CN114761808A - 使用免疫生物标志物对老年人的健康状况进行鉴定的方法 - Google Patents

使用免疫生物标志物对老年人的健康状况进行鉴定的方法 Download PDF

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CN114761808A
CN114761808A CN202080080356.4A CN202080080356A CN114761808A CN 114761808 A CN114761808 A CN 114761808A CN 202080080356 A CN202080080356 A CN 202080080356A CN 114761808 A CN114761808 A CN 114761808A
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J·M·布莱克伯恩
A·安沃
N·H·拉特
A·拉尔比
O·N·F·赛克斯
B·李
J·F·B·瓦伦祖埃拉
C·蒙特罗拉
V·童
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Agency for Science Technology and Research Singapore
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Abstract

一种通过测试从个体提取的样品中存在生物标志物来确定老年人健康状况的方法，该生物标志物是针对如下抗原的自身抗体，该抗原包括MAPK13、CD96、FKBP3、PPM1A、PHLDA1、GLRX3、FEN1和AURKA，其中该抗原还可以包括UBE2I、AAK1、YARS、ASPSCR1、CASP10、FHOD2、TCL1A和MAP4中的一种或多种，其中PHLDA1和CD96对应于健康，AURKA、FEN1、CASP10和AAK1对应于中等健康，并且UBE2I、YARS、ASPSCR1、FHOD2、TCL1A、MAP4、MAPK13、FKBP3、PPM1A和GLRX3对应于不健康。

Description

使用免疫生物标志物对老年人的健康状况进行鉴定的方法
技术领域
本发明涉及免疫生物标志物，具体是自身抗体的检测，以确定老年人的健康状况和/或衰老轨迹。
背景技术
尽管蛋白质组学研究领域取得了技术进步，但进入临床的生物标志物屈指可数，并且90％的生物标志物是蛋白质生物标志物[1]。本文所述的自身抗体生物标志物是针对抗原的自身抗体，自身抗体是由个体生成的、针对个体自身蛋白质中的一种或多种(“自身抗原”)的抗体。生物标志物[2]未能进入临床实践的一些主要原因是：
1)低敏感性和特异性
2)低预后/预测价值
3)对临床决策不重要
4)原始声明未通过验证(错误发现)
老年人的护理管理与其说取决于年龄，不如说取决于他们的共患病病史(过去和现在)对其当前健康状况的影响[3]。这些共患病对免疫系统施加了一定的压力，多年来免疫系统处理感染、癌症或慢性炎性疾病一直受到挑战[4]。
因此，本发明的目标是提供改进的一组(panel)自身抗体生物标志物，其用于评估老年人的健康状况。
发明简述
在本发明的一个方面中，提供了一种用于由从个体提取的样品确定该个体的健康的方法，包括以下步骤：
(i)测试样品中对健康是特定的生物标志物的存在；
(ii)基于对所述生物标志物的检测确定所述受试者是健康、中等健康或不健康，
其特征在于所述生物标志物是针对抗原的自身抗体，所述抗原包括AURKA、FEN1、GLRX3、PHLDA1、PPM1A、FKBP3、CD96和MAPK13。
在一个实施方式中，个体是老年人，通常至少60岁。
有利的是，自身抗体生物标志物可用于通过测量血浆抗体水平的分布表征(或诊断)老年个体的健康状况(健康、中等健康和不健康)。此外，这些自身抗体生物标志物的一个子集，特别是与健康和中等健康相关的那些，可能对非传染性疾病具有保护作用。
在一个实施方式中，使用与自身抗体生物标志物相对应的一组抗原对样品进行检测。通常，抗原是生物素化蛋白质。有利的是，生物素化确保抗原以其正确的形式折叠，以确保利用自身抗体生物标志物进行检测的准确性。
在一个实施方式中，抗原可包括UBE2I、AAK1、YARS、ASPSCR1、CASP10、FHOD2、TCL1A和MAP4中的一种或多种。
应该注意的是，不是所有的抗原产生自身抗体反映并且不能预测的先验，抗原在给定同期组群-超过1500种检测的抗原会进行先验，只有上述16个抗原的自身抗体是适合作为生物标志物以识别健康和衰老状态。
在一个实施方式中，每个生物素化蛋白质由生物素羧基载体蛋白质(BCCP)折叠标志物形成，该折叠标志物与蛋白质框内融合。
在进一步的实施方式中，生物素化蛋白质与链霉亲和素包被的基底结合。
有利的是，全长蛋白被表达为BCCP折叠标志物的融合物，当融合标签被正确折叠时，该标志物本身在体内被生物素化。相比之下，错误折叠的融合标签导致BCCP保持为“apo”(即非生物素化)形式，这样它就不能附着在链霉亲和素基底上。因此，只有正确折叠的融合蛋白才能通过附着在BCCP标志上的生物素部分附着在链霉亲和素基底上。
在一个实施方式中，基底包括玻璃载玻片、生物芯片、条、载玻片、珠、微滴板孔、表面等离子体共振支架、微流控装置、薄膜聚合物基层、水凝胶形成聚合物基层或任何其他适用于检测抗体-抗原结合的装置或技术。
在一个实施方式中，基底暴露于从人体提取的样品，由此来自样品的自身抗体生物标志物可与抗原结合。
通常，样品包括外泌体、血液、血清、血浆、尿液、唾液、羊水、脑脊液、母乳、精液或胆汁的任一种或它们的任意组合。
在一个实施方式中，在暴露于样品之后，将基底暴露于荧光标记的第二抗体，以允许来自样品、结合到所述组上的抗原的任意自身抗体的数量被确定。一般来说，第二抗体是抗人IgG，但必须认识到，其他第二抗体也可以使用，例如抗IgM、抗IgGl、抗IgG2、抗IgG3、抗IgG4或抗IgA。
在一个实施方式中，个体的健康与来自样品、与抗原特异性结合的自身抗体的相对数量或绝对数量相对应。
在一个实施方式中，该方法在体外进行。
在一个实施方式中，该方法包括检测一个或多个生物标志物的上调/下调。
在本发明的进一步方面，提供一种用于制造试剂盒的方法，所述试剂盒用于由从个体提取的样品确定老年个体的健康，所述方法包括以下步骤：
对于组中的每种抗原，用编码所述抗原的基因框内克隆生物素羧基载体蛋白质折叠标志物，并且表达得到的生物素化抗原；
将所述生物素化抗原与一种或多种链霉亲和素包被的基底上的可寻址的位置结合，从而形成抗原阵列；
由此可以通过将所述基底暴露于所述样品并且测量反应来确定与所述组上的抗原结合的来自所述样品的自身抗体的量；
其特征在于抗原包括AURKA、FEN1、GLRX3、PHLDA1、PPM1A、FKBP3、CD96和MAPK13。
在一个实施方式中，抗原可包括UBE2I、AAK1、YARS、ASPSCR1、CASP10、FHOD2、TCL1A和MAP4中的一种或多种。
本发明的进一步方面提供如下方法：其用于通过将包含本文描述的一组抗原的组合物暴露于从个体提取的样品确定该老年个体的健康并且确定来自样品的与抗原结合的自身抗体的水平。
本发明的另外进一步方面提供如下方法：其用于通过将包含本文描述的一组抗原的组合物体外暴露于从老年个体提取的样品来确定该个体的健康，并且确定来自与抗原结合的样品的自身抗体的水平。
在本发明的进一步方面中，提供一种组合物，其包含用于确定老年个体的健康的一组抗原，其特征在于，该抗原包括AURKA、FEN1、GLRX3、PHLDA1、PPM1A、FKBP3、CD96和MAPK13。
在一个实施方式中，抗原可包括UBE2I、AAK1、YARS、ASPSCR1、CASP10、FHOD2、TCL1A和MAP4中的一种或多种。
在一个实施方式中，抗原为生物素化的蛋白。
在一个实施方式中，可测量来自患者的样品中的与抗原体外结合的一种或多种自身抗体生物标志物的量以确定患者的健康状况。
在本发明的进一步方面中，提供一种组合物，其包含用于确定老年患者的健康状态的一组自身抗体生物标志物；
其中在来自患者的样品中测量一种或多种自身抗体生物标志物的水平；
其特征在于，一种或多种自身抗体生物标志物选自自身抗体，该自身抗体针对以下一种或多种抗原是特异性的：AURKA、FEN1、GLRX3、PHLDA1、PPM1A、FKBP3、CD96和MAPK13。
附图说明
对于图解本发明可能配置的附图方便地进一步描述本发明。本发明的其他配置也是可能的，因此附图的特殊性不应被理解为取代本发明之前描述的一般性。
图1图解大肠杆菌生物素羧基载体蛋白质结构域的结构。
图2图解用作载体的pPR09质粒。
图3图解与细胞周期和细胞死亡相关的蛋白质为(A)图表；(B)关联通路。
图4图解聚类分析：(A)通过对16个抗原进行tSNE聚类分析[5]表示在老年人中定义的聚类；(B)各靶蛋白抗体的表达密度；(C)针对健康状况组中的每一个的自身抗体。
图5图解同期组群选择：(A)示意图描述在研究中用于选择和分类老年个体的工作流程；(B)根据年龄和性别的老年和青年个体的分布情况；采用Kruskal-Wallis检验及Dunn校正进行统计分析；(C)用于老年个体分类的临床变量范围；(D)研究中为6个决定性临床参数选择的老年个体的特征。
发明详述
材料和方法的详细说明
基因合成与克隆。pPR09质粒(见下图2)通过标准技术构建且由c-myc标签和BCCP蛋白结构域组成，前面是多克隆位点。从合成的寡核苷酸和/或PCR产物中组装合成的基因植入物。利用Spel和Ncol克隆位点将片段克隆到pPR09中。从转化菌中纯化质粒DNA且用紫外分光光度法确定浓度。通过测序验证最终的构建物。使用限制性位点内的序列一致性为100％。制备的质粒冻干5pg保存。
重组的杆状病毒通过将杆粒与转移载体共转染到Sf9细胞系而生成，该杆粒携带强病毒多面体启动子，该转移载体携带目标蛋白质的编码序列，该Sf9细胞系从亲本草地夜蛾细胞系IPLB-Sf-21-AE衍生的克隆分离菌。由病毒系统引发的同源重组使被转染的细胞在培养温育的数天内显示出病毒细胞病变效应(CPE)的迹象。观察到最常见的CPE的平均细胞大小显著增大，是病毒子代繁殖的结果。随后称为P0的这些杆状病毒释放到培养基中，进行病毒扩增以产生更高滴度的PI病毒。
蛋白质表达。使用每孔包含6×106Sf9细胞的3ml培养基在24孔样品台中进行表达。用高滴度、低传代的重组杆状病毒的病毒原液(>107pfu/ml)感染sf9昆虫细胞。然后将被感染的细胞培养72小时以允许它们生成目标重组蛋白质。用PBS洗涤细胞，在缓冲液中重悬，然后在-80℃条件下冷冻，待裂解。根据转移载体构建体和蛋白质本身的性质，重组蛋白裂解液可以从培养的细胞或培养介质中制成颗粒。然后通过SDS-PAGE和Westernblot对Streptavidin-HRP抗体分析阳性重组蛋白质。使用该方法总共克隆和表达1557种人抗原。
阵列制造。HS(水凝胶-链霉亲和素)载玻片购自Schott且用于打印生物素化蛋白质。使用非接触式压电打印技术，将总共9纳升粗蛋白质裂解液在HS载玻片上打印四份。使用pH值7.0与7.5之间的打印缓冲液。开始洗涤和封闭之前，将载玻片离心(200xg，5分钟)干燥。用磷酸盐缓冲液中含有牛血清白蛋白或酪蛋白(浓度：0.1mg/ml)的溶液封闭打印的阵列。将pH调至7.0与7.5之间且使用冷溶液(4℃-20℃)。在两次洗涤之间不允许载玻片干燥并保护载玻片不受光照。总的来说，每个得到的“免疫组阵列”由1557个抗原组成，每个都打印四份。
实验过程。运行免疫组阵列的每一个关键实验步骤都需要第二名训练有素的人员彻底检查、精确记录和交叉检查方案中的所有步骤以减少操作者偏差。挑选样品，随机分配且相应地分配到检测架上。然后将这些样品保存在-20℃，直到实验设置完成。
1.研究同期组群
研究同期组群被分为两个年龄组：年轻对照个体(YC)和老年个体。YC组(n＝60)由18到27岁的华裔个体男性(n＝34)和女性(n＝26)组成。他们在临床上是健康的，没有报告的并存病或积极的医学治疗。老年个体的选择是在60岁及以上的华裔老年个体中进行的。通过消除种族偏见，这一初步选择增强了本研究的分析能力和结果。
基于6个临床参数(图5A)的结合，进一步选择老年个体到健康类别(健康、中等健康和不健康)：
-并存病5：反映除眼部问题外并存病总数的变量
-NADL：影响老年个体日常生活活动的残疾总数
-Wo_sfl：测量一般生活质量的参数。
-虚弱：临床综合征，其中老年个体越来越易受内部和外部压力的伤害。这是考虑到体力和认知能力的多维变量[8]
-MMSEtot：细微精神状态检查总分。它反映老年个体的认知能力[9]
-GDStot：老年抑郁量表总分。这是一种自我报告的评估，用于识别老年人的抑郁症[10]。
对老年个体健康状况的表征考虑到前面所述的6个参数，因而选择以下的组(图5A)：
-115名健康老年人，
-111名健康状况中等的老年人，
-114名不健康的老年人。
健康组之间没有显著的年龄差异，尽管由于在每个健康组中有更多的女性可以观察到性别差异(图5B、图5D)。在图5D中，粗体数字确定个人对特定类别和分数的等级。括号中的数字代表具有特定特征的个体数量。ND：不确定。平均：每个健康组中所有老年个体的平均年龄[6]。
个体整体的重新分配显示出健康的老年个体出现并发症的积累，虚弱状态增加和认知能力下降，认知能力下降与抑郁状态更多和生活质量改善关联(图5C、图5D)。
2.血清/血浆稀释
然后将样品置于+20℃的摇动培养箱中，使其解冻30分钟。当完全解冻后，每个样品以全速剧烈旋转三次，然后用微型离心机以13000克旋转减慢3分钟。将22.5pL的样品移至包含0.1％v/vTriton、0.1％w/vBSA、10％v/vPBS(20℃)的4.5mL血清分析缓冲液(SAB)中且涡流混合三次。在抽吸过程中，管是倾斜的以确保从顶部的脂质层以下采集血清，但不接触管的底部，以防存在任何沉淀物。血清/血浆稀释过程在二级生物安全柜中进行。批号记录被相应地标记，以确保正确的样品被添加到正确的管中。
3.生物标志物阵列
用镊子将阵列从存储缓冲液中取出，放入包含200mL的冷SAB(4℃)的载玻片盒和机架中且在摇床上以50rpm的转速振荡5分钟。当载玻片冲洗完毕后，阵列朝上将载玻片与早先稀释过的个体血清放置在载玻片杂交室中。所有载玻片均使用条形码扫描仪扫描到相关的批记录中，并在20℃、以50转/分的冷藏摇床中温育2小时。
4.血清结合后的阵列洗涤
蛋白阵列载玻片用30mlSAB在单个的“巴氏瓶”中冲洗两次，然后在室温下，在摇床上，以50rpm在载玻片染色盒中用200mlSAB缓冲液冲洗20分钟。所有载玻片均在相同方向按序转移。
5.与Cv3-抗IgG温育
通过与Cy3-兔抗人IgG温育在复制免疫组阵列上检测自身抗体与排列的抗原的结合。将阵列在20℃、以50rpm浸泡在杂交溶液中2小时，该杂交溶液包含SAB缓冲液稀释1000倍的Cy3-兔抗人IgG溶液。
6.与Cv3-抗IgG一起温育后洗涤
温育后，将载玻片浸入200mlSAB缓冲液中，在室温下，以50rpm3次浸泡5分钟。通过将载玻片浸泡在200毫升纯水中几分钟，除去多余的缓冲液。然后将载玻片以240g在室温下干燥2分钟。然后将载玻片保存在室温下直到扫描(最好是同一天)。用微阵列激光扫描仪(安捷伦扫描仪)以10pm分辨率测量杂交信号。根据制造商的说明检测荧光强度，其中使用Agilent特征提取软件绘制每个点。
点分割。为了获得可行的结果，采用点分割的半自动QC流程。微阵列扫描器的输出是一个原始的.tiff格式的图像文件。使用GenePix Pro7软件(Molecular Devices)对阵列上的每个点进行提取和定量。为该阵列生成GAL(GenePix Array List)文件以辅助进行图像分析。GenePix Pro7允许阵列上的每个点的自动点网格和对齐进行数据提取。在数据提取之后，为包含每个点的数值信息的每个载玻片生成GenePix Results(.GPR)文件；蛋白质ID、蛋白质名称、前景强度、背景强度等。
生物信息学分析。
1.图像分析：原始数据提取
图像分析的目的是通过测量每个探测点内所有像素的中值强度来评估血清样本中存在的自身抗体的数量。每个载玻片，即每个样品，都会生成一个原始的.tiff格式的图像文件。利用GenePix Pro7软件(Molecular Devices)对阵列上的每个点进行自动提取和量化，该软件输出阵列上每个探针点的统计数据。这包括沿着其局部背景的点内像素强度的平均值和中值。为该阵列生成GAL(GenePix Array List)文件以辅助进行图像分析。该文件包含所有探测点的信息以及它们在阵列上的位置。在数据提取之后，为每个载玻片生成GenePix Results(.GPR)文件，该文件包含每个点的信息；蛋白质ID、蛋白质名称、前景强度、背景强度等。在实验生成的数据表中，每个点的前景和背景强度都以相对荧光单位(RFU)表示。
2.数据处理和预处理
对于每一个载玻片，在每一个载玻片以4个重复阵列点上蛋白质和对照探针。在进行数据分析之前，通过以下步骤验证蛋白阵列数据的质量:
步骤1：
通过背景信号强度减去每个点的前景信号强度来计算每个点的净强度。对于每个点，背景信号强度通过原形区域计算，该原形区域是以点为中心，是点的直径的三倍。
步骤2：
移除RFU<0的复型点。
步骤3：
在阵列的点内不会见到可能超过扫描仪读取容量的饱和像素(我们的扫描仪的最大RFU是65536RFU)。因此，如果出现<2复型/s，>20％的像素中显示饱和的spot/s被删除。如果饱和点出现在该蛋白质或探针的3个或更多复型中，这些蛋白质/探针将被标记为“SAT”，并排除在下游分析之外。
步骤4：
如果只剩下1个复型点，则净强度为零。
步骤5：
计算变异系数百分比(CV％)以确定每个载玻片S.D上复型点之间的变异。
Figure BDA0003649602810000061
标记一组仅2或更小复型/s的复型点且将CV％>20％标记为“高CV”。这些复型点(即蛋白质)的平均RFU将被排除在下游分析之外。
对于CV％>20％且有3个或更多复型点剩下的蛋白质/对照，会过滤掉导致高CV％值的复型点。这是通过计算净强度的中值与每组复型点中的个体净强度之间的标准差来实现的。去掉标准偏差最高的点。重新计算CV％值并且重复此过程。
步骤6：
计算剩余复型点的净强度的平均值。
步骤7：
使用基于分位数和基于总强度的模块进行数据的复合归一化。该方法假设不同样品共享它们对照探针的共同底层分布同时考虑其中可能存在的标记点。免疫组阵列使用Cy3标记的生物素化的BSA(Cy3-BSA)在玻片上复制为阳性对照点。因此，对于任何给定的研究，它被认为是信号强度归一化的看家探针。
分位数模块采用Bolstad等人，2003[11]描述的算法。这种重组能够检测和处理任何Cy3BSA对照探针中的异常值或标记点。然后实现基于总强度的模块以获得每个样品的比例因子。这种方法假设后归一化、阳性对照应该对所有样品有共同的总强度值。这种复合方法旨在从测量的变量中归一化蛋白质阵列数据同时保持样品中的目标生物活性。步骤如下：
对所有样品中的所有cy3BSA进行基于分位数的归一化(i＝点号，j＝样品号)
1.将所有样品j中的所有Cy3-BSA装入一个i×j矩阵X中
2.对X的每列j中的点强度进行排序，得到Xson
3.取Xson的每一行i的平均值得到<Xi>
基于强度归一化
1.计算每一行i的平均值和，∑<Xi>
2.对于每个样品，k，计算所有Cy3-BSA对照的总和，∑Xk
3.对于每个样品，k，
Figure BDA0003649602810000062
数据分析来自1557种自身抗体测量的荧光信号被对数转换，以确保在任何参数分析之前归一化。对1557种自身抗体测量的每一种进行单因素方差分析ANOVA，这些测量对i)所有组(健康老年人、中等健康状态老年人、不健康老年人和年轻对照组)和ii)老年人(健康、中等健康和年轻对照组)之间以识别自身抗体，该自身抗体与剩余的相比在至少一组中显著不同(表1)。p值初始阈值0.05用来表示显著性。如图3A所示，以这种方式识别出16种抗原的自身抗体生物标志物：YARS[12]、UBE2I[13]、TCL1A[14]、PPM1A[15]、PHLDA1[16]、GLRX3[17]、FHOD2[18]、FEN1[19]、CASP10[20]、MAPK13[21]、MAP4[22]、FKBP3[23]、CD96[24]、AURKA[25]、ASPSCR1[26]和AAK[27]。在这16种抗原中，发现AURKA、FEN1、GLRX3、PHLDA1、PPM1A、FKBP3、CD96和MAPK13在两种分析中的p值均<0.02(在所有组之间和在老年人之间)。
表1
Figure BDA0003649602810000071
通路富集分析表明，16个中有4个(PHLDA1、AURKA、FEN1和UBE2I)参与细胞周期和DNA修复通路，这些通路在衰老过程中改变。
考虑到16种个体自身抗体的每一个本身都是健康状况的弱预测因子，利用tSNE进行降维以确定16种自身抗体的集体能力来区分健康群体。如图4A所示，使用tSNE降维表明，2个tSNE维度能够区分3个健康组。16种自身抗体的特异性如图4B所示。
为了识别每一种健康状态下的自身抗体，使用一系列带有Welch校正的t检验来检测每一种健康状态下的所有16种已识别的自身抗体。对于每一种自身抗体，选择三种健康状态中最佳t检验结果作为该健康状态所选择的自身抗体。这识别出PHLDA1和CD96对健康组是特异性的，AURKA、FEN1、CASP10和AAK1对中等组是特异性的，其余对不健康组是特异性的(图4C)。健康的和中等的自身抗体可能对非传染性疾病有保护作用。为每个健康状态组示出平均RFU和t检验的p值，t检验的p值表明自身抗体在区分目标健康状态组与剩余组的重要性。
本发明利用生物素羧基载体蛋白质(BCCP)折叠标志物，该标志物与编码目标蛋白的基因框内克隆，如上文EP1470229所述。大肠杆菌BCCP结构域如图1所示，其中画出的残基77-156(坐标文件lbdo)示出N-端和C-端以及在体内通过生物素连接酶连接到赖氨酸122上的单个生物素部分。
BCCP不仅作为蛋白质折叠标志物还作为蛋白质溶解度增强剂。BCCP可与目标蛋白的N-端或c-端融合。全长蛋白表达为与BCCP折叠标志物的融合物，BCCP折叠标志物在体内被生物素化但仅在蛋白质被正确折叠时被生物素化。相反，错误折叠的蛋白质驱动BCCP的错误折叠，这样它就不能被宿主生物素连接酶生物素化。因此，错误折叠的蛋白质不能特异性地附着在链霉亲和素涂层的固相支持体上。因此，只有正确折叠的蛋白质才能通过BCCP标签附着到固相支持体上。
支持体的表面化学经过精心设计且可以使用三维薄膜聚合物基层(聚乙二醇；PEG)，它保留蛋白质斑点形态并且确保在整个阵列中的斑点大小一致。PEG层抑制非特异性结合，因此减少了使用其他平台观察到的高背景。因此，用于固定选定的生物标志物的固体支持体可以抵抗非特异性的大分子吸附并且通过最小化非特异性背景结合切提供极好的信噪比和较低的检测限(即提高灵敏度)。此外，PEG层还保留排列的蛋白质和蛋白质复合物的折叠结构和功能。这对准确诊断至关重要，因为人类血清抗体通常会非特异性地与展开蛋白的疏水表面结合，从而导致在展开蛋白阵列的血清学分析中出现假阳性，此外，人类自身抗体通常与不连续表位结合，因此，对折叠蛋白或折叠错误的蛋白进行血清学检测也会导致自身抗体结合检测的假阴性。
由于结合到链霉亲和素包覆的表面上的生物素化蛋白质表现出很小的解离，因此这种相互作用提供优越的方法以将蛋白质固定在平摊表面，对蛋白质阵列、SPR和基于珠的检测等应用是理想的。使用紧凑、折叠、生物素化的、80个残基结构域的BCCP提供对例如AviTag和intein-based标签的两个显著的优势。首先，BCCP结构域被真核生物素连接酶交叉识别，使其能够在酵母、昆虫和哺乳动物细胞中有效地被生物素化而不需要共同表达大肠杆菌生物素连接酶。第二，BCCP的N端和C端与生物素酰化位点物理分离50A(如图1所示)，所以BCCP的结构域可以认为是一个隐伏跟踪，其呈现远离固相支持体表面的重组蛋白质，由此最小化非流动化导致的任意有害影响。
即使是最复杂的蛋白质的BCCP折叠标志物介导的表达成功率也超过98％。因此，该技术可以应用于高度并行化的管道从而实现高通量、高一致性的功能性验证蛋白生产。
加入BCCP允许通过测量体内生物素化程度来监测融合蛋白质折叠。这可以通过标准的印迹程序，使用SDS-PAGE或原位菌落裂解和将样品转移到膜上进行测量，然后使用链霉亲和素偶联物，例如链霉亲和素-芳香胺过氧化物酶，检测生物素化蛋白质。此外，BCCP的结构域是体内生物素化且当为蛋白质阵列制作进行多路复用蛋白质纯化时尤为有用，因为蛋白质可以被同时从细胞溶解产物纯化出来并且经由链霉抗生物素蛋白表面展现的高亲和性和特异性以单个步骤固定。
图3图解区分老年人的蛋白质，其特征是细胞周期和细胞死亡过程。(A)区分不同老年人健康状况、与16个蛋白质靶标相关的p-值。仅YARS蛋白质的血清/质粒抗体的示值读数不会也在老年人与YC个体之间进行区分(p>0.05)(B)5个蛋白质的示值读数通过通路富集分析(串联)与通路紧密关联，这些通路与细胞循环(PHLDA1、AURKA、FEN1)、DNA修复(UBE2I、FEN1)和翻译(YARS)连接。
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[15]Lin X,Duan X,Liang YY,Su Y,Wrighton KH,Long J,HuM,Davis CM,WangJ,Brunicardi FC,Shi Y,Chen YG,Meng A,Feng XH.PPM1A Functions as a SmadPhosphatase to Terminate TGFβSignaling.Cell.2016 Sep 8；166(6):1597.
[16]Chen Y,Takikawa M,Tsutsumi S,Yamaguchi Y,Okabe A,Shimada M,KawaseT,Sada A,Ezawa I,Takano Y,Nagata K,Suzuki Y,Semba K,Aburatani H,OhkiR.PHLDA1,another PHLDA family protein that inhibits Akt.Cancer Sci.2018 Nov；109(11):3532-3542.
[17]Li Bl,Chen Ml,Lu Ml,Xin-Xiang Jl,Meng-Xiong PI,Jun-WuMI.Glutaredoxin 3 promotes migration and invasion via the Notch signallingpathway in oral squamous cell carcinoma.Free Radic Res.2018 Apr；52(4):390-401.
[18]Otomo T,Tomchick DR,Otomo C,Panchal SC,Machius M,RosenMK.Structuralbasis of actin filament nucleation and processive capping by aformin homology 2 domain.Nature.2005 Feb 3；433(7025):488-94.
[19]Kathera C,Zhang J,Janardhan A,Sun H,Ali W,Zhou X,He L,GuoZ.Interacting partners of FENl and its role in the development of anticancertherapeutics.Oncotarget.2017 Apr 18；8(16):27593-27602.
[20]Horn S,Hughes MA,Schilling R,Sticht C,Tenev T,Ploesser M,Meier P,Sprick MR,MacFarlane M,Leverkus M.Caspase-10 Negatively Regulates Caspase-8-Mediated Cell Death,Switching the Response to CD95L in Favor ofNF-KBActivation and Cell Survival.Cell Rep.2017 Apr 25；19(4):785-797.
[21]Tan FL,Ooi A,Huang D,Wong JC,Qian CN,Chao C,Ooi L,Tan YM,Chung A,Cheow PC,Zhang Z,Petillo D,Yang XJ,Teh BT.p38delta/MAPK13 as a diagnosticmarker for cholangiocarcinoma and its involvement in cell motility andinvasion.Int J Cancer.2010 May 15；126(10):2353-61
[22]Kremer BE,Haystead T,Macara IG.Mammalian septins regulatemicrotubule stability through interaction with the microtubule-bindingprotein MAP4.Mol Biol Cell.2005 Oct；16(10):4648-59
[23]Zhu W,LiZ,Xiong L,Yu X,Chen X,Lin Q.FKBP3 Promotes Proliferationof Non-Small Cell Lung Cancer Cells through Regulating Spl/HDAC2/p27.Theranostics.2017 Jul 22；7(12):3078-3089
[24]Georgiev H,Ravens I,Papadogianni G,Bernhardt G.Coming of Age:CD96Emerges as Modulator oflmmune Responses.Front Immunol.2018 May 17；9:1072.
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表2
Figure BDA0003649602810000111
Figure BDA0003649602810000121
Figure BDA0003649602810000131
Figure BDA0003649602810000141
Figure BDA0003649602810000151
Figure BDA0003649602810000161
Figure BDA0003649602810000171
Figure BDA0003649602810000181
Figure BDA0003649602810000191
Figure BDA0003649602810000201
Figure BDA0003649602810000211
Figure BDA0003649602810000221
Figure BDA0003649602810000231
Figure BDA0003649602810000241
序列表
<110> 盛捷宁克斯私人有限公司
新加坡科技研究局
<120> 使用免疫生物标志物对老年人的健康状况进行鉴定的方法
<130> MJ550/6
<160> 32
<170> PatentIn version 3.5
<210> 1
<211> 1422
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products
<400> 1
atgaagaagt ttttcgactc ccggcgagag cagggcggct ctggcctggg ctccggctcc 60
agcggaggag ggggcagcac ctcgggcctg ggcagtggct acatcggaag agtcttcggc 120
atcgggcgac agcaggtcac agtggacgag gtgttggcgg aaggtggatt tgctattgta 180
tttctggtga ggacaagcaa tgggatgaaa tgtgccttga aacgcatgtt tgtcaacaat 240
gagcatgatc tccaggtgtg caagagagaa atccagataa tgagggatct ttcagggcac 300
aagaatattg tgggttacat tgattctagt atcaacaacg tgagtagcgg tgatgtatgg 360
gaagtgctca ttctgatgga cttttgtaga ggtggccagg tggtaaacct gatgaaccag 420
cgcctgcaaa caggctttac agagaatgaa gtgctccaga tattttgtga tacctgtgaa 480
gctgttgccc gcctgcatca gtgcaaaact cctattatcc accgggacct gaaggttgaa 540
aacatcctct tgcatgaccg aggccactat gtcctgtgtg actttggaag cgccaccaac 600
aaattccaga atccacaaac tgagggagtc aatgcagtag aagatgagat taagaaatac 660
acaacgctgt cctatcgagc accagaaatg gtcaacctgt acagtggcaa aatcatcact 720
acgaaggcag acatttgggc tcttggatgt ttgttgtata aattatgcta cttcactttg 780
ccatttgggg aaagtcaggt ggcaatttgt gatggaaact tcacaattcc tgataattct 840
cgatattctc aagacatgca ctgcctaatt aggtatatgt tggaaccaga ccctgacaaa 900
aggccggata tttaccaggt gtcctacttc tcatttaagc tactcaagaa agagtgccca 960
attccaaatg tacagaactc tcccattcct gcaaagcttc ctgaaccagt gaaagccagt 1020
gaggcagctg caaaaaagac ccagccaaag gccagactga cagatcccat tcccaccaca 1080
gagacttcaa ttgcaccccg ccagaggcct aaagctgggc agactcagcc gaacccagga 1140
atccttccca tccagccagc gctgacaccc cggaagaggg ccactgttca gcccccacct 1200
caggctgcag gatccagcaa tcagcctggc cttttagcca gtgttcccca accaaaaccc 1260
caagccccac ccagccagcc tctgccgcaa actcaggcca agcagccaca ggctcctccc 1320
actccacagc agacgccttc tactcaggcc cagggtctgc ccgctcaggc ccaggccaca 1380
ccccagcacc agcagcatac aataaaactt agtatgaaac tt 1422
<210> 2
<211> 1659
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 2
atggcggccc cggcaggcgg cggaggctcc gcggtgtcgg tgctggcccc gaacggccgg 60
cgccacacgg tgaaggtgac gccgagcacc gtgctgcttc aggttctgga ggacacgtgc 120
cggcggcagg acttcaaccc ctgtgaatat gatctgaagt ttcagaggag cgtgctcgac 180
ctttctctcc agtggagatt tgccaacctg cccaacaatg ccaagctgga gatggtgccc 240
gcttcccgga gccgtgaggg gcctgagaac atggttcgca tcgctttgca gctggacgat 300
ggctcgaggt tgcaggactc tttctgttca ggccagaccc tctgggagct tctcagccat 360
tttccacaga tcagggagtg cctgcagcac cccggcgggg ccaccccagt ctgcgtgtac 420
acgagggatg aggtgacggg tgaagctgcc ctgcggggca cgacgctgca gtcgctgggc 480
ctgaccgggg gcagcgccac catcaggttt gtcatgaagt gctacgaccc cgtgggcaag 540
accccaggaa gcctgggctc gtcagcgtcg gctggccagg cagccgccag cgctccactt 600
cccttggaat ctggggagct cagccgcggc gacttgagcc gtccggagga cgcggacacc 660
tcagggccct gctgcgagca cactcaggag aagcagagca caagggcacc cgcagctgcc 720
ccctttgttc ctttctcggg tgggggacag agacaggggg gccctcctgg gcccacgagg 780
cctctgacat catcttcagc taagttgccg aagtccctct ccagccctgg aggcccctcc 840
aagccaaaga agtccaagtc gggccaggat ccccagcagg agcaggagca ggagcgggag 900
cgggatcccc agcaggagca ggagcgggag cggcccgtgg accgggagcc cgtggaccgg 960
gagccggtgg tgtgccaccc cgacctggag gagcggctgc aggcctggcc agcggagctg 1020
cctgatgagt tctttgagct gacggtggac gacgtgagaa gacgcttggc ccagctcaag 1080
agtgagcgga agcgcctgga agaagccccc ttggtgacca aggccttcag ggaggcgcag 1140
ataaaggaga agctggagcg ctacccaaag gtggctctga gggtcctgtt ccccgaccgc 1200
tacgtcctac agggcttctt ccgccccagc gagacagtgg gggacttgcg agacttcgtg 1260
aggagccacc tggggaaccc cgagctgtca ttttacctgt tcatcacccc tccaaaaaca 1320
gtcctggacg accacacgca gaccctcttt caggcgaacc tcttcccggc cgctctggtg 1380
cacttgggag ccgaggagcc ggcaggtgtc tacctggagc ctggcctgct ggagcatgcc 1440
atctccccat ctgcggccga cgtgctggtg gccaggtaca tgtccagggc cgccgggtcc 1500
ccttccccat tgccagcccc tgaccctgca cctaagtctg agccagctgc tgaggagggg 1560
gcgctggtcc cccctgagcc catcccaggg acggcccagc ccgtgaagag gagcctgggc 1620
aaggtgccca agtggctgaa gctgccggcc agcaagagg 1659
<210> 3
<211> 1209
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 3
atggaccgat ctaaagaaaa ctgcatttca ggacctgtta aggctacagc tccagttgga 60
ggtccaaaac gtgttctcgt gactcagcaa tttccttgtc agaatccatt acctgtaaat 120
agtggccagg ctcagcgggt cttgtgtcct tcaaattctt cccagcgcgt tcctttgcaa 180
gcacaaaagc ttgtctccag tcacaagccg gttcagaatc agaagcagaa gcaattgcag 240
gcaaccagtg tacctcatcc tgtctccagg ccactgaata acacccaaaa gagcaagcag 300
cccctgccat cggcacctga aaataatcct gaggaggaac tggcatcaaa acagaaaaat 360
gaagaatcaa aaaagaggca gtgggctttg gaagactttg aaattggtcg ccctctgggt 420
aaaggaaagt ttggtaatgt ttatttggca agagaaaagc aaagcaagtt tattctggct 480
cttaaagtgt tatttaaagc tcagctggag aaagccggag tggagcatca gctcagaaga 540
gaagtagaaa tacagtccca ccttcggcat cctaatattc ttagactgta tggttatttc 600
catgatgcta ccagagtcta cctaattctg gaatatgcac cacttggaac agtttataga 660
gaacttcaga aactttcaaa gtttgatgag cagagaactg ctacttatat aacagaattg 720
gcaaatgccc tgtcttactg tcattcgaag agagttattc atagagacat taagccagag 780
aacttacttc ttggatcagc tggagagctt aaaattgcag attttgggtg gtcagtacat 840
gctccatctt ccaggaggac cactctctgt ggcaccctgg actacctgcc ccctgaaatg 900
attgaaggtc ggatgcatga tgagaaggtg gatctctgga gccttggagt tctttgctat 960
gaatttttag ttgggaagcc tccttttgag gcaaacacat accaagagac ctacaaaaga 1020
atatcacggg ttgaattcac attccctgac tttgtaacag agggagccag ggacctcatt 1080
tcaagactgt tgaagcataa tcccagccag aggccaatgc tcagagaagt acttgaacac 1140
ccctggatca cagcaaattc atcaaaacca tcaaattgcc aaaacaaaga atcagctagc 1200
aaacagtct 1209
<210> 4
<211> 1566
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 4
atgaaatctc aaggtcaaca ttggtattcc agttcagata aaaactgtaa agtgagcttt 60
cgtgagaagc ttctgattat tgattcaaac ctgggggtcc aagatgtgga gaacctcaag 120
tttctctgca taggattggt ccccaacaag aagctggaga agtccagctc agcctcagat 180
gtttttgaac atctcttggc agaggatctg ctgagtgagg aagacccttt cttcctggca 240
gaactcctct atatcatacg gcagaagaag ctgctgcagc acctcaactg taccaaagag 300
gaagtggagc gactgctgcc cacccgacaa agggtttctc tgtttagaaa cctgctctac 360
gaactgtcag aaggcattga ctcagagaac ttaaaggaca tgatcttcct tctgaaagac 420
tcgcttccca aaactgaaat gacctcccta agtttcctgg catttctaga gaaacaaggt 480
aaaatagatg aagataatct gacatgcctg gaggacctct gcaaaacagt tgtacctaaa 540
cttttgagaa acatagagaa atacaaaaga gagaaagcta tccagatagt gacacctcct 600
gtagacaagg aagccgagtc gtatcaagga gaggaagaac tagtttccca aacagatgtt 660
aagacattct tggaagcctt accgcaggag tcctggcaaa ataagcatgc aggtagtaat 720
ggtaacagag ccacaaatgg tgcaccaagc ctggtctcca gggggatgca aggagcatct 780
gctaacactc taaactctga aaccagcaca aagagggcag ctgtgtacag gatgaatcgg 840
aaccacagag gcctctgtgt cattgtcaac aaccacagct ttacctccct gaaggacaga 900
caaggaaccc ataaagatgc tgagatcctg agtcatgtgt tccagtggct tgggttcaca 960
gtgcatatac acaataatgt gacgaaagtg gaaatggaga tggtcctgca gaagcagaag 1020
tgcaatccag cccatgccga cggggactgc ttcgtgttct gtattctgac ccatgggaga 1080
tttggagctg tctactcttc ggatgaggcc ctcattccca ttcgggagat catgtctcac 1140
ttcacagccc tgcagtgccc tagactggct gaaaaaccta aactcttttt catccaggcc 1200
tgccaaggtg aagagataca gccttccgta tccatcgaag cagatgctct gaaccctgag 1260
caggcaccca cttccctgca ggacagtatt cctgccgagg ctgacttcct acttggtctg 1320
gccactgtcc caggctatgt atcctttcgg catgtggagg aaggcagctg gtatattcag 1380
tctctgtgta atcatctgaa gaaattggtc ccaagacatg aagacatctt atccatcctc 1440
actgctgtca acgatgatgt gagtcgaaga gtggacaaac agggaacaaa gaaacagatg 1500
ccccagcctg ctttcacact aaggaaaaaa ctagtattcc ctgtgcccct ggatgcactt 1560
tcatta 1566
<210> 5
<211> 1206
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 5
atggagaaaa aatggaaata ctgtgctgtc tattacatca tccagataca ttttgtcaag 60
ggagtttggg aaaaaacagt caacacagaa gaaaatgttt atgctacact tggctctgat 120
gtcaacctga cctgccaaac acagacagta ggcttcttcg tgcagatgca atggtccaag 180
gtcaccaata agatagacct gattgctgtc tatcatcccc aatacggctt ctactgtgcc 240
tatgggagac cctgtgagtc acttgtgact ttcacagaaa ctcctgagaa tgggtcaaaa 300
tggactctgc acttaaggaa tatgtcttgt tcagtcagtg gaaggtacga gtgtatgctt 360
gttctgtatc cagagggcat tcagactaaa atctacaacc ttctcattca gacacacgtt 420
acagcagatg aatggaacag caaccatacg atagaaatag agataaatca gactctggaa 480
ataccatgct ttcaaaatag ctcctcaaaa atttcatctg agttcaccta tgcatggtcg 540
gtggaggata atggaactca ggaaacactt atctcccaaa atcacctcat cagcaattcc 600
acattactta aagatagagt caagcttggt acagactaca gactccacct ctctccagtc 660
caaatcttcg atgatgggcg gaagttctct tgccacatta gagtcggtcc taacaaaatc 720
ttgaggagct ccaccacagt caaggttttt gctaaaccag aaatccctgt gattgtggaa 780
aataactcca cggatgtctt ggtagagaga agattcacct gcttactaaa gaatgtattt 840
cccaaagcaa atatcacatg gtttatagat ggaagttttc ttcatgatga aaaagaagga 900
atatatatta ctaatgaaga gagaaaaggc aaagatggat ttttggaact gaagtctgtt 960
ttaacaaggg tacatagtaa taaaccagcc caatcagaca acttgaccat ttggtgtatg 1020
gctctgtctc cagtcccagg aaataaagtg tggaacatct catcagaaaa gatcactttt 1080
ctcttaggtt ctgaaatttc ctcaacagac cctccactga gtgttacaga atctaccctt 1140
gacacccaac cttctccagc cagcagtgta tctcctgcaa gtaagaatgt tttcacactg 1200
agctat 1206
<210> 6
<211> 1140
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 6
atgggaattc aaggcctggc caaactaatt gctgatgtgg cccccagtgc catccgggag 60
aatgacatca agagctactt tggccgtaag gtggccattg atgcctctat gagcatttat 120
cagttcctga ttgctgttcg ccagggtggg gatgtgctgc agaatgagga gggtgagacc 180
accagccacc tgatgggcat gttctaccgc accattcgca tgatggagaa cggcatcaag 240
cccgtgtatg tctttgatgg caagccgcca cagctcaagt caggcgagct ggccaaacgc 300
agtgagcggc gggctgaggc agagaagcag ctgcagcagg ctcaggctgc tggggccgag 360
caggaggtgg aaaaattcac taagcggctg gtgaaggtca ctaagcagca caatgatgag 420
tgcaaacatc tgctgagcct catgggcatc ccttatcttg atgcacccag tgaggcagag 480
gccagctgtg ctgccctggt gaaggctggc aaagtctatg ctgcggctac cgaggacatg 540
gactgcctca ccttcggcag ccctgtgcta atgcgacacc tgactgccag tgaagccaaa 600
aagctgccaa tccaggaatt ccacctgagc cggattctgc aggagctggg cctgaaccag 660
gaacagtttg tggatctgtg catcctgcta ggcagtgact actgtgagag tatccggggt 720
attgggccca agcgggctgt ggacctcatc cagaagcaca agagcatcga ggagatcgtg 780
cggcgacttg accccaacaa gtaccctgtg ccagaaaatt ggctccacaa ggaggctcac 840
cagctcttct tggaacctga ggtgctggac ccagagtctg tggagctgaa gtggagcgag 900
ccaaatgaag aagagctgat caagttcatg tgtggtgaaa agcagttctc tgaggagcga 960
atccgcagtg gggtcaagag gctgagtaag agccgccaag gcagcaccca gggccgcctg 1020
gatgatttct tcaaggtgac cggctcactc tcttcagcta agcgcaagga gccagaaccc 1080
aagggatcca ctaagaagaa ggcaaagact ggggcagcag ggaagtttaa aaggggaaaa 1140
<210> 7
<211> 672
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 7
atggcggcgg ccgttccaca gcgggcgtgg accgtggagc agctgcgcag tgagcagctg 60
cccaagaagg acattatcaa gtttctgcag gaacacggtt cagattcgtt tcttgcagaa 120
cataaattat taggaaacat taaaaatgtg gccaagacag ctaacaagga ccacttggtt 180
acagcctata accatctttt tgaaactaag cgttttaagg gtactgaaag tataagtaaa 240
gtgtctgagc aagtaaaaaa tgtgaagctt aatgaagata aacccaaaga aaccaagtct 300
gaagagaccc tggatgaggg tccaccaaaa tatactaaat ctgttctgaa aaagggagat 360
aaaaccaact ttcccaaaaa gggagatgtt gttcactgct ggtatacagg aacactacaa 420
gatgggactg tttttgatac taatattcaa acaagtgcaa agaagaagaa aaatgccaag 480
cctttaagtt ttaaggtcgg agtaggcaaa gttatcagag gatgggatga agctctcttg 540
actatgagta aaggagaaaa ggctcgactg gagattgaac cagaatgggc ttacggaaag 600
aaaggacagc ctgatgccaa aattccacca aatgcaaaac tcacttttga agtggaatta 660
gtggatattg at 672
<210> 8
<211> 534
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 8
atggacttga ccaagagaga gtacaccatg catgaccata acacgctgct gaaggagttc 60
atcctcaaca atgaggggaa gctgaagaag ctgcaggatg atgccaagat cgcacaggat 120
gcctttgatg atgttgtgaa gtattttgga gaaaacccca agacaacacc accctctgtc 180
ttctttcctg tctttgtccg gtttgtgaaa gcatataagc aagcagaaga ggaaaatgag 240
ctgaggaaaa agcaggaaca agctctcatg gaaaaactcc tagagcaaga agctctgatg 300
gagcagcagg atccaaagtc tccttctcat aaatcaaaga ggcagcagca agagttaatt 360
gcagaattaa gaagacgaca agttaaagat aacagacatg tatatgaggg aaaagatggt 420
gccattgaag atattatcac agccttaaag aagaataata tcactaaatt tccaaatgtt 480
cactcgaggg taaggatttc ttctagcaca ccggtggtgg aggatacaca gagc 534
<210> 9
<211> 1005
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 9
atggcggcgg gggcggctga ggcagctgta gcggccgtgg aggaggtcgg ctcagccggg 60
cagtttgagg agctgctgcg cctcaaagcc aagtccctcc ttgtggtcca tttctgggca 120
ccatgggctc cacagtgtgc acagatgaac gaagttatgg cagagttagc taaagaactc 180
cctcaagttt catttgtgaa gttggaagct gaaggtgttc ctgaagtatc tgaaaaatat 240
gaaattagct ctgttcccac ttttctgttt ttcaagaatt ctcagaaaat cgaccgatta 300
gatggtgcac atgccccaga gttgaccaaa aaagttcagc gacatgcatc tagtggctcc 360
ttcctaccca gcgctaatga acatcttaaa gaagatctca accttcgctt gaagaaattg 420
actcatgctg ccccctgcat gctgtttatg aaaggaactc ctcaagaacc acgctgtggt 480
ttcagcaagc agatggtgga aattcttcac aaacataata ttcagtttag cagttttgat 540
atcttctcag atgaagaggt tcgacaggga ctcaaagcct attccagttg gcctacctat 600
cctcagctct atgtttctgg agagctcata ggaggacttg atataattaa ggagctagaa 660
gcatctgaag aactagatac aatttgtccc aaagctccca aattagagga aaggctcaaa 720
gtgctgacaa ataaagcttc tgtgatgctc tttatgaaag gaaacaaaca ggaagcaaaa 780
tgtggattca gcaaacaaat tctggaaata ctaaatagta ctggtgttga atatgaaaca 840
ttcgatatat tggaggatga agaagttcgg caaggattaa aagcttactc aaattggcca 900
acataccctc agctgtatgt gaaaggggag ctggtgggag gattggatat tgtgaaggaa 960
ctgaaagaaa atggtgaatt gctgcctata ctgagaggag aaaat 1005
<210> 10
<211> 2898
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 10
atggccaacc ttcaggagca cctgagctgc tcctcttctc cacacttacc cttcagtgaa 60
agcaaaacct tcaatggact acaagatgag ctcacagcta tggggaacca cccttctccc 120
aagctgctcg aggaccagca ggaaaagggg atggtacgaa cagagctaat cgagagcgtg 180
cacagccccg tcaccacaac agtgttgacg agcgtaagtg aggattccag ggaccagttt 240
gagaacagcg ttcttcagct aagggaacac gatgaatcag agacggcggt gtctcagggg 300
aacagcaaca cggtggacgg agagagcaca agcggaactg aagacataaa gattcagttc 360
agcaggtcag gcagtggcag tggtgggttt cttgaaggac tatttggatg cttaaggcct 420
gtatggaata tcattgggaa ggcatattcc actgattaca aattgcagca gcaagatact 480
tgggaagtgc catttgagga gatctcagag ctgcagtggc tgggtagtgg agcccaagga 540
gcggtcttct tgggcaagtt ccgggcggaa gaggtggcca tcaagaaagt gagagaacag 600
aatgagacgg atatcaagca tttgaggaag ttgaagcacc ctaacatcat cgcattcaag 660
ggtgtttgta ctcaggcccc atgttattgt attatcatgg aatactgtgc ccatggacaa 720
ctctacgagg tcttacgagc tggcaggaag atcacacctc gattgctagt agactggtcc 780
acaggaattg caagtggaat gaattatttg cacctccata aaattattca tcgtgatctc 840
aaatcaccta atgttttagt gacccacaca gatgcggtaa aaatttcaga ttttggtaca 900
tctaaggaac tcagtgacaa aagtaccaag atgtcatttg ctggcacggt cgcatggatg 960
gcgccagagg tgatacggaa tgaacctgtc tctgaaaaag ttgatatatg gtcttttgga 1020
gtggtgcttt gggagctgct gacaggagag atcccttaca aagatgtaga ttcttcagcc 1080
attatctggg gtgttggaag caacagcctc caccttccag ttccttccac ttgccctgat 1140
ggattcaaaa tccttatgaa acagacgtgg cagagtaaac ctcgaaaccg accttctttt 1200
cggcagacac tcatgcattt agacattgcc tctgcagatg tacttgccac cccacaagaa 1260
acttacttca agtctcaggc tgaatggaga gaagaagtga aaaaacattt tgagaagatc 1320
aaaagtgaag gaacttgtat acaccggtta gatgaagaac tgattcgaag gcgcagagaa 1380
gagctcaggc atgcgctgga tattcgtgaa cactatgagc ggaagcttga gcgggcgaat 1440
aatttataca tggaattgag tgccatcatg ctgcagctag aaatgcggga gaaggagctc 1500
attaagcgtg agcaagcagt ggaaaagaag tatcctggga cctacaaacg acaccctgtt 1560
cgtcctatca tccatcccaa tgccatggag aaactcatga aaaggaaagg agtgcctcac 1620
aaatctggga tgcagaccaa acggccagac ttgttgagat cagaagggat ccccaccaca 1680
gaagtggctc ccactgcatc ccctttgtcc ggaagtccca aaatgtccac ttctagcagc 1740
aagagccgat atcgaagcaa accacgccac cgccgaggga atagcagagg cagccatagt 1800
gactttgccg caatcttgaa aaaccagcca gcccaggaaa attcacccca tcccacttac 1860
ctgcaccaag ctcaatccca atacccttct cttcatcacc ataattctct gcagcagcaa 1920
taccagcagc cccctcctgc catgtcccag agtcaccatc ccagactcaa tatgcacgga 1980
caggacatag caacctgcgc caacaacctg aggtatttcg gcccagcagc agccctgcgg 2040
agcccactca gcaaccatgc tcagagacag ctgcccggct cgagccctga cctcatctcc 2100
acagccatgg ctgcagactg ctggagaagt tctgagcctg acaagggcca agctggtccc 2160
tggggctgtt gccaggctga cgcttatgac ccctgccttc agtgcaggcc agaacagtat 2220
gggtccttag acataccctc tgctgagcca gtggggagga gccctgacct ttccaagtca 2280
ccagcacata atcctctctt ggaaaacgcc cagagttctg agaaaacgga agaaaatgaa 2340
ttcagcggct gtaggtctga gtcatccctc ggcacctctc atctcggcac ccctccagcg 2400
ctacctcgaa aaacaaggcc tctgcagaag agtggagatg actcctcaga agaggaagaa 2460
ggggaagtag atagtgaagt tgaatttcca cgaagacaga ggccccatcg ctgtatcagc 2520
agctgccagt catattcaac ctttagctct gagaatttct ctgtgtctga tggagaagag 2580
ggaaatacca gtgaccactc aaacagtcct gatgagttag ctgataaact tgaagaccgc 2640
ttggcagaga agctagacga cctgctgtcc cagacgccag agattcccat tgacatatcc 2700
tcacactcgg atgggctctc tgacaaggag tgtgccgtgc gccgtgtgaa gactcagatg 2760
tctctgggca agctgtgtgt ggaggaacgt ggctatgaga accccatgca gtttgaagaa 2820
tcggactgtg actcttcaga tggggagtgt tctgatgcca cagttaggac caataaacac 2880
tacagctctg ctacctgg 2898
<210> 11
<211> 2937
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 11
atggctgacc tcagtcttgc agatgcatta acagaaccat ctccagacat tgagggagag 60
ataaagcggg acttcattgc cacactagag gcagaggcct ttgatgatgt tgtgggagaa 120
actgttggaa aaacagacta tattcctctc ctggatgttg atgagaaaac cgggaactca 180
gagtcaaaga agaaaccgtg ctcagaaact agccagattg aagatactcc atcttctaaa 240
ccaacactcc tagccaatgg tggtcatgga gtagaaggga gcgatactac agggtctcca 300
actgaattcc ttgaagagaa aatggcctac caggaatacc caaatagcca gaactggcca 360
gaagatacca acttttgttt ccaacctgag caagtggtcg atcctatcca gactgatccc 420
tttaagatgt accatgatga tgacctggca gatttggtct ttccctccag tgcgacagct 480
gatacttcaa tatttgcagg acaaaatgat cccttgaaag acagttacgg tatgtctccc 540
tgcaacacag ctgttgtacc tcaggggtgg tctgtggaag ccttaaactc tccacactca 600
gagtcctttg tttccccaga ggctgttgca gaacctcctc agccaacggc agttccctta 660
gagctagcca aggagataga aatggcatca gaagagaggc caccagcaca agcattggaa 720
ataatgatgg gactgaagac tactgacatg gcaccatcta aagaaacaga gatggccctc 780
gccaaggaca tggcactagc tacaaaaacc gaggtggcat tggctaaaga tatggaatca 840
cccaccaaat tagatgtgac actggccaag gacatgcagc catccatgga atcagatatg 900
gccctagtca aggacatgga actacccaca gaaaaagaag tggccctggt taaggatgtc 960
agatggccca cagaaacaga tgtatcttca gccaagaatg tggtactgcc cacagaaaca 1020
gaggtagccc cagccaagga tgtgacactg ttgaaagaaa cagagagggc atctcctata 1080
aaaatggact tagccccttc caaggacatg ggaccaccca aagaaaacaa gaaagaaaca 1140
gagagggcat ctcctataaa aatggacttg gctccttcca aggacatggg accacccaaa 1200
gaaaacaaga tagtcccagc caaggatttg gtattactct cagaaataga ggtggcacag 1260
gctaatgaca ttatatcatc cacagaaata tcctctgctg agaaggtggc tttgtcctca 1320
gaaacagagg tagccctggc cagggacatg acactgcccc cggaaaccaa cgtgatcttg 1380
accaaggata aagcactacc tttagaagca gaggtggccc cagtcaagga catggctcaa 1440
ctcccagaaa cagaaatagc cccggccaag gatgtggctc cgtccacagt aaaagaagtg 1500
ggcttgttga aggacatgtc tccactatca gaaacagaaa tggctctggg caaggatgtg 1560
actccacctc cagaaacaga agtagttctc atcaagaacg tatgtctgcc tccagaaatg 1620
gaggtggccc tgactgagga tcaggtccca gccctcaaaa cagaagctcc caccaccatt 1680
ggtgggttga ataaaaaacc catgagcctt gcttcaggct tagtgccagc tgccccaccc 1740
aaacgccctg ccgtcgcctc tgccaggcct tccatcttac cttcaaaaga cgtgaagcca 1800
aagcccattg cagatgcaaa ggctcctgag aagcgggcct caccatccaa gccagcttct 1860
gccccagcct ccagatctgg gtccaagagc actcagactg ttgcaaaaac cacaacagct 1920
gctgctgttg cctcaactgg cccaagcagt aggagcccct ccacgctcct gcccaagaag 1980
cccactgcca ttaagactga gggaaaacct gcagaagtca agaagatgac tgcaaagtct 2040
gtaccagctg acttgagtcg cccaaagagc acctccacca gttccatgaa gaaaaccacc 2100
actctcagtg ggacagcccc cgctgcaggg gtggttccca gccgagtcaa ggccacaccc 2160
atgccctccc ggccctccac aactcctttc atagacaaga agcccacctc ggccaaaccc 2220
agctccacca ccccccggct cagccgcctg gccaccaata cttctgctcc tgatctgaag 2280
aatgtccgct ccaaggttgg ctccacggaa aacatcaagc atcagcctgg aggaggccgg 2340
gccaaagtag agaaaaaaac agaggcagct gctacaaccc gaaagcctga atctaatgca 2400
gtcactaaaa cagccggccc aattgcaagt gcacagaaac aacctgcggg gaaagtccag 2460
atagtctcca aaaaagtgag ctacagccat attcagtcca agtgtggttc caaggacaat 2520
attaagcatg tccctggagg tggtaatgtt cagattcaga acaagaaagt ggacatctct 2580
aaggtctcct ccaagtgtgg gtctaaggct aacatcaagc acaagcctgg tggaggagat 2640
gtcaagattg aaagtcagaa gttgaacttc aaggagaagg cccaggccaa ggtgggatcc 2700
ctcgataatg tgggccacct acctgcagga ggtgctgtga agactgaggg cggtggcagc 2760
gaggctcctc tgtgtccggg tccccctgct ggggaggagc cggccatctc tgaggcagcg 2820
cctgaagctg gcgcccccac ttcagccagt ggcctcaatg gccaccccac cctgtcaggg 2880
ggtggtgacc aaagggaggc ccagaccttg gacagccaga tccaggagac aagcatc 2937
<210> 12
<211> 777
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 12
atgctggaga gtagcggctg caaagcgctg aaggagggcg tgctggagaa gcgcagcgac 60
gggttgttgc agctctggaa gaaaaagtgt tgcatcctca ccgaggaagg gctgctgctt 120
atcccgccca agcagctgca acaccagcag cagcagcaac agcagcagca gcagcagcaa 180
caacagcccg ggcaggggcc ggccgagccg tcccaaccca gtggccccgc tgtcgccagc 240
ctcgagccgc cggtcaagct caaggaactg cacttctcca acatgaagac cgtggactgt 300
gtggagcgca agggcaagta catgtacttc actgtggtga tggcagaggg caaggagatc 360
gactttcggt gcccgcaaga ccagggctgg aacgccgaga tcacgctgca gatggtgcag 420
tacaagaatc gtcaggccat cctggcggtc aaatccacgc ggcagaagca gcagcacctg 480
gtccagcagc agcccccctc gcagccgcag ccgcagccgc agctccagcc ccaaccccag 540
cctcagcctc agccgcaacc ccagccccaa tcacaacccc agcctcagcc ccaacccaag 600
cctcagcccc agcagctcca cccgtatccg catccacatc cacatccaca ctctcatcct 660
cactcgcacc cacaccctca cccgcacccg catccgcacc aaataccgca cccacaccca 720
cagccgcact cgcagccgca cgggcaccgg cttctccgca gcacctccaa ctctgcc 777
<210> 13
<211> 1146
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 13
atgggagcat ttttagacaa gccaaagatg gaaaagcata atgcccaggg gcagggtaat 60
gggttgcgat atgggctaag cagcatgcaa ggctggcgtg ttgaaatgga ggatgcacat 120
acggctgtga tcggtttgcc aagtggactt gaatcgtggt cattctttgc tgtgtatgat 180
gggcatgctg gttctcaggt tgccaaatac tgctgtgagc atttgttaga tcacatcacc 240
aataaccagg attttaaagg gtctgcagga gcaccttctg tggaaaatgt aaagaatgga 300
atcagaacag gttttctgga gattgatgaa cacatgagag ttatgtcaga gaagaaacat 360
ggtgcagata gaagtgggtc aacagctgta ggtgtcttaa tttctcccca acatacttat 420
ttcattaact gtggagactc aagaggttta ctttgtagga acaggaaagt tcatttcttc 480
acacaagatc acaaaccaag taatccgctg gagaaagaac gaattcagaa tgcaggtggc 540
tctgtaatga ttcagcgtgt gaatggctct ctggctgtat cgagggccct tggggatttt 600
gattacaaat gtgtccatgg aaaaggtcct actgagcagc ttgtctcacc agagcctgaa 660
gtccatgata ttgaaagatc tgaagaagat gatcagttca ttatccttgc atgtgatggt 720
atctgggatg ttatgggaaa tgaagagctc tgtgattttg taagatccag acttgaagtc 780
actgatgacc ttgagaaagt ttgcaatgaa gtagtcgaca cctgtttgta taagggaagt 840
cgagacaaca tgagtgtgat tttgatctgt tttccaaatg cacccaaagt atcgccagaa 900
gcagtgaaga aggaggcaga gttggacaag tacctggaat gcagagtaga agaaatcata 960
aagaagcagg gggaaggcgt ccccgactta gtccatgtga tgcgcacatt agcgagtgag 1020
aacatcccca gcctcccacc agggggtgaa ttggcaagca agaggaatgt tattgaagcc 1080
gtttacaata gactgaatcc ttacaaaaat gacgacactg actctacatc aacagatgat 1140
atgtgg 1146
<210> 14
<211> 342
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 14
atggccgagt gcccgacact cggggaggca gtcaccgacc acccggaccg cctgtgggcc 60
tgggagaagt tcgtgtattt ggacgagaag cagcacgcct ggctgccctt aaccatcgag 120
ataaaggata ggttacagtt acgggtgctc ttgcgtcggg aagacgtcgt cctggggagg 180
cctatgaccc ccacccagat aggcccaagc ctgctgccta tcatgtggca gctctaccct 240
gatggacgat accgatcctc agactccagt ttctggcgct tagtgtacca catcaagatt 300
gacggcgtgg aggacatgct tctcgagctg ctgccagatg ac 342
<210> 15
<211> 474
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 15
atgtcgggga tcgccctcag cagactcgcc caggagagga aagcatggag gaaagaccac 60
ccatttggtt tcgtggctgt cccaacaaaa aatcccgatg gcacgatgaa cctcatgaac 120
tgggagtgcg ccattccagg aaagaaaggg actccgtggg aaggaggctt gtttaaacta 180
cggatgcttt tcaaagatga ttatccatct tcgccaccaa aatgtaaatt cgaaccacca 240
ttatttcacc cgaatgtgta cccttcgggg acagtgtgcc tgtccatctt agaggaggac 300
aaggactgga ggccagccat cacaatcaaa cagatcctat taggaataca ggaacttcta 360
aatgaaccaa atatccaaga cccagctcaa gcagaggcct acacgattta ctgccaaaac 420
agagtggagt acgagaaaag ggtccgagca caagccaaga agtttgcgcc ctca 474
<210> 16
<211> 1585
<212> DNA
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 16
atgggggacg ctcccagccc tgaagagaaa ctgcacctta tcacccggaa cctgcaggag 60
gttctggggg aagagaagct gaaggagata ctgaaggagc gggaacttaa aatttactgg 120
ggaacggcaa ccacgggcaa accacatgtg gcttactttg tgcccatgtc aaagattgca 180
gacttcttaa aggcagggtg tgaggtaaca attctgtttg cggacctcca cgcatacctg 240
gataacatga aagccccatg ggaacttcta gaactccgag tcagttacta tgagaatgtg 300
atcaaagcaa tgctggagag cattggtgtg cccttggaga agctcaagtt catcaaaggc 360
actgattacc agctcagcaa agagtacaca ctagatgtgt acagactctc ctccgtggtc 420
acacagcacg attccaagaa ggctggagct gaggtggtaa agcaggtgga gcaccctttg 480
ctgagtggcc tcttataccc cggactgcag gctttggatg aagagtattt aaaagtagat 540
gcccaatttg gaggcattga tcagagaaag attttcacct ttgcagagaa gtacctccct 600
gcacttggct attcaaaacg ggtccatctg atgaatccta tggttccagg attaacaggc 660
agcaaaatga gctcttcaga agaggagtcc aagattgatc tccttgatcg gaaggaggat 720
gtgaagaaaa aactgaagaa ggccttctgt gagccaggaa atgtggagaa caatggggtt 780
ctgtccttca tcaagcatgt cctttttccc cttaagtccg agtttgtgat cctacgagat 840
gagaaatggg gtggaaacaa aacctacaca gcttacgtgg acctggaaaa ggactttgct 900
gctgaggttg tacatcctgg agacctgaag aattctgttg aagtcgcact gaacaagttg 960
ctggatccaa tccgggaaaa gtttaatacc cctgccctga aaaaactggc cagcgctgcc 1020
tacccagatc cctcaaagca gaagccaatg gccaaaggcc ctgccaagaa ttcagaacca 1080
gaggaggtca tcccatcccg gctggatatc cgtgtgggga aaatcatcac tgtggagaag 1140
cacccagatg cagacagcct gtatgtagag aagattgacg tgggggaagc tgaaccacgg 1200
actgtggtga gcggcctggt acagttcgtg cccaaggagg aactgcagga caggctggta 1260
gtggtgctgt gcaacctgaa accccagaag atgagaggag tcgagtccca aggcatgctt 1320
ctgtgtgctt ctatagaagg gataaaccgc caggttgaac ctctggaccc tccggcaggc 1380
tctgctcctg gtgagcacgt gtttgtgaag ggctatgaaa agggccaacc agatgaggag 1440
ctcaagccca agaagaaagt cttcgagaag ttgcaggctg acttcaaaat ttctgaggag 1500
tgcatcgcac agtggaagca aaccaacttc atgaccaagc tgggctccat ttcctgtaaa 1560
tcgctgaaag gggggaacat tagcc 1585
<210> 17
<211> 961
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 17
Met Lys Lys Phe Phe Asp Ser Arg Arg Glu Gln Gly Gly Ser Gly Leu
1 5 10 15
Gly Ser Gly Ser Ser Gly Gly Gly Gly Ser Thr Ser Gly Leu Gly Ser
20 25 30
Gly Tyr Ile Gly Arg Val Phe Gly Ile Gly Arg Gln Gln Val Thr Val
35 40 45
Asp Glu Val Leu Ala Glu Gly Gly Phe Ala Ile Val Phe Leu Val Arg
50 55 60
Thr Ser Asn Gly Met Lys Cys Ala Leu Lys Arg Met Phe Val Asn Asn
65 70 75 80
Glu His Asp Leu Gln Val Cys Lys Arg Glu Ile Gln Ile Met Arg Asp
85 90 95
Leu Ser Gly His Lys Asn Ile Val Gly Tyr Ile Asp Ser Ser Ile Asn
100 105 110
Asn Val Ser Ser Gly Asp Val Trp Glu Val Leu Ile Leu Met Asp Phe
115 120 125
Cys Arg Gly Gly Gln Val Val Asn Leu Met Asn Gln Arg Leu Gln Thr
130 135 140
Gly Phe Thr Glu Asn Glu Val Leu Gln Ile Phe Cys Asp Thr Cys Glu
145 150 155 160
Ala Val Ala Arg Leu His Gln Cys Lys Thr Pro Ile Ile His Arg Asp
165 170 175
Leu Lys Val Glu Asn Ile Leu Leu His Asp Arg Gly His Tyr Val Leu
180 185 190
Cys Asp Phe Gly Ser Ala Thr Asn Lys Phe Gln Asn Pro Gln Thr Glu
195 200 205
Gly Val Asn Ala Val Glu Asp Glu Ile Lys Lys Tyr Thr Thr Leu Ser
210 215 220
Tyr Arg Ala Pro Glu Met Val Asn Leu Tyr Ser Gly Lys Ile Ile Thr
225 230 235 240
Thr Lys Ala Asp Ile Trp Ala Leu Gly Cys Leu Leu Tyr Lys Leu Cys
245 250 255
Tyr Phe Thr Leu Pro Phe Gly Glu Ser Gln Val Ala Ile Cys Asp Gly
260 265 270
Asn Phe Thr Ile Pro Asp Asn Ser Arg Tyr Ser Gln Asp Met His Cys
275 280 285
Leu Ile Arg Tyr Met Leu Glu Pro Asp Pro Asp Lys Arg Pro Asp Ile
290 295 300
Tyr Gln Val Ser Tyr Phe Ser Phe Lys Leu Leu Lys Lys Glu Cys Pro
305 310 315 320
Ile Pro Asn Val Gln Asn Ser Pro Ile Pro Ala Lys Leu Pro Glu Pro
325 330 335
Val Lys Ala Ser Glu Ala Ala Ala Lys Lys Thr Gln Pro Lys Ala Arg
340 345 350
Leu Thr Asp Pro Ile Pro Thr Thr Glu Thr Ser Ile Ala Pro Arg Gln
355 360 365
Arg Pro Lys Ala Gly Gln Thr Gln Pro Asn Pro Gly Ile Leu Pro Ile
370 375 380
Gln Pro Ala Leu Thr Pro Arg Lys Arg Ala Thr Val Gln Pro Pro Pro
385 390 395 400
Gln Ala Ala Gly Ser Ser Asn Gln Pro Gly Leu Leu Ala Ser Val Pro
405 410 415
Gln Pro Lys Pro Gln Ala Pro Pro Ser Gln Pro Leu Pro Gln Thr Gln
420 425 430
Ala Lys Gln Pro Gln Ala Pro Pro Thr Pro Gln Gln Thr Pro Ser Thr
435 440 445
Gln Ala Gln Gly Leu Pro Ala Gln Ala Gln Ala Thr Pro Gln His Gln
450 455 460
Gln Gln Leu Phe Leu Lys Gln Gln Gln Gln Gln Gln Gln Pro Pro Pro
465 470 475 480
Ala Gln Gln Gln Pro Ala Gly Thr Phe Tyr Gln Gln Gln Gln Ala Gln
485 490 495
Thr Gln Gln Phe Gln Ala Val His Pro Ala Thr Gln Lys Pro Ala Ile
500 505 510
Ala Gln Phe Pro Val Val Ser Gln Gly Gly Ser Gln Gln Gln Leu Met
515 520 525
Gln Asn Phe Tyr Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
530 535 540
Gln Gln Leu Ala Thr Ala Leu His Gln Gln Gln Leu Met Thr Gln Gln
545 550 555 560
Ala Ala Leu Gln Gln Lys Pro Thr Met Ala Ala Gly Gln Gln Pro Gln
565 570 575
Pro Gln Pro Ala Ala Ala Pro Gln Pro Ala Pro Ala Gln Glu Pro Ala
580 585 590
Ile Gln Ala Pro Val Arg Gln Gln Pro Lys Val Gln Thr Thr Pro Pro
595 600 605
Pro Ala Val Gln Gly Gln Lys Val Gly Ser Leu Thr Pro Pro Ser Ser
610 615 620
Pro Lys Thr Gln Arg Ala Gly His Arg Arg Ile Leu Ser Asp Val Thr
625 630 635 640
His Ser Ala Val Phe Gly Val Pro Ala Ser Lys Ser Thr Gln Leu Leu
645 650 655
Gln Ala Ala Ala Ala Glu Ala Ser Leu Asn Lys Ser Lys Ser Ala Thr
660 665 670
Thr Thr Pro Ser Gly Ser Pro Arg Thr Ser Gln Gln Asn Val Tyr Asn
675 680 685
Pro Ser Glu Gly Ser Thr Trp Asn Pro Phe Asp Asp Asp Asn Phe Ser
690 695 700
Lys Leu Thr Ala Glu Glu Leu Leu Asn Lys Asp Phe Ala Lys Leu Gly
705 710 715 720
Glu Gly Lys His Pro Glu Lys Leu Gly Gly Ser Ala Glu Ser Leu Ile
725 730 735
Pro Gly Phe Gln Ser Thr Gln Gly Asp Ala Phe Ala Thr Thr Ser Phe
740 745 750
Ser Ala Gly Thr Ala Glu Lys Arg Lys Gly Gly Gln Thr Val Asp Ser
755 760 765
Gly Leu Pro Leu Leu Ser Val Ser Asp Pro Phe Ile Pro Leu Gln Val
770 775 780
Pro Asp Ala Pro Glu Lys Leu Ile Glu Gly Leu Lys Ser Pro Asp Thr
785 790 795 800
Ser Leu Leu Leu Pro Asp Leu Leu Pro Met Thr Asp Pro Phe Gly Ser
805 810 815
Thr Ser Asp Ala Val Ile Glu Lys Ala Asp Val Ala Val Glu Ser Leu
820 825 830
Ile Pro Gly Leu Glu Pro Pro Val Pro Gln Arg Leu Pro Ser Gln Thr
835 840 845
Glu Ser Val Thr Ser Asn Arg Thr Asp Ser Leu Thr Gly Glu Asp Ser
850 855 860
Leu Leu Asp Cys Ser Leu Leu Ser Asn Pro Thr Thr Asp Leu Leu Glu
865 870 875 880
Glu Phe Ala Pro Thr Ala Ile Ser Ala Pro Val His Lys Ala Ala Glu
885 890 895
Asp Ser Asn Leu Ile Ser Gly Phe Asp Val Pro Glu Gly Ser Asp Lys
900 905 910
Val Ala Glu Asp Glu Phe Asp Pro Ile Pro Val Leu Ile Thr Lys Asn
915 920 925
Pro Gln Gly Gly His Ser Arg Asn Ser Ser Gly Ser Ser Glu Ser Ser
930 935 940
Leu Pro Asn Leu Ala Arg Ser Leu Leu Leu Val Asp Gln Leu Ile Asp
945 950 955 960
Leu
<210> 18
<211> 553
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 18
Met Ala Ala Pro Ala Gly Gly Gly Gly Ser Ala Val Ser Val Leu Ala
1 5 10 15
Pro Asn Gly Arg Arg His Thr Val Lys Val Thr Pro Ser Thr Val Leu
20 25 30
Leu Gln Val Leu Glu Asp Thr Cys Arg Arg Gln Asp Phe Asn Pro Cys
35 40 45
Glu Tyr Asp Leu Lys Phe Gln Arg Ser Val Leu Asp Leu Ser Leu Gln
50 55 60
Trp Arg Phe Ala Asn Leu Pro Asn Asn Ala Lys Leu Glu Met Val Pro
65 70 75 80
Ala Ser Arg Ser Arg Glu Gly Pro Glu Asn Met Val Arg Ile Ala Leu
85 90 95
Gln Leu Asp Asp Gly Ser Arg Leu Gln Asp Ser Phe Cys Ser Gly Gln
100 105 110
Thr Leu Trp Glu Leu Leu Ser His Phe Pro Gln Ile Arg Glu Cys Leu
115 120 125
Gln His Pro Gly Gly Ala Thr Pro Val Cys Val Tyr Thr Arg Asp Glu
130 135 140
Val Thr Gly Glu Ala Ala Leu Arg Gly Thr Thr Leu Gln Ser Leu Gly
145 150 155 160
Leu Thr Gly Gly Ser Ala Thr Ile Arg Phe Val Met Lys Cys Tyr Asp
165 170 175
Pro Val Gly Lys Thr Pro Gly Ser Leu Gly Ser Ser Ala Ser Ala Gly
180 185 190
Gln Ala Ala Ala Ser Ala Pro Leu Pro Leu Glu Ser Gly Glu Leu Ser
195 200 205
Arg Gly Asp Leu Ser Arg Pro Glu Asp Ala Asp Thr Ser Gly Pro Cys
210 215 220
Cys Glu His Thr Gln Glu Lys Gln Ser Thr Arg Ala Pro Ala Ala Ala
225 230 235 240
Pro Phe Val Pro Phe Ser Gly Gly Gly Gln Arg Leu Gly Gly Pro Pro
245 250 255
Gly Pro Thr Arg Pro Leu Thr Ser Ser Ser Ala Lys Leu Pro Lys Ser
260 265 270
Leu Ser Ser Pro Gly Gly Pro Ser Lys Pro Lys Lys Ser Lys Ser Gly
275 280 285
Gln Asp Pro Gln Gln Glu Gln Glu Gln Glu Arg Glu Arg Asp Pro Gln
290 295 300
Gln Glu Gln Glu Arg Glu Arg Pro Val Asp Arg Glu Pro Val Asp Arg
305 310 315 320
Glu Pro Val Val Cys His Pro Asp Leu Glu Glu Arg Leu Gln Ala Trp
325 330 335
Pro Ala Glu Leu Pro Asp Glu Phe Phe Glu Leu Thr Val Asp Asp Val
340 345 350
Arg Arg Arg Leu Ala Gln Leu Lys Ser Glu Arg Lys Arg Leu Glu Glu
355 360 365
Ala Pro Leu Val Thr Lys Ala Phe Arg Glu Ala Gln Ile Lys Glu Lys
370 375 380
Leu Glu Arg Tyr Pro Lys Val Ala Leu Arg Val Leu Phe Pro Asp Arg
385 390 395 400
Tyr Val Leu Gln Gly Phe Phe Arg Pro Ser Glu Thr Val Gly Asp Leu
405 410 415
Arg Asp Phe Val Arg Ser His Leu Gly Asn Pro Glu Leu Ser Phe Tyr
420 425 430
Leu Phe Ile Thr Pro Pro Lys Thr Val Leu Asp Asp His Thr Gln Thr
435 440 445
Leu Phe Gln Ala Asn Leu Phe Pro Ala Ala Leu Val His Leu Gly Ala
450 455 460
Glu Glu Pro Ala Gly Val Tyr Leu Glu Pro Gly Leu Leu Glu His Ala
465 470 475 480
Ile Ser Pro Ser Ala Ala Asp Val Leu Val Ala Arg Tyr Met Ser Arg
485 490 495
Ala Ala Gly Ser Pro Ser Pro Leu Pro Ala Pro Asp Pro Ala Pro Lys
500 505 510
Ser Glu Pro Ala Ala Glu Glu Gly Ala Leu Val Pro Pro Glu Pro Ile
515 520 525
Pro Gly Thr Ala Gln Pro Val Lys Arg Ser Leu Gly Lys Val Pro Lys
530 535 540
Trp Leu Lys Leu Pro Ala Ser Lys Arg
545 550
<210> 19
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 19
Met Asp Arg Ser Lys Glu Asn Cys Ile Ser Gly Pro Val Lys Ala Thr
1 5 10 15
Ala Pro Val Gly Gly Pro Lys Arg Val Leu Val Thr Gln Gln Phe Pro
20 25 30
Cys Gln Asn Pro Leu Pro Val Asn Ser Gly Gln Ala Gln Arg Val Leu
35 40 45
Cys Pro Ser Asn Ser Ser Gln Arg Val Pro Leu Gln Ala Gln Lys Leu
50 55 60
Val Ser Ser His Lys Pro Val Gln Asn Gln Lys Gln Lys Gln Leu Gln
65 70 75 80
Ala Thr Ser Val Pro His Pro Val Ser Arg Pro Leu Asn Asn Thr Gln
85 90 95
Lys Ser Lys Gln Pro Leu Pro Ser Ala Pro Glu Asn Asn Pro Glu Glu
100 105 110
Glu Leu Ala Ser Lys Gln Lys Asn Glu Glu Ser Lys Lys Arg Gln Trp
115 120 125
Ala Leu Glu Asp Phe Glu Ile Gly Arg Pro Leu Gly Lys Gly Lys Phe
130 135 140
Gly Asn Val Tyr Leu Ala Arg Glu Lys Gln Ser Lys Phe Ile Leu Ala
145 150 155 160
Leu Lys Val Leu Phe Lys Ala Gln Leu Glu Lys Ala Gly Val Glu His
165 170 175
Gln Leu Arg Arg Glu Val Glu Ile Gln Ser His Leu Arg His Pro Asn
180 185 190
Ile Leu Arg Leu Tyr Gly Tyr Phe His Asp Ala Thr Arg Val Tyr Leu
195 200 205
Ile Leu Glu Tyr Ala Pro Leu Gly Thr Val Tyr Arg Glu Leu Gln Lys
210 215 220
Leu Ser Lys Phe Asp Glu Gln Arg Thr Ala Thr Tyr Ile Thr Glu Leu
225 230 235 240
Ala Asn Ala Leu Ser Tyr Cys His Ser Lys Arg Val Ile His Arg Asp
245 250 255
Ile Lys Pro Glu Asn Leu Leu Leu Gly Ser Ala Gly Glu Leu Lys Ile
260 265 270
Ala Asp Phe Gly Trp Ser Val His Ala Pro Ser Ser Arg Arg Thr Thr
275 280 285
Leu Cys Gly Thr Leu Asp Tyr Leu Pro Pro Glu Met Ile Glu Gly Arg
290 295 300
Met His Asp Glu Lys Val Asp Leu Trp Ser Leu Gly Val Leu Cys Tyr
305 310 315 320
Glu Phe Leu Val Gly Lys Pro Pro Phe Glu Ala Asn Thr Tyr Gln Glu
325 330 335
Thr Tyr Lys Arg Ile Ser Arg Val Glu Phe Thr Phe Pro Asp Phe Val
340 345 350
Thr Glu Gly Ala Arg Asp Leu Ile Ser Arg Leu Leu Lys His Asn Pro
355 360 365
Ser Gln Arg Pro Met Leu Arg Glu Val Leu Glu His Pro Trp Ile Thr
370 375 380
Ala Asn Ser Ser Lys Pro Ser Asn Cys Gln Asn Lys Glu Ser Ala Ser
385 390 395 400
Lys Gln Ser
<210> 20
<211> 521
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 20
Met Lys Ser Gln Gly Gln His Trp Tyr Ser Ser Ser Asp Lys Asn Cys
1 5 10 15
Lys Val Ser Phe Arg Glu Lys Leu Leu Ile Ile Asp Ser Asn Leu Gly
20 25 30
Val Gln Asp Val Glu Asn Leu Lys Phe Leu Cys Ile Gly Leu Val Pro
35 40 45
Asn Lys Lys Leu Glu Lys Ser Ser Ser Ala Ser Asp Val Phe Glu His
50 55 60
Leu Leu Ala Glu Asp Leu Leu Ser Glu Glu Asp Pro Phe Phe Leu Ala
65 70 75 80
Glu Leu Leu Tyr Ile Ile Arg Gln Lys Lys Leu Leu Gln His Leu Asn
85 90 95
Cys Thr Lys Glu Glu Val Glu Arg Leu Leu Pro Thr Arg Gln Arg Val
100 105 110
Ser Leu Phe Arg Asn Leu Leu Tyr Glu Leu Ser Glu Gly Ile Asp Ser
115 120 125
Glu Asn Leu Lys Asp Met Ile Phe Leu Leu Lys Asp Ser Leu Pro Lys
130 135 140
Thr Glu Met Thr Ser Leu Ser Phe Leu Ala Phe Leu Glu Lys Gln Gly
145 150 155 160
Lys Ile Asp Glu Asp Asn Leu Thr Cys Leu Glu Asp Leu Cys Lys Thr
165 170 175
Val Val Pro Lys Leu Leu Arg Asn Ile Glu Lys Tyr Lys Arg Glu Lys
180 185 190
Ala Ile Gln Ile Val Thr Pro Pro Val Asp Lys Glu Ala Glu Ser Tyr
195 200 205
Gln Gly Glu Glu Glu Leu Val Ser Gln Thr Asp Val Lys Thr Phe Leu
210 215 220
Glu Ala Leu Pro Gln Glu Ser Trp Gln Asn Lys His Ala Gly Ser Asn
225 230 235 240
Gly Asn Arg Ala Thr Asn Gly Ala Pro Ser Leu Val Ser Arg Gly Met
245 250 255
Gln Gly Ala Ser Ala Asn Thr Leu Asn Ser Glu Thr Ser Thr Lys Arg
260 265 270
Ala Ala Val Tyr Arg Met Asn Arg Asn His Arg Gly Leu Cys Val Ile
275 280 285
Val Asn Asn His Ser Phe Thr Ser Leu Lys Asp Arg Gln Gly Thr His
290 295 300
Lys Asp Ala Glu Ile Leu Ser His Val Phe Gln Trp Leu Gly Phe Thr
305 310 315 320
Val His Ile His Asn Asn Val Thr Lys Val Glu Met Glu Met Val Leu
325 330 335
Gln Lys Gln Lys Cys Asn Pro Ala His Ala Asp Gly Asp Cys Phe Val
340 345 350
Phe Cys Ile Leu Thr His Gly Arg Phe Gly Ala Val Tyr Ser Ser Asp
355 360 365
Glu Ala Leu Ile Pro Ile Arg Glu Ile Met Ser His Phe Thr Ala Leu
370 375 380
Gln Cys Pro Arg Leu Ala Glu Lys Pro Lys Leu Phe Phe Ile Gln Ala
385 390 395 400
Cys Gln Gly Glu Glu Ile Gln Pro Ser Val Ser Ile Glu Ala Asp Ala
405 410 415
Leu Asn Pro Glu Gln Ala Pro Thr Ser Leu Gln Asp Ser Ile Pro Ala
420 425 430
Glu Ala Asp Phe Leu Leu Gly Leu Ala Thr Val Pro Gly Tyr Val Ser
435 440 445
Phe Arg His Val Glu Glu Gly Ser Trp Tyr Ile Gln Ser Leu Cys Asn
450 455 460
His Leu Lys Lys Leu Val Pro Arg Met Leu Lys Phe Leu Glu Lys Thr
465 470 475 480
Met Glu Ile Arg Gly Arg Lys Arg Thr Val Trp Gly Ala Lys Gln Ile
485 490 495
Ser Ala Thr Ser Leu Pro Thr Ala Ile Ser Ala Gln Thr Pro Arg Pro
500 505 510
Pro Met Arg Arg Trp Ser Ser Val Ser
515 520
<210> 21
<211> 585
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 21
Met Glu Lys Lys Trp Lys Tyr Cys Ala Val Tyr Tyr Ile Ile Gln Ile
1 5 10 15
His Phe Val Lys Gly Val Trp Glu Lys Thr Val Asn Thr Glu Glu Asn
20 25 30
Val Tyr Ala Thr Leu Gly Ser Asp Val Asn Leu Thr Cys Gln Thr Gln
35 40 45
Thr Val Gly Phe Phe Val Gln Met Gln Trp Ser Lys Val Thr Asn Lys
50 55 60
Ile Asp Leu Ile Ala Val Tyr His Pro Gln Tyr Gly Phe Tyr Cys Ala
65 70 75 80
Tyr Gly Arg Pro Cys Glu Ser Leu Val Thr Phe Thr Glu Thr Pro Glu
85 90 95
Asn Gly Ser Lys Trp Thr Leu His Leu Arg Asn Met Ser Cys Ser Val
100 105 110
Ser Gly Arg Tyr Glu Cys Met Leu Val Leu Tyr Pro Glu Gly Ile Gln
115 120 125
Thr Lys Ile Tyr Asn Leu Leu Ile Gln Thr His Val Thr Ala Asp Glu
130 135 140
Trp Asn Ser Asn His Thr Ile Glu Ile Glu Ile Asn Gln Thr Leu Glu
145 150 155 160
Ile Pro Cys Phe Gln Asn Ser Ser Ser Lys Ile Ser Ser Glu Phe Thr
165 170 175
Tyr Ala Trp Ser Val Glu Asn Ser Ser Thr Asp Ser Trp Val Leu Leu
180 185 190
Ser Lys Gly Ile Lys Glu Asp Asn Gly Thr Gln Glu Thr Leu Ile Ser
195 200 205
Gln Asn His Leu Ile Ser Asn Ser Thr Leu Leu Lys Asp Arg Val Lys
210 215 220
Leu Gly Thr Asp Tyr Arg Leu His Leu Ser Pro Val Gln Ile Phe Asp
225 230 235 240
Asp Gly Arg Lys Phe Ser Cys His Ile Arg Val Gly Pro Asn Lys Ile
245 250 255
Leu Arg Ser Ser Thr Thr Val Lys Val Phe Ala Lys Pro Glu Ile Pro
260 265 270
Val Ile Val Glu Asn Asn Ser Thr Asp Val Leu Val Glu Arg Arg Phe
275 280 285
Thr Cys Leu Leu Lys Asn Val Phe Pro Lys Ala Asn Ile Thr Trp Phe
290 295 300
Ile Asp Gly Ser Phe Leu His Asp Glu Lys Glu Gly Ile Tyr Ile Thr
305 310 315 320
Asn Glu Glu Arg Lys Gly Lys Asp Gly Phe Leu Glu Leu Lys Ser Val
325 330 335
Leu Thr Arg Val His Ser Asn Lys Pro Ala Gln Ser Asp Asn Leu Thr
340 345 350
Ile Trp Cys Met Ala Leu Ser Pro Val Pro Gly Asn Lys Val Trp Asn
355 360 365
Ile Ser Ser Glu Lys Ile Thr Phe Leu Leu Gly Ser Glu Ile Ser Ser
370 375 380
Thr Asp Pro Pro Leu Ser Val Thr Glu Ser Thr Leu Asp Thr Gln Pro
385 390 395 400
Ser Pro Ala Ser Ser Val Ser Pro Ala Arg Tyr Pro Ala Thr Ser Ser
405 410 415
Val Thr Leu Val Asp Val Ser Ala Leu Arg Pro Asn Thr Thr Pro Gln
420 425 430
Pro Ser Asn Ser Ser Met Thr Thr Arg Gly Phe Asn Tyr Pro Trp Thr
435 440 445
Ser Ser Gly Thr Asp Thr Lys Lys Ser Val Ser Arg Ile Pro Ser Glu
450 455 460
Thr Tyr Ser Ser Ser Pro Ser Gly Ala Gly Ser Thr Leu His Asp Asn
465 470 475 480
Val Phe Thr Ser Thr Ala Arg Ala Phe Ser Glu Val Pro Thr Thr Ala
485 490 495
Asn Gly Ser Thr Lys Thr Asn His Val His Ile Thr Gly Ile Val Val
500 505 510
Asn Lys Pro Lys Asp Gly Met Ser Trp Pro Val Ile Val Ala Ala Leu
515 520 525
Leu Phe Cys Cys Met Ile Leu Phe Gly Leu Gly Val Arg Lys Trp Cys
530 535 540
Gln Tyr Gln Lys Glu Ile Met Glu Arg Pro Pro Pro Phe Lys Pro Pro
545 550 555 560
Pro Pro Pro Ile Lys Tyr Thr Cys Ile Gln Glu Pro Asn Glu Ser Asp
565 570 575
Leu Pro Tyr His Glu Met Glu Thr Leu
580 585
<210> 22
<211> 380
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 22
Met Gly Ile Gln Gly Leu Ala Lys Leu Ile Ala Asp Val Ala Pro Ser
1 5 10 15
Ala Ile Arg Glu Asn Asp Ile Lys Ser Tyr Phe Gly Arg Lys Val Ala
20 25 30
Ile Asp Ala Ser Met Ser Ile Tyr Gln Phe Leu Ile Ala Val Arg Gln
35 40 45
Gly Gly Asp Val Leu Gln Asn Glu Glu Gly Glu Thr Thr Ser His Leu
50 55 60
Met Gly Met Phe Tyr Arg Thr Ile Arg Met Met Glu Asn Gly Ile Lys
65 70 75 80
Pro Val Tyr Val Phe Asp Gly Lys Pro Pro Gln Leu Lys Ser Gly Glu
85 90 95
Leu Ala Lys Arg Ser Glu Arg Arg Ala Glu Ala Glu Lys Gln Leu Gln
100 105 110
Gln Ala Gln Ala Ala Gly Ala Glu Gln Glu Val Glu Lys Phe Thr Lys
115 120 125
Arg Leu Val Lys Val Thr Lys Gln His Asn Asp Glu Cys Lys His Leu
130 135 140
Leu Ser Leu Met Gly Ile Pro Tyr Leu Asp Ala Pro Ser Glu Ala Glu
145 150 155 160
Ala Ser Cys Ala Ala Leu Val Lys Ala Gly Lys Val Tyr Ala Ala Ala
165 170 175
Thr Glu Asp Met Asp Cys Leu Thr Phe Gly Ser Pro Val Leu Met Arg
180 185 190
His Leu Thr Ala Ser Glu Ala Lys Lys Leu Pro Ile Gln Glu Phe His
195 200 205
Leu Ser Arg Ile Leu Gln Glu Leu Gly Leu Asn Gln Glu Gln Phe Val
210 215 220
Asp Leu Cys Ile Leu Leu Gly Ser Asp Tyr Cys Glu Ser Ile Arg Gly
225 230 235 240
Ile Gly Pro Lys Arg Ala Val Asp Leu Ile Gln Lys His Lys Ser Ile
245 250 255
Glu Glu Ile Val Arg Arg Leu Asp Pro Asn Lys Tyr Pro Val Pro Glu
260 265 270
Asn Trp Leu His Lys Glu Ala His Gln Leu Phe Leu Glu Pro Glu Val
275 280 285
Leu Asp Pro Glu Ser Val Glu Leu Lys Trp Ser Glu Pro Asn Glu Glu
290 295 300
Glu Leu Ile Lys Phe Met Cys Gly Glu Lys Gln Phe Ser Glu Glu Arg
305 310 315 320
Ile Arg Ser Gly Val Lys Arg Leu Ser Lys Ser Arg Gln Gly Ser Thr
325 330 335
Gln Gly Arg Leu Asp Asp Phe Phe Lys Val Thr Gly Ser Leu Ser Ser
340 345 350
Ala Lys Arg Lys Glu Pro Glu Pro Lys Gly Ser Thr Lys Lys Lys Ala
355 360 365
Lys Thr Gly Ala Ala Gly Lys Phe Lys Arg Gly Lys
370 375 380
<210> 23
<211> 223
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 23
Met Ala Ala Ala Val Pro Gln Arg Ala Trp Thr Val Glu Gln Leu Arg
1 5 10 15
Ser Glu Gln Leu Pro Lys Lys Asp Ile Ile Lys Phe Leu Gln Glu His
20 25 30
Gly Ser Asp Ser Phe Leu Ala Glu His Lys Leu Leu Gly Asn Ile Lys
35 40 45
Asn Val Ala Lys Thr Ala Asn Lys Asp His Leu Val Thr Ala Tyr Asn
50 55 60
His Leu Phe Glu Thr Lys Arg Phe Lys Gly Thr Glu Ser Ile Ser Lys
65 70 75 80
Val Ser Glu Gln Val Lys Asn Val Lys Leu Asn Glu Asp Lys Pro Lys
85 90 95
Glu Thr Lys Ser Glu Glu Thr Leu Asp Glu Gly Pro Pro Lys Tyr Thr
100 105 110
Lys Ser Val Leu Lys Lys Gly Asp Lys Thr Asn Phe Pro Lys Lys Gly
115 120 125
Asp Val Val His Cys Trp Tyr Thr Gly Thr Leu Gln Asp Gly Thr Val
130 135 140
Phe Asp Thr Asn Ile Gln Thr Ser Ala Lys Lys Lys Lys Asn Ala Lys
145 150 155 160
Pro Leu Ser Phe Lys Val Gly Val Gly Lys Val Ile Arg Gly Trp Asp
165 170 175
Glu Ala Leu Leu Thr Met Ser Lys Gly Glu Lys Ala Arg Leu Glu Ile
180 185 190
Glu Pro Glu Trp Ala Tyr Gly Lys Lys Gly Gln Pro Asp Ala Lys Ile
195 200 205
Pro Pro Asn Ala Lys Leu Thr Phe Glu Val Glu Leu Val Asp Ile
210 215 220
<210> 24
<211> 1086
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 24
Met Gly Asn Ala Gly Ser Met Asp Ser Gln Gln Thr Asp Phe Arg Ala
1 5 10 15
His Asn Val Pro Leu Lys Leu Pro Met Pro Glu Pro Gly Glu Leu Glu
20 25 30
Glu Arg Phe Ala Ile Val Leu Asn Ala Met Asn Leu Pro Pro Asp Lys
35 40 45
Ala Arg Leu Leu Arg Gln Tyr Asp Asn Glu Lys Lys Trp Glu Leu Ile
50 55 60
Cys Asp Gln Glu Arg Phe Gln Val Lys Asn Pro Pro His Thr Tyr Ile
65 70 75 80
Gln Lys Leu Lys Gly Tyr Leu Asp Pro Ala Val Thr Arg Lys Lys Phe
85 90 95
Arg Arg Arg Val Gln Glu Ser Thr Gln Val Leu Arg Glu Leu Glu Ile
100 105 110
Ser Leu Arg Thr Asn His Ile Gly Trp Val Arg Glu Phe Leu Asn Glu
115 120 125
Glu Asn Lys Gly Leu Asp Val Leu Val Glu Tyr Leu Ser Phe Ala Gln
130 135 140
Tyr Ala Val Thr Phe Asp Phe Glu Ser Val Glu Ser Thr Val Glu Ser
145 150 155 160
Ser Val Asp Lys Ser Lys Pro Trp Ser Arg Ser Ile Glu Asp Leu His
165 170 175
Arg Gly Ser Asn Leu Pro Ser Pro Val Gly Asn Ser Val Ser Arg Ser
180 185 190
Gly Arg His Ser Ala Leu Arg Tyr Asn Thr Leu Pro Ser Arg Arg Thr
195 200 205
Leu Lys Asn Ser Arg Leu Val Ser Lys Lys Asp Asp Val His Val Cys
210 215 220
Ile Met Cys Leu Arg Ala Ile Met Asn Tyr Gln Tyr Gly Phe Asn Met
225 230 235 240
Val Met Ser His Pro His Ala Val Asn Glu Ile Ala Leu Ser Leu Asn
245 250 255
Asn Lys Asn Pro Arg Thr Lys Ala Leu Val Leu Glu Leu Leu Ala Ala
260 265 270
Val Cys Leu Val Arg Gly Gly His Glu Ile Ile Leu Ser Ala Phe Asp
275 280 285
Asn Phe Lys Glu Val Cys Gly Glu Lys Gln Arg Phe Glu Lys Leu Met
290 295 300
Glu His Phe Arg Asn Glu Asp Asn Asn Ile Asp Phe Met Val Ala Ser
305 310 315 320
Met Gln Phe Ile Asn Ile Val Val His Ser Val Glu Asp Met Asn Phe
325 330 335
Arg Val His Leu Gln Tyr Glu Phe Thr Lys Leu Gly Leu Asp Glu Tyr
340 345 350
Leu Asp Lys Leu Lys His Thr Glu Ser Asp Lys Leu Gln Val Gln Ile
355 360 365
Gln Ala Tyr Leu Asp Asn Val Phe Asp Val Gly Ala Leu Leu Glu Asp
370 375 380
Ala Glu Thr Lys Asn Ala Ala Leu Glu Arg Val Glu Glu Leu Glu Glu
385 390 395 400
Asn Ile Ser His Leu Ser Glu Lys Leu Gln Asp Thr Glu Asn Glu Ala
405 410 415
Met Ser Lys Ile Val Glu Leu Glu Lys Gln Leu Met Gln Arg Asn Lys
420 425 430
Glu Leu Asp Val Val Arg Glu Ile Tyr Lys Asp Ala Asn Thr Gln Val
435 440 445
His Thr Leu Arg Lys Met Val Lys Glu Lys Glu Glu Ala Ile Gln Arg
450 455 460
Gln Ser Thr Leu Glu Lys Lys Ile His Glu Leu Glu Lys Gln Gly Thr
465 470 475 480
Ile Lys Ile Gln Lys Lys Gly Asp Gly Asp Ile Ala Ile Leu Pro Val
485 490 495
Val Ala Ser Gly Thr Leu Ser Met Gly Ser Glu Val Val Ala Gly Asn
500 505 510
Ser Val Gly Pro Thr Met Gly Ala Ala Ser Ser Gly Pro Leu Pro Pro
515 520 525
Pro Pro Pro Pro Leu Pro Pro Ser Ser Asp Thr Pro Glu Thr Val Gln
530 535 540
Asn Gly Pro Val Thr Pro Pro Met Pro Pro Pro Pro Pro Pro Pro Pro
545 550 555 560
Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro Gly
565 570 575
Pro Ala Ala Glu Thr Val Pro Ala Pro Pro Leu Ala Pro Pro Leu Pro
580 585 590
Ser Ala Pro Pro Leu Pro Gly Thr Ser Ser Pro Thr Val Val Phe Asn
595 600 605
Ser Gly Leu Ala Ala Val Lys Ile Lys Lys Pro Ile Lys Thr Lys Phe
610 615 620
Arg Met Pro Val Phe Asn Trp Val Ala Leu Lys Pro Asn Gln Ile Asn
625 630 635 640
Gly Thr Val Phe Asn Glu Ile Asp Asp Glu Arg Ile Leu Glu Asp Leu
645 650 655
Asn Val Asp Glu Phe Glu Glu Ile Phe Lys Thr Lys Ala Gln Gly Pro
660 665 670
Ala Ile Asp Leu Ser Ser Ser Lys Gln Lys Ile Pro Gln Lys Gly Ser
675 680 685
Asn Lys Val Thr Leu Leu Glu Ala Asn Arg Ala Lys Asn Leu Ala Ile
690 695 700
Thr Leu Arg Lys Ala Gly Lys Thr Ala Asp Glu Ile Cys Lys Ala Ile
705 710 715 720
His Val Phe Asp Leu Lys Thr Leu Pro Val Asp Phe Val Glu Cys Leu
725 730 735
Met Arg Phe Leu Pro Thr Glu Asn Glu Val Lys Val Leu Arg Leu Tyr
740 745 750
Glu Arg Glu Arg Lys Pro Leu Glu Asn Leu Ser Asp Glu Asp Arg Phe
755 760 765
Met Met Gln Phe Ser Lys Ile Glu Arg Leu Met Gln Lys Met Thr Ile
770 775 780
Met Ala Phe Ile Gly Asn Phe Ala Glu Ser Ile Gln Met Leu Thr Pro
785 790 795 800
Gln Leu His Ala Ile Ile Ala Ala Ser Val Ser Ile Lys Ser Ser Gln
805 810 815
Lys Leu Lys Lys Ile Leu Glu Ile Ile Leu Ala Leu Gly Asn Tyr Met
820 825 830
Asn Ser Ser Lys Arg Gly Ala Val Tyr Gly Phe Lys Leu Gln Ser Leu
835 840 845
Asp Leu Leu Leu Asp Thr Lys Ser Thr Asp Arg Lys Gln Thr Leu Leu
850 855 860
His Tyr Ile Ser Asn Val Val Lys Glu Lys Tyr His Gln Val Ser Leu
865 870 875 880
Phe Tyr Asn Glu Leu His Tyr Val Glu Lys Ala Ala Ala Val Ser Leu
885 890 895
Glu Asn Val Leu Leu Asp Val Lys Glu Leu Gln Arg Gly Met Asp Leu
900 905 910
Thr Lys Arg Glu Tyr Thr Met His Asp His Asn Thr Leu Leu Lys Glu
915 920 925
Phe Ile Leu Asn Asn Glu Gly Lys Leu Lys Lys Leu Gln Asp Asp Ala
930 935 940
Lys Ile Ala Gln Asp Ala Phe Asp Asp Val Val Lys Tyr Phe Gly Glu
945 950 955 960
Asn Pro Lys Thr Thr Pro Pro Ser Val Phe Phe Pro Val Phe Val Arg
965 970 975
Phe Val Lys Ala Tyr Lys Gln Ala Glu Glu Glu Asn Glu Leu Arg Lys
980 985 990
Lys Gln Glu Gln Ala Leu Met Glu Lys Leu Leu Glu Gln Glu Ala Leu
995 1000 1005
Met Glu Gln Gln Asp Pro Lys Ser Pro Ser His Lys Ser Lys Arg
1010 1015 1020
Gln Gln Gln Glu Leu Ile Ala Glu Leu Arg Arg Arg Gln Val Lys
1025 1030 1035
Asp Asn Arg His Val Tyr Glu Gly Lys Asp Gly Ala Ile Glu Asp
1040 1045 1050
Ile Ile Thr Val Leu Lys Thr Val Pro Phe Thr Ala Arg Thr Ala
1055 1060 1065
Lys Arg Gly Ser Arg Phe Phe Cys Glu Pro Val Leu Thr Glu Glu
1070 1075 1080
Tyr His Tyr
1085
<210> 25
<211> 335
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 25
Met Ala Ala Gly Ala Ala Glu Ala Ala Val Ala Ala Val Glu Glu Val
1 5 10 15
Gly Ser Ala Gly Gln Phe Glu Glu Leu Leu Arg Leu Lys Ala Lys Ser
20 25 30
Leu Leu Val Val His Phe Trp Ala Pro Trp Ala Pro Gln Cys Ala Gln
35 40 45
Met Asn Glu Val Met Ala Glu Leu Ala Lys Glu Leu Pro Gln Val Ser
50 55 60
Phe Val Lys Leu Glu Ala Glu Gly Val Pro Glu Val Ser Glu Lys Tyr
65 70 75 80
Glu Ile Ser Ser Val Pro Thr Phe Leu Phe Phe Lys Asn Ser Gln Lys
85 90 95
Ile Asp Arg Leu Asp Gly Ala His Ala Pro Glu Leu Thr Lys Lys Val
100 105 110
Gln Arg His Ala Ser Ser Gly Ser Phe Leu Pro Ser Ala Asn Glu His
115 120 125
Leu Lys Glu Asp Leu Asn Leu Arg Leu Lys Lys Leu Thr His Ala Ala
130 135 140
Pro Cys Met Leu Phe Met Lys Gly Thr Pro Gln Glu Pro Arg Cys Gly
145 150 155 160
Phe Ser Lys Gln Met Val Glu Ile Leu His Lys His Asn Ile Gln Phe
165 170 175
Ser Ser Phe Asp Ile Phe Ser Asp Glu Glu Val Arg Gln Gly Leu Lys
180 185 190
Ala Tyr Ser Ser Trp Pro Thr Tyr Pro Gln Leu Tyr Val Ser Gly Glu
195 200 205
Leu Ile Gly Gly Leu Asp Ile Ile Lys Glu Leu Glu Ala Ser Glu Glu
210 215 220
Leu Asp Thr Ile Cys Pro Lys Ala Pro Lys Leu Glu Glu Arg Leu Lys
225 230 235 240
Val Leu Thr Asn Lys Ala Ser Val Met Leu Phe Met Lys Gly Asn Lys
245 250 255
Gln Glu Ala Lys Cys Gly Phe Ser Lys Gln Ile Leu Glu Ile Leu Asn
260 265 270
Ser Thr Gly Val Glu Tyr Glu Thr Phe Asp Ile Leu Glu Asp Glu Glu
275 280 285
Val Arg Gln Gly Leu Lys Ala Tyr Ser Asn Trp Pro Thr Tyr Pro Gln
290 295 300
Leu Tyr Val Lys Gly Glu Leu Val Gly Gly Leu Asp Ile Val Lys Glu
305 310 315 320
Leu Lys Glu Asn Gly Glu Leu Leu Pro Ile Leu Arg Gly Glu Asn
325 330 335
<210> 26
<211> 966
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 26
Met Ala Asn Phe Gln Glu His Leu Ser Cys Ser Ser Ser Pro His Leu
1 5 10 15
Pro Phe Ser Glu Ser Lys Thr Phe Asn Gly Leu Gln Asp Glu Leu Thr
20 25 30
Ala Met Gly Asn His Pro Ser Pro Lys Leu Leu Glu Asp Gln Gln Glu
35 40 45
Lys Gly Met Val Arg Thr Glu Leu Ile Glu Ser Val His Ser Pro Val
50 55 60
Thr Thr Thr Val Leu Thr Ser Val Ser Glu Asp Ser Arg Asp Gln Phe
65 70 75 80
Glu Asn Ser Val Leu Gln Leu Arg Glu His Asp Glu Ser Glu Thr Ala
85 90 95
Val Ser Gln Gly Asn Ser Asn Thr Val Asp Gly Glu Ser Thr Ser Gly
100 105 110
Thr Glu Asp Ile Lys Ile Gln Phe Ser Arg Ser Gly Ser Gly Ser Gly
115 120 125
Gly Phe Leu Glu Gly Leu Phe Gly Cys Leu Arg Pro Val Trp Asn Ile
130 135 140
Ile Gly Lys Ala Tyr Ser Thr Asp Tyr Lys Leu Gln Gln Gln Asp Thr
145 150 155 160
Trp Glu Val Pro Phe Glu Glu Ile Ser Glu Leu Gln Trp Leu Gly Ser
165 170 175
Gly Ala Gln Gly Ala Val Phe Leu Gly Lys Phe Arg Ala Glu Glu Val
180 185 190
Ala Ile Lys Lys Val Arg Glu Gln Asn Glu Thr Asp Ile Lys His Leu
195 200 205
Arg Lys Leu Lys His Pro Asn Ile Ile Ala Phe Lys Gly Val Cys Thr
210 215 220
Gln Ala Pro Cys Tyr Cys Ile Ile Met Glu Tyr Cys Ala His Gly Gln
225 230 235 240
Leu Tyr Glu Val Leu Arg Ala Gly Arg Lys Ile Thr Pro Arg Leu Leu
245 250 255
Val Asp Trp Ser Thr Gly Ile Ala Ser Gly Met Asn Tyr Leu His Leu
260 265 270
His Lys Ile Ile His Arg Asp Leu Lys Ser Pro Asn Val Leu Val Thr
275 280 285
His Thr Asp Ala Val Lys Ile Ser Asp Phe Gly Thr Ser Lys Glu Leu
290 295 300
Ser Asp Lys Ser Thr Lys Met Ser Phe Ala Gly Thr Val Ala Trp Met
305 310 315 320
Ala Pro Glu Val Ile Arg Asn Glu Pro Val Ser Glu Lys Val Asp Ile
325 330 335
Trp Ser Phe Gly Val Val Leu Trp Glu Leu Leu Thr Gly Glu Ile Pro
340 345 350
Tyr Lys Asp Val Asp Ser Ser Ala Ile Ile Trp Gly Val Gly Ser Asn
355 360 365
Ser Leu His Leu Pro Val Pro Ser Thr Cys Pro Asp Gly Phe Lys Ile
370 375 380
Leu Met Lys Gln Thr Trp Gln Ser Lys Pro Arg Asn Arg Pro Ser Phe
385 390 395 400
Arg Gln Thr Leu Met His Leu Asp Ile Ala Ser Ala Asp Val Leu Ala
405 410 415
Thr Pro Gln Glu Thr Tyr Phe Lys Ser Gln Ala Glu Trp Arg Glu Glu
420 425 430
Val Lys Lys His Phe Glu Lys Ile Lys Ser Glu Gly Thr Cys Ile His
435 440 445
Arg Leu Asp Glu Glu Leu Ile Arg Arg Arg Arg Glu Glu Leu Arg His
450 455 460
Ala Leu Asp Ile Arg Glu His Tyr Glu Arg Lys Leu Glu Arg Ala Asn
465 470 475 480
Asn Leu Tyr Met Glu Leu Ser Ala Ile Met Leu Gln Leu Glu Met Arg
485 490 495
Glu Lys Glu Leu Ile Lys Arg Glu Gln Ala Val Glu Lys Lys Tyr Pro
500 505 510
Gly Thr Tyr Lys Arg His Pro Val Arg Pro Ile Ile His Pro Asn Ala
515 520 525
Met Glu Lys Leu Met Lys Arg Lys Gly Val Pro His Lys Ser Gly Met
530 535 540
Gln Thr Lys Arg Pro Asp Leu Leu Arg Ser Glu Gly Ile Pro Thr Thr
545 550 555 560
Glu Val Ala Pro Thr Ala Ser Pro Leu Ser Gly Ser Pro Lys Met Ser
565 570 575
Thr Ser Ser Ser Lys Ser Arg Tyr Arg Ser Lys Pro Arg His Arg Arg
580 585 590
Gly Asn Ser Arg Gly Ser His Ser Asp Phe Ala Ala Ile Leu Lys Asn
595 600 605
Gln Pro Ala Gln Glu Asn Ser Pro His Pro Thr Tyr Leu His Gln Ala
610 615 620
Gln Ser Gln Tyr Pro Ser Leu His His His Asn Ser Leu Gln Gln Gln
625 630 635 640
Tyr Gln Gln Pro Pro Pro Ala Met Ser Gln Ser His His Pro Arg Leu
645 650 655
Asn Met His Gly Gln Asp Ile Ala Thr Cys Ala Asn Asn Leu Arg Tyr
660 665 670
Phe Gly Pro Ala Ala Ala Leu Arg Ser Pro Leu Ser Asn His Ala Gln
675 680 685
Arg Gln Leu Pro Gly Ser Ser Pro Asp Leu Ile Ser Thr Ala Met Ala
690 695 700
Ala Asp Cys Trp Arg Ser Ser Glu Pro Asp Lys Gly Gln Ala Gly Pro
705 710 715 720
Trp Gly Cys Cys Gln Ala Asp Ala Tyr Asp Pro Cys Leu Gln Cys Arg
725 730 735
Pro Glu Gln Tyr Gly Ser Leu Asp Ile Pro Ser Ala Glu Pro Val Gly
740 745 750
Arg Ser Pro Asp Leu Ser Lys Ser Pro Ala His Asn Pro Leu Leu Glu
755 760 765
Asn Ala Gln Ser Ser Glu Lys Thr Glu Glu Asn Glu Phe Ser Gly Cys
770 775 780
Arg Ser Glu Ser Ser Leu Gly Thr Ser His Leu Gly Thr Pro Pro Ala
785 790 795 800
Leu Pro Arg Lys Thr Arg Pro Leu Gln Lys Ser Gly Asp Asp Ser Ser
805 810 815
Glu Glu Glu Glu Gly Glu Val Asp Ser Glu Val Glu Phe Pro Arg Arg
820 825 830
Gln Arg Pro His Arg Cys Ile Ser Ser Cys Gln Ser Tyr Ser Thr Phe
835 840 845
Ser Ser Glu Asn Phe Ser Val Ser Asp Gly Glu Glu Gly Asn Thr Ser
850 855 860
Asp His Ser Asn Ser Pro Asp Glu Leu Ala Asp Lys Leu Glu Asp Arg
865 870 875 880
Leu Ala Glu Lys Leu Asp Asp Leu Leu Ser Gln Thr Pro Glu Ile Pro
885 890 895
Ile Asp Ile Ser Ser His Ser Asp Gly Leu Ser Asp Lys Glu Cys Ala
900 905 910
Val Arg Arg Val Lys Thr Gln Met Ser Leu Gly Lys Leu Cys Val Glu
915 920 925
Glu Arg Gly Tyr Glu Asn Pro Met Gln Phe Glu Glu Ser Asp Cys Asp
930 935 940
Ser Ser Asp Gly Glu Cys Ser Asp Ala Thr Val Arg Thr Asn Lys His
945 950 955 960
Tyr Ser Ser Ala Thr Trp
965
<210> 27
<211> 1152
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 27
Met Ala Asp Leu Ser Leu Ala Asp Ala Leu Thr Glu Pro Ser Pro Asp
1 5 10 15
Ile Glu Gly Glu Ile Lys Arg Asp Phe Ile Ala Thr Leu Glu Ala Glu
20 25 30
Ala Phe Asp Asp Val Val Gly Glu Thr Val Gly Lys Thr Asp Tyr Ile
35 40 45
Pro Leu Leu Asp Val Asp Glu Lys Thr Gly Asn Ser Glu Ser Lys Lys
50 55 60
Lys Pro Cys Ser Glu Thr Ser Gln Ile Glu Asp Thr Pro Ser Ser Lys
65 70 75 80
Pro Thr Leu Leu Ala Asn Gly Gly His Gly Val Glu Gly Ser Asp Thr
85 90 95
Thr Gly Ser Pro Thr Glu Phe Leu Glu Glu Lys Met Ala Tyr Gln Glu
100 105 110
Tyr Pro Asn Ser Gln Asn Trp Pro Glu Asp Thr Asn Phe Cys Phe Gln
115 120 125
Pro Glu Gln Val Val Asp Pro Ile Gln Thr Asp Pro Phe Lys Met Tyr
130 135 140
His Asp Asp Asp Leu Ala Asp Leu Val Phe Pro Ser Ser Ala Thr Ala
145 150 155 160
Asp Thr Ser Ile Phe Ala Gly Gln Asn Asp Pro Leu Lys Asp Ser Tyr
165 170 175
Gly Met Ser Pro Cys Asn Thr Ala Val Val Pro Gln Gly Trp Ser Val
180 185 190
Glu Ala Leu Asn Ser Pro His Ser Glu Ser Phe Val Ser Pro Glu Ala
195 200 205
Val Ala Glu Pro Pro Gln Pro Thr Ala Val Pro Leu Glu Leu Ala Lys
210 215 220
Glu Ile Glu Met Ala Ser Glu Glu Arg Pro Pro Ala Gln Ala Leu Glu
225 230 235 240
Ile Met Met Gly Leu Lys Thr Thr Asp Met Ala Pro Ser Lys Glu Thr
245 250 255
Glu Met Ala Leu Ala Lys Asp Met Ala Leu Ala Thr Lys Thr Glu Val
260 265 270
Ala Leu Ala Lys Asp Met Glu Ser Pro Thr Lys Leu Asp Val Thr Leu
275 280 285
Ala Lys Asp Met Gln Pro Ser Met Glu Ser Asp Met Ala Leu Val Lys
290 295 300
Asp Met Glu Leu Pro Thr Glu Lys Glu Val Ala Leu Val Lys Asp Val
305 310 315 320
Arg Trp Pro Thr Glu Thr Asp Val Ser Ser Ala Lys Asn Val Val Leu
325 330 335
Pro Thr Glu Thr Glu Val Ala Pro Ala Lys Asp Val Thr Leu Leu Lys
340 345 350
Glu Thr Glu Arg Ala Ser Pro Ile Lys Met Asp Leu Ala Pro Ser Lys
355 360 365
Asp Met Gly Pro Pro Lys Glu Asn Lys Lys Glu Thr Glu Arg Ala Ser
370 375 380
Pro Ile Lys Met Asp Leu Ala Pro Ser Lys Asp Met Gly Pro Pro Lys
385 390 395 400
Glu Asn Lys Ile Val Pro Ala Lys Asp Leu Val Leu Leu Ser Glu Ile
405 410 415
Glu Val Ala Gln Ala Asn Asp Ile Ile Ser Ser Thr Glu Ile Ser Ser
420 425 430
Ala Glu Lys Val Ala Leu Ser Ser Glu Thr Glu Val Ala Leu Ala Arg
435 440 445
Asp Met Thr Leu Pro Pro Glu Thr Asn Val Ile Leu Thr Lys Asp Lys
450 455 460
Ala Leu Pro Leu Glu Ala Glu Val Ala Pro Val Lys Asp Met Ala Gln
465 470 475 480
Leu Pro Glu Thr Glu Ile Ala Pro Ala Lys Asp Val Ala Pro Ser Thr
485 490 495
Val Lys Glu Val Gly Leu Leu Lys Asp Met Ser Pro Leu Ser Glu Thr
500 505 510
Glu Met Ala Leu Gly Lys Asp Val Thr Pro Pro Pro Glu Thr Glu Val
515 520 525
Val Leu Ile Lys Asn Val Cys Leu Pro Pro Glu Met Glu Val Ala Leu
530 535 540
Thr Glu Asp Gln Val Pro Ala Leu Lys Thr Glu Ala Pro Leu Ala Lys
545 550 555 560
Asp Gly Val Leu Thr Leu Ala Asn Asn Val Thr Pro Ala Lys Asp Val
565 570 575
Pro Pro Leu Ser Glu Thr Glu Ala Thr Pro Val Pro Ile Lys Asp Met
580 585 590
Glu Ile Ala Gln Thr Gln Lys Gly Ile Ser Glu Asp Ser His Leu Glu
595 600 605
Ser Leu Gln Asp Val Gly Gln Ser Ala Ala Pro Thr Phe Met Ile Ser
610 615 620
Pro Glu Thr Val Thr Gly Thr Gly Lys Lys Cys Ser Leu Pro Ala Glu
625 630 635 640
Glu Asp Ser Val Leu Glu Lys Leu Gly Glu Arg Lys Pro Cys Asn Ser
645 650 655
Gln Pro Ser Glu Leu Ser Ser Glu Thr Ser Gly Ile Ala Arg Pro Glu
660 665 670
Glu Gly Arg Pro Val Val Ser Gly Thr Gly Asn Asp Ile Thr Thr Pro
675 680 685
Pro Asn Lys Glu Leu Pro Pro Ser Pro Glu Lys Lys Thr Lys Pro Leu
690 695 700
Ala Thr Thr Gln Pro Ala Lys Thr Ser Thr Ser Lys Ala Lys Thr Gln
705 710 715 720
Pro Thr Ser Leu Pro Lys Gln Pro Ala Pro Thr Thr Ile Gly Gly Leu
725 730 735
Asn Lys Lys Pro Met Ser Leu Ala Ser Gly Leu Val Pro Ala Ala Pro
740 745 750
Pro Lys Arg Pro Ala Val Ala Ser Ala Arg Pro Ser Ile Leu Pro Ser
755 760 765
Lys Asp Val Lys Pro Lys Pro Ile Ala Asp Ala Lys Ala Pro Glu Lys
770 775 780
Arg Ala Ser Pro Ser Lys Pro Ala Ser Ala Pro Ala Ser Arg Ser Gly
785 790 795 800
Ser Lys Ser Thr Gln Thr Val Ala Lys Thr Thr Thr Ala Ala Ala Val
805 810 815
Ala Ser Thr Gly Pro Ser Ser Arg Ser Pro Ser Thr Leu Leu Pro Lys
820 825 830
Lys Pro Thr Ala Ile Lys Thr Glu Gly Lys Pro Ala Glu Val Lys Lys
835 840 845
Met Thr Ala Lys Ser Val Pro Ala Asp Leu Ser Arg Pro Lys Ser Thr
850 855 860
Ser Thr Ser Ser Met Lys Lys Thr Thr Thr Leu Ser Gly Thr Ala Pro
865 870 875 880
Ala Ala Gly Val Val Pro Ser Arg Val Lys Ala Thr Pro Met Pro Ser
885 890 895
Arg Pro Ser Thr Thr Pro Phe Ile Asp Lys Lys Pro Thr Ser Ala Lys
900 905 910
Pro Ser Ser Thr Thr Pro Arg Leu Ser Arg Leu Ala Thr Asn Thr Ser
915 920 925
Ala Pro Asp Leu Lys Asn Val Arg Ser Lys Val Gly Ser Thr Glu Asn
930 935 940
Ile Lys His Gln Pro Gly Gly Gly Arg Ala Lys Val Glu Lys Lys Thr
945 950 955 960
Glu Ala Ala Ala Thr Thr Arg Lys Pro Glu Ser Asn Ala Val Thr Lys
965 970 975
Thr Ala Gly Pro Ile Ala Ser Ala Gln Lys Gln Pro Ala Gly Lys Val
980 985 990
Gln Ile Val Ser Lys Lys Val Ser Tyr Ser His Ile Gln Ser Lys Cys
995 1000 1005
Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly Asn Val
1010 1015 1020
Gln Ile Gln Asn Lys Lys Val Asp Ile Ser Lys Val Ser Ser Lys
1025 1030 1035
Cys Gly Ser Lys Ala Asn Ile Lys His Lys Pro Gly Gly Gly Asp
1040 1045 1050
Val Lys Ile Glu Ser Gln Lys Leu Asn Phe Lys Glu Lys Ala Gln
1055 1060 1065
Ala Lys Val Gly Ser Leu Asp Asn Val Gly His Leu Pro Ala Gly
1070 1075 1080
Gly Ala Val Lys Thr Glu Gly Gly Gly Ser Glu Ala Pro Leu Cys
1085 1090 1095
Pro Gly Pro Pro Ala Gly Glu Glu Pro Ala Ile Ser Glu Ala Ala
1100 1105 1110
Pro Glu Ala Gly Ala Pro Thr Ser Ala Ser Gly Leu Asn Gly His
1115 1120 1125
Pro Thr Leu Ser Gly Gly Gly Asp Gln Arg Glu Ala Gln Thr Leu
1130 1135 1140
Asp Ser Gln Ile Gln Glu Thr Ser Ile
1145 1150
<210> 28
<211> 401
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 28
Met Arg Arg Ala Pro Ala Ala Glu Arg Leu Leu Glu Leu Gly Phe Pro
1 5 10 15
Pro Arg Cys Gly Arg Gln Glu Pro Pro Phe Pro Leu Gly Val Thr Arg
20 25 30
Gly Trp Gly Arg Trp Pro Ile Gln Lys Arg Arg Glu Gly Ala Arg Pro
35 40 45
Val Pro Phe Ser Glu Arg Ser Gln Glu Asp Gly Arg Gly Pro Ala Ala
50 55 60
Arg Ser Ser Gly Thr Leu Trp Arg Ile Arg Thr Arg Leu Ser Leu Cys
65 70 75 80
Arg Asp Pro Glu Pro Pro Pro Pro Leu Cys Leu Leu Arg Val Ser Leu
85 90 95
Leu Cys Ala Leu Arg Ala Gly Gly Arg Gly Ser Arg Trp Gly Glu Asp
100 105 110
Gly Ala Arg Leu Leu Leu Leu Pro Pro Ala Arg Ala Ala Gly Asn Gly
115 120 125
Glu Ala Glu Pro Ser Gly Gly Pro Ser Tyr Ala Gly Arg Met Leu Glu
130 135 140
Ser Ser Gly Cys Lys Ala Leu Lys Glu Gly Val Leu Glu Lys Arg Ser
145 150 155 160
Asp Gly Leu Leu Gln Leu Trp Lys Lys Lys Cys Cys Ile Leu Thr Glu
165 170 175
Glu Gly Leu Leu Leu Ile Pro Pro Lys Gln Leu Gln His Gln Gln Gln
180 185 190
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro Gly Gln Gly
195 200 205
Pro Ala Glu Pro Ser Gln Pro Ser Gly Pro Ala Val Ala Ser Leu Glu
210 215 220
Pro Pro Val Lys Leu Lys Glu Leu His Phe Ser Asn Met Lys Thr Val
225 230 235 240
Asp Cys Val Glu Arg Lys Gly Lys Tyr Met Tyr Phe Thr Val Val Met
245 250 255
Ala Glu Gly Lys Glu Ile Asp Phe Arg Cys Pro Gln Asp Gln Gly Trp
260 265 270
Asn Ala Glu Ile Thr Leu Gln Met Val Gln Tyr Lys Asn Arg Gln Ala
275 280 285
Ile Leu Ala Val Lys Ser Thr Arg Gln Lys Gln Gln His Leu Val Gln
290 295 300
Gln Gln Pro Pro Ser Gln Pro Gln Pro Gln Pro Gln Leu Gln Pro Gln
305 310 315 320
Pro Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro Gln Ser Gln Pro Gln
325 330 335
Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Gln Leu His Pro Tyr Pro
340 345 350
His Pro His Pro His Pro His Ser His Pro His Ser His Pro His Pro
355 360 365
His Pro His Pro His Pro His Gln Ile Pro His Pro His Pro Gln Pro
370 375 380
His Ser Gln Pro His Gly His Arg Leu Leu Arg Ser Thr Ser Asn Ser
385 390 395 400
Ala
<210> 29
<211> 382
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 29
Met Gly Ala Phe Leu Asp Lys Pro Lys Met Glu Lys His Asn Ala Gln
1 5 10 15
Gly Gln Gly Asn Gly Leu Arg Tyr Gly Leu Ser Ser Met Gln Gly Trp
20 25 30
Arg Val Glu Met Glu Asp Ala His Thr Ala Val Ile Gly Leu Pro Ser
35 40 45
Gly Leu Glu Ser Trp Ser Phe Phe Ala Val Tyr Asp Gly His Ala Gly
50 55 60
Ser Gln Val Ala Lys Tyr Cys Cys Glu His Leu Leu Asp His Ile Thr
65 70 75 80
Asn Asn Gln Asp Phe Lys Gly Ser Ala Gly Ala Pro Ser Val Glu Asn
85 90 95
Val Lys Asn Gly Ile Arg Thr Gly Phe Leu Glu Ile Asp Glu His Met
100 105 110
Arg Val Met Ser Glu Lys Lys His Gly Ala Asp Arg Ser Gly Ser Thr
115 120 125
Ala Val Gly Val Leu Ile Ser Pro Gln His Thr Tyr Phe Ile Asn Cys
130 135 140
Gly Asp Ser Arg Gly Leu Leu Cys Arg Asn Arg Lys Val His Phe Phe
145 150 155 160
Thr Gln Asp His Lys Pro Ser Asn Pro Leu Glu Lys Glu Arg Ile Gln
165 170 175
Asn Ala Gly Gly Ser Val Met Ile Gln Arg Val Asn Gly Ser Leu Ala
180 185 190
Val Ser Arg Ala Leu Gly Asp Phe Asp Tyr Lys Cys Val His Gly Lys
195 200 205
Gly Pro Thr Glu Gln Leu Val Ser Pro Glu Pro Glu Val His Asp Ile
210 215 220
Glu Arg Ser Glu Glu Asp Asp Gln Phe Ile Ile Leu Ala Cys Asp Gly
225 230 235 240
Ile Trp Asp Val Met Gly Asn Glu Glu Leu Cys Asp Phe Val Arg Ser
245 250 255
Arg Leu Glu Val Thr Asp Asp Leu Glu Lys Val Cys Asn Glu Val Val
260 265 270
Asp Thr Cys Leu Tyr Lys Gly Ser Arg Asp Asn Met Ser Val Ile Leu
275 280 285
Ile Cys Phe Pro Asn Ala Pro Lys Val Ser Pro Glu Ala Val Lys Lys
290 295 300
Glu Ala Glu Leu Asp Lys Tyr Leu Glu Cys Arg Val Glu Glu Ile Ile
305 310 315 320
Lys Lys Gln Gly Glu Gly Val Pro Asp Leu Val His Val Met Arg Thr
325 330 335
Leu Ala Ser Glu Asn Ile Pro Ser Leu Pro Pro Gly Gly Glu Leu Ala
340 345 350
Ser Lys Arg Asn Val Ile Glu Ala Val Tyr Asn Arg Leu Asn Pro Tyr
355 360 365
Lys Asn Asp Asp Thr Asp Ser Thr Ser Thr Asp Asp Met Trp
370 375 380
<210> 30
<211> 114
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 30
Met Ala Glu Cys Pro Thr Leu Gly Glu Ala Val Thr Asp His Pro Asp
1 5 10 15
Arg Leu Trp Ala Trp Glu Lys Phe Val Tyr Leu Asp Glu Lys Gln His
20 25 30
Ala Trp Leu Pro Leu Thr Ile Glu Ile Lys Asp Arg Leu Gln Leu Arg
35 40 45
Val Leu Leu Arg Arg Glu Asp Val Val Leu Gly Arg Pro Met Thr Pro
50 55 60
Thr Gln Ile Gly Pro Ser Leu Leu Pro Ile Met Trp Gln Leu Tyr Pro
65 70 75 80
Asp Gly Arg Tyr Arg Ser Ser Asp Ser Ser Phe Trp Arg Leu Val Tyr
85 90 95
His Ile Lys Ile Asp Gly Val Glu Asp Met Leu Leu Glu Leu Leu Pro
100 105 110
Asp Asp
<210> 31
<211> 158
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 31
Met Ser Gly Ile Ala Leu Ser Arg Leu Ala Gln Glu Arg Lys Ala Trp
1 5 10 15
Arg Lys Asp His Pro Phe Gly Phe Val Ala Val Pro Thr Lys Asn Pro
20 25 30
Asp Gly Thr Met Asn Leu Met Asn Trp Glu Cys Ala Ile Pro Gly Lys
35 40 45
Lys Gly Thr Pro Trp Glu Gly Gly Leu Phe Lys Leu Arg Met Leu Phe
50 55 60
Lys Asp Asp Tyr Pro Ser Ser Pro Pro Lys Cys Lys Phe Glu Pro Pro
65 70 75 80
Leu Phe His Pro Asn Val Tyr Pro Ser Gly Thr Val Cys Leu Ser Ile
85 90 95
Leu Glu Glu Asp Lys Asp Trp Arg Pro Ala Ile Thr Ile Lys Gln Ile
100 105 110
Leu Leu Gly Ile Gln Glu Leu Leu Asn Glu Pro Asn Ile Gln Asp Pro
115 120 125
Ala Gln Ala Glu Ala Tyr Thr Ile Tyr Cys Gln Asn Arg Val Glu Tyr
130 135 140
Glu Lys Arg Val Arg Ala Gln Ala Lys Lys Phe Ala Pro Ser
145 150 155
<210> 32
<211> 528
<212> PRT
<213> Artificial Sequence
<220>
<223> A synthetic gene insert was assembled from synthetic
oligonucleotides and/or PCR products.
<400> 32
Met Gly Asp Ala Pro Ser Pro Glu Glu Lys Leu His Leu Ile Thr Arg
1 5 10 15
Asn Leu Gln Glu Val Leu Gly Glu Glu Lys Leu Lys Glu Ile Leu Lys
20 25 30
Glu Arg Glu Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr Gly Lys Pro
35 40 45
His Val Ala Tyr Phe Val Pro Met Ser Lys Ile Ala Asp Phe Leu Lys
50 55 60
Ala Gly Cys Glu Val Thr Ile Leu Phe Ala Asp Leu His Ala Tyr Leu
65 70 75 80
Asp Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val Ser Tyr
85 90 95
Tyr Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile Gly Val Pro Leu
100 105 110
Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp Tyr Gln Leu Ser Lys Glu
115 120 125
Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Val Val Thr Gln His Asp
130 135 140
Ser Lys Lys Ala Gly Ala Glu Val Val Lys Gln Val Glu His Pro Leu
145 150 155 160
Leu Ser Gly Leu Leu Tyr Pro Gly Leu Gln Ala Leu Asp Glu Glu Tyr
165 170 175
Leu Lys Val Asp Ala Gln Phe Gly Gly Ile Asp Gln Arg Lys Ile Phe
180 185 190
Thr Phe Ala Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Val
195 200 205
His Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys Met Ser
210 215 220
Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp
225 230 235 240
Val Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu
245 250 255
Asn Asn Gly Val Leu Ser Phe Ile Lys His Val Leu Phe Pro Leu Lys
260 265 270
Ser Glu Phe Val Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn Lys Thr
275 280 285
Tyr Thr Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala Glu Val Val
290 295 300
His Pro Gly Asp Leu Lys Asn Ser Val Glu Val Ala Leu Asn Lys Leu
305 310 315 320
Leu Asp Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys Lys Leu
325 330 335
Ala Ser Ala Ala Tyr Pro Asp Pro Ser Lys Gln Lys Pro Met Ala Lys
340 345 350
Gly Pro Ala Lys Asn Ser Glu Pro Glu Glu Val Ile Pro Ser Arg Leu
355 360 365
Asp Ile Arg Val Gly Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala
370 375 380
Asp Ser Leu Tyr Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg
385 390 395 400
Thr Val Val Ser Gly Leu Val Gln Phe Val Pro Lys Glu Glu Leu Gln
405 410 415
Asp Arg Leu Val Val Val Leu Cys Asn Leu Lys Pro Gln Lys Met Arg
420 425 430
Gly Val Glu Ser Gln Gly Met Leu Leu Cys Ala Ser Ile Glu Gly Ile
435 440 445
Asn Arg Gln Val Glu Pro Leu Asp Pro Pro Ala Gly Ser Ala Pro Gly
450 455 460
Glu His Val Phe Val Lys Gly Tyr Glu Lys Gly Gln Pro Asp Glu Glu
465 470 475 480
Leu Lys Pro Lys Lys Lys Val Phe Glu Lys Leu Gln Ala Asp Phe Lys
485 490 495
Ile Ser Glu Glu Cys Ile Ala Gln Trp Lys Gln Thr Asn Phe Met Thr
500 505 510
Lys Leu Gly Ser Ile Ser Cys Lys Ser Leu Lys Gly Gly Asn Ile Ser
515 520 525

Claims (19)

1.一种用于由从个体提取的样品确定该个体的健康的方法，包括以下步骤：
(i)测试样品中对健康是特定的生物标志物的存在；
(ii)基于对所述生物标志物的检测确定所述受试者是健康、中等健康或不健康，
其特征在于所述生物标志物是针对抗原的自身抗体，所述抗原包括AURKA、FEN1、GLRX3、PHLDA1、PPM1A、FKBP3、CD96和MAPK13。
2.根据权利要求1的方法，其中所述抗原进一步包括UBE2I、AAK1、YARS、ASPSCR1、CASP10、FHOD2、TCL1A和MAP4中的一种或多种。
3.根据权利要求1或2的方法，其中将所述抗原暴露于从人提取的样品，从而来自所述样品的自身抗体生物标志物可以与所述抗原结合。
4.根据权利要求3的方法，其中所述抗原随后暴露于荧光标记的二抗以允许确定与所述抗原结合的来自所述样品的任何自身抗体的量。
5.根据权利要求4的方法，其中所述人的健康状况对应于与所述抗原特异性结合的来自所述样品的自身抗体的相对量或绝对量。
6.根据前述权利要求中任一项的方法，其中所述样品包括外泌体、血液、血清、血浆、尿液、唾液、羊水、脑脊液、母乳、精液或胆汁的任一种或它们的任意组合。
7.根据前述权利要求中任一项的方法，其中所述步骤在体外进行。
8.根据前述权利要求中任一项的方法，其中所述方法包括检测一种或多种生物标志物的上调/下调。
9.根据前述权利要求中任一项的方法，其中PHLDA1和CD96对应于健康，AURKA、FEN1、CASP10和AAK1对应于中等健康，并且UBE2I、YARS、ASPSCR1、FHOD2、TCL1A、MAP4、MAPK13、FKBP3、PPM1A和GLRX3对应于不健康。
10.根据前述权利要求中任一项的方法，其中所述个体是老年人。
11.根据前述权利要求中任一项的方法，其中所述抗原是生物素化蛋白质。
12.根据权利要求11的方法，其中每种生物素化蛋白质由与蛋白质框内融合的生物素羧基载体蛋白质折叠标志物构成。
13.根据权利要求11或12的方法，其中所述生物素化蛋白质与链霉亲和素包被的基底结合。
14.根据权利要求13的方法，其中所述基底包含形成水凝胶的聚合物基层。
15.一种用于制造试剂盒的方法，所述试剂盒用于由从老年个体提取的样品确定该个体的健康，所述方法包括以下步骤：
对于组中的每种抗原，用编码所述抗原的基因框内克隆生物素羧基载体蛋白质折叠标志物，并且表达得到的生物素化抗原；
将所述生物素化抗原与一种或多种链霉亲和素包被的基底上的可寻址的位置结合，从而形成抗原阵列；
由此可以通过将所述基底暴露于所述样品并且测量反应来确定与所述组上的抗原结合的来自所述样品的自身抗体的量；
其特征在于所述抗原包括UBE2I、AAK1、YARS、ASPSCR1、CASP10、FHOD2、TCL1A、MAP4、MAPK13、CD96、FKBP3、PPM1A、PHLDA1、GLRX3、FEN1和AURKA。
16.一种组合物，其包含一组用于确定老年个体的健康的抗原，其特征在于所述抗原包括MAPK13、CD96、FKBP3、PPM1A、PHLDA1、GLRX3、FEN1和AURKA，并且任选地包括UBE2I、AAK1、YARS、ASPSCR1、CASP10、FHOD2、TCL1A和MAP4中的一种或多种。
17.根据权利要求16的组合物，其中所述抗原是生物素化蛋白质。
18.根据权利要求16-17中任一项的组合物，其中能够测量来自患者的样品中的与所述抗原体外结合的一种或多种自身抗体生物标志物的量以确定患者的健康状况。
19.一种组合物，其包含一组用于确定老年患者的健康状况的自身抗体生物标志物：
其中在来自所述患者的样品中测量一种或多种自身抗体生物标志物的水平；
其特征在于所述一种或多种自身抗体生物标志物选自对以下抗原的一种或多种具有特异性的自身抗体：MAPK13、CD96、FKBP3、PPM1A、PHLDA1、GLRX3、FEN1和AURKA；
并且任选地包含以下抗原的一种或多种：UBE2I、AAK1、YARS、ASPSCR1、CASP10、FHOD2、TCL1A和MAP4。
CN202080080356.4A 2019-09-25 2020-09-23 使用免疫生物标志物对老年人的健康状况进行鉴定的方法 Pending CN114761808A (zh)

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