CN113801205A - 一种提高甲型流感病毒h1三聚体蛋白抗原性的改造方法 - Google Patents

一种提高甲型流感病毒h1三聚体蛋白抗原性的改造方法 Download PDF

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CN113801205A
CN113801205A CN202111069035.7A CN202111069035A CN113801205A CN 113801205 A CN113801205 A CN 113801205A CN 202111069035 A CN202111069035 A CN 202111069035A CN 113801205 A CN113801205 A CN 113801205A
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邓磊
刘德建
郑何平
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Abstract

本发明属于生物技术领域，具体涉及引入二硫键改造甲型流感病毒血凝素H1蛋白质结构，进而促使H1胞外区形成稳定的三聚体状态，增强H1胞外区抗原性和特异性免疫保护作用。方法包括：将H1胞外区的信号肽序列进行置换和添加标签后插入到pFastBac1载体中，获得重组质粒；依据蛋白质结构信息和辅助软件分析，在重组质粒的H1胞外域基因序列中引入半胱氨酸突变获得多种H1突变体质粒；利用重组杆状病毒感染悬浮培养昆虫细胞表达多种重组H1蛋白；利用分子生物学实验方法筛选获得H1突变体，能够形成更稳定的三聚体状态和更好的抗原性。小鼠免疫实验证实优化后的H1突变体提供了更强的体内特异性免疫保护作用。

Description

一种提高甲型流感病毒H1三聚体蛋白抗原性的改造方法
技术领域
本发明属于生物材料技术领域，具体涉及提高甲型流感病毒H1三聚体蛋白抗原性的改造方法。
背景技术
流感是一种人类呼吸道传染病，主要由甲型流感和乙型流感病毒引起。通常每年会有季节性流行，据世界卫生组织(WHO)估计，流行性感冒每年导致约10亿例感染，其中严重病例300-500万例，死亡病例30-50万例。接种疫苗仍然是预防流感的最有效方式。
由于流感病毒的持续性抗原漂移，多价季节性流感疫苗每年都需要根据预测的流行毒株更新疫苗毒株，但是对来年流行毒株的预测可能不是十分准确，导致疫苗毒株和正在流行的毒株的匹配性不好，降低疫苗的有效性。血凝素HA是流感病毒最主要的表面糖蛋白，是适应性免疫反应的主要靶点。HA先被合成为单体形式的前体蛋白HA0，然后三个HA0组装为三聚体形式。当融合肽被宿主的相关蛋白酶切割后，HA0融合前状态的空间构象发生细微变化并且一级结构氨基酸序列分为HA1和HA2。亚稳态的HA融合前形式对低pH条件敏感。HA1残基构成包含受体结合位点的HA高度突变的头部区域，HA2和一部分HA1构成负责与细胞膜膜融合过程紧密相关的H1保守的柄部区域。天然H1包含6个单体内二硫键(4个HA1-HA1，1个HA2-HA2和1个HA1-HA2)。
2009年大流行H1N1毒株的HA具有解离为单体的倾向，疫苗的低水平免疫反应和免疫保护作用与抗原不稳定性相关。将HA与外源基序融合表达可以增强HA抗原三聚化。Yang等人报道，体外表达的携带外源三聚体基序的pdm09 H1重组蛋白切除外源三聚体基序后在溶液中分解成单体形式，在结晶条件下又重新形成三聚体H1。然而，外源三聚体基序免疫原性往往较强，容易诱导较强脱靶免疫反应，因此，引入外源三聚体基序有降低靶抗原特异性免疫保护作用的风险。
发明内容
针对现有技术存在的问题，本发明利用的昆虫细胞蜂毒素信号肽序列，蛋白分泌后信号肽会被完全切除，在C末端只添加4个氨基酸残基组成的FactorXa切割位点和6×His亲和标签，没有引入任何外源三聚体基序。基于H1的结构信息，设计H1单体间和/单体内二硫键突变，提高重组H1的三聚体构象的稳定性和免疫原性。
为实现上述发明目的，本发明提供了一种提高甲型流感病毒H1三聚体蛋白抗原性的改造方法，所述方法具体包括：
将甲型流感病毒H1的信号肽序列进行置换成昆虫细胞蜂毒素信号肽序列，并在H1胞外基因序列的羧基端添加切割位点和亲和纯化标签序列，依据昆虫细胞宿主优化密码子，合成在pFastBac1载体中，获得带有甲型流感病毒H1序列的重组质粒；
在所述带有甲型流感病毒H1序列的重组质粒的H1胞外域基因序列引入单体间或/和单体内半胱氨酸突变获得H1突变体的pFastBac1重组质粒，所述突变包括点突变和插入氨基酸突变；
利用bac-to-bac昆虫杆状病毒表达系统表达所述H1突变体并纯化获得H1三聚体重组蛋白。
进一步的，所述切割位点包括：Factor Xa切割位点、凝血酶裂解位点、HRV 3C蛋白酶切位点、TEV蛋白酶切位点、肠激酶切位点、SUMO蛋白酶切位点；
所述标签序列包括：6×His标签、HA标签、FLAG标签、谷胱甘肽巯基转移酶标签、麦芽糖结合蛋白标签、NusA标签、SUMO标签、Strep-tag标签。
进一步的，所述甲型流感病毒H1包括但不限于毒株A/Washington/5/2011(H1N1)、毒株A/New Caledonia/20/1999(H1N1)和与所述毒株A/Washington/5/2011(H1N1)、毒株A/New Caledonia/20/1999(H1N1)的氨基酸序列具有89％以上同源性的毒株。
进一步的，所述毒株A/Washington/5/2011(H1N1)的突变位点包括单体间点突变K68C-R76C、L20C-K47C、G4C-N117C，插入氨基酸9C-K121C、9C10G-K121C、N124C-G134C；并以插入氨基酸9C-K121C、9C10G-K121C的质粒为模板再次引入单体内点突变，包括L318C-H111C、V321C-G13C、K308C-T93C。
进一步的，所述毒株A/New Caledonia/20/1999(H1N1)的突变位点包括单体间点突变K68C-R76C、L24C-G47C、G4C-N117C，插入氨基酸9C-K121C、9C10G-K121C、E97C-K58C；并以点突变L24C-G47C的质粒为模板再次引入单体内点突变，包括L321C-H111C、T319C-I48C、V23C-E105C、R311C-T93C。
进一步的，利用bac-to-bac昆虫杆状病毒表达系统表达所述H1突变体并纯化获得H1三聚体重组蛋白过程具体包括：
将编码所述H1突变体的pFastBac1重组质粒进行转化DH10Bac菌株后，蓝白斑筛选获得重组bacmid，将bacmid转染静置培养的昆虫细胞sf9制备P1代重组杆状病毒，将0.5mlP1代感染20ml生长期状态1×106/ml密度sf9制备病毒滴度更高的P2代重组杆状病毒，利用P2重组杆状病毒感染较大体积sf9细胞培养物表达H1突变体重组蛋白；
搜集上清液，采用聚丙烯酰胺凝胶电泳法、考马斯亮蓝染色法、Western blot方法检测并确认H1的表达情况和三聚体状态，离心上清液后，通过亲和层析柱和尺寸排阻色谱柱纯化获得H1突变体重组蛋白；
所述将编码H1突变体的DNA片段重组的真核表达载体包括但不限于pCDNA3.1、pCAGGS质粒载体，表达重组蛋白的细胞株包括但不限于HEK293、CHO和MDCK细胞株。
基于同一发明构思的，本发明实施例还提供了重组甲型流感病毒H1三聚体抗原，所述H1三聚体抗原由上述改造方法制备获得；
所述重组H1三聚体抗原的氨基酸序列如SEQ ID NO:3-14和SEQ ID NO:17-26所示；
所述重组H1三聚体抗原的氨基酸序列还包括与如SEQ ID NO:3-14和SEQ ID NO:17-26所示氨基酸序列具有89％以上同源性的氨基酸序列。
基于同一发明构思的，本发明实施例还提供了一种重组甲型流感病毒疫苗，所述甲型流感病毒疫苗包括上述重组甲型流感病毒H1三聚体抗原。
进一步的，所述疫苗还包括疫苗载体，所述疫苗载体包括：DNA载体、mRNA载体、水凝胶载体、脱溶剂纳米颗粒载体、蛋白质自组装纳米颗粒载体、腺病毒载体、慢病毒载体、植物病毒载体、减毒人感染病毒载体、人感染复制缺陷型病毒载体，以及破伤风类毒素、白喉类毒素、白喉毒素的无毒变异体、B群脑膜炎球菌外膜蛋白复合体、白介素、病原相关分子模式、损伤相关分子模式融合蛋白载体。
有益效果：
本发明依据蛋白质结构信息和辅助软件分析结果，以野生型H1作为模板设计了多个单体间二硫键突变体，获得的H1能够形成稳定的三聚体，且能够提高H1广谱中和抗体CT149、CR9114、CR6261、C179和3I14的结合能力；本发明进行通过设计多个单体内或单体间的二硫键突变体，能够进一步提高三聚体抗原的稳定性以及广谱单抗的结合能力，其应用至小鼠体内时，能够诱导形成高滴度H1特异性抗体、H1交叉反应性抗体和H2交叉反应性抗体，且能提供更有效特异性免疫保护作用。
附图说明
图1为本发明实施例提供的Westernblot检测Wash11野生型和引入单体间二硫键突变体重组蛋白的表达分析图；
图2为本发明实施例提供的Westernblot检测NC99野生型和引入单体间二硫键突变体重组蛋白的表达分析图；
图3为本发明实施例提供的ELISA检测Wash11 WT、Wash11#1、#2、#3、#4、#5和#6分别与CT149、CR9114、CR6261、C179和3I14等识别HA柄部表位广谱中和单克隆抗体的结合能力对比图；
图4为本发明实施例提供的ELISA检测NC99 WT和NC99#1、#2、#3、#4、#5和#6分别与CT149、CR9114、CR6261、C179和3I14等识别HA柄部表位广谱中和单克隆抗体的结合能力对比图；
图5为本发明实施例提供的Western blot检测Wash11和NC99引入单体内二硫键突变体重组蛋白的表达分析图；
图6为本发明实施例提供的ELISA检测Wash11#4、Wash11#4-1、Wash11#4-2和Wash11#4-3与CT149、CR9114、CR6261和3I14等识别HA柄部表位广谱中和单克隆抗体的结合能力对比图；
图7为本发明实施例提供的ELISA检测Wash11#5、Wash11#5-1、Wash11#5-2和Wash11#5-3与CT149、CR9114、CR6261和3I14等识别HA柄部表位广谱中和单克隆抗体的结合能力对比图；
图8为本发明实施例提供的ELISA检测NC99#2、NC99#2-1、NC99#2-2、NC99#2-3和NC99#2-4与CT149、CR9114、CR6261和3I14等识别HA柄部表位广谱中和单克隆抗体的结合能力对比图；
图9为本发明实施例提供的ELISA检测Wash11 WT、Wash11#4、Wash11#5、NC99 WT和NC99#2的小鼠二免血清与同源HA抗原结合的特异性抗体滴度，每条曲线代表一只小鼠，浅灰色曲线代表阴性对照组的小鼠血清，黑色曲线为用于PR8 H1N1病毒的攻毒保护研究的小鼠血清，深灰色曲线为用于X179AH1N1病毒的攻毒保护研究的小鼠血清；
图10为本发明实施例提供的ELISA检测Wash11 WT、Wash11#4、Wash11#5、NC99 WT和NC99#2的小鼠二免血清与Wash11、NC99野生型和单体间二硫键突变体的交叉反应性抗体滴度；
图11为本发明实施例提供的ELISA检测Wash11 WT、Wash11#4、Wash11#5、NC99 WT和NC99#2的小鼠二免血清与异型的H2、H5和H7的交叉反应性抗体滴度；
图12为本发明实施例提供的Wash11 WT、Wash11#4和Wash11#5的流感病毒挑战研究中对5倍致死剂量PR8和X179A H1N1病毒攻毒后14天内小鼠体重变化和存活率；
图13为本发明实施例提供的NC99 WT和NC99#2的流感病毒挑战研究中对5倍致死剂量PR8和X179A H1N1病毒攻毒后14天内小鼠体重变化和存活率；
图14为本发明实施例提供的ELISA检测Wash11#5、Wash11#5-1、Wash11#5-2、Wash11#5-3、NC99#2、NC99#2-1、NC99#2-2、NC99#2-3和NC99#2-4的小鼠二免血清与同源HA抗原结合的特异性抗体滴度，每条曲线代表一只小鼠，浅灰色曲线代表阴性对照组的小鼠血清，黑色曲线为用于PR8 H1N1病毒的攻毒保护研究的小鼠血清，深灰色曲线为用于X179AH1N1病毒的攻毒保护研究的小鼠血清；
图15为本发明实施例提供的ELISA检测Wash11#5、Wash11#5-1、Wash11#5-2和Wash11#5-3的小鼠二免血清与Wash11 WT、NC99 WT和异型的H2、H5和H7的交叉反应性抗体滴度；
图16为本发明实施例提供的ELISA检测NC99#2、NC99#2-1、NC99#2-2、NC99#2-3和NC99#2-4的小鼠二免血清与Wash11 WT、NC99 WT和异型的H2、H5和H7的交叉反应性抗体滴度；
图17为本发明实施例提供的Wash11#5、Wash11#5-1、Wash11#5-2和Wash11#5-3的流感病毒挑战研究中对10倍致死剂量X179A和PR8 H1N1病毒攻毒后14天内小鼠体重变化和存活率；
图18为本发明实施例提供的NC99#2、NC99#2-1、NC99#2-2、NC99#2-3和NC99#2-4的流感病毒挑战研究中对10倍致死剂量X179A和PR8 H1N1病毒攻毒后14天内小鼠体重变化和存活率。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合具体实施例进行详细描述，但本发明的保护范围并不限于以下具体实施例。
除非另有定义，下文中所使用的所有专业术语与本领域技术人员通常理解含义相同。本文中所使用的专业术语只是为了描述具体实施例的目的，并不是旨在限制本发明的保护范围。
除非另有特别说明，本发明中用到的各种原材料、试剂、仪器和设备等均可通过市场购买得到或者可通过现有方法制备得到。
在本发明实施例中，主要通过通过迭代设计的策略构建稳定的可溶性H1三聚体免疫原；首先通过R327Q突变稳定H1的融合前构象，基于已解析的H1晶体结构，通过结构生物学分析设计可能的突变组合，引入H1单体间的二硫键，分别通过变性非还原和变性还原条件的SDS-PAGE和Western Blot分析H1单体间二硫键突变体的三聚体和单体表达情况，通过ELISA分析H1单体间二硫键突变体与广谱中和抗原的结合能力评价其抗原性。选择相较于野生型H1三聚体形成和抗原性良好的单体间二硫键突变体作为模板，基于结构生物学知识设计可能的突变组合，引入H1单体内二硫键，抗原性评价方法同H1单体间二硫键突变体评价方法。
在本发明实施例中，昆虫细胞sf9购买于ATCC。利用昆虫细胞表达多种识别H1柄部区域的广谱中和性单克隆抗体(bnAb)，包括CT149(抗原表位分布在两个相邻HA单体表面)、CR9114(抗原表位分布在一个HA单体表面)、CR6261(抗原表位分布在一个HA单体表面)、C179(抗原表位分布在一个HA单体表面)和3I14(抗原表位分布在两个相邻HA单体表面)等，利用蛋白G树脂亲和层析的方法纯化各种抗体。甲型流感病毒A/Puerto Rico/8/1934(H1N1)、A/reassortant/NYMC X-179(H1N1)和低致病性禽流感病毒A/Chicken/Jiangsu/7/2002(H9N2)获赠于湖南师范大学生命科学学院常海燕博士课题组。乙型流感病毒B/Anhui-Tunxi/1528/2014获赠于中国疾病预防控制中心。甲型流感病毒A/New Jersey/8/1976(HswN1)(H1N1)、A/Hong Kong/8/68(H3N2)、A/Victoria/3/75(H3N2)和A/equine/Kentucky/1/91(H3N8)病毒购买于ATCC。A/Guizhou/54/1989(H3N2)和乙型流感病毒B/Brisbane/3/2007(Yamagatalineage)购买于Sino Biological。试验小鼠为6-8周龄SPF级BALB/c雌鼠。将A/Washington/5/2011(H1N1；毒株名称缩写：Wash11),A/New Caledonia/20/1999(H1N1；毒株名称缩写：NC99),A/Shanghai/202/1957(H2N2),A/Vietnam/1203/2004(H5N1)和A/Shanghai/1/2013(H7N9)病毒H1的信号肽序列置换为昆虫细胞蜂毒素信号肽序列，在H1胞外域基因序列的羧基端添加FactorXa切割位点和6×His标签序列，按照昆虫细胞宿主优化密码子，将H1胞外域基因序列插入到pFastBac1载体的多克隆位点中构建重组质粒。
按照
Figure BDA0003259759290000071
Site-Directed Mutagenesis Kit(NEB,E0554S)说明书操作进行突变处理，具体包括：
在pFastBac1-Wash11胞外域质粒引入点突变R327Q，帮助稳定H1的融合前构象。尝试的单体间二硫键突变组合包括：K68C-R76C及其附近位点：G67C-R76C,E69C-R76C,K68C-K75C,G67C-K75C,E69C-K75C,K68C-I77C,G67C-I77C,E69C-I77C，L20C-K47C及其附近位点：V19C-K47C,E21C-K47C,L20C-D46C,V19C-D46C,E21C-D46C,L20C-I48C,V19C-I48C,E21C-I48C；G4C-N117C及其附近位点：F3C-N117C,A5C-N117C,G4C-K116C,F3C-K116C,5C-K116C,G4C-L118C,F3C-L118C,A5C-L118C；Insert9C-K121C,Insert9C10G-K121C及其附近位点：G8C-K121C,F9C-K121C,G8C-E120C,F9C-E120C,Insert9C-E120C,Insert9C10G-E120C,G8C-V122C,F9C-V122C,Insert9C-V122C,Insert9C10G-V122C；N124C-G134C及其附近位点：R123C-G134C，Q125C-G134C，N124C-I133C，R123C-I133C,Q125C-I133C,N124C-N135C,R123C-N135C,Q125C-N135C。以其中的点突变K68C-R76C(Wash11#1)，L20C-K47C(Wash11#2)，G4C-N117C(Wash11#3)，和插入氨基酸9C-K121C(Wash11#4)，9C10G-K121C(Wash11#5)，N124C-G134C(Wash11#6)为例；接着分别以Wash11#4和Wash11#5为模板分别引入3对半胱氨酸单体内突变组合：L318C-H111C(Wash11#4-1和Wash11#5-1)、V321C-G13C(Wash11#4-2和Wash11#5-2)和K308C-T93C(Wash11#4-3和Wash11#5-3)，共获得12个以A/Washington/5/2011(H1N1)毒株为模板的突变质粒。应该理解的是，其他单体间突变位点在本毒株和与本毒株的氨基酸序列具有高于89％同源性的其他2009年大流行之后流行的H1毒株中应用时，能够产生增强H1三聚体的稳定性和免疫保护效果。
在pFastBac1-NC99胞外域质粒引入点突变R327Q，帮助稳定H1的融合前构象，软件分析单体间二硫键突变组合包括：K68C-R76C及其附近位点：G67C-R76C,E69C-R76C,K68C-R75C,G67C-R75C,E69C-R75C,K68C-M77C,G67C-M77C,E69C-M77C；L24C-G47C及其附近位点：V23C-G47C,E25C-G47C,L24C-N46C,V23C-N46C,E25C-N46C,L24C-I48C,V23C-I48C,E25C-I48C；G4C-N117C及其附近位点：F3C-N117C,A5C-N117C,G4C-K116C,F3C-K116C,5C-K116C,G4C-L118C,F3C-L118C,A5C-L118C；Insert9C-K121C,Insert9C10G-K121C及其附近位点：G8C-K121C,F9C-K121C,G8C-E120C,F9C-E120C,Insert9C-E120C,Insert9C10G-E120C,G8C-V122C,F9C-V122C,Insert9C-V122C,Insert9C10G-V122C；K58C-E97C及其附近位点：E57C-E97C,M59C-E97C,K58C-A96C,E57C-A96C,M59C-A96C,K58C-L98C,E57C-L98C,M59C-L98C。以其中的点突变：K68C-R76C(NC99#1)，L24C-G47C(NC99#2)，G4C-N117C(NC99#3)，插入9C-K121C(NC99#4)，插入9C10G-K121C(NC99#5)，E97C-K58C(NC99#6)为例；接着以NC99#2为模板引入4对半胱氨酸突变组合：L321C-H111C(NC99#2-1)、T319C-I48C(NC99#2-2)、V23C-E105C(NC99#2-3)和R311C-T93C(NC99#2-4)，共获得10个以A/New Caledonia/20/1999(H1N1)毒株为模板的突变质粒。应该理解的是，其他单体间突变位点在本毒株和与本毒株的氨基酸序列具有高于89％的同源性的其他2009年大流行之前流行的H1毒株中应用时，能够增强H1的稳定性和免疫保护效果。
H1的表达与纯化：
将上述获得13个以A/Washington/5/2011(H1N1)毒株H1为模板的突变体质粒，和11个以A/New Caledonia/20/1999(H1N1)毒株H1为模板的突变体质粒进行表达纯化，步骤具体包括：
将编码所述H1突变体的pFastBac1重组质粒转化到DH10Bac感受态细胞，通过蓝白斑实验筛选阳性的重组Bacmid，利用
Figure BDA0003259759290000091
II转染试剂(Gibco)将阳性重组Bacmid分别转染至sf9昆虫细胞，27℃条件下，静置培养72小时；培养上清液接种sf9细胞，27℃，110rpm/min条件下，震荡培养72h后离心收集培养上清液；利用His标签的单克隆抗体通过Western Blot实验检测H1的表达情况；将培养上清液接种sf9细胞，在27℃，110rpm/min条件下扩大培养，72小时后离心收集培养上清液，经0.45μm滤器过滤后，利用Ni Sepharose6Fast Flow(GE Healthcare)树脂和Superdex 200Increase 10/300GL(GE Healthcare)凝胶柱纯化H1重组蛋白，利用30kDa超滤管(Millipore)将H1重组蛋白洗脱液置换为PBS，分装后-80℃保存。
经上述方法共获得22种突变体蛋白，具体包括：Wash11#1、Wash11#2、Wash11#3、Wash11#4、Wash11#5、Wash11#6、Wash11#4-1、Wash11#4-2、Wash11#4-3、Wash11#5-1、Wash11#5-2、Wash11#5-3、NC99#1、NC99#2、NC99#3、NC99#4、NC99#5、NC99#6、NC99#2-1、NC99#2-2、NC99#2-3、NC99#2-4突变体蛋白，并对上述突变体蛋白进行氨基酸序列检测，具体详见序列表SEQ ID NO:3-14和SEQ ID NO:17-26所示，A/Washington/5/2011(H1N1)野生型H1的氨基酸序列如序列表SEQ ID NO:1，引入R327Q突变的氨基酸序列如序列表SEQ IDNO:2，A/New Caledonia/20/1999(H1N1)野生型H1的氨基酸序列如序列表SEQ ID NO:15，引入R327Q突变的氨基酸序列如序列表SEQ ID NO:16。
SDS-PAGE和WesternBlot
在SDS变性非还原和SDS、DTT变性还原条件下对H1野生型和突变体蛋白进行10％聚丙烯酰胺SDS-PAGE电泳分析，将蛋白样品转印到PVDF膜上，10％脱脂牛奶(TBS中含0.05％的吐温-20，TBST)室温封闭2小时，His标签鼠源单克隆抗原室温孵育2h，TBST洗涤3遍后，室温孵育羊抗鼠二抗1h，TBST洗涤3遍后，使用ECL化学发色液(Bio-Rad)进行显影曝光。其结果如图1、2、6所示，以Wash11 H1野生型作为模板设计6个单体间二硫键突变体中，Wash11#1、Wash11#2、Wash11#3、Wash11#4和Wash11#5有H1三聚体形成，其中Wash11#4和Wash11#5表达量较高，Wash11#1、Wash11#2和Wash11#3表达量较低，Wash11#6没有表达成功(图1)；以NC99 H1野生型做模板设计的6个单体间二硫键突变体均形成三聚体蛋白(图2)；以Wash11#4和Wash11#5为模板设计3组突变组合引入H1单体内二硫键，以NC99#2为模板设计4对突变组合引入H1单体内二硫键，其中Wash11#4-1、Wash11#4-2、Wash11#5-1、Wash11#5-2、NC99#2-1、NC99#2-2、NC99#2-3有三聚体形成，Wash11#4-3、Wash11#5-3、NC99#2-4没有三聚体形成(图5)。
ELISA检测H1突变体与广谱中和抗原的结合能力：
为了检测H1突变体与广谱中和抗原CT149、CR9114、CR6261、C179和3I14的结合能力，将100ng/孔的可溶性野生型或突变体H1蛋白包被到反应板中，4℃过夜；PBST(PBS含0.05％吐温-20)洗涤孔板后，用封闭液(PBS含5％脱脂牛奶)室温封闭1.5h,用PBST洗涤后，加入封闭液倍比稀释的H1广谱中和抗原(初始浓度10μg/ml或5μg/ml,5倍倍比稀释)，室温孵育1h；用PBST洗涤3遍后，羊抗人抗原1：5000稀释后室温孵育1h,用PBST洗3遍后，避光加入TMB单组份显色液，37℃孵育10min后加入等体积的1M H2SO4终止反应。用酶标仪测定OD450 nm处的吸光值。
以突变体蛋白Wash11#4、Wash11#5为例，Wash11野生型H1做对比，以突变体蛋白NC99#2为例，NC99野生型H1做对比，采用上述实验进行引入单体间二硫键H1广谱中和抗体反应能力的大小，其结果如图3和4所示。由图可知，相较于Wash11、NC99野生型H1,单体间二硫键突变体Wash11#4和Wash11#5、NC99#2和NC99#3提高了H1与广谱中和抗原CT149、CR9114、CR6261、C179和3I14的结合能力。
以突变体蛋白Wash11#4-1、Wash11#4-2、Wash11#4-3，Wash11#5-1、Wash11#5-2、Wash11#5-3为例，并以Wash11#4和Wash11#5做对比，以突变体蛋白NC99#2-1、NC99#2-2、NC99#2-3和NC99#2-4为例，并以NC99#2做对比，采用上述实验进行引入单体内二硫键H1广谱中和抗体反应能力的大小，其结果如图6、7、8所示。相较于突变体Wash11#4，Wash11#4-1、Wash11#4-2和Wash11#4-3与CT149、CR9114、CR6261和3I14的结合能力无显著提升；相较于Wash11单体间二硫键突变体Wash11#5，Wash11#5-2和Wash11#5-3显著提高了与CT149、CR9114和CR6261的结合能力；相较于突变体NC99#2，NC99#2-3显著提高了与CT149、CR9114和CR6261的结合能力。
单体间二硫键突变体小鼠免疫和流感病毒攻毒实验：
将6-8周龄SPF级BALB/c雌鼠随机分为5-6只/笼。将各种H1重组蛋白或PBS与弗氏不完全佐剂按照体积比1：1混合乳化后，按照10μg免疫原每只的剂量腹腔注射接种小鼠。初次免疫前2天，初次免疫后21天和增强免疫后14天采集血清检测特异性抗原滴度。将50μl 5倍半致死剂量(50％letH1ldose，LD50)的H1N1流感病毒PR8和X179A通过滴鼻方式攻毒，并记录感染后14天内各组小鼠的体重变化和存活率。
ELISA检测小鼠血清的H1特异性抗原和交叉反应性抗原滴度：
小鼠血清用PBS 10倍倍比稀释，其他操作方法同上。
如图12所示，滴鼻攻毒5倍致死剂量PR8 H1N1，免疫Wash11#5的小鼠感染后第7天体重降低到原始值的80％左右，之后体重逐渐回复，最终存活率为80％；免疫Wash11 WT和Wash11#4的小鼠攻毒后体重逐渐降低，最终全部死亡，在5倍致死剂量X179AH1N1病毒的攻击下，免疫Wash11 WT，Wash11#4和Wash11#5的小鼠体重未出现下降，并全部存活。如图13所示，在5倍致死剂量PR8 H1N1病毒的攻击下，免疫NC99#2的小鼠体重未出现下降，并全部存活；免疫NC99 WT的小鼠攻毒后体重逐渐降低，最终全部死亡，5×LD50 X179AH1N1攻毒后，免疫NC99 WT的小鼠感染后第6天体重降低到原始值的85％左右，之后体重逐渐回复，最终存活率为33％；免疫NC99#2的小鼠感染后第6天体重降低到原始值的88％左右，之后体重逐渐回复，最终存活率为66％。由此可知，H1单体间二硫键突变体对小鼠的保护效果优于野生型。
由图9-11可知，Wash11和NC99野生型和引入了单体间二硫键的H1突变体抗原均在小鼠体内诱导了高滴度的抗原特异性抗体反应，该抗体反应能够交叉结合不同毒株的H1、H2、H5和H7。引入了单体间二硫键的H1突变体诱导的H5和H7交叉反应性抗体滴度水平略低于Wash11和NC99野生型H1，各组均未检测到His标签的特异性抗体。
如表1公开的Wash11/NC99 WT&interCC Mutant的二免小鼠血清中和效价(100TCID50/Well,DPI3)，可知Wash11 WT,Wash11#4和Wash11#5的免疫血清均不能中和PR8H1N1病毒，对pdm09重配病毒X179AH1N1病毒的中和效价均高于1280，Wash11#4和Wash11#5免疫血清对NJ76 H1N1病毒的中和效价分别为40和320，显著高于Wash11 WT中和效价20；NC99 WT免疫血清对PR8和X179AH1N1病毒的中和效价小于10，NC99#2对二者的中和效价分别为20和10，NC99 WT和NC99#2对NJ76 H1N1的中和效价均为10；另外，所有H1的免疫血清均不能中和H3N2，H3N8，H9N2和IBV。
表1 流感病毒体外微量中和试验测定Wash11 WT、Wash11#4、Wash11#5、NC99 WT和NC99#2的二免小鼠血清的中和效价
Figure BDA0003259759290000131
Figure BDA0003259759290000141
如表2公开的Wash11/NC99 WT&interCC Mutant的二免小鼠血清血凝抑制效价(血凝抑制实验使用4×HA的灭活流感病毒PR8 H1N1、X179A H1N1和NJ76 H1N1)，可知Wash11WT、Wash11#4和Wash11#5的免疫血清均不能抑制PR8 H1N1病毒的血凝现象，对pdm09重配病毒X179AH1N1的血凝抑制效价分别为512、1024和2048，对NJ76 H1N1的血凝抑制效价分别为32、32和64；NC99 WT和NC99#2的二免小鼠血清均不能抑制PR8 H1N1、X179AH1N1和NJ76H1N1的血凝现象。
表2 血凝抑制试验测定Wash11 WT、Wash11#4、Wash11#5、NC99 WT和NC99#2二免小鼠血清的血凝抑制效价
Wash11 WT Wash11#4 Wash11#5 NC99 WT NC99#2 PBS
PR8 H1N1 <8 <8 <8 <8 <8 <8
X179A H1N1 512 1024 2048 <8 <8 <8
NJ76 H1N1 32 32 64 <8 <8 <8
小鼠接种单体内二硫键突变体抗原后进行流感病毒攻毒实验：
将6-8周龄SPF级BALB/c雌鼠随机分为5-6只/笼。将各种H1重组蛋白或PBS与弗氏不完全佐剂按照体积比1：1混合乳化后，按照10μg免疫原每只的剂量腹腔注射接种小鼠。初次免疫前2天，初次免疫后21天和增强免疫后14天采集血清检测特异性抗原滴度。将50μl10倍半致死剂量(50％lethal dose，LD50)的H1N1流感病毒PR8和X179A通过滴鼻方式攻毒，并记录感染后14天内各组小鼠的体重变化和存活率。
ELISA检测小鼠血清的H1特异性抗原和交叉反应性抗原滴度：
小鼠血清用PBS 10倍倍比稀释，其他操作方法同上。
如图17所示，在10倍致死剂量X179A H1N1病毒的攻击下，免疫Wash11#5、Wash11#5-2和Wash11#5-3的小鼠感染后平均体重维持在原始值的90％以上，最终存活率分别为100％、100％和80％，免疫Wash11#5-1的小鼠感染后第5天平均体重降至原始值的78％左右，最终存活率为83％；在10倍致死剂量PR8 H1N1病毒的攻击下，免疫Wash11#5的小鼠感染后第9天体重降至原始值的78％左右，之后平均体重逐渐回复，最终存活率为40％；免疫Wash11#5-1的小鼠感染后第8天体重降至原始值的73％左右，之后平均体重逐渐回复，最终存活率为20％；免疫Wash11#5-2和Wash11#5-3的小鼠攻毒后体重逐渐降低，最终全部死亡。如图18所示，在10倍致死剂量X179AH1N1病毒的攻击下，免疫NC99#2的小鼠感染后第6天平均体重降至原始值的75％左右，之后体重逐渐回复，最终存活率为33％；免疫NC99#2-1的小鼠感染后第8天平均体重降至原始值的73％左右，之后体重逐渐回复，最终存活率为60％；免疫NC99#2-3的小鼠感染后第8天体重降至原始值的82％左右，之后体重逐渐回复，最终存活率为100％；免疫NC99#2-2和NC99#2-4的小鼠全部死亡；10×LD50 PR8 H1N1攻毒后，免疫NC99#2的小鼠感染后第8天体重降低到原始值的78％左右，之后体重逐渐回复，最终存活率为33％；免疫NC99#2-3的小鼠感染后第8天平均体重降低到原始值的72％左右，之后体重逐渐回复，最终存活率为25％；免疫NC99#2-1和NC99#2-2和NC99#2-4的小鼠攻毒后体重逐渐降低，最终全部死亡。
由图14-16可知，Wash11#5和NC99#2和单体内二硫键突变体均在小鼠体内诱导了高滴度H1特异性抗体，H1不同毒株的交叉反应性抗体和H2交叉反应性抗体，Wash11单体内二硫键突变体Wash11#5-1和Wash11#5-2诱导的H2和H7交叉反应性抗体水平高于Wash11#5。
如表3公开的Wash11#5/NC99#2&intraCC Mutant小鼠二免血清中和效价(200TCID50/Well,DPI3)，可知Wash11#5,Wash11#5-2和Wash11#5-3的免疫血清均不能中和PR8 H1N1病毒，Wash11#5-1的免疫血清对PR8 H1N1病毒的中和效价为10；Wash11#5和Wash11#5-3的免疫血清对pdm09重配病毒X179AH1N1病毒的中和效价为1280，Wash11#5-1和Wash11#5-2的免疫血清对pdm09重配病毒X179A H1N1病毒的中和效价高于1280；Wash11#5，Wash11#5-1，Wash11#5-2，Wash11#5-3和Wash11#5-4的免疫血清对NJ76 H1N1病毒的中和效价分别为160，80，160和40；NC99#2，NC99#2-2和NC99#2-3对PR8 H1N1病毒的中和效价为20，NC99#2-1和NC99#2-4不能中和PR8 H1N1病毒；NC99#2和NC99#2-1-NC99#2-3对X179AH1N1病毒的中和效价为10，NC99#2-4不能中和X179A H1N1病毒；NC99#2和NC99#2-1-NC99#2-4对NJ76 H1N1的中和效价均为10。
表3 流感病毒体外微量中和试验测定Wash11#5、Wash11#5-1、Wash11#5-2、Wash11#5-3、NC99#2、NC99#2-1、NC99#2-2、NC99#2-3和NC99#2-4的二免小鼠血清的中和效价
Figure BDA0003259759290000161
如表4公开的Wash11#5/NC99#2&intraCC Mutant小鼠二免血清血凝抑制效价(4*HA)，可知Wash11#5和Wash11#5-1-Wash11#5-3的免疫血清均不能抑制PR8 H1N1病毒的血凝现象，对pdm09重配病毒X179AH1N1病毒的血凝抑制效价分别为512、1024、2048和256，对NJ76 H1N1病毒的血凝抑制效价分别为64、32、32和16；NC99#2和NC99#2-1-NC99#2-4的免疫血清均不能抑制PR8 H1N1、X179AH1N1和NJ76 H1N1病毒的血凝现象。
表4 血凝抑制试验测定Wash11#5、Wash11#5-1、Wash11#5-2、Wash11#5-3、NC99#2、NC99#2-1、NC99#2-2、NC99#2-3和NC99#2-4的二免小鼠血清的血凝抑制效价
Figure BDA0003259759290000171
以上所述实施例，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明的技术范围内，根据本发明的技术方案及其构思加以等同替换或改变，都应涵盖在本发明的保护范围内。
序列表
<110> 湖南大学
<120> 一种提高甲型流感病毒H1三聚体蛋白抗原性的改造方法
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 543
<212> PRT
<213> Artificial Sequence
<400> 1
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly
340 345 350
Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly
355 360 365
Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala
370 375 380
Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn Lys Val
385 390 395 400
Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys
405 410 415
Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val
420 425 430
Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val
435 440 445
Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys
450 455 460
Asn Leu Tyr Glu Lys Val Arg Asn Gln Leu Lys Asn Asn Ala Lys Glu
465 470 475 480
Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys
485 490 495
Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu
500 505 510
Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser
515 520 525
Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 2
<211> 543
<212> PRT
<213> Artificial Sequence
<400> 2
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly
340 345 350
Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly
355 360 365
Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala
370 375 380
Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn Lys Val
385 390 395 400
Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys
405 410 415
Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val
420 425 430
Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val
435 440 445
Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys
450 455 460
Asn Leu Tyr Glu Lys Val Arg Asn Gln Leu Lys Asn Asn Ala Lys Glu
465 470 475 480
Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys
485 490 495
Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu
500 505 510
Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser
515 520 525
Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 3
<211> 543
<212> PRT
<213> Artificial Sequence
<400> 3
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly
340 345 350
Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly
355 360 365
Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala
370 375 380
Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn Lys Val
385 390 395 400
Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Cys
405 410 415
Glu Phe Asn His Leu Glu Lys Cys Ile Glu Asn Leu Asn Lys Lys Val
420 425 430
Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val
435 440 445
Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys
450 455 460
Asn Leu Tyr Glu Lys Val Arg Asn Gln Leu Lys Asn Asn Ala Lys Glu
465 470 475 480
Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys
485 490 495
Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu
500 505 510
Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser
515 520 525
Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 4
<211> 543
<212> PRT
<213> Artificial Sequence
<400> 4
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Cys Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly
340 345 350
Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly
355 360 365
Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala
370 375 380
Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Cys Ile Thr Asn Lys Val
385 390 395 400
Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys
405 410 415
Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val
420 425 430
Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val
435 440 445
Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys
450 455 460
Asn Leu Tyr Glu Lys Val Arg Asn Gln Leu Lys Asn Asn Ala Lys Glu
465 470 475 480
Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys
485 490 495
Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu
500 505 510
Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser
515 520 525
Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 5
<211> 543
<212> PRT
<213> Artificial Sequence
<400> 5
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Gln Gly Leu Phe Cys
340 345 350
Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly
355 360 365
Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala
370 375 380
Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn Lys Val
385 390 395 400
Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys
405 410 415
Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val
420 425 430
Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val
435 440 445
Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys
450 455 460
Cys Leu Tyr Glu Lys Val Arg Asn Gln Leu Lys Asn Asn Ala Lys Glu
465 470 475 480
Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys
485 490 495
Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu
500 505 510
Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser
515 520 525
Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 6
<211> 544
<212> PRT
<213> Artificial Sequence
<400> 6
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly
340 345 350
Ala Ile Ala Gly Cys Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp
355 360 365
Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala
370 375 380
Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn Lys
385 390 395 400
Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly
405 410 415
Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys
420 425 430
Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu
435 440 445
Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val
450 455 460
Lys Asn Leu Tyr Glu Cys Val Arg Asn Gln Leu Lys Asn Asn Ala Lys
465 470 475 480
Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr
485 490 495
Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser
500 505 510
Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu
515 520 525
Ser Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 7
<211> 545
<212> PRT
<213> Artificial Sequence
<400> 7
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly
340 345 350
Ala Ile Ala Gly Cys Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val
355 360 365
Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr
370 375 380
Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn
385 390 395 400
Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val
405 410 415
Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys
420 425 430
Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu
435 440 445
Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn
450 455 460
Val Lys Asn Leu Tyr Glu Cys Val Arg Asn Gln Leu Lys Asn Asn Ala
465 470 475 480
Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn
485 490 495
Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr
500 505 510
Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu
515 520 525
Glu Ser Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His
530 535 540
His
545
<210> 8
<211> 543
<212> PRT
<213> Artificial Sequence
<400> 8
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly
340 345 350
Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly
355 360 365
Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala
370 375 380
Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn Lys Val
385 390 395 400
Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys
405 410 415
Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val
420 425 430
Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val
435 440 445
Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys
450 455 460
Asn Leu Tyr Glu Lys Val Arg Cys Gln Leu Lys Asn Asn Ala Lys Glu
465 470 475 480
Ile Cys Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys
485 490 495
Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu
500 505 510
Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser
515 520 525
Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 9
<211> 544
<212> PRT
<213> Artificial Sequence
<400> 9
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Cys Arg Asn Val Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly
340 345 350
Ala Ile Ala Gly Cys Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp
355 360 365
Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala
370 375 380
Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn Lys
385 390 395 400
Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly
405 410 415
Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys
420 425 430
Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu
435 440 445
Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr Cys Asp Ser Asn Val
450 455 460
Lys Asn Leu Tyr Glu Cys Val Arg Asn Gln Leu Lys Asn Asn Ala Lys
465 470 475 480
Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr
485 490 495
Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser
500 505 510
Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu
515 520 525
Ser Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 10
<211> 544
<212> PRT
<213> Artificial Sequence
<400> 10
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Leu Arg Asn Cys Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly
340 345 350
Ala Ile Ala Gly Cys Phe Ile Glu Gly Cys Trp Thr Gly Met Val Asp
355 360 365
Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala
370 375 380
Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn Lys
385 390 395 400
Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly
405 410 415
Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys
420 425 430
Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu
435 440 445
Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val
450 455 460
Lys Asn Leu Tyr Glu Cys Val Arg Asn Gln Leu Lys Asn Asn Ala Lys
465 470 475 480
Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr
485 490 495
Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser
500 505 510
Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu
515 520 525
Ser Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 11
<211> 544
<212> PRT
<213> Artificial Sequence
<400> 11
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Cys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly
340 345 350
Ala Ile Ala Gly Cys Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp
355 360 365
Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala
370 375 380
Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn Lys
385 390 395 400
Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly
405 410 415
Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys
420 425 430
Val Asp Asp Gly Phe Leu Asp Ile Trp Cys Tyr Asn Ala Glu Leu Leu
435 440 445
Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val
450 455 460
Lys Asn Leu Tyr Glu Cys Val Arg Asn Gln Leu Lys Asn Asn Ala Lys
465 470 475 480
Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr
485 490 495
Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser
500 505 510
Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu
515 520 525
Ser Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 12
<211> 545
<212> PRT
<213> Artificial Sequence
<400> 12
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Cys Arg Asn Val Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly
340 345 350
Ala Ile Ala Gly Cys Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val
355 360 365
Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr
370 375 380
Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn
385 390 395 400
Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val
405 410 415
Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys
420 425 430
Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu
435 440 445
Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr Cys Asp Ser Asn
450 455 460
Val Lys Asn Leu Tyr Glu Cys Val Arg Asn Gln Leu Lys Asn Asn Ala
465 470 475 480
Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn
485 490 495
Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr
500 505 510
Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu
515 520 525
Glu Ser Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His
530 535 540
His
545
<210> 13
<211> 545
<212> PRT
<213> Artificial Sequence
<400> 13
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Leu Arg Asn Cys Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly
340 345 350
Ala Ile Ala Gly Cys Gly Phe Ile Glu Gly Cys Trp Thr Gly Met Val
355 360 365
Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr
370 375 380
Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn
385 390 395 400
Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val
405 410 415
Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys
420 425 430
Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu
435 440 445
Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn
450 455 460
Val Lys Asn Leu Tyr Glu Cys Val Arg Asn Gln Leu Lys Asn Asn Ala
465 470 475 480
Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn
485 490 495
Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr
500 505 510
Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu
515 520 525
Glu Ser Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His
530 535 540
His
545
<210> 14
<211> 545
<212> PRT
<213> Artificial Sequence
<400> 14
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys
50 55 60
Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
130 135 140
Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala
145 150 155 160
Gly Ala Lys Gly Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
165 170 175
Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys
180 185 190
Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr Ala Asp
195 200 205
Gln Gln Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Thr
210 215 220
Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys
225 230 235 240
Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu
245 250 255
Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro
260 265 270
Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile
275 280 285
Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
290 295 300
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr
305 310 315 320
Ile Gly Lys Cys Pro Lys Tyr Val Cys Ser Thr Lys Leu Arg Leu Ala
325 330 335
Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly
340 345 350
Ala Ile Ala Gly Cys Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val
355 360 365
Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr
370 375 380
Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn
385 390 395 400
Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val
405 410 415
Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys
420 425 430
Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Cys Tyr Asn Ala Glu Leu
435 440 445
Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn
450 455 460
Val Lys Asn Leu Tyr Glu Cys Val Arg Asn Gln Leu Lys Asn Asn Ala
465 470 475 480
Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn
485 490 495
Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr
500 505 510
Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu
515 520 525
Glu Ser Thr Arg Ile Tyr Gln Ile Glu Gly Arg His His His His His
530 535 540
His
545
<210> 15
<211> 542
<212> PRT
<213> Artificial Sequence
<400> 15
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu
50 55 60
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
130 135 140
Pro Asn His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly
145 150 155 160
Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly
165 170 175
Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu
180 185 190
Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asn Gln
195 200 205
Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser
210 215 220
His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
225 230 235 240
Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
245 250 255
Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
260 265 270
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser
275 280 285
Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly
290 295 300
Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile
305 310 315 320
Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr
325 330 335
Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp
355 360 365
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
370 375 380
Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn
385 390 395 400
Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu
405 410 415
Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp
420 425 430
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
435 440 445
Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
450 455 460
Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
465 470 475 480
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met
485 490 495
Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
500 505 510
Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met
515 520 525
Gly Val Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 16
<211> 542
<212> PRT
<213> Artificial Sequence
<400> 16
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu
50 55 60
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
130 135 140
Pro Asn His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly
145 150 155 160
Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly
165 170 175
Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu
180 185 190
Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asn Gln
195 200 205
Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser
210 215 220
His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
225 230 235 240
Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
245 250 255
Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
260 265 270
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser
275 280 285
Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly
290 295 300
Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile
305 310 315 320
Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr
325 330 335
Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp
355 360 365
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
370 375 380
Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn
385 390 395 400
Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu
405 410 415
Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp
420 425 430
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
435 440 445
Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
450 455 460
Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
465 470 475 480
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met
485 490 495
Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
500 505 510
Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met
515 520 525
Gly Val Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 17
<211> 542
<212> PRT
<213> Artificial Sequence
<400> 17
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu
50 55 60
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
130 135 140
Pro Asn His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly
145 150 155 160
Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly
165 170 175
Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu
180 185 190
Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asn Gln
195 200 205
Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser
210 215 220
His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
225 230 235 240
Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
245 250 255
Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
260 265 270
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser
275 280 285
Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly
290 295 300
Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile
305 310 315 320
Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr
325 330 335
Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp
355 360 365
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
370 375 380
Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn
385 390 395 400
Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Cys Glu
405 410 415
Phe Asn Lys Leu Glu Arg Cys Met Glu Asn Leu Asn Lys Lys Val Asp
420 425 430
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
435 440 445
Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
450 455 460
Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
465 470 475 480
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met
485 490 495
Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
500 505 510
Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met
515 520 525
Gly Val Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 18
<211> 542
<212> PRT
<213> Artificial Sequence
<400> 18
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Cys Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu
50 55 60
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
130 135 140
Pro Asn His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly
145 150 155 160
Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly
165 170 175
Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu
180 185 190
Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asn Gln
195 200 205
Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser
210 215 220
His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
225 230 235 240
Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
245 250 255
Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
260 265 270
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser
275 280 285
Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly
290 295 300
Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile
305 310 315 320
Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr
325 330 335
Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp
355 360 365
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
370 375 380
Gln Lys Ser Thr Gln Asn Ala Ile Asn Cys Ile Thr Asn Lys Val Asn
385 390 395 400
Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu
405 410 415
Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp
420 425 430
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
435 440 445
Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
450 455 460
Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
465 470 475 480
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met
485 490 495
Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
500 505 510
Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met
515 520 525
Gly Val Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 19
<211> 542
<212> PRT
<213> Artificial Sequence
<400> 19
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu
50 55 60
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
130 135 140
Pro Asn His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly
145 150 155 160
Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly
165 170 175
Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu
180 185 190
Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asn Gln
195 200 205
Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser
210 215 220
His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
225 230 235 240
Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
245 250 255
Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
260 265 270
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser
275 280 285
Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly
290 295 300
Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile
305 310 315 320
Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr
325 330 335
Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Gln Gly Leu Phe Cys Ala
340 345 350
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp
355 360 365
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
370 375 380
Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn
385 390 395 400
Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu
405 410 415
Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp
420 425 430
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
435 440 445
Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Cys
450 455 460
Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
465 470 475 480
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met
485 490 495
Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
500 505 510
Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met
515 520 525
Gly Val Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 20
<211> 543
<212> PRT
<213> Artificial Sequence
<400> 20
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu
50 55 60
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
130 135 140
Pro Asn His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly
145 150 155 160
Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly
165 170 175
Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu
180 185 190
Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asn Gln
195 200 205
Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser
210 215 220
His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
225 230 235 240
Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
245 250 255
Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
260 265 270
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser
275 280 285
Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly
290 295 300
Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile
305 310 315 320
Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr
325 330 335
Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Cys Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly
355 360 365
Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala
370 375 380
Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val
385 390 395 400
Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys
405 410 415
Glu Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val
420 425 430
Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val
435 440 445
Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys
450 455 460
Asn Leu Tyr Glu Cys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu
465 470 475 480
Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys
485 490 495
Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu
500 505 510
Glu Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser
515 520 525
Met Gly Val Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 21
<211> 544
<212> PRT
<213> Artificial Sequence
<400> 21
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu
50 55 60
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
130 135 140
Pro Asn His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly
145 150 155 160
Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly
165 170 175
Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu
180 185 190
Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asn Gln
195 200 205
Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser
210 215 220
His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
225 230 235 240
Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
245 250 255
Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
260 265 270
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser
275 280 285
Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly
290 295 300
Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile
305 310 315 320
Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr
325 330 335
Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Cys Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp
355 360 365
Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala
370 375 380
Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys
385 390 395 400
Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly
405 410 415
Lys Glu Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys
420 425 430
Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu
435 440 445
Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
450 455 460
Lys Asn Leu Tyr Glu Cys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys
465 470 475 480
Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu
485 490 495
Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser
500 505 510
Glu Glu Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu
515 520 525
Ser Met Gly Val Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 22
<211> 542
<212> PRT
<213> Artificial Sequence
<400> 22
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu
50 55 60
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
130 135 140
Pro Asn His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly
145 150 155 160
Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly
165 170 175
Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu
180 185 190
Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asn Gln
195 200 205
Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser
210 215 220
His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
225 230 235 240
Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
245 250 255
Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
260 265 270
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser
275 280 285
Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly
290 295 300
Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile
305 310 315 320
Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr
325 330 335
Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp
355 360 365
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
370 375 380
Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn
385 390 395 400
Ser Val Ile Glu Cys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu
405 410 415
Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp
420 425 430
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Cys Leu Leu Val Leu
435 440 445
Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
450 455 460
Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
465 470 475 480
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met
485 490 495
Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
500 505 510
Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met
515 520 525
Gly Val Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 23
<211> 542
<212> PRT
<213> Artificial Sequence
<400> 23
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Cys Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu
50 55 60
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
130 135 140
Pro Asn His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly
145 150 155 160
Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly
165 170 175
Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu
180 185 190
Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asn Gln
195 200 205
Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser
210 215 220
His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
225 230 235 240
Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
245 250 255
Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
260 265 270
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser
275 280 285
Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly
290 295 300
Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile
305 310 315 320
Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr
325 330 335
Gly Cys Arg Asn Ile Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp
355 360 365
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
370 375 380
Gln Lys Ser Thr Gln Asn Ala Ile Asn Cys Ile Thr Asn Lys Val Asn
385 390 395 400
Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu
405 410 415
Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp
420 425 430
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
435 440 445
Leu Glu Asn Glu Arg Thr Leu Asp Phe Cys Asp Ser Asn Val Lys Asn
450 455 460
Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
465 470 475 480
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met
485 490 495
Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
500 505 510
Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met
515 520 525
Gly Val Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 24
<211> 542
<212> PRT
<213> Artificial Sequence
<400> 24
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Cys Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu
50 55 60
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
130 135 140
Pro Asn His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly
145 150 155 160
Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly
165 170 175
Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu
180 185 190
Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asn Gln
195 200 205
Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser
210 215 220
His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
225 230 235 240
Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
245 250 255
Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
260 265 270
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser
275 280 285
Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly
290 295 300
Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile
305 310 315 320
Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Cys
325 330 335
Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp
355 360 365
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
370 375 380
Gln Lys Ser Thr Gln Asn Ala Ile Asn Cys Cys Thr Asn Lys Val Asn
385 390 395 400
Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu
405 410 415
Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp
420 425 430
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
435 440 445
Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
450 455 460
Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
465 470 475 480
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met
485 490 495
Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
500 505 510
Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met
515 520 525
Gly Val Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 25
<211> 542
<212> PRT
<213> Artificial Sequence
<400> 25
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Cys Cys Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu
50 55 60
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
130 135 140
Pro Asn His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly
145 150 155 160
Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly
165 170 175
Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu
180 185 190
Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asn Gln
195 200 205
Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser
210 215 220
His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
225 230 235 240
Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
245 250 255
Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
260 265 270
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser
275 280 285
Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly
290 295 300
Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile
305 310 315 320
Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr
325 330 335
Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp
355 360 365
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
370 375 380
Gln Lys Ser Thr Gln Asn Ala Ile Asn Cys Ile Thr Asn Lys Val Asn
385 390 395 400
Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu
405 410 415
Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp
420 425 430
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
435 440 445
Leu Glu Asn Cys Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
450 455 460
Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
465 470 475 480
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met
485 490 495
Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
500 505 510
Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met
515 520 525
Gly Val Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540
<210> 26
<211> 542
<212> PRT
<213> Artificial Sequence
<400> 26
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn
20 25 30
Ser Thr Asp Thr Val Asp Thr Val Cys Glu Lys Asn Val Thr Val Thr
35 40 45
His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu
50 55 60
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly
65 70 75 80
Trp Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser
85 90 95
Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr
100 105 110
Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
115 120 125
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
130 135 140
Pro Asn His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly
145 150 155 160
Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly
165 170 175
Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu
180 185 190
Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asn Gln
195 200 205
Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser
210 215 220
His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
225 230 235 240
Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
245 250 255
Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
260 265 270
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser
275 280 285
Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly
290 295 300
Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile
305 310 315 320
Gly Glu Cys Pro Lys Tyr Val Cys Ser Ala Lys Leu Arg Met Val Thr
325 330 335
Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp
355 360 365
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
370 375 380
Gln Lys Ser Thr Gln Asn Ala Ile Asn Cys Ile Thr Asn Lys Val Asn
385 390 395 400
Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu
405 410 415
Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp
420 425 430
Asp Gly Phe Leu Asp Ile Trp Cys Tyr Asn Ala Glu Leu Leu Val Leu
435 440 445
Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
450 455 460
Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
465 470 475 480
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met
485 490 495
Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
500 505 510
Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met
515 520 525
Gly Val Tyr Gln Ile Glu Gly Arg His His His His His His
530 535 540

Claims (9)

1.一种提高甲型流感病毒H1三聚体蛋白抗原性的改造方法，其特征在于，所述方法具体包括：
将甲型流感病毒H1的信号肽序列进行置换成昆虫细胞蜂毒素信号肽序列，并在H1胞外基因序列的羧基端添加切割位点和亲和纯化标签序列，依据昆虫细胞宿主优化密码子，合成在pFastBac1载体中，获得带有甲型流感病毒H1序列的重组质粒；
在所述带有甲型流感病毒H1序列的重组质粒的H1胞外域基因序列引入单体间或/和单体内半胱氨酸突变获得H1突变体的pFastBac1重组质粒，所述突变包括点突变和插入氨基酸突变；
利用bac-to-bac昆虫杆状病毒表达系统表达所述H1突变体并纯化获得H1三聚体重组蛋白。
2.根据权利要求1所述的提高甲型流感病毒H1三聚体蛋白抗原性的改造方法，其特征在于，所述切割位点包括：FactorXa切割位点、凝血酶裂解位点、HRV3C蛋白酶切位点、TEV蛋白酶切位点、肠激酶切位点、SUMO蛋白酶切位点；
所述标签序列包括：6×His标签、HA标签、FLAG标签、谷胱甘肽巯基转移酶标签、麦芽糖结合蛋白标签、NusA标签、SUMO标签、Strep-tag标签。
3.根据权利要求1所述的提高甲型流感病毒H1三聚体蛋白抗原性的改造方法，其特征在于，所述甲型流感病毒H1包括但不限于毒株A/Washington/5/2011(H1N1)、毒株A/NewCaledonia/20/1999(H1N1)和与所述毒株A/Washington/5/2011(H1N1)、毒株A/NewCaledonia/20/1999(H1N1)的氨基酸序列具有89％以上同源性的毒株。
4.根据权利要求3所述的提高甲型流感病毒H1三聚体蛋白抗原性的改造方法，其特征在于，所述毒株A/Washington/5/2011(H1N1)的突变位点包括单体间点突变K68C-R76C、L20C-K47C、G4C-N117C，插入氨基酸9C-K121C、9C10G-K121C、N124C-G134C；并以插入氨基酸9C-K121C、9C10G-K121C的质粒为模板再次引入单体内点突变，包括L318C-H111C、V321C-G13C、K308C-T93C。
5.根据权利要求3所述的提高甲型流感病毒H1三聚体蛋白抗原性的改造方法，其特征在于，所述毒株A/NewCaledonia/20/1999(H1N1)的突变位点包括单体间点突变K68C-R76C、L24C-G47C、G4C-N117C，插入氨基酸9C-K121C、9C10G-K121C、E97C-K58C；并以点突变L24C-G47C的质粒为模板再次引入单体内点突变，包括L321C-H111C、T319C-I48C、V23C-E105C、R311C-T93C。
6.根据权利要求1所述的提高甲型流感病毒H1三聚体蛋白抗原性的改造方法，其特征在于，利用bac-to-bac昆虫杆状病毒表达系统表达所述H1突变体并纯化获得H1三聚体重组蛋白过程具体包括：
将编码所述H1突变体的pFastBac1重组质粒进行转化DH10Bac菌株后，蓝白斑筛选获得重组bacmid，将bacmid转染静置培养的昆虫细胞sf9制备P1代重组杆状病毒，将0.5mlP1代感染20ml生长期状态1×106/ml密度sf9制备病毒滴度更高的P2代重组杆状病毒，利用P2重组杆状病毒感染较大体积sf9细胞培养物表达H1突变体重组蛋白；
搜集上清液，采用聚丙烯酰胺凝胶电泳法、考马斯亮蓝染色法、Western blot方法检测并确认H1的表达情况和三聚体状态，离心上清液后，通过亲和层析柱和尺寸排阻色谱柱纯化获得H1突变体重组蛋白；
所述将编码H1突变体的DNA片段重组的真核表达载体包括但不限于pCDNA3.1、pCAGGS质粒载体，表达重组蛋白的细胞株包括但不限于HEK293、CHO和MDCK细胞株。
7.重组甲型流感病毒H1三聚体抗原，其特征在于，所述H1三聚体抗原由权利要求1-6任意所述的改造方法制备获得；
所述重组H1三聚体抗原的氨基酸序列如SEQ ID NO:3-14和SEQ ID NO:17-26所示；
所述重组H1三聚体抗原的氨基酸序列还包括与如SEQ ID NO:3-14和SEQ ID NO:17-26所示氨基酸序列具有89％以上同源性的氨基酸序列。
8.一种重组甲型流感病毒疫苗，其特征在于，所述甲型流感病毒疫苗包括权利要求1-6任意所述改造方法制备获得或权利要求7任意所述的重组甲型流感病毒H1三聚体抗原。
9.根据权利要求8所述的甲型流感病毒疫苗，其特征在于，所述疫苗还包括疫苗载体，所述疫苗载体包括：DNA载体、mRNA载体、水凝胶载体、脱溶剂纳米颗粒载体、蛋白质自组装纳米颗粒载体、腺病毒载体、慢病毒载体、植物病毒载体、减毒人感染病毒载体、人感染复制缺陷型病毒载体，以及破伤风类毒素、白喉类毒素、白喉毒素的无毒变异体、B群脑膜炎球菌外膜蛋白复合体、白介素、病原相关分子模式、损伤相关分子模式融合蛋白载体。
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