，温度4℃，转速13500rpm，时长20min。开机预冷，超声破碎后的细胞液配平置于转子中离心。

(5)收样：收集上清置于冰水中，保持低温状态，弃沉淀，即得到粗酶液。

活性评估

4-硝基苯基-β-D-半乳糖苷(pNP-β-D-半乳糖)为底物时，可用来快速检测重组表达的β-半乳糖苷酶是否具有酶解的活性，其原理如图5所示，因为半乳糖苷酶断裂β键型，使非还原端的半乳糖残基基团被释放，从而另一端的对硝基苯酚(pNP)也被释放，生成游离的pNP产物，而在碱性溶液中，pNP的水溶液呈现出肉眼可见的黄色，因此根据颜色是否有变化即可快速鉴定重组的β-半乳糖苷酶是否具有活性。pNP-β-D-Galactose购买于阿拉丁试剂(上海)有限公司。

制备的粗酶液加入反应体系，反应体系如下表3所示，总体积为10μL。底物为4-硝基苯基-β-D-半乳糖苷，终浓度为2mM，将反应置于中性缓冲溶液中，置于37℃恒温培养箱中，反应20min，同时设置阴性对照和阳性对照。阴性对照为不插入目的基因的空载体pRSF-Duet的BL21(DE3、LacZ^-)的粗酶液以排除大肠杆菌本身对底物pNP-β-D-半乳糖的背景干扰。

表3重组蛋白β-半乳糖苷酶对4-硝基苯基-β-D-半乳糖苷的活性测试

以pNP-β-半乳糖为底物利用酶标仪对释放出游离的pNP在405nm处测定吸光度，根据吸光值的强弱判断重组β-半乳糖苷酶的酶活评估。确定所有实验组控制表达条件(表达时间，表达菌株的生长状态)一致。表达产物的区别如图6所示。

如图6所示，表达的重组蛋白活性表明除了融合酶Linker 3比LbGalAB略低，其他融合的β-半乳糖苷酶的酶活性比LbGalAB异二聚体的活性高。根据现有理论，随着肽链Linker长度的增加，融合蛋白的活性会增强，因为Linker长度可以提供亚基之间合适的空间距离，从而影响蛋白的活性，但是从图6看，并不是Linker长度越长，融合蛋白的活性越强，本申请Linker5连接的β-半乳糖苷酶融合蛋白的活性最高。

表达的融合蛋白在乳糖中具有分解乳糖的作用，通过对人乳、牛乳以及山羊乳的乳样进行处理，以水代替重组β-半乳糖苷酶的实验组为阴性对照，通过HPLC检测样品中的乳糖峰，对比酶解前、后的乳样发现其中的乳糖峰(保留时间为7-8min之间)发生了明显变化，人乳、牛乳以及山羊乳中乳糖都几乎被酶解完全(A：酶切前乳样中低聚糖的峰型图；B：酶切后乳样中低聚糖的峰型图)，如图7所示。

本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。

序列表

<110> 南京农业大学

<120> β-半乳糖苷酶融合蛋白及其制备方法与用途

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aaacatatga ctaccaaccg gggtcgg 27

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<212> DNA

<213> 人工序列(Artificial Sequence)

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ccatggacca tcatcatcat catcataaca ccaccaaact gaacgttatc ttcggtgacg 60

ttaccctggg tgtttctggt ccgggtttcc actacatctt cgcttacgac cgtggtggtc 120

tggaatctct ggttcaggac ggtaaagaat ggctgtaccg taccccgatg ccggctctgt 180

ggcgtgctac caccgacaac gaccgtggca atggcttctc taccaaatcg gctcagtggc 240

tgggtgctga cctgttctct tcttgcgacc acatctctgt tgctatcgac ggtcagtcta 300

tcccgctgcc gatcgctccg gaaaacaacc gttactctga ccacgaaacc gctaccaccg 360

ttgctgttac cttcacctac accaccccga ccaccccggc taccaccatc gctgttacct 420

acaccgttgc tgcttctggt gctatgaccg ttgctgttca ctacgctggt aaagctgacc 480

tgccggaact gccggctctg ggtctgcgtc tggttatgcc gaccccggcg ctcggcttcg 540

cttaccaggg cctgtctggt gaaacctacc cggaccgtaa agctggtggt cgtaaaggta 600

tccaccaggt tgctggtctg ccggttaccc cgtacctggt tccgcaggaa tgcggtatgc 660

acgttgacaa ccagtgggtt accgttaccc gtggtactac ccagaacaac gctgacgctg 720

accacgacgc tttctctctg aaagttcgtc agacccagca ccacttcgct ttctcttgcc 780

tgccgtacac cccgaccgaa ctggaaaacg ctacccacca ggaagaactg ccggtcccgc 840

gtcgtaccgt tctgaccatc tacggtgctg ttcgtggtgt tggtggtatc gactcttggg 900

gttctgacgc tgaagctccg taccacatcg ctggtgactc tgaccacgac ttctctttcg 960

aaatcgctgg tccgaccccg gtttaaggat cc 992

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<212> DNA

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catatgaaag ctgacctgac ctggctggac gacccgcaga ccttccgtat caaccagctg 60

ccggctcact ctgaccaccg tggttacgct tctgttgaag aagctaccgc tcagcactct 120

tctctggttc agtctctgga cggtacttgg cagttcgctt tcgctccgga cccggttcac 180

aggttcgaag gtttctacca gccggactac gaccgttctg ctttcgaccg tctgaccgtt 240

ccgggtcaca tcgaactggc tggttacggt cagatccagt acatcaacac cgcttacccg 300

tgggaaggtc accactaccg tcgtccggct tactctatgg gtgctgacca gccggaaaaa 360

ggtatgttct ctaccgaccc gcagaacacc gttggtgctt acgttaaaca cttcaccctg 420

aacccggctc tggcgaatca gcgtgtaagc atcgaatttg acggtgttga acaggctatg 480

ttcctgtggc tgaacggtca gttcgttggt tacgctgaag actctttctc tcgttctgaa 540

tttgacctga ccccgtacct gcaggctggt cagaacctgc tggctgttga agttttcaaa 600

cactctaccg ctgctttcct ggaagaccag gacatgttcc gtttctctgg tatcttccgt 660

tctgttcgtc tggttgctaa accggaactg cacgttgaag acctgaccat ccgtgctggt 720

ctggacgacg ctttccagac cggtgacctg aaagttcgtc tgcagctgac cgctgcttct 780

cagctgtctg gtactgctac cgctcagctg ctgacagctg acggccagga agtatgggcc 840

accgaacagc cggctgcttc taccctggac ctggctgctg ctatcgacca cgttcacctg 900

tgggaccacc acgacccgta cctgtaccag ctgcgtatca ccctgaaaga cgttgctggt 960

caggttgttg aagttgttcc gtacccggtt ggtttccgtc gtatcgaact gaaagacaaa 1020

gttatgtgcc tgaacggtca gcgtctgatc ctgaacggtg ttaaccgtca cgaatgggac 1080

gctcaccgtg gtcgtgctgt tactatggct gacatgaccc aggacctgca gaccttccac 1140

gacaaccaca tcaacgctgt tcgtacctgc cactacccgg accaggacgc ttggtactac 1200

ctgtgcgacc agcagggtat ctacatgatg gctgaaaaca acctggaaac ccacggtact 1260

tggcagaaaa tgggtgctgt tgaaccgtct tacaacgttc cgggttctct gccgcagtgg 1320

cagctggctg ttctggaccg tgctaaatct aactacgaaa tgttcaaaaa ccacccggct 1380

gttctgttct ggtctctggg taacgaatct tacgctggtg acaacatcgc tgctatggac 1440

gctttctacc accacgctga cccgacccgt ctgacccact acgaaggtgt ttgccgtaac 1500

cgtgtttacg aagaccgtat ctctgacatg gaatctatga tgtacgaccc gccgcgtgct 1560

atcgaagact acctgaaaaa cgacccgcag aaaccgttcg ttaactgcga atacatgcac 1620

gacatgggta actctctggg tggtatggct tcttacgacg ctctgatcga ccagtacccg 1680

atgtaccagg gtggtttcat ctgggacttc atcgaccagg ctctgtgggt taaagacgaa 1740

gttaccggtc agccggttct gcgttacggt ggtgacttcg acgaccgtca ctctgactac 1800

gaattttctg gtgacggtct gctgttcgct gaccgtaccc cgaaaccggc tctgcaggaa 1860

gttgactact actacggtca gcacgactaa ctcgag 1896

<210> 10

<211> 958

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 10

Met Asp His His His His His His Asn Thr Thr Lys Leu Asn Val Ile

1 5 10 15

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

20 25 30

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

35 40 45

Glu Trp Leu Tyr Arg Thr Pro Met Pro Ala Leu Trp Arg Ala Thr Thr

50 55 60

Asp Asn Asp Arg Gly Asn Gly Phe Ser Thr Lys Ser Ala Gln Trp Leu

65 70 75 80

Gly Ala Asp Leu Phe Ser Ser Cys Asp His Ile Ser Val Ala Ile Asp

85 90 95

Gly Gln Ser Ile Pro Leu Pro Ile Ala Pro Glu Asn Asn Arg Tyr Ser

100 105 110

Asp His Glu Thr Ala Thr Thr Val Ala Val Thr Phe Thr Tyr Thr Thr

115 120 125

Pro Thr Thr Pro Ala Thr Thr Ile Ala Val Thr Tyr Thr Val Ala Ala

130 135 140

Ser Gly Ala Met Thr Val Ala Val His Tyr Ala Gly Lys Ala Asp Leu

145 150 155 160

Pro Glu Leu Pro Ala Leu Gly Leu Arg Leu Val Met Pro Thr Pro Ala

165 170 175

Leu Gly Phe Ala Tyr Gln Gly Leu Ser Gly Glu Thr Tyr Pro Asp Arg

180 185 190

Lys Ala Gly Gly Arg Lys Gly Ile His Gln Val Ala Gly Leu Pro Val

195 200 205

Thr Pro Tyr Leu Val Pro Gln Glu Cys Gly Met His Val Asp Asn Gln

210 215 220

Trp Val Thr Val Thr Arg Gly Thr Thr Gln Asn Asn Ala Asp Ala Asp

225 230 235 240

His Asp Ala Phe Ser Leu Lys Val Arg Gln Thr Gln His His Phe Ala

245 250 255

Phe Ser Cys Leu Pro Tyr Thr Pro Thr Glu Leu Glu Asn Ala Thr His

260 265 270

Gln Glu Glu Leu Pro Val Pro Arg Arg Thr Val Leu Thr Ile Tyr Gly

275 280 285

Ala Val Arg Gly Val Gly Gly Ile Asp Ser Trp Gly Ser Asp Ala Glu

290 295 300

Ala Pro Tyr His Ile Ala Gly Asp Ser Asp His Asp Phe Ser Phe Glu

305 310 315 320

Ile Ala Gly Pro Thr Pro Val Gly Ser His Met Lys Ala Asp Leu Thr

325 330 335

Trp Leu Asp Asp Pro Gln Thr Phe Arg Ile Asn Gln Leu Pro Ala His

340 345 350

Ser Asp His Arg Gly Tyr Ala Ser Val Glu Glu Ala Thr Ala Gln His

355 360 365

Ser Ser Leu Val Gln Ser Leu Asp Gly Thr Trp Gln Phe Ala Phe Ala

370 375 380

Pro Asp Pro Val His Arg Phe Glu Gly Phe Tyr Gln Pro Asp Tyr Asp

385 390 395 400

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

405 410 415

Gly Tyr Gly Gln Ile Gln Tyr Ile Asn Thr Ala Tyr Pro Trp Glu Gly

420 425 430

His His Tyr Arg Arg Pro Ala Tyr Ser Met Gly Ala Asp Gln Pro Glu

435 440 445

Lys Gly Met Phe Ser Thr Asp Pro Gln Asn Thr Val Gly Ala Tyr Val

450 455 460

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

465 470 475 480

Glu Phe Asp Gly Val Glu Gln Ala Met Phe Leu Trp Leu Asn Gly Gln

485 490 495

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

500 505 510

Thr Pro Tyr Leu Gln Ala Gly Gln Asn Leu Leu Ala Val Glu Val Phe

515 520 525

Lys His Ser Thr Ala Ala Phe Leu Glu Asp Gln Asp Met Phe Arg Phe

530 535 540

Ser Gly Ile Phe Arg Ser Val Arg Leu Val Ala Lys Pro Glu Leu His

545 550 555 560

Val Glu Asp Leu Thr Ile Arg Ala Gly Leu Asp Asp Ala Phe Gln Thr

565 570 575

Gly Asp Leu Lys Val Arg Leu Gln Leu Thr Ala Ala Ser Gln Leu Ser

580 585 590

Gly Thr Ala Thr Ala Gln Leu Leu Thr Ala Asp Gly Gln Glu Val Trp

595 600 605

Ala Thr Glu Gln Pro Ala Ala Ser Thr Leu Asp Leu Ala Ala Ala Ile

610 615 620

Asp His Val His Leu Trp Asp His His Asp Pro Tyr Leu Tyr Gln Leu

625 630 635 640

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

645 650 655

Tyr Pro Val Gly Phe Arg Arg Ile Glu Leu Lys Asp Lys Val Met Cys

660 665 670

Leu Asn Gly Gln Arg Leu Ile Leu Asn Gly Val Asn Arg His Glu Trp

675 680 685

Asp Ala His Arg Gly Arg Ala Val Thr Met Ala Asp Met Thr Gln Asp

690 695 700

Leu Gln Thr Phe His Asp Asn His Ile Asn Ala Val Arg Thr Cys His

705 710 715 720

Tyr Pro Asp Gln Asp Ala Trp Tyr Tyr Leu Cys Asp Gln Gln Gly Ile

725 730 735

Tyr Met Met Ala Glu Asn Asn Leu Glu Thr His Gly Thr Trp Gln Lys

740 745 750

Met Gly Ala Val Glu Pro Ser Tyr Asn Val Pro Gly Ser Leu Pro Gln

755 760 765

Trp Gln Leu Ala Val Leu Asp Arg Ala Lys Ser Asn Tyr Glu Met Phe

770 775 780

Lys Asn His Pro Ala Val Leu Phe Trp Ser Leu Gly Asn Glu Ser Tyr

785 790 795 800

Ala Gly Asp Asn Ile Ala Ala Met Asp Ala Phe Tyr His His Ala Asp

805 810 815

Pro Thr Arg Leu Thr His Tyr Glu Gly Val Cys Arg Asn Arg Val Tyr

820 825 830

Glu Asp Arg Ile Ser Asp Met Glu Ser Met Met Tyr Asp Pro Pro Arg

835 840 845

Ala Ile Glu Asp Tyr Leu Lys Asn Asp Pro Gln Lys Pro Phe Val Asn

850 855 860

Cys Glu Tyr Met His Asp Met Gly Asn Ser Leu Gly Gly Met Ala Ser

865 870 875 880

Tyr Asp Ala Leu Ile Asp Gln Tyr Pro Met Tyr Gln Gly Gly Phe Ile

885 890 895

Trp Asp Phe Ile Asp Gln Ala Leu Trp Val Lys Asp Glu Val Thr Gly

900 905 910

Gln Pro Val Leu Arg Tyr Gly Gly Asp Phe Asp Asp Arg His Ser Asp

915 920 925

Tyr Glu Phe Ser Gly Asp Gly Leu Leu Phe Ala Asp Arg Thr Pro Lys

930 935 940

Pro Ala Leu Gln Glu Val Asp Tyr Tyr Tyr Gly Gln His Asp

945 950 955

<210> 11

<211> 961

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 11

Met Asp His His His His His His Asn Thr Thr Lys Leu Asn Val Ile

1 5 10 15

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

20 25 30

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

35 40 45

Glu Trp Leu Tyr Arg Thr Pro Met Pro Ala Leu Trp Arg Ala Thr Thr

50 55 60

Asp Asn Asp Arg Gly Asn Gly Phe Ser Thr Lys Ser Ala Gln Trp Leu

65 70 75 80

Gly Ala Asp Leu Phe Ser Ser Cys Asp His Ile Ser Val Ala Ile Asp

85 90 95

Gly Gln Ser Ile Pro Leu Pro Ile Ala Pro Glu Asn Asn Arg Tyr Ser

100 105 110

Asp His Glu Thr Ala Thr Thr Val Ala Val Thr Phe Thr Tyr Thr Thr

115 120 125

Pro Thr Thr Pro Ala Thr Thr Ile Ala Val Thr Tyr Thr Val Ala Ala

130 135 140

Ser Gly Ala Met Thr Val Ala Val His Tyr Ala Gly Lys Ala Asp Leu

145 150 155 160

Pro Glu Leu Pro Ala Leu Gly Leu Arg Leu Val Met Pro Thr Pro Ala

165 170 175

Leu Gly Phe Ala Tyr Gln Gly Leu Ser Gly Glu Thr Tyr Pro Asp Arg

180 185 190

Lys Ala Gly Gly Arg Lys Gly Ile His Gln Val Ala Gly Leu Pro Val

195 200 205

Thr Pro Tyr Leu Val Pro Gln Glu Cys Gly Met His Val Asp Asn Gln

210 215 220

Trp Val Thr Val Thr Arg Gly Thr Thr Gln Asn Asn Ala Asp Ala Asp

225 230 235 240

His Asp Ala Phe Ser Leu Lys Val Arg Gln Thr Gln His His Phe Ala

245 250 255

Phe Ser Cys Leu Pro Tyr Thr Pro Thr Glu Leu Glu Asn Ala Thr His

260 265 270

Gln Glu Glu Leu Pro Val Pro Arg Arg Thr Val Leu Thr Ile Tyr Gly

275 280 285

Ala Val Arg Gly Val Gly Gly Ile Asp Ser Trp Gly Ser Asp Ala Glu

290 295 300

Ala Pro Tyr His Ile Ala Gly Asp Ser Asp His Asp Phe Ser Phe Glu

305 310 315 320

Ile Ala Gly Pro Thr Pro Val Gly Ser Gly Ser Gly His Met Lys Ala

325 330 335

Asp Leu Thr Trp Leu Asp Asp Pro Gln Thr Phe Arg Ile Asn Gln Leu

340 345 350

Pro Ala His Ser Asp His Arg Gly Tyr Ala Ser Val Glu Glu Ala Thr

355 360 365

Ala Gln His Ser Ser Leu Val Gln Ser Leu Asp Gly Thr Trp Gln Phe

370 375 380

Ala Phe Ala Pro Asp Pro Val His Arg Phe Glu Gly Phe Tyr Gln Pro

385 390 395 400

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

405 410 415

Glu Leu Ala Gly Tyr Gly Gln Ile Gln Tyr Ile Asn Thr Ala Tyr Pro

420 425 430

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

435 440 445

Gln Pro Glu Lys Gly Met Phe Ser Thr Asp Pro Gln Asn Thr Val Gly

450 455 460

Ala Tyr Val Lys His Phe Thr Leu Asn Pro Ala Leu Ala Asn Gln Arg

465 470 475 480

Val Ser Ile Glu Phe Asp Gly Val Glu Gln Ala Met Phe Leu Trp Leu

485 490 495

Asn Gly Gln Phe Val Gly Tyr Ala Glu Asp Ser Phe Ser Arg Ser Glu

500 505 510

Phe Asp Leu Thr Pro Tyr Leu Gln Ala Gly Gln Asn Leu Leu Ala Val

515 520 525

Glu Val Phe Lys His Ser Thr Ala Ala Phe Leu Glu Asp Gln Asp Met

530 535 540

Phe Arg Phe Ser Gly Ile Phe Arg Ser Val Arg Leu Val Ala Lys Pro

545 550 555 560

Glu Leu His Val Glu Asp Leu Thr Ile Arg Ala Gly Leu Asp Asp Ala

565 570 575

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

580 585 590

Gln Leu Ser Gly Thr Ala Thr Ala Gln Leu Leu Thr Ala Asp Gly Gln

595 600 605

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

610 615 620

Ala Ala Ile Asp His Val His Leu Trp Asp His His Asp Pro Tyr Leu

625 630 635 640

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

645 650 655

Val Val Pro Tyr Pro Val Gly Phe Arg Arg Ile Glu Leu Lys Asp Lys

660 665 670

Val Met Cys Leu Asn Gly Gln Arg Leu Ile Leu Asn Gly Val Asn Arg

675 680 685

His Glu Trp Asp Ala His Arg Gly Arg Ala Val Thr Met Ala Asp Met

690 695 700

Thr Gln Asp Leu Gln Thr Phe His Asp Asn His Ile Asn Ala Val Arg

705 710 715 720

Thr Cys His Tyr Pro Asp Gln Asp Ala Trp Tyr Tyr Leu Cys Asp Gln

725 730 735

Gln Gly Ile Tyr Met Met Ala Glu Asn Asn Leu Glu Thr His Gly Thr

740 745 750

Trp Gln Lys Met Gly Ala Val Glu Pro Ser Tyr Asn Val Pro Gly Ser

755 760 765

Leu Pro Gln Trp Gln Leu Ala Val Leu Asp Arg Ala Lys Ser Asn Tyr

770 775 780

Glu Met Phe Lys Asn His Pro Ala Val Leu Phe Trp Ser Leu Gly Asn

785 790 795 800

Glu Ser Tyr Ala Gly Asp Asn Ile Ala Ala Met Asp Ala Phe Tyr His

805 810 815

His Ala Asp Pro Thr Arg Leu Thr His Tyr Glu Gly Val Cys Arg Asn

820 825 830

Arg Val Tyr Glu Asp Arg Ile Ser Asp Met Glu Ser Met Met Tyr Asp

835 840 845

Pro Pro Arg Ala Ile Glu Asp Tyr Leu Lys Asn Asp Pro Gln Lys Pro

850 855 860

Phe Val Asn Cys Glu Tyr Met His Asp Met Gly Asn Ser Leu Gly Gly

865 870 875 880

Met Ala Ser Tyr Asp Ala Leu Ile Asp Gln Tyr Pro Met Tyr Gln Gly

885 890 895

Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Leu Trp Val Lys Asp Glu

900 905 910

Val Thr Gly Gln Pro Val Leu Arg Tyr Gly Gly Asp Phe Asp Asp Arg

915 920 925

His Ser Asp Tyr Glu Phe Ser Gly Asp Gly Leu Leu Phe Ala Asp Arg

930 935 940

Thr Pro Lys Pro Ala Leu Gln Glu Val Asp Tyr Tyr Tyr Gly Gln His

945 950 955 960

Asp

<210> 12

<211> 963

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 12

Met Asp His His His His His His Asn Thr Thr Lys Leu Asn Val Ile

1 5 10 15

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

20 25 30

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

35 40 45

Glu Trp Leu Tyr Arg Thr Pro Met Pro Ala Leu Trp Arg Ala Thr Thr

50 55 60

Asp Asn Asp Arg Gly Asn Gly Phe Ser Thr Lys Ser Ala Gln Trp Leu

65 70 75 80

Gly Ala Asp Leu Phe Ser Ser Cys Asp His Ile Ser Val Ala Ile Asp

85 90 95

Gly Gln Ser Ile Pro Leu Pro Ile Ala Pro Glu Asn Asn Arg Tyr Ser

100 105 110

Asp His Glu Thr Ala Thr Thr Val Ala Val Thr Phe Thr Tyr Thr Thr

115 120 125

Pro Thr Thr Pro Ala Thr Thr Ile Ala Val Thr Tyr Thr Val Ala Ala

130 135 140

Ser Gly Ala Met Thr Val Ala Val His Tyr Ala Gly Lys Ala Asp Leu

145 150 155 160

Pro Glu Leu Pro Ala Leu Gly Leu Arg Leu Val Met Pro Thr Pro Ala

165 170 175

Leu Gly Phe Ala Tyr Gln Gly Leu Ser Gly Glu Thr Tyr Pro Asp Arg

180 185 190

Lys Ala Gly Gly Arg Lys Gly Ile His Gln Val Ala Gly Leu Pro Val

195 200 205

Thr Pro Tyr Leu Val Pro Gln Glu Cys Gly Met His Val Asp Asn Gln

210 215 220

Trp Val Thr Val Thr Arg Gly Thr Thr Gln Asn Asn Ala Asp Ala Asp

225 230 235 240

His Asp Ala Phe Ser Leu Lys Val Arg Gln Thr Gln His His Phe Ala

245 250 255

Phe Ser Cys Leu Pro Tyr Thr Pro Thr Glu Leu Glu Asn Ala Thr His

260 265 270

Gln Glu Glu Leu Pro Val Pro Arg Arg Thr Val Leu Thr Ile Tyr Gly

275 280 285

Ala Val Arg Gly Val Gly Gly Ile Asp Ser Trp Gly Ser Asp Ala Glu

290 295 300

Ala Pro Tyr His Ile Ala Gly Asp Ser Asp His Asp Phe Ser Phe Glu

305 310 315 320

Ile Ala Gly Pro Thr Pro Val Gly Ser Gly Gly Ser Gly Gly His Met

325 330 335

Lys Ala Asp Leu Thr Trp Leu Asp Asp Pro Gln Thr Phe Arg Ile Asn

340 345 350

Gln Leu Pro Ala His Ser Asp His Arg Gly Tyr Ala Ser Val Glu Glu

355 360 365

Ala Thr Ala Gln His Ser Ser Leu Val Gln Ser Leu Asp Gly Thr Trp

370 375 380

Gln Phe Ala Phe Ala Pro Asp Pro Val His Arg Phe Glu Gly Phe Tyr

385 390 395 400

Gln Pro Asp Tyr Asp Arg Ser Ala Phe Asp Arg Leu Thr Val Pro Gly

405 410 415

His Ile Glu Leu Ala Gly Tyr Gly Gln Ile Gln Tyr Ile Asn Thr Ala

420 425 430

Tyr Pro Trp Glu Gly His His Tyr Arg Arg Pro Ala Tyr Ser Met Gly

435 440 445

Ala Asp Gln Pro Glu Lys Gly Met Phe Ser Thr Asp Pro Gln Asn Thr

450 455 460

Val Gly Ala Tyr Val Lys His Phe Thr Leu Asn Pro Ala Leu Ala Asn

465 470 475 480

Gln Arg Val Ser Ile Glu Phe Asp Gly Val Glu Gln Ala Met Phe Leu

485 490 495

Trp Leu Asn Gly Gln Phe Val Gly Tyr Ala Glu Asp Ser Phe Ser Arg

500 505 510

Ser Glu Phe Asp Leu Thr Pro Tyr Leu Gln Ala Gly Gln Asn Leu Leu

515 520 525

Ala Val Glu Val Phe Lys His Ser Thr Ala Ala Phe Leu Glu Asp Gln

530 535 540

Asp Met Phe Arg Phe Ser Gly Ile Phe Arg Ser Val Arg Leu Val Ala

545 550 555 560

Lys Pro Glu Leu His Val Glu Asp Leu Thr Ile Arg Ala Gly Leu Asp

565 570 575

Asp Ala Phe Gln Thr Gly Asp Leu Lys Val Arg Leu Gln Leu Thr Ala

580 585 590

Ala Ser Gln Leu Ser Gly Thr Ala Thr Ala Gln Leu Leu Thr Ala Asp

595 600 605

Gly Gln Glu Val Trp Ala Thr Glu Gln Pro Ala Ala Ser Thr Leu Asp

610 615 620

Leu Ala Ala Ala Ile Asp His Val His Leu Trp Asp His His Asp Pro

625 630 635 640

Tyr Leu Tyr Gln Leu Arg Ile Thr Leu Lys Asp Val Ala Gly Gln Val

645 650 655

Val Glu Val Val Pro Tyr Pro Val Gly Phe Arg Arg Ile Glu Leu Lys

660 665 670

Asp Lys Val Met Cys Leu Asn Gly Gln Arg Leu Ile Leu Asn Gly Val

675 680 685

Asn Arg His Glu Trp Asp Ala His Arg Gly Arg Ala Val Thr Met Ala

690 695 700

Asp Met Thr Gln Asp Leu Gln Thr Phe His Asp Asn His Ile Asn Ala

705 710 715 720

Val Arg Thr Cys His Tyr Pro Asp Gln Asp Ala Trp Tyr Tyr Leu Cys

725 730 735

Asp Gln Gln Gly Ile Tyr Met Met Ala Glu Asn Asn Leu Glu Thr His

740 745 750

Gly Thr Trp Gln Lys Met Gly Ala Val Glu Pro Ser Tyr Asn Val Pro

755 760 765

Gly Ser Leu Pro Gln Trp Gln Leu Ala Val Leu Asp Arg Ala Lys Ser

770 775 780

Asn Tyr Glu Met Phe Lys Asn His Pro Ala Val Leu Phe Trp Ser Leu

785 790 795 800

Gly Asn Glu Ser Tyr Ala Gly Asp Asn Ile Ala Ala Met Asp Ala Phe

805 810 815

Tyr His His Ala Asp Pro Thr Arg Leu Thr His Tyr Glu Gly Val Cys

820 825 830

Arg Asn Arg Val Tyr Glu Asp Arg Ile Ser Asp Met Glu Ser Met Met

835 840 845

Tyr Asp Pro Pro Arg Ala Ile Glu Asp Tyr Leu Lys Asn Asp Pro Gln

850 855 860

Lys Pro Phe Val Asn Cys Glu Tyr Met His Asp Met Gly Asn Ser Leu

865 870 875 880

Gly Gly Met Ala Ser Tyr Asp Ala Leu Ile Asp Gln Tyr Pro Met Tyr

885 890 895

Gln Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Leu Trp Val Lys

900 905 910

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

915 920 925

Asp Arg His Ser Asp Tyr Glu Phe Ser Gly Asp Gly Leu Leu Phe Ala

930 935 940

Asp Arg Thr Pro Lys Pro Ala Leu Gln Glu Val Asp Tyr Tyr Tyr Gly

945 950 955 960

Gln His Asp

<210> 13

<211> 963

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 13

Met Asp His His His His His His Asn Thr Thr Lys Leu Asn Val Ile

1 5 10 15

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

20 25 30

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

35 40 45

Glu Trp Leu Tyr Arg Thr Pro Met Pro Ala Leu Trp Arg Ala Thr Thr

50 55 60

Asp Asn Asp Arg Gly Asn Gly Phe Ser Thr Lys Ser Ala Gln Trp Leu

65 70 75 80

Gly Ala Asp Leu Phe Ser Ser Cys Asp His Ile Ser Val Ala Ile Asp

85 90 95

Gly Gln Ser Ile Pro Leu Pro Ile Ala Pro Glu Asn Asn Arg Tyr Ser

100 105 110

Asp His Glu Thr Ala Thr Thr Val Ala Val Thr Phe Thr Tyr Thr Thr

115 120 125

Pro Thr Thr Pro Ala Thr Thr Ile Ala Val Thr Tyr Thr Val Ala Ala

130 135 140

Ser Gly Ala Met Thr Val Ala Val His Tyr Ala Gly Lys Ala Asp Leu

145 150 155 160

Pro Glu Leu Pro Ala Leu Gly Leu Arg Leu Val Met Pro Thr Pro Ala

165 170 175

Leu Gly Phe Ala Tyr Gln Gly Leu Ser Gly Glu Thr Tyr Pro Asp Arg

180 185 190

Lys Ala Gly Gly Arg Lys Gly Ile His Gln Val Ala Gly Leu Pro Val

195 200 205

Thr Pro Tyr Leu Val Pro Gln Glu Cys Gly Met His Val Asp Asn Gln

210 215 220

Trp Val Thr Val Thr Arg Gly Thr Thr Gln Asn Asn Ala Asp Ala Asp

225 230 235 240

His Asp Ala Phe Ser Leu Lys Val Arg Gln Thr Gln His His Phe Ala

245 250 255

Phe Ser Cys Leu Pro Tyr Thr Pro Thr Glu Leu Glu Asn Ala Thr His

260 265 270

Gln Glu Glu Leu Pro Val Pro Arg Arg Thr Val Leu Thr Ile Tyr Gly

275 280 285

Ala Val Arg Gly Val Gly Gly Ile Asp Ser Trp Gly Ser Asp Ala Glu

290 295 300

Ala Pro Tyr His Ile Ala Gly Asp Ser Asp His Asp Phe Ser Phe Glu

305 310 315 320

Ile Ala Gly Pro Thr Pro Val Gly Ser Gly Gly Gly Ser Gly His Met

325 330 335

Lys Ala Asp Leu Thr Trp Leu Asp Asp Pro Gln Thr Phe Arg Ile Asn

340 345 350

Gln Leu Pro Ala His Ser Asp His Arg Gly Tyr Ala Ser Val Glu Glu

355 360 365

Ala Thr Ala Gln His Ser Ser Leu Val Gln Ser Leu Asp Gly Thr Trp

370 375 380

Gln Phe Ala Phe Ala Pro Asp Pro Val His Arg Phe Glu Gly Phe Tyr

385 390 395 400

Gln Pro Asp Tyr Asp Arg Ser Ala Phe Asp Arg Leu Thr Val Pro Gly

405 410 415

His Ile Glu Leu Ala Gly Tyr Gly Gln Ile Gln Tyr Ile Asn Thr Ala

420 425 430

Tyr Pro Trp Glu Gly His His Tyr Arg Arg Pro Ala Tyr Ser Met Gly

435 440 445

Ala Asp Gln Pro Glu Lys Gly Met Phe Ser Thr Asp Pro Gln Asn Thr

450 455 460

Val Gly Ala Tyr Val Lys His Phe Thr Leu Asn Pro Ala Leu Ala Asn

465 470 475 480

Gln Arg Val Ser Ile Glu Phe Asp Gly Val Glu Gln Ala Met Phe Leu

485 490 495

Trp Leu Asn Gly Gln Phe Val Gly Tyr Ala Glu Asp Ser Phe Ser Arg

500 505 510

Ser Glu Phe Asp Leu Thr Pro Tyr Leu Gln Ala Gly Gln Asn Leu Leu

515 520 525

Ala Val Glu Val Phe Lys His Ser Thr Ala Ala Phe Leu Glu Asp Gln

530 535 540

Asp Met Phe Arg Phe Ser Gly Ile Phe Arg Ser Val Arg Leu Val Ala

545 550 555 560

Lys Pro Glu Leu His Val Glu Asp Leu Thr Ile Arg Ala Gly Leu Asp

565 570 575

Asp Ala Phe Gln Thr Gly Asp Leu Lys Val Arg Leu Gln Leu Thr Ala

580 585 590

Ala Ser Gln Leu Ser Gly Thr Ala Thr Ala Gln Leu Leu Thr Ala Asp

595 600 605

Gly Gln Glu Val Trp Ala Thr Glu Gln Pro Ala Ala Ser Thr Leu Asp

610 615 620

Leu Ala Ala Ala Ile Asp His Val His Leu Trp Asp His His Asp Pro

625 630 635 640

Tyr Leu Tyr Gln Leu Arg Ile Thr Leu Lys Asp Val Ala Gly Gln Val

645 650 655

Val Glu Val Val Pro Tyr Pro Val Gly Phe Arg Arg Ile Glu Leu Lys

660 665 670

Asp Lys Val Met Cys Leu Asn Gly Gln Arg Leu Ile Leu Asn Gly Val

675 680 685

Asn Arg His Glu Trp Asp Ala His Arg Gly Arg Ala Val Thr Met Ala

690 695 700

Asp Met Thr Gln Asp Leu Gln Thr Phe His Asp Asn His Ile Asn Ala

705 710 715 720

Val Arg Thr Cys His Tyr Pro Asp Gln Asp Ala Trp Tyr Tyr Leu Cys

725 730 735

Asp Gln Gln Gly Ile Tyr Met Met Ala Glu Asn Asn Leu Glu Thr His

740 745 750

Gly Thr Trp Gln Lys Met Gly Ala Val Glu Pro Ser Tyr Asn Val Pro

755 760 765

Gly Ser Leu Pro Gln Trp Gln Leu Ala Val Leu Asp Arg Ala Lys Ser

770 775 780

Asn Tyr Glu Met Phe Lys Asn His Pro Ala Val Leu Phe Trp Ser Leu

785 790 795 800

Gly Asn Glu Ser Tyr Ala Gly Asp Asn Ile Ala Ala Met Asp Ala Phe

805 810 815

Tyr His His Ala Asp Pro Thr Arg Leu Thr His Tyr Glu Gly Val Cys

820 825 830

Arg Asn Arg Val Tyr Glu Asp Arg Ile Ser Asp Met Glu Ser Met Met

835 840 845

Tyr Asp Pro Pro Arg Ala Ile Glu Asp Tyr Leu Lys Asn Asp Pro Gln

850 855 860

Lys Pro Phe Val Asn Cys Glu Tyr Met His Asp Met Gly Asn Ser Leu

865 870 875 880

Gly Gly Met Ala Ser Tyr Asp Ala Leu Ile Asp Gln Tyr Pro Met Tyr

885 890 895

Gln Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Leu Trp Val Lys

900 905 910

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

915 920 925

Asp Arg His Ser Asp Tyr Glu Phe Ser Gly Asp Gly Leu Leu Phe Ala

930 935 940

Asp Arg Thr Pro Lys Pro Ala Leu Gln Glu Val Asp Tyr Tyr Tyr Gly

945 950 955 960

Gln His Asp

<210> 14

<211> 963

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 14

Met Asp His His His His His His Asn Thr Thr Lys Leu Asn Val Ile

1 5 10 15

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

20 25 30

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

35 40 45

Glu Trp Leu Tyr Arg Thr Pro Met Pro Ala Leu Trp Arg Ala Thr Thr

50 55 60

Asp Asn Asp Arg Gly Asn Gly Phe Ser Thr Lys Ser Ala Gln Trp Leu

65 70 75 80

Gly Ala Asp Leu Phe Ser Ser Cys Asp His Ile Ser Val Ala Ile Asp

85 90 95

Gly Gln Ser Ile Pro Leu Pro Ile Ala Pro Glu Asn Asn Arg Tyr Ser

100 105 110

Asp His Glu Thr Ala Thr Thr Val Ala Val Thr Phe Thr Tyr Thr Thr

115 120 125

Pro Thr Thr Pro Ala Thr Thr Ile Ala Val Thr Tyr Thr Val Ala Ala

130 135 140

Ser Gly Ala Met Thr Val Ala Val His Tyr Ala Gly Lys Ala Asp Leu

145 150 155 160

Pro Glu Leu Pro Ala Leu Gly Leu Arg Leu Val Met Pro Thr Pro Ala

165 170 175

Leu Gly Phe Ala Tyr Gln Gly Leu Ser Gly Glu Thr Tyr Pro Asp Arg

180 185 190

Lys Ala Gly Gly Arg Lys Gly Ile His Gln Val Ala Gly Leu Pro Val

195 200 205

Thr Pro Tyr Leu Val Pro Gln Glu Cys Gly Met His Val Asp Asn Gln

210 215 220

Trp Val Thr Val Thr Arg Gly Thr Thr Gln Asn Asn Ala Asp Ala Asp

225 230 235 240

His Asp Ala Phe Ser Leu Lys Val Arg Gln Thr Gln His His Phe Ala

245 250 255

Phe Ser Cys Leu Pro Tyr Thr Pro Thr Glu Leu Glu Asn Ala Thr His

260 265 270

Gln Glu Glu Leu Pro Val Pro Arg Arg Thr Val Leu Thr Ile Tyr Gly

275 280 285

Ala Val Arg Gly Val Gly Gly Ile Asp Ser Trp Gly Ser Asp Ala Glu

290 295 300

Ala Pro Tyr His Ile Ala Gly Asp Ser Asp His Asp Phe Ser Phe Glu

305 310 315 320

Ile Ala Gly Pro Thr Pro Val Gly Ser Gly Gly Gly Gly Ser His Met

325 330 335

Lys Ala Asp Leu Thr Trp Leu Asp Asp Pro Gln Thr Phe Arg Ile Asn

340 345 350

Gln Leu Pro Ala His Ser Asp His Arg Gly Tyr Ala Ser Val Glu Glu

355 360 365

Ala Thr Ala Gln His Ser Ser Leu Val Gln Ser Leu Asp Gly Thr Trp

370 375 380

Gln Phe Ala Phe Ala Pro Asp Pro Val His Arg Phe Glu Gly Phe Tyr

385 390 395 400

Gln Pro Asp Tyr Asp Arg Ser Ala Phe Asp Arg Leu Thr Val Pro Gly

405 410 415

His Ile Glu Leu Ala Gly Tyr Gly Gln Ile Gln Tyr Ile Asn Thr Ala

420 425 430

Tyr Pro Trp Glu Gly His His Tyr Arg Arg Pro Ala Tyr Ser Met Gly

435 440 445

Ala Asp Gln Pro Glu Lys Gly Met Phe Ser Thr Asp Pro Gln Asn Thr

450 455 460

Val Gly Ala Tyr Val Lys His Phe Thr Leu Asn Pro Ala Leu Ala Asn

465 470 475 480

Gln Arg Val Ser Ile Glu Phe Asp Gly Val Glu Gln Ala Met Phe Leu

485 490 495

Trp Leu Asn Gly Gln Phe Val Gly Tyr Ala Glu Asp Ser Phe Ser Arg

500 505 510

Ser Glu Phe Asp Leu Thr Pro Tyr Leu Gln Ala Gly Gln Asn Leu Leu

515 520 525

Ala Val Glu Val Phe Lys His Ser Thr Ala Ala Phe Leu Glu Asp Gln

530 535 540

Asp Met Phe Arg Phe Ser Gly Ile Phe Arg Ser Val Arg Leu Val Ala

545 550 555 560

Lys Pro Glu Leu His Val Glu Asp Leu Thr Ile Arg Ala Gly Leu Asp

565 570 575

Asp Ala Phe Gln Thr Gly Asp Leu Lys Val Arg Leu Gln Leu Thr Ala

580 585 590

Ala Ser Gln Leu Ser Gly Thr Ala Thr Ala Gln Leu Leu Thr Ala Asp

595 600 605

Gly Gln Glu Val Trp Ala Thr Glu Gln Pro Ala Ala Ser Thr Leu Asp

610 615 620

Leu Ala Ala Ala Ile Asp His Val His Leu Trp Asp His His Asp Pro

625 630 635 640

Tyr Leu Tyr Gln Leu Arg Ile Thr Leu Lys Asp Val Ala Gly Gln Val

645 650 655

Val Glu Val Val Pro Tyr Pro Val Gly Phe Arg Arg Ile Glu Leu Lys

660 665 670

Asp Lys Val Met Cys Leu Asn Gly Gln Arg Leu Ile Leu Asn Gly Val

675 680 685

Asn Arg His Glu Trp Asp Ala His Arg Gly Arg Ala Val Thr Met Ala

690 695 700

Asp Met Thr Gln Asp Leu Gln Thr Phe His Asp Asn His Ile Asn Ala

705 710 715 720

Val Arg Thr Cys His Tyr Pro Asp Gln Asp Ala Trp Tyr Tyr Leu Cys

725 730 735

Asp Gln Gln Gly Ile Tyr Met Met Ala Glu Asn Asn Leu Glu Thr His

740 745 750

Gly Thr Trp Gln Lys Met Gly Ala Val Glu Pro Ser Tyr Asn Val Pro

755 760 765

Gly Ser Leu Pro Gln Trp Gln Leu Ala Val Leu Asp Arg Ala Lys Ser

770 775 780

Asn Tyr Glu Met Phe Lys Asn His Pro Ala Val Leu Phe Trp Ser Leu

785 790 795 800

Gly Asn Glu Ser Tyr Ala Gly Asp Asn Ile Ala Ala Met Asp Ala Phe

805 810 815

Tyr His His Ala Asp Pro Thr Arg Leu Thr His Tyr Glu Gly Val Cys

820 825 830

Arg Asn Arg Val Tyr Glu Asp Arg Ile Ser Asp Met Glu Ser Met Met

835 840 845

Tyr Asp Pro Pro Arg Ala Ile Glu Asp Tyr Leu Lys Asn Asp Pro Gln

850 855 860

Lys Pro Phe Val Asn Cys Glu Tyr Met His Asp Met Gly Asn Ser Leu

865 870 875 880

Gly Gly Met Ala Ser Tyr Asp Ala Leu Ile Asp Gln Tyr Pro Met Tyr

885 890 895

Gln Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Leu Trp Val Lys

900 905 910

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

915 920 925

Asp Arg His Ser Asp Tyr Glu Phe Ser Gly Asp Gly Leu Leu Phe Ala

930 935 940

Asp Arg Thr Pro Lys Pro Ala Leu Gln Glu Val Asp Tyr Tyr Tyr Gly

945 950 955 960

Gln His Asp

<210> 15

<211> 966

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 15

Met Asp His His His His His His Asn Thr Thr Lys Leu Asn Val Ile

1 5 10 15

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

20 25 30

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

35 40 45

Glu Trp Leu Tyr Arg Thr Pro Met Pro Ala Leu Trp Arg Ala Thr Thr

50 55 60

Asp Asn Asp Arg Gly Asn Gly Phe Ser Thr Lys Ser Ala Gln Trp Leu

65 70 75 80

Gly Ala Asp Leu Phe Ser Ser Cys Asp His Ile Ser Val Ala Ile Asp

85 90 95

Gly Gln Ser Ile Pro Leu Pro Ile Ala Pro Glu Asn Asn Arg Tyr Ser

100 105 110

Asp His Glu Thr Ala Thr Thr Val Ala Val Thr Phe Thr Tyr Thr Thr

115 120 125

Pro Thr Thr Pro Ala Thr Thr Ile Ala Val Thr Tyr Thr Val Ala Ala

130 135 140

Ser Gly Ala Met Thr Val Ala Val His Tyr Ala Gly Lys Ala Asp Leu

145 150 155 160

Pro Glu Leu Pro Ala Leu Gly Leu Arg Leu Val Met Pro Thr Pro Ala

165 170 175

Leu Gly Phe Ala Tyr Gln Gly Leu Ser Gly Glu Thr Tyr Pro Asp Arg

180 185 190

Lys Ala Gly Gly Arg Lys Gly Ile His Gln Val Ala Gly Leu Pro Val

195 200 205

Thr Pro Tyr Leu Val Pro Gln Glu Cys Gly Met His Val Asp Asn Gln

210 215 220

Trp Val Thr Val Thr Arg Gly Thr Thr Gln Asn Asn Ala Asp Ala Asp

225 230 235 240

His Asp Ala Phe Ser Leu Lys Val Arg Gln Thr Gln His His Phe Ala

245 250 255

Phe Ser Cys Leu Pro Tyr Thr Pro Thr Glu Leu Glu Asn Ala Thr His

260 265 270

Gln Glu Glu Leu Pro Val Pro Arg Arg Thr Val Leu Thr Ile Tyr Gly

275 280 285

Ala Val Arg Gly Val Gly Gly Ile Asp Ser Trp Gly Ser Asp Ala Glu

290 295 300

Ala Pro Tyr His Ile Ala Gly Asp Ser Asp His Asp Phe Ser Phe Glu

305 310 315 320

Ile Ala Gly Pro Thr Pro Val Gly Ser Gly Gly Gly Ser Gly Gly Gly

325 330 335

Ser His Met Lys Ala Asp Leu Thr Trp Leu Asp Asp Pro Gln Thr Phe

340 345 350

Arg Ile Asn Gln Leu Pro Ala His Ser Asp His Arg Gly Tyr Ala Ser

355 360 365

Val Glu Glu Ala Thr Ala Gln His Ser Ser Leu Val Gln Ser Leu Asp

370 375 380

Gly Thr Trp Gln Phe Ala Phe Ala Pro Asp Pro Val His Arg Phe Glu

385 390 395 400

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

405 410 415

Val Pro Gly His Ile Glu Leu Ala Gly Tyr Gly Gln Ile Gln Tyr Ile

420 425 430

Asn Thr Ala Tyr Pro Trp Glu Gly His His Tyr Arg Arg Pro Ala Tyr

435 440 445

Ser Met Gly Ala Asp Gln Pro Glu Lys Gly Met Phe Ser Thr Asp Pro

450 455 460

Gln Asn Thr Val Gly Ala Tyr Val Lys His Phe Thr Leu Asn Pro Ala

465 470 475 480

Leu Ala Asn Gln Arg Val Ser Ile Glu Phe Asp Gly Val Glu Gln Ala

485 490 495

Met Phe Leu Trp Leu Asn Gly Gln Phe Val Gly Tyr Ala Glu Asp Ser

500 505 510

Phe Ser Arg Ser Glu Phe Asp Leu Thr Pro Tyr Leu Gln Ala Gly Gln

515 520 525

Asn Leu Leu Ala Val Glu Val Phe Lys His Ser Thr Ala Ala Phe Leu

530 535 540

Glu Asp Gln Asp Met Phe Arg Phe Ser Gly Ile Phe Arg Ser Val Arg

545 550 555 560

Leu Val Ala Lys Pro Glu Leu His Val Glu Asp Leu Thr Ile Arg Ala

565 570 575

Gly Leu Asp Asp Ala Phe Gln Thr Gly Asp Leu Lys Val Arg Leu Gln

580 585 590

Leu Thr Ala Ala Ser Gln Leu Ser Gly Thr Ala Thr Ala Gln Leu Leu

595 600 605

Thr Ala Asp Gly Gln Glu Val Trp Ala Thr Glu Gln Pro Ala Ala Ser

610 615 620

Thr Leu Asp Leu Ala Ala Ala Ile Asp His Val His Leu Trp Asp His

625 630 635 640

His Asp Pro Tyr Leu Tyr Gln Leu Arg Ile Thr Leu Lys Asp Val Ala

645 650 655

Gly Gln Val Val Glu Val Val Pro Tyr Pro Val Gly Phe Arg Arg Ile

660 665 670

Glu Leu Lys Asp Lys Val Met Cys Leu Asn Gly Gln Arg Leu Ile Leu

675 680 685

Asn Gly Val Asn Arg His Glu Trp Asp Ala His Arg Gly Arg Ala Val

690 695 700

Thr Met Ala Asp Met Thr Gln Asp Leu Gln Thr Phe His Asp Asn His

705 710 715 720

Ile Asn Ala Val Arg Thr Cys His Tyr Pro Asp Gln Asp Ala Trp Tyr

725 730 735

Tyr Leu Cys Asp Gln Gln Gly Ile Tyr Met Met Ala Glu Asn Asn Leu

740 745 750

Glu Thr His Gly Thr Trp Gln Lys Met Gly Ala Val Glu Pro Ser Tyr

755 760 765

Asn Val Pro Gly Ser Leu Pro Gln Trp Gln Leu Ala Val Leu Asp Arg

770 775 780

Ala Lys Ser Asn Tyr Glu Met Phe Lys Asn His Pro Ala Val Leu Phe

785 790 795 800

Trp Ser Leu Gly Asn Glu Ser Tyr Ala Gly Asp Asn Ile Ala Ala Met

805 810 815

Asp Ala Phe Tyr His His Ala Asp Pro Thr Arg Leu Thr His Tyr Glu

820 825 830

Gly Val Cys Arg Asn Arg Val Tyr Glu Asp Arg Ile Ser Asp Met Glu

835 840 845

Ser Met Met Tyr Asp Pro Pro Arg Ala Ile Glu Asp Tyr Leu Lys Asn

850 855 860

Asp Pro Gln Lys Pro Phe Val Asn Cys Glu Tyr Met His Asp Met Gly

865 870 875 880

Asn Ser Leu Gly Gly Met Ala Ser Tyr Asp Ala Leu Ile Asp Gln Tyr

885 890 895

Pro Met Tyr Gln Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Leu

900 905 910

Trp Val Lys Asp Glu Val Thr Gly Gln Pro Val Leu Arg Tyr Gly Gly

915 920 925

Asp Phe Asp Asp Arg His Ser Asp Tyr Glu Phe Ser Gly Asp Gly Leu

930 935 940

Leu Phe Ala Asp Arg Thr Pro Lys Pro Ala Leu Gln Glu Val Asp Tyr

945 950 955 960

Tyr Tyr Gly Gln His Asp

965

Claims

1.一种β-半乳糖苷酶融合蛋白，其特征在于，所述融合蛋白通过使用柔性肽接头连接两个β-半乳糖苷酶催化亚基刚性单元。

2.根据权利要求1所述的β-半乳糖苷酶融合蛋白，其特征在于，所述β-半乳糖苷酶催化亚基刚性单元为同型亚基或异型亚基。

3.根据权利要求2所述的β-半乳糖苷酶融合蛋白，其特征在于，所述β-半乳糖苷酶催化亚基来源于短乳酸杆菌Lactobacillus brevis。

4.根据权利要求1所述的β-半乳糖苷酶融合蛋白，其特征在于，所述柔性肽接头为由几个氨基酸相互连接形成的含有多个肽键的一条柔性链状结构。

5.根据权利要求4所述的β-半乳糖苷酶融合蛋白，其特征在于，所述柔性肽接头选自甘氨酸、丝氨酸、甲硫氨酸、缬氨酸、赖氨酸、天冬氨酸、天冬酰胺或丙氨酸中的一种或多种。

6.如权利要求4所述的β-半乳糖苷酶融合蛋白，其特征在于，所述柔性肽接头的分子式含有(GxSy)n的重复序列，其中，x、y、n为大于等于1的整数。

7.如权利要求6所述的β-半乳糖苷酶融合蛋白，其特征在于，所述柔性肽接头选自以下组：

Linker1：GSHM；

Linker2：GSGSGHM；

Linker3：GSGGSGGHM；

Linker4：GSGGGSGHM；

Linker5：GSGGGGSHM；

Linker6：GSGGGSGGGSHM。

8.如权利要求7所述的β-半乳糖苷酶融合蛋白，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID NO：10-15所示。

9.一种如权利要求1-8任一所述的β-半乳糖苷酶融合蛋白的制备方法，其特征在于，包括如下步骤：

设计含有酶切位点的DNA序列，两个亚基的N端、C端携带不同的酶切位点基因序列，对肽链部分的一端或两端设计相应的酶切位点；

合成引物序列，以单亚基的DNA序列为模板，引物部分包括酶切位点、柔性肽接头的DNA序列、以及可与模板互补的DNA片段；

使用引物进行PCR扩增，在原亚基模板上扩增出携带有柔性肽接头DNA序列的长片段；

对扩增产物进行酶切酶连，通过酶切位点分别连接两个亚基的N端和C端，在亚基之间插入柔性肽接头；所述柔性肽接头为通过相关酶切位点酶切连接、翻译后形成的氨基酸；

构建重组载体并在原核生物中表达。

10.一种如权利要求1-8任一所述的β-半乳糖苷酶融合蛋白在催化水解β-1,4糖苷键相连的非还原端半乳糖基团中的应用。