阅读说明：本技术 一种利用亲和免疫介质纯化蛋黄抗体的方法 (Method for purifying yolk antibody by using affinity immune medium ) 是由 杨严俊 张秀芳 李俊华 常翠华 苏宇杰 顾璐萍 于 2020-07-10 设计创作，主要内容包括：本发明公开了一种利用亲和免疫介质纯化蛋黄抗体的方法,属于食品技术领域。本发明利用一种硅藻土亲和蛋白标签开发了一种新型的抗体亲和免疫纯化方法。该亲和纯化技术采用廉价的硅藻土助滤剂作为载体,以带有核糖体蛋白L2标签抗原蛋白为偶联配基,利用了核糖体蛋白L2对硅藻土的特异性吸附作用,构建了一种成本低、效率高的亲和免疫纯化方法。高效低成本的特异性蛋黄抗体亲和纯化技术的开发不仅能有效提高特异性蛋黄免疫球蛋白分离提取效率,也能进一步提高蛋黄抗体的附加值和扩大应用领域,这对于提高特异性蛋黄免疫球蛋白在高端生物抗体市场中竞争力具有重要意义。(The invention discloses a method for purifying yolk antibodies by using an affinity immune medium, belonging to the technical field of food. The invention develops a novel antibody affinity immune purification method by utilizing a diatomite affinity protein label. The affinity purification technology adopts a cheap diatomite filter aid as a carrier, takes an antigen protein with a ribosomal protein L2 label as a coupling ligand, and utilizes the specific adsorption effect of the ribosomal protein L2 on diatomite to construct an affinity immune purification method with low cost and high efficiency. The development of the high-efficiency low-cost affinity purification technology for the specific egg yolk immunoglobulin can not only effectively improve the separation and extraction efficiency of the specific egg yolk immunoglobulin, but also further improve the additional value of the egg yolk antibody and expand the application field, which has important significance for improving the competitiveness of the specific egg yolk immunoglobulin in the high-end biological antibody market.)

一种利用亲和免疫介质纯化蛋黄抗体的方法

技术领域

本发明涉及一种利用亲和免疫介质纯化蛋黄抗体的方法，属于食品技术领域。

背景技术

目前，对于抗体的纯化，主要是利用静电性相互作用的离子交换色谱法、利用疏水性相互作用的疏水性色谱法及利用对于抗体的亲和相互作用的蛋白A色谱法等，但是现在通用的抗体的纯化方法均存在操作冗杂、价格昂贵等缺点。

蛋黄免疫球蛋白是一种重要的生物活性抗体，在动物疾病免疫防治、减少或停用动物日粮中亚治疗量的抗生素已经表现出较好的替代效果。部分国内外厂家已经成功的实现了蛋黄免疫球蛋白在生物医药及日用食品领域的应用，开发了能对口腔溃疡有辅助治疗作用的免疫球蛋白口腔喷剂，具有抗龋齿功效的口香糖，具有抗幽门螺杆菌(预防胃炎及胃溃疡)特性的饮料，具有抑菌止痒功效白色念珠菌特异抗体护理液及抗流感病毒牙膏等多款产品。

一直以来，生物蛋白中的色谱或非色谱纯化方法不得不在分离效果、成本和易用性之间进行权衡。由于传统的水相稀释-膜柱分离方法在抗体提取中，尤其是提取的蛋黄抗体中特异性抗体含量低，致使其在工商业、科研、分析检测、理论研究和疾病诊断与防治等方面应用中存在一定的局限性。近年来，亲和免疫层析技术用于分离抗体的技术得到了重视，有报道亲和免疫层析技术由于可从蛋黄样品粗提澄清液中简单过柱处理即可分离出所需的高纯度的特异蛋黄免疫球蛋白。然而亲和免疫层析也存在一些问题，如存在载体发展不足导致价格昂贵(琼脂糖颗粒等)、配基纯度要求过高不易制备和洗脱过程中易造成目的物质失活等缺点。

新种类载体的研究开发及偶联配基的高纯度制备是制约高纯度抗体高效分离技术发展的瓶颈。

发明内容

为了解决上述问题，本发明利用一种硅藻土亲和蛋白标签开发了一种新型的抗体亲和免疫纯化方法。该亲和纯化技术采用廉价的硅藻土助滤剂作为载体，以带有核糖体蛋白L2标签抗原蛋白为偶联配基，利用了核糖体蛋白L2对硅藻土的特异性吸附作用，构建了一种成本低、效率高的亲和免疫纯化方法。高效低成本的特异性蛋黄抗体亲和纯化技术的开发不仅能有效提高特异性蛋黄免疫球蛋白分离提取效率，也能进一步提高蛋黄抗体的附加值和扩大应用领域，这对于提高特异性蛋黄免疫球蛋白在高端生物抗体市场中竞争力具有重要意义。

本发明提供了一种用于抗体纯化的亲和免疫介质，所述亲和免疫介质为吸附融合蛋白的硅藻土，所述融合蛋白为将来源于致病菌或病毒抗原蛋白与核糖体L2蛋白的融合得到的。

在本发明的一种实施方式中，所述核糖体L2蛋白含有如SEQ ID NO.2所示的多肽序列。

在本发明的一种实施方式中，编码所述核糖体L2蛋白含有如SEQ ID NO.7所示的核苷酸序列。

在本发明的一种实施方式中，所述核糖体L2蛋白的氨基酸序列如SEQ ID NO.1所示。

在本发明的一种实施方式中，编码所述核糖体L2蛋白的核苷酸序列如SEQ IDNO.6所示。

在本发明的一种实施方式中，编码所述L2蛋白的基因的获得，可以通过设计PCR引物，从大肠杆菌HB101菌株、BL21(DE3)菌株、TOP10菌等含有L2蛋白菌株进行扩增得到相关基因；也可以按照其氨基酸序列，依据表达细胞的基因密码子偏好，合成相应的基因序列。

本发明提供了一种用于抗体纯化的亲和免疫介质的制备方法，包括如下步骤：(1)合成编码核糖体L2蛋白基因序列；(2)将编码来源于致病菌或病毒的抗原蛋白基因与编码核糖体L2蛋白的基因融合后克隆至带有信号肽的分泌型表达载体中，构建重组表达载体；(3)将所述重组表达载体转化表达宿主，并对表达宿主进行培养表达；(4)将培养结束后所得的发酵液离心，取上清液；(5)将所述上清液与硅藻土混合，使得上清液中的融合蛋白吸附在硅藻土上，得到亲和免疫介质；

或者，包括如下步骤：(1)合成编码核糖体L2蛋白基因序列；(2)将编码来源于致病菌或病毒的抗原蛋白基因与编码核糖体L2蛋白的基因融合后克隆至不含信号肽的胞内表达载体中，构建重组表达载体；(3)将所述重组表达载体转化表达宿主，并对表达宿主进行培养表达；(4)培养结束后破碎宿主细胞，将细胞破碎液离心并取上清液；(5)将所述上清液与硅藻土混合，使得上清液中的融合蛋白吸附在硅藻土上，得到亲和免疫介质。

在本发明的一种实施方式中，所述表达宿主为细菌或真菌。

在本发明的一种实施方式中，所述表达宿主为大肠杆菌。

本发明还提供了一种纯化蛋黄抗体方法，所述方法为：以上述固定有相应抗原蛋白的亲和免疫介质为吸附剂，对蛋黄抗体进行纯化。

本发明还提供了一种蛋黄抗体纯化方法，将含有抗体的清液与固定有相应抗原蛋白的亲和免疫介质混合，静置并抽离清液后，用去离子水和低离子强度盐溶液进行清洗亲和免疫介质，洗去非特异性吸附杂蛋白，再用较高离子强度的盐溶液对亲和免疫介质进行特异性抗体的洗脱，最后经过超滤脱盐得到脱盐后的高纯度特异性抗体。

在本发明的一种实施方式中，所述蛋黄抗体为尿素酶UerB蛋黄抗体、鞭毛鞘膜蛋白HpaA蛋黄抗体、空泡细胞毒素VacA蛋黄抗体和/或细胞毒素CagA蛋黄抗体。

在本发明的一种实施方式中，所述含有抗体的清液的制备方法为：从经过抗原蛋白免疫蛋鸡所产鸡蛋中分离得到蛋黄，将蛋黄与去离子水或缓冲液混合，静置后，抽取上清过滤得到。

在本发明的一种实施方式中，所述含有抗体的清液的制备方法为：从经过抗原蛋白免疫蛋鸡所产鸡蛋中分离得到蛋黄，与4-10倍的经过机械冷却至0-5℃的去离子水或pH值在5.5-7.0缓冲液进行混合，静置3-16小时后抽取上清，经过陶瓷膜微滤后得到清液，与固定有相应抗原亲和免疫介质混合0.5-1小时，抽离清液后，用去离子水和低离子强度盐溶液进行清洗，洗去非特异性吸附杂蛋白，然后再用较高离子强度的盐溶液进行特异性抗体的洗脱，最后经过超滤脱盐得到脱盐后的高纯度特异性蛋黄免疫球蛋白。

在本发明的一种实施方式中，所述抗原蛋白为来自于幽门螺杆菌的尿素酶UerB、鞭毛鞘膜蛋白HpaA、空泡细胞毒素VacA和/或细胞毒素CagA。

在本发明的一种实施方式中，所述尿素酶UerB氨基酸序列如SEQ ID NO.3所示，所述鞭毛鞘膜蛋白HpaA氨基酸序列如SEQ ID NO.4所示，空泡细胞毒素VacA氨基酸序列如SEQID NO.5所示，所述细胞毒素CagA氨基酸序列如SEQ ID NO.11所示。

在本发明的一种实施方式中，编码所述尿素酶UerB基因的核苷酸序列如SEQ IDNO.8所示，编码所述鞭毛鞘膜蛋白HpaA基因的核苷酸序列如SEQ ID NO.9所示，编码所述空泡细胞毒素VacA基因的核苷酸序列如SEQ ID NO.10所示，编码所述细胞毒素CagA基因的核苷酸序列如SEQ ID NO.12所示。

本发明还提供了上述的免疫亲和介质或上述的制备方法或上述的纯化蛋黄抗体的方法在纯化蛋黄抗体中的应用。

本发明的有益效果：

(1)本发明提供了一种全新的纯化蛋黄抗体的方法，采用了廉价硅藻土材料制备亲和免疫介质，因此利用本发明提供的亲和免疫介质进行抗体纯化，解决了亲和免疫基质价格昂贵的问题；采用本发明的方法，既提高了抗体的提取效率和提取纯度，又降低了生产的成本，是一种可行的工业化大规模生产方法。

(2)本发明提供的亲和免疫介质的制备方法利用了核糖体L2蛋白对廉价硅藻土材料的特殊亲和吸附能力，将目的蛋白或多肽与核糖体L2蛋白融合后即可采用常规方法进行重组表达，特异性抗原融合蛋白能通过一步吸附固定化在硅藻土基质上，固定过程不使用任何有毒试剂，固定过程安全、绿色、快速，能有效提高亲和免疫介质的生产效率。

(3)本发明的核糖体蛋白L2能作为重组酶在硅藻土固定化的连接臂，为活性蛋白的固定化提供了更多选择。

附图说明

图1：不同氯化钠浓度下特异性尿素酶蛋黄抗体电泳洗脱图；其中，1-4条带分别为0.25，0.50，0.75和1mol/L氯化钠浓度梯度洗脱下硅藻土亲和介质中流出特异性尿素酶蛋黄抗体成分。

具体实施方式

以下对本发明的优选实施例进行说明，应当理解实施例是为了更好地解释本发明，不用于限制本发明。

下述实施例中涉及的硅藻土购自临江益瑞石硅藻土有限公司。

下述实施例中涉及的培养基如下：

ZYM-5052培养基：每升培养基含蛋白胨10g，酵母提取物5g，磷酸氢二钠4.46，磷酸二氢钾1.7，氯化铵1.33，硫酸钠0.354，甘油2.5，葡萄糖0.25，乳糖1，硫酸镁24.65，痕量元素混合物。

LB培养基：每升培养基含蛋白10g,酵母提取物5g，氯化钠5g NaCl,用1mol/L NaOH调pH至7.4用去离子水定容至1升。

下述实施例中涉及的检测方法如下：

蛋黄免疫球蛋白纯度检测：采用常规聚丙烯酰胺凝胶电泳法，浓缩胶4％，分离胶8％，浓缩电压80V，分离电压120V。用考马斯亮蓝对分离胶进行染色，脱色拍照后采用Quality One软件进行条带光密度分析。

实施例1：融合蛋白表达载体和宿主菌的构建

融合蛋白表达载体的制备：

化学合成编码核糖体L2蛋白的基因(核苷酸序列如SEQ ID NO.6所示)和编码来自于幽门螺杆菌的尿素酶UerB的基因(核苷酸序列如SEQ ID NO.8所示)，通过融合PCR将编码L2蛋白的基因和编码来自于幽门螺杆菌尿素酶的基因UerB连接，获得目的基因融合蛋白；将目的基因与pET28a质粒经限制性内切酶BamHI/XhoI酶切后进行连接，得到连接产物；将连接产物转化大肠杆菌E coli BL21(DE3)中，得到转化产物；将转化产物接入ZYM-5052培养基中进行自诱导培养，先37℃培养4小时，再接着在20-28℃下培养12-16小时；培养后提取质粒进行酶切验证以及测序验证，验证正确即获得重组大肠杆菌E coli BL21(DE3)/融合蛋白以及重组质粒pET28a/融合蛋白。

实施例2：硅藻土亲和免疫介质的制备

(1)将实施例1得到的重组大肠杆菌E coli BL21(DE3)/融合蛋白接种于LB培养基上，37℃下过夜培养，得到种子液；将上述种子液以2％的接种量接种于ZYM-5052培养基，37℃、200r/min培养4小时后，在再28度下培养12小时使培养基中菌液浓度度OD达到1.8后收集菌液，离心收集菌体细胞；

(2)将步骤(1)得到的菌体细胞，用pH7.0的10mM/L的磷酸盐缓冲液重悬后，采用超声波或高压均质进行低温细胞破碎，经滤布过滤后，得到澄清的含有融合蛋白的细胞破碎液；

(3)将澄清的细胞破碎液与所选定的硅藻土进行搅拌混合1小时，再通过300目滤布过滤截留硅藻土；

(4)用10倍的去离子水清洗硅藻土基质，再用1.0mol/L的NaCl清洗硅藻土去除非特异性吸附杂蛋白，再用去离子水清洗硅藻土，最后得到固定有抗原蛋白-蛋白L2融合蛋白的硅藻土亲和介质。

实施例3：特异性尿素酶蛋黄抗体的纯化

(1)将经过来自于幽门螺杆菌的尿素酶免疫的蛋鸡所产鸡蛋蛋黄的分离：鸡蛋清洗、干燥后，采用分蛋器分离得到蛋黄；

(2)蛋黄免疫球蛋白粗提：将分离得到的蛋黄与水以料液比1∶4混合，料液pH用柠檬酸调至7.0，温度4℃下静置16小时，抽取静置上清，经陶瓷微滤后得到澄清的粗提液；

(3)特异性蛋黄抗体纯化：将步骤(2)中澄清粗提液与实施例2中得到的固定有特异性抗原蛋白的硅藻土亲和介质混合，搅拌混合1小时后，抽去水相溶液，先用去离子水按1:10料水比清洗硅藻土，再用0.1mol/L的NaCl溶液对硅藻土进行再次清洗，直至无非特异性吸附杂蛋白流出，接着采用1.0mol/L的NaCl溶液洗脱吸附的特异性蛋黄抗体，再经过截留分子量为5万道尔顿的聚砜超滤膜进行超滤脱盐，最后得到脱盐的抗尿素酶特异性蛋黄免疫球蛋白。对得到的特异性蛋黄免疫球蛋白纯度进行测试，结果显示：纯度为93％。

实施例4：特异性鞭毛鞘膜蛋白(HpaA)蛋黄抗体的纯化

具体实施方式同实施例1-3，区别在于，将抗原调整为鞭毛鞘膜蛋白HpaA(编码所述鞭毛鞘膜蛋白HpaA基因的核苷酸序列如SEQ ID NO.9所示)，将抗体调整为：抗鞭毛鞘膜蛋白蛋黄抗体，所述抗体的制备方法为：

(1)鸡蛋清洗、干燥后，采用分蛋器分离得到蛋黄；

(2)蛋黄免疫球蛋白粗提：将分离得到的蛋黄与水以料液比1∶6混合，料液pH用柠檬酸调至6.5，温度4℃下静置16小时，抽取静置上清，经陶瓷微滤后得到澄清的粗提液；

(3)特异性蛋黄抗体纯化：将步骤(2)中澄清粗提液与按实施例2方法得到的固定有特异性鞭毛鞘膜蛋白抗原蛋白的硅藻土亲和介质混合，搅拌混合1小时后，抽去水相溶液，先用去离子水按1:20料水比清洗硅藻土，再用0.15mol/L的NaCl溶液对硅藻土进行再次清洗，直至无非特异性吸附杂蛋白流出，接着采用1.0mol/L的NaCl溶液洗脱吸附的特异性蛋黄抗体，再经过截留分子量为5万道尔顿的聚砜超滤膜进行超滤脱盐，对得到的特异性蛋黄免疫球蛋白纯度进行测试，结果显示：纯度为94％。

实施例5：特异性空泡细胞毒素(VacA)蛋黄抗体的纯化

具体实施方式同实施例1-3，区别在于，将抗原调整为空泡细胞毒素VacA(编码空泡细胞毒素VacA基因的核苷酸序列如SEQ ID NO.10所示)，将抗体调整为：抗空泡细胞毒素VacA蛋黄抗体，所述抗体的制备方法为：

(1)鸡蛋清洗、干燥后，采用分蛋器分离得到蛋黄；

(2)蛋黄免疫球蛋白粗提：将分离得到的蛋黄与水以料液比1∶10混合，料液pH用柠檬酸调至5.5，温度4℃下静置16小时，抽取静置上清，经陶瓷微滤后得到澄清的粗提液；

(3)特异性蛋黄抗体纯化：将步骤(2)中澄清粗提液与按实施例2方法得到的固定有特异性鞭毛鞘膜蛋白抗原蛋白的硅藻土亲和介质混合，搅拌混合1小时后，抽去水相溶液，先用去离子水按1:20料水比清洗硅藻土，再用0.2mol/L的NaCl溶液对硅藻土进行再次清洗，直至无非特异性吸附杂蛋白流出，接着采用1.0mol/L的NaCl溶液洗脱吸附的特异性蛋黄抗体，再经过截留分子量为5万道尔顿的聚砜超滤膜进行超滤脱盐，对得到的特异性蛋黄免疫球蛋白纯度进行测试，结果显示：纯度为94％。

实施例6：不同浓度的盐溶液对蛋黄抗体纯化的影响

具体实施方式同实施例3，区别在于，分别调整NaCl浓度为0.25，0.50，0.75和1mol/L洗脱液，采用不同浓度NaCl溶液洗脱下蛋黄抗体洗脱特征如图1所示，结果显示，采用1mol/L氯化钠浓度梯度洗脱下，硅藻土亲和介质纯化抗体的效果最佳。

虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

SEQUENCE LISTING

<110> 尤丽康（江苏）生物医药有限公司

<120> 一种利用亲和免疫介质纯化蛋黄抗体的方法

<130> BAA200495A

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 273

<212> PRT

<213> 人工序列

<400> 1

Met Ala Val Val Lys Cys Lys Pro Thr Ser Pro Gly Arg Arg His Val

1 5 10 15

Val Lys Val Val Asn Pro Glu Leu His Lys Gly Lys Pro Phe Ala Pro

20 25 30

Leu Leu Glu Lys Asn Ser Lys Ser Gly Gly Arg Asn Asn Asn Gly Arg

35 40 45

Ile Thr Thr Arg His Ile Gly Gly Gly His Lys Gln Ala Tyr Arg Ile

50 55 60

Val Asp Phe Lys Arg Asn Lys Asp Gly Ile Pro Ala Val Val Glu Arg

65 70 75 80

Leu Glu Tyr Asp Pro Asn Arg Ser Ala Asn Ile Ala Leu Val Leu Tyr

85 90 95

Lys Asp Gly Glu Arg Arg Tyr Ile Leu Ala Pro Lys Gly Leu Lys Ala

100 105 110

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

115 120 125

Thr Leu Pro Met Arg Asn Ile Pro Val Gly Ser Thr Val His Asn Val

130 135 140

Glu Met Lys Pro Gly Lys Gly Gly Gln Leu Ala Arg Ser Ala Gly Thr

145 150 155 160

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

165 170 175

Arg Ser Gly Glu Met Arg Lys Val Glu Ala Asp Cys Arg Ala Thr Leu

180 185 190

Gly Glu Val Gly Asn Ala Glu His Met Leu Arg Val Leu Gly Lys Ala

195 200 205

Gly Ala Ala Arg Trp Arg Gly Val Arg Pro Thr Val Arg Gly Thr Ala

210 215 220

Met Asn Pro Val Asp His Pro His Gly Gly Gly Glu Gly Arg Asn Phe

225 230 235 240

Gly Lys His Pro Val Thr Pro Trp Gly Val Gln Thr Lys Gly Lys Lys

245 250 255

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

260 265 270

Lys

<210> 2

<211> 71

<212> PRT

<213> 人工序列

<400> 2

Met Ala Arg Gly Lys Ala Gly Ala Ala Arg Trp Arg Gly Val Arg Pro

1 5 10 15

Thr Val Arg Gly Thr Ala Met Asn Pro Val Asp His Pro His Gly Gly

20 25 30

Gly Glu Gly Arg Asn Phe Gly Lys His Pro Val Thr Pro Trp Gly Val

35 40 45

Gln Thr Lys Gly Lys Lys Thr Arg Ser Asn Lys Arg Thr Asp Lys Phe

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Ile Val Arg Arg Arg Ser Lys

65 70

<210> 3

<211> 569

<212> PRT

<213> 人工序列

<400> 3

Met Lys Lys Ile Ser Arg Lys Glu Tyr Val Ser Met Tyr Gly Pro Thr

1 5 10 15

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

20 25 30

Glu His Asp Tyr Thr Ile Tyr Gly Glu Glu Leu Lys Phe Gly Gly Gly

35 40 45

Lys Thr Leu Arg Glu Gly Met Ser Gln Ser Asn Asn Pro Ser Lys Glu

50 55 60

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

65 70 75 80

Ile Tyr Lys Ala Asp Ile Gly Ile Lys Asp Gly Lys Ile Ala Gly Ile

85 90 95

Gly Lys Gly Gly Asn Lys Asp Met Gln Asp Gly Val Lys Asn Asn Leu

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Thr Gly Pro Ala Asp Gly Thr Asn Ala Thr Thr Ile Thr Pro Gly Arg

165 170 175

Arg Asn Leu Lys Phe Met Leu Arg Ala Ala Glu Glu Tyr Ser Met Asn

180 185 190

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

195 200 205

Asp Gln Ile Glu Ala Gly Ala Ile Gly Phe Lys Ile His Glu Asp Trp

210 215 220

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

225 230 235 240

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

245 250 255

Cys Val Glu Asp Thr Met Ala Ala Ile Ala Gly Arg Thr Met His Thr

260 265 270

Phe His Thr Glu Gly Ala Gly Gly Gly His Ala Pro Asp Ile Ile Lys

275 280 285

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

290 295 300

Pro Phe Thr Val Asn Thr Glu Ala Glu His Met Asp Met Leu Met Val

305 310 315 320

Cys His His Leu Asp Lys Ser Ile Lys Glu Asp Val Gln Phe Ala Asp

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Lys Glu Phe Gly Arg Leu Lys Glu Glu Lys Gly Asp Asn Asp Asn Phe

385 390 395 400

Arg Ile Lys Arg Tyr Leu Ser Lys Tyr Thr Ile Asn Pro Ala Ile Ala

405 410 415

His Gly Ile Ser Glu Tyr Val Gly Ser Val Glu Val Gly Lys Val Ala

420 425 430

Asp Leu Val Leu Trp Ser Pro Ala Phe Phe Gly Val Lys Pro Asn Met

435 440 445

Ile Ile Lys Gly Gly Phe Ile Ala Leu Ser Gln Met Gly Asp Ala Asn

450 455 460

Ala Ser Ile Pro Thr Pro Gln Pro Val Tyr Tyr Arg Glu Met Phe Ala

465 470 475 480

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

485 490 495

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

500 505 510

Val Leu Pro Val Lys Asn Cys Arg Asn Ile Thr Lys Lys Asp Met Gln

515 520 525

Phe Asn Asp Thr Thr Ala His Ile Glu Val Asn Pro Glu Thr Tyr His

530 535 540

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

545 550 555 560

Ser Leu Ala Gln Leu Phe Ser Ile Phe

565

<210> 4

<211> 260

<212> PRT

<213> 人工序列

<400> 4

Met Arg Ala Asn Asn His Phe Lys Asp Phe Ala Trp Lys Lys Cys Leu

1 5 10 15

Leu Gly Ala Ser Val Val Ala Leu Leu Val Gly Cys Ser Pro His Ile

20 25 30

Ile Glu Thr Asn Glu Val Ala Leu Lys Leu Asn Tyr His Pro Ala Ser

35 40 45

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

50 55 60

Phe Gln Tyr Ser Asp Asn Ile Ala Lys Glu Tyr Glu Asn Lys Phe Lys

65 70 75 80

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

85 90 95

Lys Val Ile Ser Val Asp Ser Ser Asp Lys Asp Asp Leu Ser Phe Ser

100 105 110

Gln Lys Lys Glu Gly Tyr Leu Ala Val Ala Met Asn Gly Glu Ile Val

115 120 125

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

130 135 140

Leu Leu Phe Ser Thr Gly Leu Asp Lys Met Glu Gly Val Leu Ile Pro

145 150 155 160

Ala Gly Phe Val Lys Val Thr Ile Leu Glu Pro Met Ser Gly Glu Ser

165 170 175

Leu Asp Ser Phe Thr Met Asp Leu Ser Glu Leu Asp Ile Gln Glu Lys

180 185 190

Phe Leu Lys Thr Thr His Ser Ser His Ser Gly Gly Leu Val Ser Thr

195 200 205

Met Val Lys Gly Thr Asp Asn Ser Asn Asp Ala Ile Lys Ser Ala Leu

210 215 220

Asn Lys Ile Phe Ala Asn Ile Met Gln Glu Ile Asp Lys Lys Leu Thr

225 230 235 240

Gln Lys Asn Leu Glu Ser Tyr Gln Lys Asp Ala Lys Glu Leu Lys Asn

245 250 255

Lys Arg Asn Arg

260

<210> 5

<211> 1290

<212> PRT

<213> 人工序列

<400> 5

Met Glu Ile Gln Gln Thr His Arg Lys Ile Asn Arg Pro Leu Val Ser

1 5 10 15

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

20 25 30

Ala Ala Phe Phe Thr Thr Val Ile Ile Pro Ala Ile Val Gly Gly Ile

35 40 45

Ala Thr Gly Ala Ala Val Gly Thr Val Ser Gly Leu Leu Ser Trp Gly

50 55 60

Leu Lys Gln Ala Glu Glu Ala Asn Lys Thr Pro Asp Lys Pro Asp Lys

65 70 75 80

Val Trp Arg Ile Gln Ala Gly Arg Gly Phe Asn Asn Phe Pro His Lys

85 90 95

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

100 105 110

Trp Asp Trp Gly Asn Ala Ala Arg His Tyr Trp Val Lys Gly Gly Gln

115 120 125

Trp Asn Lys Leu Glu Val Asp Met Lys Asp Ala Val Gly Thr Tyr Lys

130 135 140

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

145 150 155 160

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

165 170 175

Ser Tyr Lys Asp Ser Ala Asp Arg Thr Thr Arg Val Asp Phe Asn Ala

180 185 190

Lys Asn Ile Leu Ile Asp Asn Phe Leu Glu Ile Asn Asn Arg Val Gly

195 200 205

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

210 215 220

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

225 230 235 240

Gly Ala Thr Leu Asn Leu Ala Ser Ser Ser Val Lys Leu Met Gly Asn

245 250 255

Val Trp Met Gly Arg Leu Gln Tyr Val Gly Ala Tyr Leu Ala Pro Ser

260 265 270

Tyr Ser Thr Ile Asn Thr Ser Lys Val Thr Gly Glu Val Asn Phe Asn

275 280 285

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

290 295 300

Ser Asn Lys Thr His Ile Gly Thr Leu Asp Leu Trp Gln Ser Ala Gly

305 310 315 320

Leu Asn Ile Ile Ala Pro Pro Glu Gly Gly Tyr Lys Asp Lys Pro Lys

325 330 335

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

340 345 350

Asn Ser Ala Gln Asn Asn Asn Asn Thr Gln Val Ile Asn Pro Pro Asn

355 360 365

Ser Ala Gln Lys Thr Glu Ile Gln Pro Thr Gln Val Ile Asn Gly Pro

370 375 380

Phe Ala Gly Gly Lys Asp Thr Val Val Asn Ile Asn Arg Ile Asn Thr

385 390 395 400

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

405 410 415

Thr Asn Ala Ala His Leu His Ile Gly Lys Gly Gly Ile Asn Leu Ser

420 425 430

Asn Gln Ala Ser Gly Arg Ser Leu Leu Val Glu Asn Leu Thr Gly Asn

435 440 445

Ile Thr Val Asp Gly Pro Leu Arg Val Asn Asn Gln Val Gly Gly Tyr

450 455 460

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

465 470 475 480

Thr Lys Asn Gly Thr Ala Thr Phe Asn Asn Asp Ile Ser Leu Gly Arg

485 490 495

Phe Val Asn Leu Lys Val Asp Ala His Thr Ala Asn Phe Lys Gly Ile

500 505 510

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

515 520 525

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

530 535 540

Ile Lys Asn Phe Asn Ile Asn Glu Leu Leu Val Lys Thr Asn Gly Val

545 550 555 560

Ser Val Gly Glu Tyr Thr His Phe Ser Glu Asp Ile Gly Ser Gln Ser

565 570 575

Arg Ile Asn Thr Val Arg Leu Glu Thr Gly Thr Arg Ser Ile Phe Ser

580 585 590

Gly Gly Val Lys Phe Lys Ser Gly Glu Lys Leu Val Ile Asp Glu Phe

595 600 605

Tyr Tyr Ser Pro Trp Asn Tyr Phe Asp Ala Arg Asn Ile Lys Asn Val

610 615 620

Glu Ile Thr Arg Lys Phe Ala Ser Ser Thr Pro Glu Asn Pro Trp Gly

625 630 635 640

Thr Ser Lys Leu Met Phe Asn Asn Leu Thr Leu Gly Gln Asn Ala Val

645 650 655

Met Asp Tyr Ser Gln Phe Ser Asn Leu Thr Ile Gln Gly Asp Phe Ile

660 665 670

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

675 680 685

Thr Leu Asn Val Gly Asn Ala Ala Ala Met Met Phe Asn Asn Asp Ile

690 695 700

Asp Ser Ala Thr Gly Phe Tyr Lys Pro Leu Ile Lys Ile Asn Ser Ala

705 710 715 720

Gln Asp Leu Ile Lys Asn Thr Glu His Val Leu Leu Lys Ala Lys Ile

725 730 735

Ile Gly Tyr Gly Asn Val Ser Thr Gly Thr Asn Gly Ile Ser Asn Val

740 745 750

Asn Leu Glu Glu Gln Phe Lys Glu Arg Leu Ala Leu Tyr Asn Asn Asn

755 760 765

Asn Arg Met Asp Thr Cys Val Val Arg Asn Thr Asp Asp Ile Lys Ala

770 775 780

Cys Gly Met Ala Ile Gly Asn Gln Ser Met Val Asn Asn Pro Asp Asn

785 790 795 800

Tyr Lys Tyr Leu Ile Gly Lys Ala Trp Lys Asn Ile Gly Ile Ser Lys

805 810 815

Thr Ala Asn Gly Ser Lys Ile Ser Val Tyr Tyr Leu Gly Asn Ser Thr

820 825 830

Pro Thr Glu Asn Gly Gly Asn Thr Thr Asn Leu Pro Thr Asn Thr Thr

835 840 845

Asn Asn Ala Arg Ser Ala Asn Tyr Ala Leu Val Lys Asn Ala Pro Phe

850 855 860

Ala His Ser Ala Thr Pro Asn Leu Val Ala Ile Asn Gln His Asp Phe

865 870 875 880

Gly Thr Ile Glu Ser Val Phe Glu Leu Ala Asn Arg Ser Lys Asp Ile

885 890 895

Asp Thr Leu Tyr Thr His Ser Gly Ala Lys Gly Arg Asp Leu Leu Gln

900 905 910

Thr Leu Leu Ile Asp Ser His Asp Ala Gly Tyr Ala Arg Gln Met Ile

915 920 925

Asp Asn Thr Ser Thr Gly Glu Ile Thr Lys Gln Leu Asn Ala Ala Thr

930 935 940

Thr Thr Leu Asn Asn Ile Ala Ser Leu Glu His Lys Thr Ser Ser Leu

945 950 955 960

Gln Thr Leu Ser Leu Ser Asn Ala Met Ile Leu Asn Ser Arg Leu Val

965 970 975

Asn Leu Ser Arg Lys His Thr Asn Asn Ile Asp Ser Phe Ala Lys Arg

980 985 990

Leu Gln Ala Leu Lys Asp Gln Arg Phe Ala Ser Leu Glu Ser Ala Ala

995 1000 1005

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

1010 1015 1020

Val Trp Ala Asn Ala Ile Gly Gly Ala Ser Leu Asn Asn Gly Ser

1025 1030 1035

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

1040 1045 1050

Asn Gly Gln Val Glu Ala Ile Val Gly Gly Phe Gly Ser Tyr Gly

1055 1060 1065

Tyr Ser Ser Phe Ser Asn Arg Ala Asn Ser Leu Asn Ser Gly Ala

1070 1075 1080

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

1085 1090 1095

His Glu Phe Asp Phe Glu Ala Gln Gly Ala Leu Gly Ser Asp Gln

1100 1105 1110

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

1115 1120 1125

Ser Tyr Asn Tyr Leu Ala Tyr Ser Ala Ala Thr Arg Ala Ser Tyr

1130 1135 1140

Gly Tyr Asp Phe Ala Phe Phe Lys Asn Ala Leu Val Leu Lys Pro

1145 1150 1155

Ser Val Gly Val Ser Tyr Asn His Leu Gly Ser Thr Asn Phe Lys

1160 1165 1170

Ser Asn Ser Thr Asn Lys Val Ala Leu Ser Asn Gly Ser Ser Ser

1175 1180 1185

Gln His Leu Phe Asn Ala Ser Ala Asn Val Glu Ala Arg Tyr Tyr

1190 1195 1200

Tyr Gly Asp Thr Ser Tyr Phe Tyr Met Asn Ala Gly Val Leu Gln

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

Val Met Met Gly Gly Glu Leu Gln Leu Ala Lys Glu Val Phe Leu

1250 1255 1260

Asn Leu Gly Phe Val Tyr Leu His Asn Leu Ile Ser Asn Ile Gly

1265 1270 1275

His Phe Ala Ser Asn Leu Gly Met Arg Tyr Ser Phe

1280 1285 1290

<210> 6

<211> 819

<212> DNA

<213> 人工序列

<400> 6

atggcagttg ttaaatgtaa accgacatct ccgggtcgtc gccacgtagt taaagtggtt 60

aaccctgagc tgcacaaggg caaacctttt gctccgttgc tggaaaaaaa cagcaaatcc 120

ggtggtcgta acaacaatgg ccgtatcacc actcgtcata tcggtggtgg ccacaagcag 180

gcttaccgta ttgttgactt caaacgcaac aaagacggta tcccggcagt tgttgaacgt 240

cttgagtacg atccgaaccg ttccgcgaac atcgcgctgg ttctgtacaa agacggtgaa 300

cgccgttaca tcctggcccc taaaggcctg aaagctggcg accagattca gtctggcgtt 360

gatgctgcaa tcaaaccagg taacaccctg ccgatgcgca acatcccggt tggttctact 420

gttcataacg tagaaatgaa accaggtaaa ggcggtcagc tggcacgttc cgctggtact 480

tacgttcaga tcgttgctcg tgatggtgct tatgtcaccc tgcgtctgcg ttctggtgaa 540

atgcgtaaag tagaagcaga ctgccgtgca actctgggcg aagttggcaa tgctgagcat 600

atgctgcgcg ttctgggtaa agcaggtgct gcacgctggc gtggtgttcg tccgaccgtt 660

cgcggtaccg cgatgaaccc ggtagaccac ccacatggtg gtggtgaagg tcgtaacttt 720

ggtaagcacc cggtaactcc gtggggcgtt cagaccaaag gtaagaagac ccgcagcaac 780

aagcgtactg ataaattcat cgtacgtcgc cgtagcaaa 819

<210> 7

<211> 213

<212> DNA

<213> 人工序列

<400> 7

atggcacgtg gtaaagcagg tgctgcacgc tggcgtggtg ttcgtccgac cgttcgcggt 60

accgcgatga acccggtaga ccacccacat ggtggtggtg aaggtcgtaa ctttggtaag 120

cacccggtaa ctccgtgggg cgttcagacc aaaggtaaga agacccgcag caacaagcgt 180

actgataaat tcatcgtacg tcgccgtagc aaa 213

<210> 8

<211> 1710

<212> DNA

<213> 人工序列

<400> 8

atgaaaaaga ttagcagaaa agaatatgtt tctatgtatg gccctactac aggcgataaa 60

gtgagattgg gcgatacaga cttgatcgct gaagtagaac atgactacac catttatggc 120

gaagagctta aattcggtgg cggtaaaacc ctaagagaag gcatgagcca atctaacaat 180

cctagcaaag aagaactgga tttaatcatc actaacgctt taatcgtgga ttacaccggt 240

atttataaag cggatattgg tattaaagat ggcaaaatcg ctggcattgg taaaggcggt 300

aacaaagaca tgcaagatgg cgttaaaaac aatcttagcg tgggtcctgc tactgaagcc 360

ttagccggtg aaggtttgat cgtaactgct ggtggtattg acacacacat ccacttcatc 420

tccccccaac aaatccctac agcttttgca agcggtgtaa caaccatgat tggtggcgga 480

actggccctg ctgatggcac taacgcaacc actatcactc caggcagaag aaatttaaaa 540

ttcatgctca gagcggctga agaatattct atgaactttg gtttcttggc taaaggtaac 600

gtttctaacg atgcgagctt agccgatcaa attgaagctg gtgcgattgg ctttaaaatc 660

cacgaagact ggggtaccac tccttctgca atcaatcatg cgttagatgt tgcagacaaa 720

tacgatgtgc aagtcgctat ccacacagac actttgaatg aagccggttg cgtggaagac 780

actatggcag ccattgccgg acgcactatg cacactttcc acactgaagg cgctggcggc 840

ggacacgctc ctgatattat taaagtggcc ggtgaacaca acattctacc tgcttccact 900

aaccccacta tccctttcac cgtgaataca gaagccgaac acatggacat gcttatggtg 960

tgccaccact tggataaaag cattaaagaa gatgttcagt tcgctgattc aaggatccgc 1020

cctcaaacca ttgcggctga agacactttg catgacatgg ggattttctc aatcaccagt 1080

tctgactctc aagctatggg tcgtgtgggt gaagttatca ccagaacttg gcaaacagct 1140

gacaaaaaca aaaaagaatt tggccgcttg aaagaagaaa aaggcgataa cgacaacttc 1200

aggatcaaac gctacttgtc taaatacacc attaacccag cgatcgctca tgggattagc 1260

gagtatgtcg gttctgtaga agtgggcaaa gtggctgact tggtattgtg gagtccagca 1320

ttctttggtg tgaaacccaa catgatcatc aaaggcggat tcattgcatt gagtcaaatg 1380

ggtgatgcga acgcttctat ccctacccca caaccggttt attacagaga aatgttcgct 1440

catcatggta aagctaaata cgatgcaaac atcacttttg tgtctcaagc ggcttatgac 1500

aaaggcatta aagaagaatt agggcttgaa agacaagtgt tgccggtaaa aaattgcaga 1560

aacatcacta aaaaagacat gcaattcaac gacactaccg ctcacattga agtcaatcct 1620

gaaacttacc atgtgttcgt ggatggcaaa gaagtaactt ctaaaccagc caataaagtg 1680

agcttggctc aactctttag cattttctag 1710

<210> 9

<211> 783

<212> DNA

<213> 人工序列

<400> 9

atgagagcaa ataatcattt taaagatttt gcatggaaaa aatgcctttt aggcgcgagc 60

gtggtggctt tgttggtggg atgcagcccg catattattg aaaccaatga agtcgctttg 120

aaattgaatt accatccagc tagcgagaaa gttcaagcgt tagatgaaaa gatcttgctt 180

ttaaggccag cttttcaata cagcgataat attgctaaag agtatgaaaa caaattcaag 240

aatcaaacca cgcttaaggt tgaagagatc ttgcaaaatc aaggctataa ggttattagc 300

gtagatagca gcgataaaga cgatctttct ttttcgcaaa aaaaagaagg gtatttggcc 360

gtcgctatga atggcgaaat tgttttacgc cccgatccta aaaggaccat acagaaaaaa 420

tcagaacccg ggttattatt ctccactggt ttggacaaaa tggaaggggt tttaatcccg 480

gctgggtttg tcaaggttac catactagag cctatgagtg gggaatcttt agattctttt 540

acgatggatt tgagcgagtt ggacattcaa gaaaaattct taaaaaccac ccattcaagc 600

catagcgggg ggttagttag cactatggtt aagggaacgg ataattctaa tgacgcgatc 660

aagagcgctt tgaataagat ttttgcaaat atcatgcaag aaatagacaa aaagctcact 720

caaaagaatt tagaatctta tcaaaaagac gccaaggaat tgaaaaacaa gagaaaccga 780

taa 783

<210> 10

<211> 3873

<212> DNA

<213> 人工序列

<400> 10

atggaaatac aacaaacaca ccgcaaaatc aatcgccctc tggtttctct tgctttagta 60

ggagcgttag tcagcatcac accgcaacaa agtcatgccg cctttttcac aaccgtgatc 120

attccagcca ttgttggggg gatcgctaca ggcgctgctg taggaacggt ctcagggctt 180

cttagctggg ggctcaaaca agccgaagaa gccaataaaa ccccagataa acccgataaa 240

gtttggcgca ttcaagcagg aagaggcttc aataattttc ctcacaagga atacgactta 300

tacaaatccc ttttatccag taagattgat ggaggctggg attgggggaa tgccgctagg 360

cattattggg tcaaaggcgg gcaatggaac aagcttgaag tggatatgaa agacgctgta 420

gggacttata aactttcagg ccttatcaac tttactggtg gggatttaga tgtcaatatg 480

caaaaagcca ctttgcgctt gggccaattc aatggcaatt ctttcacaag ctataaggat 540

agtgctgatc gcaccacgag agtggatttc aacgctaaaa atatcttaat tgataatttt 600

ttagaaatca ataatcgtgt gggttctgga gccgggagga aagccagctc tacggtttta 660

actttgcaag cttcagaagg gatcactagc agtaaaaacg ctgaaatttc tctttatgat 720

ggtgccacgc tcaatttggc ttcaagcagt gttaaattaa tgggtaatgt gtggatgggc 780

cgtttgcaat acgtgggagc gtatctggcc ccttcataca gcacgataaa cacttcaaaa 840

gtgacagggg aagtgaattt taaccatctc actgtgggcg atcacaacgc tgctcaagca 900

ggcattatcg ctagtaacaa gactcatatt ggcacattgg atttgtggca aagcgcgggg 960

ctaaacatta tcgcccctcc agaaggcggt tataaggata aacctaagga taaacctagt 1020

aacaccacgc aaaataatgc taacaacaac caacaaaaca gcgctcaaaa caataataac 1080

actcaggtca ttaacccacc caacagcgcg caaaaaacag aaattcaacc cacgcaagtc 1140

attaatgggc cttttgctgg cggcaaagac acggtggtca atatcaaccg catcaacact 1200

aacgctgatg gcacgattag agtgggaggg tataaagctt ctcttaccac caatgcggct 1260

catttgcata tcggcaaagg cggtatcaat ctgtccaatc aagcgagcgg gcgttcttta 1320

ttggtggaaa atctaaccgg gaatatcacc gttgatgggc ctttaagagt gaataaccaa 1380

gtgggtggtt atgctcttgc aggatcaaac gcgaattttg agtttaaggc tggcacggat 1440

accaaaaacg gcacagccac ttttaataac gatattagtt tgggaagatt tgtgaattta 1500

aaagtggatg ctcatacagc taattttaaa ggtattgata cgggtaatgg tggtttcaac 1560

accttggatt ttagtggcgt tacagacaaa gtcaatatca acaagctcat cacagcttcc 1620

actaatgtgg ccattaaaaa cttcaacatt aatgaattgt tggttaagac caatggggtg 1680

agtgtggggg aatacactca ttttagcgaa gatataggca gtcaatcgcg catcaacacc 1740

gtgcgtttag aaactggcac taggtcaatc ttttctgggg gtgtcaaatt taaaagcggc 1800

gaaaaattgg ttatagatga gttttactat agcccttgga attattttga cgctaggaat 1860

attaaaaatg ttgaaatcac cagaaaattc gcttcttcaa ccccagaaaa cccttggggc 1920

acatcaaaac tcatgtttaa taatctaacc ctgggtcaaa atgcggtcat ggactatagt 1980

caattttcaa atttaaccat tcagggggat tttatcaaca atcaaggcac tatcaactat 2040

ctggtccgag gcgggaaagt ggcaacctta aatgtaggca atgcagcagc tatgatgttt 2100

aataatgata tagacagcgc gaccggattt tacaaaccgc tcatcaagat taacagcgct 2160

caagatctca ttaaaaatac agagcatgtt ttattgaaag cgaaaatcat tggttatggt 2220

aatgtttcta caggtaccaa tggcattagt aatgttaatc tagaagagca attcaaagag 2280

cgcctagccc tttataacaa caataaccgc atggatactt gtgtggtgcg aaatactgat 2340

gacattaaag catgcggtat ggctatcggc aatcaaagca tggtgaacaa ccctgacaat 2400

tacaagtatc ttatcggtaa agcatggaaa aatataggca tcagtaaaac ggctaacggc 2460

tctaaaattt cggtgtatta tttaggcaat tctacgccta ctgagaatgg tggcaatacc 2520

accaacttac ccacaaacac cactaataat gcgcgttctg ctaactacgc tctcgtgaag 2580

aacgctcctt tcgctcacag cgccactcct aatttagtcg ctatcaatca gcatgatttt 2640

ggcactattg agagcgtgtt tgaattggct aaccgctcta aagatattga cacgctttat 2700

actcattcag gtgcaaaagg tagggatctc ttgcaaacct tattgattga tagccatgat 2760

gcgggttacg ctagacaaat gattgataac acaagcaccg gtgaaatcac caagcaattg 2820

aatgcggcca ctaccacttt aaacaacata gccagtttag agcataaaac cagcagctta 2880

caaaccttga gcttgagcaa tgcgatgatt ttaaattctc gtttagtcaa tctctccagg 2940

aagcacacca acaatattga ctcgttcgct aagcgcttac aagctttaaa agatcaaaga 3000

ttcgcttctt tagaaagcgc ggcggaagtg ttgtatcaat ttgcccctaa atatgaaaaa 3060

cctaccaatg tttgggctaa cgctattggg ggagcgagct tgaataatgg ttctaacgct 3120

tcattgtatg gcacaagtgc gggcgtagat gcttacctta acgggcaagt ggaagctatt 3180

gtgggcggtt ttggaagcta tggttatagt tcttttagta atcgtgcgaa ctctcttaac 3240

tctggggcca ataacactaa ttttggcgtg tatagccgta tctttgctaa tcagcacgaa 3300

tttgactttg aagctcaagg ggcgctaggg agtgatcaat caagcttgaa tttcaaaagt 3360

gctttattgc aagatttgaa tcaaagctat aattacttag cctatagcgc tgcaacaaga 3420

gcgagctatg gttatgactt tgcgtttttt aagaacgctt tagtgttaaa accaagcgtg 3480

ggcgtgagct ataaccattt aggttcaacc aactttaaaa gcaacagcac taataaagtg 3540

gctttgagta atggctctag cagtcagcat ctattcaacg ctagcgctaa tgtggaagcg 3600

cgctattatt atggagacac ttcatacttc tatatgaacg ctggagtttt acaagaattt 3660

gctaactttg gttctagcaa tgcggtgtct ttaaacacct ttaaagtgaa tgccgctcgc 3720

aaccctttaa atacccatgc cagagtgatg atgggtgggg aattgcaatt agctaaagaa 3780

gtgtttttga atttgggctt tgtttatttg cacaatttga tttccaatat aggccatttc 3840

gcttccaatt taggaatgag gtatagtttc taa 3873

<210> 11

<211> 1247

<212> PRT

<213> 人工序列

<400> 11

Met Thr Asn Glu Thr Ile Asn Gln Gln Pro Gln Thr Glu Ala Ala Phe

1 5 10 15

Asn Pro Gln Gln Phe Ile Asn Asn Leu Gln Val Ala Phe Leu Lys Val

20 25 30

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

35 40 45

Lys Asn Asp Arg Asp Asn Arg Gln Ala Phe Asp Gly Ile Ser Gln Leu

50 55 60

Arg Glu Glu Tyr Ser Asn Lys Ala Ile Lys Asn Pro Thr Lys Lys Asn

65 70 75 80

Gln Tyr Phe Ser Asp Phe Ile Asn Lys Ser Asn Asp Leu Ile Asn Lys

85 90 95

Asp Asn Leu Ile Asp Ile Gly Ser Ser Ile Lys Ser Phe Gln Lys Phe

100 105 110

Gly Thr Gln Arg Tyr Arg Ile Phe Thr Ser Trp Val Ser His Gln Asn

115 120 125

Asp Pro Ser Lys Ile Asn Thr Arg Ser Ile Arg Asn Phe Met Glu Asn

130 135 140

Ile Ile Gln Pro Pro Ile Pro Asp Asp Lys Glu Lys Ala Glu Phe Leu

145 150 155 160

Lys Ser Ala Lys Gln Ser Phe Ala Gly Ile Ile Ile Gly Asn Gln Ile

165 170 175

Arg Thr Asp Gln Lys Phe Met Gly Val Phe Asp Glu Phe Leu Lys Glu

180 185 190

Arg Gln Glu Ala Glu Lys Asn Gly Glu Pro Thr Gly Gly Asp Trp Leu

195 200 205

Asp Ile Phe Leu Ser Phe Val Phe Asn Lys Glu Gln Ser Ser Asp Val

210 215 220

Lys Glu Ala Ile Asn Gln Glu Pro Val Pro His Val Gln Pro Asp Ile

225 230 235 240

Ala Thr Thr Thr Thr His Ile Gln Gly Leu Pro Pro Glu Ser Arg Asp

245 250 255

Leu Leu Asp Glu Arg Gly Asn Phe Ser Lys Phe Thr Leu Gly Asp Met

260 265 270

Glu Met Leu Asp Val Glu Gly Val Ala Asp Ile Asp Pro Asn Tyr Lys

275 280 285

Phe Asn Gln Leu Leu Ile His Asn Asn Ala Leu Ser Ser Val Leu Met

290 295 300

Gly Ser His Asn Gly Ile Glu Pro Glu Lys Val Ser Leu Leu Tyr Ala

305 310 315 320

Gly Asn Gly Gly Phe Gly Ala Lys His Asp Trp Asn Ala Thr Val Gly

325 330 335

Tyr Lys Asn Gln Gln Gly Asp Asn Val Ala Thr Leu Ile Asn Val His

340 345 350

Met Lys Asn Gly Ser Gly Leu Val Ile Ala Gly Gly Glu Lys Gly Ile

355 360 365

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

370 375 380

Gln Arg Ala Leu Ser Gln Glu Glu Ile Arg Asn Lys Ile Asp Phe Met

385 390 395 400

Glu Phe Leu Ala Gln Asn Asn Ala Lys Leu Asp Asn Leu Ser Glu Lys

405 410 415

Glu Lys Glu Lys Phe Gln Asn Glu Ile Glu Asp Phe Gln Lys Asp Ser

420 425 430

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

435 440 445

Lys Lys Asp Pro Lys His Ser Ala Leu Ile Thr Glu Phe Gly Lys Gly

450 455 460

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

465 470 475 480

Leu Asp Arg Glu Lys Asn Val Thr Leu Gln Gly Asn Leu Lys His Asp

485 490 495

Ser Val Met Phe Val Asn Tyr Ser Asn Phe Lys Tyr Thr Asn Ala Ser

500 505 510

Lys Ser Pro Asp Lys Gly Val Gly Val Thr Asn Gly Val Ser His Leu

515 520 525

Asp Ala Gly Phe Ser Lys Val Ala Val Phe Asn Leu Pro Asp Leu Asn

530 535 540

Asn Leu Ala Ile Thr Ser Phe Val Arg Arg Asn Leu Glu Asn Lys Leu

545 550 555 560

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

565 570 575

Phe Leu Ser Ser Asn Lys Glu Leu Val Gly Lys Ala Leu Asn Phe Asn

580 585 590

Lys Ala Val Ala Asp Ala Lys Asn Thr Gly Asn Tyr Asp Glu Val Lys

595 600 605

Lys Ala Gln Lys Asp Leu Glu Lys Ser Leu Arg Lys Arg Glu His Leu

610 615 620

Glu Lys Glu Val Glu Lys Lys Leu Glu Ser Lys Ser Gly Asn Lys Asn

625 630 635 640

Lys Met Glu Ala Lys Ala Gln Ala Asn Ser Gln Lys Asp Lys Ile Phe

645 650 655

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

660 665 670

Ser Gln Asn Leu Lys Gly Ile Lys Arg Glu Leu Ser Asp Lys Leu Glu

675 680 685

Lys Ile Asn Lys Asp Leu Lys Asp Phe Ser Lys Ser Phe Asp Glu Phe

690 695 700

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

705 710 715 720

Ala Leu Lys Gly Ser Val Lys Asp Leu Gly Ile Asn Pro Glu Trp Ile

725 730 735

Ser Lys Val Glu Asn Leu Asn Ala Ala Leu Asn Glu Phe Lys Asn Gly

740 745 750

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

755 760 765

Asn Ser Val Lys Asp Val Ile Ile Asn Gln Lys Ile Thr Asp Lys Val

770 775 780

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

785 790 795 800

Ser Arg Val Glu Gln Ala Leu Ala Asp Leu Lys Asn Phe Ser Lys Glu

805 810 815

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

820 825 830

Lys Ser Glu Ile Tyr Gln Ser Val Lys Asn Gly Val Asn Gly Thr Leu

835 840 845

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

850 855 860

Phe Ser Asp Ile Lys Lys Glu Leu Asn Glu Lys Phe Lys Asn Phe Asn

865 870 875 880

Asn Asn Asn Asn Asn Gly Leu Glu Asn Glu Pro Ile Tyr Ala Lys Val

885 890 895

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

900 905 910

Ala Gln Val Ala Lys Lys Val Asn Ala Lys Ile Asp Arg Leu Asn Gln

915 920 925

Ala Ala Ser Gly Leu Gly Gly Val Gly Gln Ala Gly Phe Pro Leu Lys

930 935 940

Arg His Asp Lys Val Asp Asp Leu Ser Lys Val Gly Arg Ser Val Ser

945 950 955 960

Pro Glu Pro Ile Tyr Ala Thr Ile Asp Asp Leu Gly Gly Pro Phe Pro

965 970 975

Leu Lys Arg His Asp Lys Val Asp Asp Leu Ser Lys Val Gly Arg Ser

980 985 990

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

995 1000 1005

Phe Pro Leu Lys Arg His Asp Lys Val Asp Asp Leu Ser Lys Val

1010 1015 1020

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

1025 1030 1035

Leu Gly Gly Pro Phe Pro Leu Lys Arg His Asp Lys Val Asp Asp

1040 1045 1050

Leu Ser Lys Val Gly Leu Ser Arg Asn Gln Glu Leu Ala Gln Lys

1055 1060 1065

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

1070 1075 1080

Phe Ser Asn Leu Glu Gln Thr Ile Asp Lys Leu Lys Asp Ser Thr

1085 1090 1095

Lys Tyr Asn Ser Val Asn Leu Trp Val Glu Ser Ala Lys Lys Val

1100 1105 1110

Pro Ala Ser Leu Ser Ala Lys Leu Asp Asn Tyr Ala Thr Asn Ser

1115 1120 1125

His Thr Arg Ile Asn Ser Asn Ile Gln Asn Gly Ala Ile Asn Glu

1130 1135 1140

Lys Ala Thr Gly Met Leu Thr Gln Lys Asn Pro Glu Trp Leu Lys

1145 1150 1155

Leu Val Asn Asp Lys Ile Val Ala His Asn Val Gly Ser Val Pro

1160 1165 1170

Leu Ser Glu Tyr Asp Lys Ile Gly Phe Asn Gln Lys Asn Met Lys

1175 1180 1185

Asp Tyr Ser Asp Ser Phe Lys Phe Ser Thr Lys Leu Asn Asn Ala

1190 1195 1200

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

1205 1210 1215

Phe Ser Thr Gly Tyr Tyr Ser Leu Ala Arg Glu Asn Ala Glu His

1220 1225 1230

Gly Ile Lys Asn Val Asn Thr Lys Gly Gly Phe Gln Lys Ser

1235 1240 1245

<210> 12

<211> 3744

<212> DNA

<213> 人工序列

<400> 12

atgactaacg aaaccattaa ccaacaacca caaaccgaag cggcttttaa cccgcagcaa 60

tttatcaaca atcttcaagt ggcttttctt aaagttgata acgctgtcgc ttcatacgat 120

cctgatcaaa aaccaatcgt tgataagaat gatagggata acaggcaagc ttttgatgga 180

atctcgcaat taagggaaga atactccaat aaagcgatca aaaatcctac caaaaagaat 240

cagtattttt cagactttat caataagagc aatgatttaa tcaacaaaga caatctcatt 300

gatataggtt cttccataaa aagctttcag aaatttggga ctcagcgtta ccgaattttc 360

acaagttggg tgtcccatca aaacgatccg tctaaaatca acacccgatc gatccgaaat 420

tttatggaaa atatcataca accccctatc cctgatgaca aagaaaaagc agagtttttg 480

aaatctgcca aacaatcttt tgcaggaatc attataggga atcaaatccg aacggatcaa 540

aagttcatgg gcgtgtttga tgaattcttg aaagaaaggc aagaagcaga aaaaaatgga 600

gagcctactg gtggggattg gttggatatt tttttatcat ttgtatttaa caaagaacaa 660

tcttctgatg tcaaagaagc aatcaatcaa gaaccagttc cccatgtcca accagatata 720

gccactacca ccacccacat acaaggctta ccgcctgaat ctagggattt gcttgatgaa 780

aggggtaatt tttctaaatt cactcttggc gatatggaaa tgttagatgt tgagggcgtc 840

gccgacattg atcctaatta caagttcaat caattattga ttcacaataa cgctctgtct 900

tctgtgttaa tggggagtca taatggcata gaacctgaaa aagtttcatt attgtatgcg 960

ggcaatggtg gttttggagc caagcacgat tggaacgcca ccgttggtta taaaaaccaa 1020

caaggcgaca atgtggctac actcattaat gtgcatatga aaaacggcag tggcttagtc 1080

atagcaggtg gtgagaaagg gattaacaac cctagttttt atctctacaa agaagaccaa 1140

ctcacaggct cacaacgagc attgagtcaa gaagagatcc gaaacaaaat agatttcatg 1200

gaatttcttg cacaaaacaa tgctaaatta gacaacttga gcgagaaaga gaaagaaaaa 1260

ttccaaaatg agattgaaga ttttcaaaaa gactctaagg cttatttaga cgccctaggg 1320

aatgatcgta ttgcctttgt ttctaaaaaa gacccaaaac attcagcttt aattactgag 1380

tttggtaagg gggatttgag ctacactctc aaagattatg ggaaaaaagc agatagagct 1440

ttagataggg agaaaaatgt tactcttcaa ggtaacctaa aacatgatag cgtgatgttt 1500

gttaattatt ctaatttcaa atacaccaac gcctccaaga gtcctgataa gggtgtaggc 1560

gttacaaatg gcgtttccca tttagacgca ggctttagca aggtagctgt ctttaatttg 1620

cctgatttaa ataatctcgc tatcactagt ttcgtaaggc ggaatttaga gaataaacta 1680

gtcactgaag gattgtccct acaagaagct aataagctta tcaaagattt tttgagcagc 1740

aacaaagaat tggttggaaa agctttaaac ttcaataaag ctgtagctga cgctaaaaac 1800

acaggcaact atgatgaagt gaaaaaagct cagaaagatc ttgaaaaatc tctaaggaaa 1860

cgagagcatt tagagaaaga agtagagaaa aaattggaga gcaaaagcgg caacaaaaat 1920

aaaatggaag cgaaagctca agctaacagc caaaaagata agatttttgc gttgatcaat 1980

aaagaggcta atagggacgc aagagcaatc gcttactctc agaatcttaa aggcatcaaa 2040

agggaattgt ctgataaact tgaaaaaatc aacaaggatt tgaaagactt tagtaaatct 2100

tttgatgaat tcaaaaatgg caaaaataag gattttagca aggcagaaga aacgctaaaa 2160

gcccttaaag gctcggtgaa agatttaggt atcaatccgg aatggatttc aaaagttgaa 2220

aaccttaatg cagctttgaa tgaattcaaa aatggcaaaa ataaggattt cagcaaggta 2280

acgcaagcaa aaagcgacct tgaaaattcc gttaaagatg tgatcatcaa tcaaaagata 2340

acggataaag ttgacaatct caatcaagcg gtatcaatgg ctaaagcaac gggtgatttc 2400

agtagggtag agcaagcgtt agccgatctc aaaaacttct caaaggagca attggctcaa 2460

caaactcaaa aaaatgaaag tttcaatgtt ggaaaaaaat ctgaaatata tcaatccgtt 2520

aagaatggtg tgaacggaac cctagtcggt aatgggttat ctggaataga ggccacagct 2580

ctcgccaaaa atttttcgga tatcaagaaa gaattgaatg agaaatttaa aaatttcaat 2640

aacaataaca ataatggact cgaaaacgaa cccatttatg ctaaagttaa taaaaagaaa 2700

acaggacaag tagctagccc tgaagaaccc atttacgctc aagttgctaa aaaggtgaat 2760

gcaaaaattg accgactcaa tcaagcagca agtggtttgg gtggtgtagg gcaagcgggc 2820

ttccctttga aaaggcatga taaagttgat gatctcagta aggtagggcg atcagttagc 2880

cctgaaccca tttatgctac gattgatgat ctcggcggac ctttcccttt gaaaaggcat 2940

gataaagttg atgatctcag taaggtaggg cgatcagtta gccctgaacc catttatgct 3000

acgattgatg atctcggcgg acctttccct ttgaaaaggc atgataaagt tgatgatctc 3060

agtaaggtag ggcgatcagt tagccctgaa cccatttatg ctacgattga tgatctcggc 3120

ggacctttcc ctttgaaaag gcatgataaa gttgatgatc tcagtaaggt agggctttca 3180

aggaatcaag aattggctca gaaaattgac aatctcagtc aagcggtatc agaagctaaa 3240

gcaggttttt ttagcaatct agagcaaacg atagacaagc tcaaagattc tacaaaatac 3300

aattctgtga atctatgggt tgaaagtgca aaaaaagtgc ctgctagttt gtcagcgaaa 3360

ctagacaatt acgctactaa cagccacaca cgcattaata gcaatatcca aaatggagca 3420

atcaatgaaa aagcgaccgg tatgctaacg caaaaaaacc ctgagtggct caagctcgtg 3480

aatgataaga tagttgcgca taatgtggga agcgttcctt tgtcagagta tgataaaatt 3540

ggcttcaacc agaagaatat gaaagattat tctgattcgt tcaagttttc caccaagttg 3600

aacaatgctg taaaagacgt taagtctagc tttacgcaat ttttagccaa tgcattttct 3660

acaggatatt actccttggc gagggaaaat gcggagcatg gaatcaaaaa tgttaataca 3720

aaaggtggtt tccaaaaatc ttaa 3744