CN110468201B - Escc频繁突变基因的靶向测序及其在获得判断escc预后的生物标记物中的应用 - Google Patents

Abstract

本发明涉及高通量生物测序技术领域，尤其涉及针对食管鳞状细胞癌(ESCC)的频繁突变基因的靶向测序，及其在获得ESCC预后生物标记物中的应用。

Description

ESCC频繁突变基因的靶向测序及其在获得判断ESCC预后的生 物标记物中的应用

技术领域

本发明涉及高通量生物测序技术领域，尤其涉及针对食管鳞状细胞癌(ESCC)的频繁突变基因的靶向测序，及其在获得判断ESCC预后的生物标记物中的应用。

背景技术

食管鳞状细胞癌(ESCC)是最常见的食管癌组织类型，我国90％以上的食管癌属于食管鳞状细胞癌。我国也是世界上食管鳞状细胞癌发病率最高的国家，每年有25万新发病例，是我国发病率位居第4位的恶性肿瘤，造成约15万例死亡，在肿瘤相关的死亡中列第4位，严重威胁广大患者生命健康。尽管在早期诊断及手术、放化疗等治疗方面取得了一定进展，由于局部复发及远处转移率高，食管鳞状细胞癌的5年总生存率不足15％，且目前用于预测食管鳞状细胞癌预后的临床指标尚不精确。因此寻找新的预后标记，进一步精确预测食管鳞状细胞癌的预后，是制定个性化治疗策略从而改善生存的第一步。

大量的临床数据及基础研究的结果显示，食管鳞状细胞癌是一种高度异质性的肿瘤，不同类型的食管鳞状细胞癌患者之间或者同一患者体内的不同原发癌灶或者原发癌灶与转移癌灶在肿留细胞的增殖、分化、侵袭和转移能力以及对药物的治疗反应存在差异。包括不同种族、不同地域之间遗传背景的差异、多阶段多基因多位点的基因突变谱差异、基因表达谱差异，以及个体间细胞分化差异等在内的多重因素导致了ESCC异质性的形成。着眼于ESCC异质性的个体化治疗方案的制定影响着ESCC患者远期生存以及预后。基因芯片技术等高通量测序技术的出现和迅猛发展，为进一步研究ESCC患者的治疗提供理论和技术基础，基于基因组学的不同ESCC的异质性研究，不仅能够更加清楚地反应不同乳腺癌所具有的各异的临床表现和预后，同时还为ESCC精准治疗提供依据。

二代测序(NGS)技术的最新进展使得可以同时检测多种癌症相关的突变。为了提高对食管鳞状细胞癌(ESCC)遗传基础的了解，已经实施了几项ESCC的大规模基因组分析。这些分析鉴定了ESCC的基因谱和显著相关的突变基因。此外，在ESCC患者中发现了高频率发生的C→T突变，尤其是发生在CpG二核苷酸中的C→T突变。

这些全面的基因组分析主要通过基因组规模或外显子组规模的NGS技术进行。利用大规模的NGS研究鉴定ESCC的全基因组突变概况使得靶向测序的临床应用成为可能。然而，目前关于ESCC患者的预后和潜在治疗性生物标志物的基因组信息仍然是有限的。

发明概述

本发明关注与ESCC患者预后相关的基因组信息，利用靶向测序方法寻找与ESCC预后显著相关的基因组信息，为潜在性的治疗手段提供更多有用的临床信息。

本发明构建一个判断ESCC致病性的多基因组合(Panel)，该多基因组合(Panel)可以为ESCC患者提供预后判断和潜在性治疗的生物标志物。该多基因组合(Panel)含有频繁突变的ESCC基因，包括FAT1，FAT2，FAT3，FAT4，AJUBA，NOTCH1，NOTCH2，NOTCH3，FBXW7，DAAM2，CTNNB1，SFRP4，DVL3，PIK3CA，PTEN，ERBB4，NFE2L2，CDKN2A，CHEK2，KEAP1，RB1，TP53，KMT2D，KMT2C，KDM6A，CREBBP，ASH1L，NSD1，PBRM1，EP300，SETD1B，APC，BRCA1，BRCA2，SYNE1，CSMD3，LRP1B，PTCH1，CUL3，RNF213，FAM135B，ADAM29，ZNF750，EGFR。通过对该多基因组合(Panel)的靶向测序能够获得ESCC疾病诊断及治疗方面的有益信息。

相较于现有技术中已有的全基因组规模或外显子组规模测序而言，靶向测序更适合应用于临床，其时间成本和经济花费更加得以优化。并且，由于大规模测序覆盖度相对较低，因此全基因组或全外显子组的测序的敏感性和特异性比较有限，这也阻碍了全基因组测序或全外显子组测序应用于临床。本发明通过对特定的多基因组合的靶向测序提高了检测ESCC相关Panel的敏感性和特异性。

本发明利用Ion Torrent二代测序平台针对此44个频繁突变基因进行靶向测序。经过构建随机生存森林模型，对测序结果进行log-rank检验生存分析，并利用Cox比例风险回归模型进行平行分析。研究结果表明，肿瘤分化程度/组织学分级、KMT2C基因突变和ZNF750基因突变为ESCC的三个预后因素。本发明涉及ESCC预后判断的方法，所述方法涉及检测肿瘤分化程度/组织学分级、KMT2C基因突变和ZNF750基因突变。其中所述KMT2C突变包括在SEQ ID NO：2所示序列结构上含有如下所示突变中的一种或多种突变：V752I，A1685S，A2204T，V2790I，P3465L和P3308L，所述ZNF750突变包括在SEQ ID NO：4所示序列结构上含有如下所示突变中的一种或多种突变：K101fs，Y25C，S70X，P9S，W207X和H13Q，优选为Y25C和H13Q；更为优选地，所述生物标记为A1685S的KMT2C突变和Y25C的ZNF750突变组合。进一步地，其中所述ESCC为I-II期的食管鳞状细胞癌。

发明详述

本发明构建了靶向测序的多基因组合(Panel)，该多基因组合用于检测I-II期食管鳞状细胞癌(ESCC)。该多基因组合(Panel)含有44个与ESCC发生相关的频繁突变基因，具体为FAT1，FAT2，FAT3，FAT4，AJUBA，NOTCH1，NOTCH2，NOTCH3，FBXW7，DAAM2，CTNNB1，SFRP4，DVL3，PIK3CA，PTEN，ERBB4，NFE2L2，CDKN2A，CHEK2，KEAP1，RB1，TP53，KMT2D，KMT2C，KDM6A，CREBBP，ASH1L，NSD1，PBRM1，EP300，SETD1B，APC，BRCA1，BRCA2，SYNE1，CSMD3，LRP1B，PTCH1，CUL3，RNF213，FAM135B，ADAM29，ZNF750，EGFR基因。该多基因组合(Panel)为I-II期ESCC患者的临床诊断及其治疗提供帮助。

具体而言，本发明根据COSMIC数据库筛选高频突变的基因，随后利用IT-PGM二代测序平台对119个ESCC FFPE样品进行测序，每个扩增子的覆盖度至少为250×，IT-PGM平台检测到的突变已证明在10％等位基因频率下具有可靠的敏感性。因此，如果研究中等位基因频率低于10％，筛选获得致病性的突变。

通过对本发明所设计的的panel靶向测序，对测序数据进行生物信息学分析，结果表明肿瘤分化程度，KMT2C突变和ZNF750突变与ESCC患者的总生存期(overall survival，OS)相关。美国癌症联合委员会(AJCC)癌症分期系统的第七版和第八版中也已记载，肿瘤分化程度/组织学分级为判断预后的金标准之一，并且将肿瘤分化程度/组织学分级作为划分ESCC患者为不同进展组的标准。其中，与预后相关的KMT2C突变包括V752I，A1685S，A2204T，V2790I，P3465L和P3308L。与预后相关的ZNF750突变包括K101fs，Y25C，S70X，P9S，W207X和H13Q，优选为Y25C和H13Q。

其中，用于检测I-II期食管鳞状细胞癌(ESCC)的多基因组合(Panel)的手段，包括针对靶基因的引物及其相关试剂盒等。

KMT2C(赖氨酸N-甲基转移酶2C)也被称为骨髓/淋巴或混合谱系白血病蛋白3(MLL3)，其编码MLL家族成员中的酶，该酶在赖氨酸4(H3K4)上甲基化组蛋白H3以促进基因组可接近性(accessibility)和转录。人KMT2C的mRNA以及其蛋白序列分别如序列表中的SEQ ID NO：1和2所示。KMT2C基因突变在多种癌症(包括小细胞肺癌，胃癌，结直肠癌和非霍奇金淋巴瘤)中普遍存在。已发表的研究报道了ESCC中KMT2C基因频繁突变。已有研究表明KMT2C的下调在ESCC的进展中是非常重要的。然而现有技术中没有证据表明KMT2C突变，尤其是具体的KMT2C突变类型与ESCC患者的预后有关。本发明首次证明具体的KMT2C突变类型提示ESCC患者具有不良的预后。在本研究的所有患者中，有40.3％(48/119)的患者具有KMT2C突变，包括V752I，A1685S，A2204T，V2790I，P3465L和P3308L。

ZNF750(锌指蛋白750)基因编码具有核定位位点和C2H2锌指结构域的蛋白质。该蛋白作用于p63/TP63的下游并激活晚期表皮分化基因的表达。人ZNF750的mRNA以及其蛋白序列分别如序列表中的SEQ ID NO：3和4所示。ESCC组织中ZNF750基因突变显著，ZNF750为ESCC的肿瘤抑制基因。本发明首次发现，具体的ZNF750突变与ESCC患者生存率低有关，其中所述的具体ZNF750突变包括K101fs，Y25C，S70X，P9S，W207X和H13Q。

本研究探讨了ESCC病例中频繁发生突变的基因，并将病理特征(肿瘤分化程度)和基因特征(ZNF750和KMT2C基因突变)结合，建立随机森林方法(RSF)模型预测ESCC患者的预后；同时平行地进行Cox比例风险回归模型进行预后因素分析。本研究为I-II期ESCC患者提供了评估预后和选择有效治疗的生物标志物。

在本发明中，考虑肿瘤分化程度，KMT2C和ZNF750基因突变，建立随机生存森林方法(RSF)模型预测ESCC患者的预后。RSF为统计学习理论模型，它利用bootsrap重抽样方法从原始样本中抽取多个样本，对每个bootsrap样本进行决策树建模，然后组合多棵决策树的预测，通过投票得出最终预测结果。RSF具有很高的预测准确率，对异常值和噪声具有很好的容忍度，且不容易出现过拟合。传统的生存分析方法中，通常用到的方法都依靠很强的限制性假设(例如比例危险率函数的假定)，有或者需对协变量识别其交互作用以便其做回归分析，档期变数大时，要确定变数之间的交互作用就更加繁冗，如靠专业知识来判断其交互作用又会缩小其探索的空间，这些问题在随机生存森林的分析方法中都得到解决。随机生存森林的算法如下：(1)从原使数据中随机抽取ntree个bootstrap样本；(2)对每个bootstrap样本生成树。在此过程中，对于每个节点随机抽取mtry个协变量，以此用于选取最佳分裂变量。对于选取最佳分裂变量依据生存分裂准则，每个节点的分裂都是尽可能的最大化左右子节点的生存差异性；(3)完整的生成一个树直到叶节点的样本数不少于nodesize；(4)通过森林中ntree棵树的信息来估计整体的累积危险力；(5)通过out-of-bag(OOB)数据来计算误差。

本发明通过随机生存森林模型对测序结果以及预后因素进行分析，同时还利用Cox比例风险回归模型进行验证，其中的C-index，即一致性指数(index of concordance)，用来评价模型的预测能力，它估计了预测结果与实际观察到的结果相一致的概率。一致性指数的计算方法为：把所研究的资料中的所有研究对象随机地两两组成对子。以生存分析为例，对于一对病人，如果生存时间较长的一位的预测生存时间也长于另一位的预测生存时间，或预测的生存概率高的一位的生存时间长于生存概率低的另一位，则称之为预测结果与实际结果一致。一致性指数(C-index)为介于0.5和1之间的数值，以量化预测模型的判别能力，0.5为完全不一致，说明该模型没有预测作用，1为完全一致，说明该模型预测结果与实际完全一致。在实际应用中，很难找到完全一致的预测模型。

本发明中所采用的测序技术——Ion Torrent测序技术，其以半导体芯片技术为基础，利用碱基互补配对的天然原理，通过离子传感器捕获DNA合成时碱基延伸释放H+而导致局部的PH变化，并即刻将化学信号转化为数字信号，以此实时判读碱基，最终获得每个DNA片段的碱基序列。相比于其他测序技术，Ion Torrent测序技术突破了传统上依靠荧光标记以及光学图像采集数据原理的技术限制，使得其在设备成本、测序速度、测序费用上具有更好的优势。

研究结果表明，肿瘤分化程度为ESCC患者的预后因素，该研究结果与现有研究的结果一致。本发明的研究结果还表明，KMT2C和ZNF750突变与ESCC患者存活率显著相关，其中KMT2C突变包括在SEQ ID NO：2所示序列结构上含有如下所示突变中的一种或多种突变：V752I，A1685S，A2204T，V2790I，P3465L和P3308L；ZNF750突变包括在SEQ ID NO：4所示序列结构上含有如下所示突变中的一种或多种突变：K101fs，Y25C，S70X，P9S，W207X和H13Q，优选为Y25C和H13Q。

附图说明

图1：119个ESCC样品测序的靶区域中映射读数(mapped reads)的值以及读取深度，多基因组合(panel)测序一致性以及百分比读数。

图2：119例ESCC样本中通过靶向测序鉴定的44个基因的体细胞突变。A.每个检测样品的体细胞突变数目；B.主要的临床病理特征(上)，以及通过突变类型用不同颜色标记的44种基因(底部)；列对应于检测的样本。

图3：119个ESCC样本中于靶向区域鉴定的突变标签。

图4：在靶向测序中鉴定有PIK3CA，EGFR，KMT2C和ZNF750蛋白改变的示意图。

图5：KMT2C基因突变，ZNF750基因突变和肿瘤分化程度/组织学分级关于队列总生存的Kaplan-Meier曲线(p＜0.05，log-rank检验)。

图6：随机生存森林(RSF)分析。A.基于Cox回归分析KMT2C突变，ZNF750突变和肿瘤分化程度/组织学分级的列线图。B.列线图的校准曲线，C.淋巴管浸润状态，KMT2C突变和ZNF750突变的变量重要性(Variable Importance，VIMP)，D.KMT2C突变，ZNF750突变，肿瘤分化程度/组织学分级及三个特征构建的RSF模型预测误差。

实施例

1、病例收集以及基因组DNA提取

收集于2004年至2009年间在中国医学科学院肿瘤医院进行ESCC根治性切除术的病例119例，其中T1N0 18例，T1N1 6例，T2N0 46例，T2N1 15例，T3N0 34例。所有患者均在2004年至2009年间接受根治性切除术，并且不需要进行放疗和化疗。随访结束日期为2012年6月，通过电话随访或临床资料咨询获得随访资料。所有患者的中位生存时间为66个月。淋巴管浸润(LVI)的组织学诊断标准是根据位于内皮内衬，淋巴管或血管间隙内的肿瘤细胞团进行诊断确定。病理诊断由两位病理医生独立完成。表1第1-3列总结了入选本实验的所有患者的一般临床资料信息。

表1.119例ESCC患者的一般临床资料

*基因与相应的临床病理特征相关(Fisher精确检验，p＜0.05).

利用QIAamp DNA Mini试剂盒(Qiagen，Hilgen，德国)对119例ESCC患者的FFPE组织样本进行基因组DNA的提取。对所有病例的H&E切片进行回顾，以确定用于显微切割的80％恶性细胞的最小区域。

设计进行靶向测序的多基因组合(Panel)

首先，根据已发表的大规模基因组研究和COSMIC数据库，获得在ESCC中反复改变的44个基因中的538个突变。44个基因包括FAT1，FAT2，FAT3，FAT4，AJUBA，NOTCH1，NOTCH2，NOTCH3，FBXW7，DAAM2，CTNNB1，SFRP4，DVL3，PIK3CA，PTEN，ERBB4，NFE2L2，CDKN2A，CHEK2，KEAP1，RB1，TP53，KMT2D，KMT2C，KDM6A，CREBBP，ASH1L，NSD1，PBRM1，EP300，SETD1B，APC，BRCA1，BRCA2，SYNE1，CSMD3，LRP1B，PTCH1，CUL3，RNF213，FAM135B，ADAM29，ZNF750，EGFR编码基因。在选择感兴趣的突变之后，使用Ion Ampliseq Designer来设计涵盖针对IonTorrent测序技术的靶突变多重引物。定制设计的多基因组合(Panel)为含有332个引物对的引物池，其中涵盖538个变体中的466个，并用于产生目标扩增子文库(表2)。设计中扩增子覆盖的碱基总数为39.56kb。

表2由Ampliseq Designer设计的332个扩增子的位置

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扩增子文库的制备

利用Qubit DNA高灵敏度测定试剂盒(Life Technologies，CA，USA)进行DNA浓度的定量。15ng DNA作为模板，利用IonAmpliSeq HiFi Master Mix试剂盒(Ion AmpliSeq试剂盒版本2.0b；Life Technologies，CA，USA)产生定制设计的扩增子文库。FuPa试剂(LifeTechnologies，CA，USA)用于部分性地消化引物序列并对扩增子进行磷酸化，并将IonXpress条码适配(Life Technologies，CA，USA)连接到扩增子上。连接后，用1.5倍样本体积的Agencourt AMP试剂(Beckman Coulter，CA，USA)和新鲜制备的70％乙醇纯化文库。使用Ion Library Quantitation试剂盒(Life Technologies，CA，USA)通过qPCR确定每个IonAmpliSeq^TM文库的浓度。

油包水PCR和靶向测序

利用Ion PGM模板OT2试剂盒v2(Life Technologies)和Ion One Touch 2系统(Life Technologies，CA，USA)将文库DNA克隆扩增到IonSpheres(ISPs；LifeTechnologies，CA，USA)上。对富集的ISP进行测序，使用Ion 318芯片对合并的文库进行测序8到10个样本。测序在PGM测序仪上进行，使用的测序试剂盒产自Life Technologies，CA，USA。

数据处理

利用Torrent Suite软件(TS)4.2.1版(Life Technologies，CA，USA)人类参考基因组(hg19)为参比基因组，对测序产生的reads进行比对，并产生测序质控参数。由平均测序深度，多基因组合(panel)均一性和靶区富集度这三个指标来评估扩增子测序的总体性能。利用Variant Caller插件(Life Technologies，CA，USA，4.0版本)识别突变，并利用覆盖分析插件(Life Technologies，CA，USA，4.0版本)评估目标区域覆盖度。通过基因组浏览器(Integrative Genomics Viewer，IGV)对突变进行可视化，并且比较正向和反向reads中突变的分布。用ANNOVAR软件对突变的位置，氨基酸变化和功能分类进行注释。ANNOVAR的注释还将突变与来自COSMIC(78版本)，dbSNP(138版本)和dbNSFP数据库的癌症特定数据相关联。利用dbNSFP数据库通过使用SIFT，PloyPhen-2，FATHMM和MutationTaster算法对基因突变所致的蛋白质功能改变进行预测。根据来自cBioProtal网站的OncoKB数据库注释突变的临床意义和治疗含义。在千人基因组计划数据库(东亚人)中人群等位基因频率(MAF)＞1％的突变为常见SNP，其被归类为胚系突变。平均覆盖率未能达到250x的扩增子在进步一分析中会被去除。等位基因频率低于10％的突变被过滤掉。进一步地，在突变水平和基因水平对错义突变，无义突变，移码插入、缺失突变和剪接位点突变进行统计分析。利用cBioProtal的MutationMapper显示基因组坐标以注释变体。

统计学分析

通过Fisher精确检验对检验基因突变与临床病理特征之间的关联性进行统计学分析。利用Kaplan-Meier曲线估计生存概率并绘制存活曲线。Log-rank检验用于检验单因素的生存概率差异(p＜0.05)。采用Cox多因素比例风险回归模型分析多因素总生存率(Overall Survival，OS)的相关性，并计算各预后特征的危险比(HR)和及其95％置信区间(CI)。利用R语言的survival包绘制Kaplan-Meier曲线并进行log-rank检验，并且构建Cox多因素比例风险回归模型。利用R语言的rms包绘制列线图(Nomograms)。

利用R语言的randomForestSRC包构建随机生存森林(RSF)模型预测患者预后。采用log-rank分裂法构造20个随机的重抽样生存树。通过平均所有树，获得关于事件时间的变量重要性(Variable Importance，VIMP)的可靠测量。用基于一致性指数(C-index)计算的预测错误率来衡量RSF模型的预测精度：RSF预测错误率＝1-一致性指数。利用R软件“SomaticSignatures”绘制Mutation signature。

结果

靶向测序的特征

本研究中通过靶向测序对119例ESCC患者的标本进行检测评估。单个样本平均测序reads个数为384348个(范围从201039个到1141517个)。样品平均测序深度为1036.9×(范围从521.2×到3155.0×)，Panel平均均一性(panel uniformity)为94.89％(87.84％～97.11％)，平均靶区富集度为96.83％(90.15％～98.73％)(图1)。332个扩增子中，有22个扩增子的平均测序覆盖率低于250×(表3)。进一步分析剩余的310个扩增子覆盖区域以确定突变。

表3.平均测序覆盖率低于250×的扩增子

扩增子名称	基因名称
		AMPL7153100997	LRP1B
AMPL7153034876	CSMD3
		AMPL7157182742	BRCA2
AMPL7152999795	NOTCH1
		AMPL7158162405	NOTCH2
AMPL7154413603	RB1
		AMPL7160130171	KMT2D
AMPL7158179993	NOTCH1
		AMPL7160162396	KDM6A
AMPL7153667979	CDKN2A
		AMPL7154632675	KMT2D
AMPL7160162367	BRCA2
		AMPL7158327362	RB1
AMPL7160145663	CSMD3
		AMPL7160162395	KDM6A
AMPL7160162347	CSMD3
		AMPL7160162280	FAT4
AMPL7160162345	CSMD3
		AMPL7160145673	EP300
AMPL7160162337	CSMD3
		AMPL7156927713	KDM6A
AMPL7159800849	LRP1B

每个样品的平均突变数为3个(范围从个0到13个)(图2A)。所有ESCC样品中，目标区域上的主要突变特征是在CpG二核苷酸中，尤其是在ApCpG三核苷酸中发生的C到T转换。GpCpT三核苷酸中C到A的颠换也占很大的比例(图3)。

靶向测序的体细胞突变

通过检测共发现389个体细胞突变(MAF＜1％)，包括21个重复性突变，以及255个仅发生一次的突变(表4)。最常见的突变基因为

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检测到112个致病性(本申请中即为ESCC致癌性)或可能致癌性突变，160个意义不明的突变，和4个良性或可能良性的突变。112个致病性或可能致癌性突变中，EGFRp.L858R，BRCA2p.R2842H，PIK3CA p.E545K，PIK3CA p.E545Q，PIK3CA p.H1047R，PTENp.E43X，PTEN p.P95L，PTCH1 p.Y224fs被认为是可能可以作为潜在的靶向治疗的靶点。

21个重复发生的突变中，有17个致病性或可能致癌性突变，3个意义不明的变体和1个良性突变。在致病性或可能致癌性突变中，EGFR p.L858R(1.7％)，BRCA2p.R2842H(1.7％)，PIK3CA p.E542K(3.4％)，PIK3CAp.E545K(3.4％)和PIK3CAp.H1047R(1.7％％)在不同的患者中可能可以作为潜在的靶向治疗靶点。21个重复发生的突变包括一个剪接位点突变(TP53，c.673-2A＞G)，一个无义突变(TP53，c.C190T，p.R64X)，一个移码缺失(EP300，c.5546delC，p.S1849fs)，和18个错义突变，其中16个错义突变通过四种算法中的三种被验证均会导致蛋白质功能改变(表5)。

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在仅发生一次的255个突变中，有95个致病性或可能致癌性突变，157个意义不明的突变和2个良性突变。在致病性或可能致癌性突变中，PTEN p.E43X，PTEN p.R130X，PIK3CA p.E545Q，PIK3CA p.Q546K，PTCH1 p.Y224fs和其他突变可能可以作为靶向治疗的潜在治疗靶点(表6)。这255个突变包括6个剪接位点突变，27个无义突变，21个移码突变和201个错义突变，其中101个错义突变通过四种算法中的三种被验证均会导致蛋白质功能改变。276个突变中已有122个突变在COSMIC数据库中报道了，其中有154个突变未报道。

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突变与临床病理特征的相关性

在淋巴结转移病例中脉管瘤栓(LVI)的比例(23.8％，5/21)显著高于非淋巴结转移病例(5.1％，5/09)(95％CI：1.1679～27.8839，P＝0.015)。脉管瘤栓(LVI)的患者中ZNF750基因突变的比例(40％，4/10)显著高于无LVI患者(1.8％，2/109)(95％CI：3.868～425.2775，P＝0.0004)，淋巴结转移患者中ZNF750基因突变比例(19.0％，4/21)明显高于无淋巴结转移者(2.0％，2/98)(95％CI：1.4412～129.8545，P＝0.0088)。有吸烟史的患者TP53基因突变比例(46.4％，32/69)显著低于非吸烟史组(68.2％，30/44)(95％CI：0.1680～0.9515，P＝0.0327)。分化良好组中APC基因突变患者的比例(21.7％，5/23)明显低于中分化组(4.6％，3/72)(95％CI：0.0228～0.9128，P＝0.01878)(表1第4列)。

突变的预后值和临床病理特征

ESCC患者的肿瘤分化程度，KMT2C基因，ZNF750基因突变与总生存期OS显著相关(p＝7e-04，p＝0.0346，p＝0.000473)(图5)。KMT2C基因突变患者占40.3％(48/119)，其中41.7％(20/48)预后不良，KMT2C基因的突变位点包括V752I，A1685S，A2204T，V2790I，P3465L和P3308L。KMT2C突变型与野生型的风险比为1.815(95％CI：1.035～3.182，P＝0.0376)。ZNF750基因突变患者占5.04％(6/119)，其中83.3％(5/6)预后不良，ZNF750基因突变所对应的蛋白结构上的改变包括K101fs，Y25C，S70X，P9S，W207X和H13Q。ZNF750突变型与野生型的风险比为4.648(95％CI：1.809～11.94，P＝0.00141)。KMT2C基因突变和ZNF750基因突变发生在不同的患者中，仅有1例总生存期OS为16个月的患者同时携带KMT2C基因突变(A1685S)和ZNF750基因突变(Y25C)。

在Cox多因素回归分析中，KMT2C基因突变与总生存期OS相关(HR＝1.781，95％CI：0.985-3.218，P＝0.056)。ZNF750基因突变和肿瘤分化程度分别与OS显著相关(HR＝4.274，95％CI：1.523-11.99，p＝0.006；HR＝1.719，95％CI：1.027-2.878，p＝0.04)。基于Cox多因素回归模型构建列线图，3年和5年OS的预测C指数为0.673(图6A)。图6B为本发明技术方案中所涉及Cox模型的校准曲线。

利用KMT2C基因突变，ZNF750基因突变或肿瘤分化程度/组织学分级构建RSF模型，预测误差分别为0.453，0.540和0.490。利用肿瘤分化程度、KMT2C基因突变、和ZNF750基因突变共同构建RSF模型，预测误差为0.344(图6C)。肿瘤分化程度，KMT2C基因和ZNF750基因突变的VIMP分别为0.131，0.092，0.015(图6D)。

本申请中描述了本发明的优选实施方式，包括发明人所知道的实施本发明的最佳方式。对于阅读了前述说明书的本领域普通技术人员来说，那些优选实施方式的变通方式可以是显而易见的。本发明人期望本领域技术人员适当地使用这种变化，并且发明人旨在以与本文具体描述的不同的方式实施本发明。因此，本发明包括随后附具的权利要求中所述主题的所有适用法律准许的改变和等同实施。而且，本发明包括以上所述要素的所有可能的变通方式的任意组合，除非在本申请中另有说明或者与上下文明显矛盾。

SEQUENCE LISTING

<110> 中国医学科学院肿瘤医院

<120> ESCC频繁突变基因的靶向测序及其在获得判断ESCC预后的生物标记物中的应

用

<130> C18P1930

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 16872

<212> DNA

<213> 人类

<400> 1

gaggtgcgcg cgcccgcgcc gatgtgtgtg agtgcgtgtc ctgctcgctc catgttgccg 60

cctctcccgg tacctgctgc tgctcccggg gctgcgggaa atgcgagagg ctgagccggg 120

gaggaggaac ccgagcagca gcggcggcgg cggcggccgc ggcggcggga gccccccagg 180

aggaggaccg ggatccatgt gtctttcctg gtgactagga tgtcgtcgga ggaggacaag 240

agcgtggagc agccgcagcc gccgccacca ccccccgagg agcctggagc cccggccccg 300

agccccgcag ccgcagacaa aagacctcgg ggccggcctc gcaaagatgg cgcttcccct 360

ttccagagag ccagaaagaa acctcgaagt agggggaaaa ctgcagtgga agatgaggac 420

agcatggatg ggctggagac aacagaaaca gaaacgattg tggaaacaga aatcaaagaa 480

caatctgcag aagaggatgc tgaagcagaa gtggataaca gcaaacagct aattccaact 540

cttcagcgat ctgtgtctga ggaatcggca aactccctgg tctctgttgg tgtagaagcc 600

aaaatcagtg aacagctctg cgctttttgt tactgtgggg aaaaaagttc cttaggacaa 660

ggagacttaa aacaattcag aataacgcct ggatttatct tgccatggag aaaccaacct 720

tctaacaaga aggacattga tgacaacagc aatggaacct atgagaaaat gcaaaactca 780

gcaccacgaa aacaaagagg acagagaaaa gaacgatctc ctcagcagaa tatagtatct 840

tgtgtaagtg taagcaccca gacagcttca gatgatcaag ctggtaaact gtgggatgaa 900

ctcagtctgg ttgggcttcc agatgccatt gatatccaag ccttatttga ttctacaggc 960

acttgttggg ctcatcaccg ttgtgtggag tggtcactag gagtatgcca gatggaagaa 1020

ccattgttag tgaacgtgga caaagctgtt gtctcaggga gcacagaacg atgtgcattt 1080

tgtaagcacc ttggagccac tatcaaatgc tgtgaagaga aatgtaccca gatgtatcat 1140

tatccttgtg ctgcaggagc cggcaccttt caggatttca gtcacatctt cctgctttgt 1200

ccagaacaca ttgaccaagc tcctgaaaga tcgaaggaag atgcaaactg tgcagtgtgc 1260

gacagcccgg gagacctctt agatcagttc ttttgtacta cttgtggtca gcactatcat 1320

ggaatgtgcc tggatatagc ggttactcca ttaaaacgtg caggttggca atgtcctgag 1380

tgcaaagtgt gccagaactg caaacaatcg ggagaagata gcaagatgct agtgtgtgat 1440

acgtgtgaca aagggtatca tactttttgt cttcaaccag ttatgaaatc agtaccaacc 1500

aatggctgga aatgcaaaaa ttgcagaata tgtatagagt gtggcacacg gtctagttct 1560

cagtggcacc acaattgcct gatatgtgac aattgttacc aacagcagga taacttatgt 1620

cccttctgtg ggaagtgtta tcatccagaa ttgcagaaag acatgcttca ttgtaatatg 1680

tgcaaaaggt gggttcacct agagtgtgac aaaccaacag atcatgaact ggatactcag 1740

ctcaaagaag agtatatctg catgtattgt aaacacctgg gagctgagat ggatcgttta 1800

cagccaggtg aggaagtgga gatagctgag ctcactacag attataacaa tgaaatggaa 1860

gttgaaggcc ctgaagatca aatggtattc tcagagcagg cagctaataa agatgtcaac 1920

ggtcaggagt ccactcctgg aattgttcca gatgcggttc aagtccacac tgaagagcaa 1980

cagaagagtc atccctcaga aagtcttgac acagatagtc ttcttattgc tgtatcatcc 2040

caacatacag tgaatactga attggaaaaa cagatttcta atgaagttga tagtgaagac 2100

ctgaaaatgt cttctgaagt gaagcatatt tgtggcgaag atcaaattga agataaaatg 2160

gaagtgacag aaaacattga agtcgttaca caccagatca ctgtgcagca agaacaactg 2220

cagttgttag aggaacctga aacagtggta tccagagaag aatcaaggcc tccaaaatta 2280

gtcatggaat ctgtcactct tccactagaa accttagtgt ccccacatga ggaaagtatt 2340

tcattatgtc ctgaggaaca gttggttata gaaaggctac aaggagaaaa ggaacagaaa 2400

gaaaattctg aactttctac tggattgatg gactctgaaa tgactcctac aattgagggt 2460

tgtgtgaaag atgtttcata ccaaggaggc aaatctataa agttatcatc tgagacagag 2520

tcatcatttt catcatcagc agacataagc aaggcagatg tgtcttcctc cccaacacct 2580

tcttcagact tgccttcgca tgacatgctg cataattacc cttcagctct tagttcctct 2640

gctggaaaca tcatgccaac aacttacatc tcagtcactc caaaaattgg catgggtaaa 2700

ccagctatta ctaagagaaa attttctcct ggtagacctc ggtccaaaca gggggcttgg 2760

agtacccata atacagtgag cccaccttcc tggtccccag acatttcaga aggtcgggaa 2820

atttttaaac ccaggcagct tcctggcagt gccatttgga gcatcaaagt gggccgtggg 2880

tctggatttc caggaaagcg gagacctcga ggtgcaggac tgtcggggcg aggtggccga 2940

ggcaggtcaa agctgaaaag tggaatcgga gctgttgtat tacctggggt gtctactgca 3000

gatatttcat caaataagga tgatgaagaa aactctatgc acaatacagt tgtgttgttt 3060

tctagcagtg acaagttcac tttgaatcag gatatgtgtg tagtttgtgg cagttttggc 3120

caaggagcag aaggaagatt acttgcctgt tctcagtgtg gtcagtgtta ccatccatac 3180

tgtgtcagta ttaagatcac taaagtggtt cttagcaaag gttggaggtg tcttgagtgc 3240

actgtgtgtg aggcctgtgg gaaggcaact gacccaggaa gactcctgct gtgtgatgac 3300

tgtgacataa gttatcacac ctactgccta gaccctccat tgcagacagt tcccaaagga 3360

ggctggaagt gcaaatggtg tgtttggtgc agacactgtg gagcaacatc tgcaggtcta 3420

agatgtgaat ggcagaacaa ttacacacag tgcgctcctt gtgcaagctt atcttcctgt 3480

ccagtctgct atcgaaacta tagagaagaa gatcttattc tgcaatgtag acaatgtgat 3540

agatggatgc atgcagtttg tcagaactta aatactgagg aagaagtgga aaatgtagca 3600

gacattggtt ttgattgtag catgtgcaga ccctatatgc ctgcgtctaa tgtgccttcc 3660

tcagactgct gtgaatcttc acttgtagca caaattgtca caaaagtaaa agagctagac 3720

ccacccaaga cttataccca ggatggtgtg tgtttgactg aatcagggat gactcagtta 3780

cagagcctca cagttacagt tccaagaaga aaacggtcaa aaccaaaatt gaaattgaag 3840

attataaatc agaatagcgt ggccgtcctt cagacccctc cagacatcca atcagagcat 3900

tcaagggatg gtgaaatgga tgatagtcga gaaggagaac ttatggattg tgatggaaaa 3960

tcagaatcta gtcctgagcg ggaagctgtg gatgatgaaa ctaagggagt ggaaggaaca 4020

gatggtgtca aaaagagaaa aaggaaacca tacagaccag gtattggtgg atttatggtg 4080

cggcaaagaa gtcgaactgg gcaagggaaa accaaaagat ctgtgatcag aaaagattcc 4140

tcaggctcta tttccgagca gttaccttgc agagatgatg gctggagtga gcagttacca 4200

gatactttag ttgatgaatc tgtttctgtt actgaaagca ctgaaaaaat aaagaagaga 4260

taccgaaaaa ggaaaaataa gcttgaagaa actttccctg cctatttaca agaagctttc 4320

tttggaaaag atcttctaga tacaagtaga caaagcaaga taagtttaga taatctgtca 4380

gaagatggag ctcagctttt atataaaaca aacatgaaca caggtttctt ggatccttcc 4440

ttagatccac tacttagttc atcctcggct ccaacaaaat ctggaactca cggtcctgct 4500

gatgacccat tagctgatat ttctgaagtt ttaaacacag atgatgacat tcttggaata 4560

atttcagatg atctagcaaa atcagttgat cattcagata ttggtcctgt cactgatgat 4620

ccttcctctt tgcctcagcc aaatgtcaat cagagttcac gaccattaag tgaagaacag 4680

ctagatggga tcctcagtcc tgaactagac aaaatggtca cagatggagc aattcttgga 4740

aaattatata aaattccaga gcttggcgga aaagatgttg aagacttatt tacagctgta 4800

cttagtcctg cgaacactca gccaactcca ttgccacagc ctcccccacc aacacagctg 4860

ttgccaatac acaatcagga tgctttttca cggatgcctc tcatgaatgg ccttattgga 4920

tccagtcctc atctcccaca taattctttg ccacctggaa gcggactggg aactttctct 4980

gcaattgcac aatcctctta tcctgatgcc agggataaaa attcagcctt taatccaatg 5040

gcaagtgatc ctaacaactc ttggacatca tcagctccca ctgtggaagg agaaaatgac 5100

acaatgtcga atgcccagag aagcacgctt aagtgggaga aagaggaggc tctgggtgaa 5160

atggcaactg ttgccccagt tctctacacc aatattaatt tccccaactt aaaggaagaa 5220

ttccctgatt ggactactag agtgaagcaa attgccaaat tgtggagaaa agcaagctca 5280

caagaaagag caccatatgt gcaaaaagcc agagataaca gagctgcttt acgcattaat 5340

aaagtacaga tgtcaaatga ttccatgaaa aggcagcaac agcaagatag cattgatccc 5400

agctctcgta ttgattcgga gctttttaaa gatcctttaa agcaaagaga atcagaacat 5460

gaacaggaat ggaaatttag acagcaaatg cgtcagaaaa gtaagcagca agctaaaatt 5520

gaagccacac agaaacttga acaggtgaaa aatgagcagc agcagcagca acaacagcaa 5580

tttggttctc agcatcttct ggtgcagtct ggttcagata caccaagtag tgggatacag 5640

agtcccttga cacctcagcc tggcaatgga aatatgtctc ctgcacagtc attccataaa 5700

gaactgttta caaaacagcc acccagtacc cctacgtcta catcttcaga tgatgtgttt 5760

gtaaagccac aagctccacc tcctcctcca gccccatccc ggattcccat ccaggatagt 5820

ctttctcagg ctcagacttc tcagccaccc tcaccgcaag tgttttcacc tgggtcctct 5880

aactcacgac caccatctcc aatggatcca tatgcaaaaa tggttggtac ccctcgacca 5940

cctcctgtgg gccatagttt ttccagaaga aattctgctg caccagtgga aaactgtaca 6000

cctttatcat cggtatctag gccccttcaa atgaatgaga caacagcaaa taggccatcc 6060

cctgtcagag atttatgttc ttcttccacg acaaataatg acccctatgc aaaacctcca 6120

gacacaccta ggcctgtgat gacagatcaa tttcccaaat ccttgggcct atcccggtct 6180

cctgtagttt cagaacaaac tgcaaaaggc cctatagcag ctggaaccag tgatcacttt 6240

actaaaccat ctcctagggc agatgtgttt caaagacaaa ggatacctga ctcatatgca 6300

cgacccttgt tgacacctgc acctcttgat agtggtcctg gaccttttaa gactccaatg 6360

caacctcctc catcctctca ggatccttat ggatcagtgt cacaggcatc aaggcgattg 6420

tctgttgacc cttatgaaag gcctgctttg acaccaagac ctatagataa tttttctcat 6480

aatcagtcaa atgatccata tagtcagcct ccccttaccc cacatccagc agtgaatgaa 6540

tcttttgccc atccttcaag ggctttttcc cagcctggaa ccatatcaag gccaacatct 6600

caggacccat actcccaacc cccaggaact ccacgacctg ttgtagattc ttattcccaa 6660

tcttcaggaa cagctaggtc caatacagac ccttactctc aacctcctgg aactccccgg 6720

cctactactg ttgacccata tagtcagcag ccccaaaccc caagaccatc tacacaaact 6780

gacttgtttg ttacacctgt aacaaatcag aggcattctg atccatatgc tcatcctcct 6840

ggaacaccaa gacctggaat ttctgtccct tactctcagc caccagcaac accaaggcca 6900

aggatttcag agggttttac taggtcctca atgacaagac cagtcctcat gccaaatcag 6960

gatcctttcc tgcaagcagc acaaaaccga ggaccagctt tacctggccc gttggtaagg 7020

ccacctgata catgttccca gacacctagg ccccctggac ctggtctttc agacacattt 7080

agccgtgttt ccccatctgc tgcccgtgat ccctatgatc agtctccaat gactccaaga 7140

tctcagtctg actcttttgg aacaagtcaa actgcccatg atgttgctga tcagccaagg 7200

cctggatcag aggggagctt ctgtgcatct tcaaactctc caatgcactc ccaaggccag 7260

cagttctctg gtgtctccca acttcctgga cctgtgccaa cttcaggagt aactgataca 7320

cagaatactg taaatatggc ccaagcagat acagagaaat tgagacagcg gcagaagtta 7380

cgtgaaatca ttctccagca gcaacagcag aagaagattg caggtcgaca ggagaagggg 7440

tcacaggact cacccgcagt gcctcatcca gggcctcttc aacactggca accagagaat 7500

gttaaccagg ctttcaccag acccccacct ccctatcctg ggaacattag gtctcctgtt 7560

gcccctcctt taggacctag atatgctgtt ttcccaaaag atcagcgtgg accctatcct 7620

cctgatgttg ctagtatggg gatgagacct catggattta gatttggatt tccaggaggt 7680

agtcatggta ccatgccgag tcaagagcgc ttccttgtgc ctcctcagca aatacaggga 7740

tctggagttt ctccacagct aagaagatca gtatctgtag atatgcctag gcctttaaat 7800

aactcacaaa tgaataatcc agttggactt cctcagcatt tttcaccaca gagcttgcca 7860

gttcagcagc acaacatact gggccaagca tatattgaac tgagacatag ggctcctgac 7920

ggaaggcaac ggctgccttt cagtgctcca cctggcagcg ttgtagaggc atcttctaat 7980

ctgagacatg gaaacttcat tccccggcca gactttccgg gccctagaca cacagacccc 8040

atgcgacgac ctccccaggg tctacctaat cagctacctg tgcacccaga tttggaacaa 8100

gtgccaccat ctcaacaaga gcaaggtcat tctgtccatt catcttctat ggtcatgagg 8160

actctgaacc atccactagg tggtgaattt tcagaagctc ctttgtcaac atctgtaccg 8220

tctgaaacaa cgtctgataa tttacagata accacccagc cttctgatgg tctagaggaa 8280

aaacttgatt ctgatgaccc ttctgtgaag gaactggatg ttaaagacct tgagggggtt 8340

gaagtcaaag acttagatga tgaagatctt gaaaacttaa atttagatac agaggatggc 8400

aaggtagttg aattggatac tttagataat ttggaaacta atgatcccaa cctggatgac 8460

ctcttaaggt caggagagtt tgatatcatt gcatatacag atccagaact tgacatggga 8520

gataagaaaa gcatgtttaa tgaggaacta gaccttccaa ttgatgataa gttagataat 8580

cagtgtgtat ctgttgaacc aaaaaaaaag gaacaagaaa acaaaactct ggttctctct 8640

gataaacatt caccacagaa aaaatccact gttaccaatg aggtaaaaac ggaagtactg 8700

tctccaaatt ctaaggtgga atccaaatgt gaaactgaaa aaaatgatga gaataaagat 8760

aatgttgaca ctccttgctc acaggcttct gctcactcag acctaaatga tggagaaaag 8820

acttctttgc atccttgtga tccagatcta tttgagaaaa gaaccaatcg agaaactgct 8880

ggccccagtg caaatgtcat tcaggcatcc actcaactac ctgctcaaga tgtaataaac 8940

tcttgtggca taactggatc aactccagtt ctctcaagtt tacttgctaa tgagaaatct 9000

gataattcag acattaggcc atcggggtct ccaccaccac caactctgcc ggcctcccca 9060

tccaatcatg tgtcaagttt gcctcctttc atagcaccgc ctggccgtgt tttggataat 9120

gccatgaatt ctaatgtgac agtagtctct agggtaaacc atgttttttc tcagggtgtg 9180

caggtaaacc cagggctcat tccaggtcaa tcaacagtta accacagtct ggggacagga 9240

aaacctgcaa ctcaaactgg gcctcaaaca agtcagtctg gtaccagtag catgtctgga 9300

ccccaacagc taatgattcc tcaaacatta gcacagcaga atagagagag gccccttctt 9360

ctagaagaac agcctctact tctacaggat cttttggatc aagaaaggca agaacagcag 9420

cagcaaagac agatgcaagc catgattcgt cagcgatcag aaccgttctt ccctaatatt 9480

gattttgatg caattacaga tcctataatg aaagccaaaa tggtggccct taaaggtata 9540

aataaagtga tggcacaaaa caatctgggc atgccaccaa tggtgatgag caggttccct 9600

tttatgggcc aggtggtaac tggaacacag aacagtgaag gacagaacct tggaccacag 9660

gccattcctc aggatggcag tataacacat cagatttcta ggcctaatcc tccaaatttt 9720

ggtccaggct ttgtcaatga ttcacagcgt aagcagtatg aagagtggct ccaggagacc 9780

caacagctgc ttcaaatgca gcagaagtat cttgaagaac aaattggtgc tcacagaaaa 9840

tctaagaagg ccctttcagc taaacaacgt actgccaaga aagctgggcg tgaatttcca 9900

gaggaagatg cagaacaact caagcatgtt actgaacagc aaagcatggt tcagaaacag 9960

ctagaacaga ttcgtaaaca acagaaagaa catgctgaat tgattgaaga ttatcggatc 10020

aaacagcagc agcaatgtgc aatggcccca cctaccatga tgcccagtgt ccagccccag 10080

ccacccctaa ttccaggtgc cactccaccc accatgagcc aacccacctt tcccatggtg 10140

ccacagcagc ttcagcacca gcagcacaca acagttattt ctggccatac tagccctgtt 10200

agaatgccca gtttacctgg atggcaaccc aacagtgctc ctgcccacct gcccctcaat 10260

cctcctagaa ttcagccccc aattgcccag ttaccaataa aaacttgtac accagcccca 10320

gggacagtct caaatgcaaa tccacagagt ggaccaccac ctcgggtaga atttgatgac 10380

aacaatccct ttagtgaaag ttttcaagaa cgggaacgta aggaacgttt acgagaacag 10440

caagagagac aacggatcca actcatgcag gaggtagata gacaaagagc tttgcagcag 10500

aggatggaaa tggagcagca tggtatggtg ggctctgaga taagtagtag taggacatct 10560

gtgtcccaga ttcccttcta cagttccgac ttaccttgtg attttatgca acctctagga 10620

ccccttcagc agtctccaca acaccaacag caaatggggc aggttttaca gcagcagaat 10680

atacaacaag gatcaattaa ttcaccctcc acccaaactt tcatgcagac taatgagcga 10740

aggcaggtag gccctccttc atttgttcct gattcaccat caatccctgt tggaagccca 10800

aatttttctt ctgtgaagca gggacatgga aatctttctg ggaccagctt ccagcagtcc 10860

ccagtgaggc cttcttttac acctgcttta ccagcagcac ctccagtagc taatagcagt 10920

ctcccatgtg gccaagattc tactataacc catggacaca gttatccggg atcaacccaa 10980

tcgctcattc agttgtattc tgatataatc ccagaggaaa aagggaaaaa gaaaagaaca 11040

agaaagaaga aaagagatga tgatgcagaa tccaccaagg ctccatcaac tccccattca 11100

gatataactg ccccaccgac tccaggcatc tcagaaacta cctctactcc tgcagtgagc 11160

acacccagtg agcttcctca acaagccgac caagagtcgg tggaaccagt cggcccatcc 11220

actcccaata tggcagcagg ccagctatgt acagaattag agaacaaact gcccaatagt 11280

gatttctcac aagcaactcc aaatcaacag acgtatgcaa attcagaagt agacaagctc 11340

tccatggaaa cccctgccaa aacagaagag ataaaactgg aaaaggctga gacagagtcc 11400

tgcccaggcc aagaggagcc taaattggag gaacagaatg gtagtaaggt agaaggaaac 11460

gctgtagcct gtcctgtctc ctcagcacag agtcctcccc attctgctgg ggcccctgct 11520

gccaaaggag actcagggaa tgaacttctg aaacacttgt tgaaaaataa aaagtcatct 11580

tctcttttga atcaaaaacc tgagggcagt atttgttcag aagatgactg tacaaaggat 11640

aataaactag ttgagaagca gaacccagct gaaggactgc aaactttggg ggctcaaatg 11700

caaggtggtt ttggatgtgg caaccagttg ccaaaaacag atggaggaag tgaaaccaag 11760

aaacagcgaa gcaaacggac tcagaggacg ggtgagaaag cagcacctcg ctcaaagaaa 11820

aggaaaaagg acgaagagga gaaacaagct atgtactcta gcactgacac gtttacccac 11880

ttgaaacagc agaataattt aagtaatcct ccaacacccc ctgcctctct tcctcctaca 11940

ccacctccta tggcttgtca gaagatggcc aatggttttg caacaactga agaacttgct 12000

ggaaaagccg gagtgttagt gagccatgaa gttaccaaaa ctctaggacc taaaccattt 12060

cagctgccct tcagacccca ggacgacttg ttggcccgag ctcttgctca gggccccaag 12120

acagttgatg tgccagcctc cctcccaaca ccacctcata acaatcagga agaattaagg 12180

atacaggatc actgtggtga tcgagatact cctgacagtt ttgttccctc atcctctcct 12240

gagagtgtgg ttggggtaga agtgagcagg tatccagatc tgtcattggt caaggaggag 12300

cctccagaac cggtgccgtc ccccatcatt ccaattcttc ctagcactgc tgggaaaagt 12360

tcagaatcaa gaaggaatga catcaaaact gagccaggca ctttatattt tgcgtcacct 12420

tttggtcctt ccccaaatgg tcccagatca ggtcttatat ctgtagcaat tactctgcat 12480

cctacagctg ctgagaacat tagcagtgtt gtggctgcat tttccgacct tcttcacgtc 12540

cgaatcccta acagctatga ggttagcagt gctccagatg tcccatccat gggtttggtc 12600

agtagccaca gaatcaaccc gggtttggag tatcgacagc atttacttct ccgtgggcct 12660

ccgccaggat ctgcaaaccc tcccagatta gtgagctctt accggctgaa gcagcctaat 12720

gtaccatttc ctccaacaag caatggtctt tctggatata aggattctag tcatggtatt 12780

gcagaaagcg cagcactcag accacagtgg tgttgtcatt gtaaagtggt tattcttgga 12840

agtggtgtgc ggaaatcttt caaagatctg acccttttga acaaggattc ccgagaaagc 12900

accaagaggg tagagaagga cattgtcttc tgtagtaata actgctttat tctttattca 12960

tcaactgcac aagcgaaaaa ctcagaaaac aaggaatcca ttccttcatt gccacaatca 13020

cctatgagag aaacgccttc caaagcattt catcagtaca gcaacaacat ctccactttg 13080

gatgtgcact gtctccccca gctcccagag aaagcttctc cccctgcctc accacccatc 13140

gccttccctc ctgcttttga agcagcccaa gtcgaggcca agccagatga gctgaaggtg 13200

acagtcaagc tgaagcctcg gctaagagct gtccatggtg ggtttgaaga ttgcaggccg 13260

ctcaataaaa aatggagagg aatgaaatgg aagaagtgga gcattcatat tgtaatccct 13320

aaggggacat ttaaaccacc ttgtgaggat gaaatagatg aatttctaaa gaaattgggc 13380

acttccctta aacctgatcc tgtgcccaaa gactatcgga aatgttgctt ttgtcatgaa 13440

gaaggtgatg gattgacaga tggaccagca aggctactca accttgactt ggatctgtgg 13500

gtccacttga actgcgctct gtggtccacg gaggtctatg agactcaggc tggtgcctta 13560

ataaatgtgg agctagctct gaggagaggc ctacaaatga aatgtgtctt ctgtcacaag 13620

acgggtgcca ctagtggatg ccacagattt cgatgcacca acatttatca cttcacttgc 13680

gccattaaag cacaatgcat gttttttaag gacaaaacta tgctttgccc catgcacaaa 13740

ccaaagggaa ttcatgagca agaattaagt tactttgcag tcttcaggag ggtctatgtt 13800

cagcgtgatg aggtgcgaca gattgctagc atcgtgcaac gaggagaacg ggaccatacc 13860

tttcgcgtgg gtagcctcat cttccacaca attggtcagc tgcttccaca gcagatgcaa 13920

gcattccatt ctcctaaagc actcttccct gtgggctatg aagccagccg gctgtactgg 13980

agcactcgct atgccaatag gcgctgccgc tacctgtgct ccattgagga gaaggatggg 14040

cgcccagtgt ttgtcatcag gattgtggaa caaggccatg aagacctggt tctaagtgac 14100

atctcaccta aaggtgtctg ggataagatt ttggagcctg tggcatgtgt gagaaaaaag 14160

tctgaaatgc tccagctttt cccagcgtat ttaaaaggag aggatctgtt tggcctgacc 14220

gtctctgcag tggcacgcat agcggaatca cttcctgggg ttgaggcatg tgaaaattat 14280

accttccgat acggccgaaa tcctctcatg gaacttcctc ttgccgttaa ccccacaggt 14340

tgtgcccgtt ctgaacctaa aatgagtgcc catgtcaaga ggtttgtgtt aaggcctcac 14400

accttaaaca gcaccagcac ctcaaagtca tttcagagca cagtcactgg agaactgaac 14460

gcaccttata gtaaacagtt tgttcactcc aagtcatcgc agtaccggaa gatgaaaact 14520

gaatggaaat ccaatgtgta tctggcacgg tctcggattc aggggctggg cctgtatgct 14580

gctcgagaca ttgagaaaca caccatggtc attgagtaca tcgggactat cattcgaaac 14640

gaagtagcca acaggaaaga gaagctttat gagtctcaga accgtggtgt gtacatgttc 14700

cgcatggata acgaccatgt gattgacgcg acgctcacag gagggcccgc aaggtatatc 14760

aaccattcgt gtgcacctaa ttgtgtggct gaagtggtga cttttgagag aggacacaaa 14820

attatcatca gctccagtcg gagaatccag aaaggagaag agctctgcta tgactataag 14880

tttgactttg aagatgacca gcacaagatt ccgtgtcact gtggagctgt gaactgccgg 14940

aagtggatga actgaaatgc attccttgct agctcagcgg gcggcttgtc cctaggaaga 15000

ggcgattcaa cacaccattg gaattttgca gacagaaaga gatttttgtt ttctgtttta 15060

tgactttttg aaaaagcttc tgggagttct gatttcctca gtcctttagg ttaaagcagc 15120

gccaggagga agctgacaga agcagcgttc ctgaagtggc cgaggttaaa cggaatcaca 15180

gaatggtcca gcacttttgc ttttttttct tttccttttc tttttttttt gtttgttttt 15240

tgttttgttt ttcccttgtg ggtgggtttc attgttttgg ttttctagtc tcactaagga 15300

gaaactttta ctggggcaaa gagccgatgg ctgccctgcc ccgggcaggg gccttcctat 15360

gaatgtaaga ctgaaatcac cagcgagggg gacagagagt gctggccacg gccttattaa 15420

aaaggggcag gccctctaac ttcaaaatgt ttttaaataa agtagacacc actgaacaag 15480

gaatgtactg aaatgacttc cttagggata gagctaaggg ataataactt gcactaaata 15540

catttaaata cttgattcca tgagtcagtt tattgtagtt tttgatttct gtaaaataag 15600

agaaactttt gtatttatta ttgaataagt gaatgaagct atttttaaat aaagttagaa 15660

gaaagccaag ctgctgctgt tacctgcaga actaacaaac cctgttactt tgtacagata 15720

tgtaaatatt ttgagaaaaa atacagtata aaaatagtta ttgaccaaat gctaccaggc 15780

tctgcagcag ctcgggggct tataaaatgt tcatagggat gttacaatat aattttgtgt 15840

tataaaatat gccattataa ttatgtaata accaaaattt caacctagag tgttgggggt 15900

tttttggaaa ccgcagtcta ttagtactca atggttttat acaccttact tctgacagag 15960

cggggcgtat gctacgacta caacttttat agctgttttg gtaatttaaa ctaatttttt 16020

catattatat tgttgcatcc ctacttcttc agtcaggttt ttttgtgctt acaatttgtg 16080

ataactgtga ataactgctt aaaaatacac ccaaatggag gctgaatttt ttcttcagca 16140

aaagtagttt tgattagaac tttgtttcag ccacagagaa tcatgtaaac gtaataggat 16200

catgtagcag aaacttaaat ctaacccttt agccttctat ttaacacaaa aatttgaaaa 16260

agttaaaaaa aaaaaggaga tgtgattatg cttacagctg caggactctg gcaatagggt 16320

ttttggaaga tgtaatttta aaatgtgttt gtatgaactg tttgtttaca tttctttaat 16380

aaaaaaaaca ctgttttgtg tttgcttgta gaaacttaat cagcattttg aaccaggtta 16440

gctttttatt ttgtacttaa aattctggta ctgacacttc acaggctaag tataaaatga 16500

agttttgtgt gcacaattca agtggactgt aaactgttgg tatattcagt gatgcagttc 16560

tgaacttgta tatggcatga tgtattttta tcttacagaa taaatcaatt gtatatattt 16620

ttctcttgat aaatagctgt atgaaatttg tttcctgaat atttttcttc tcttgtacaa 16680

tatcctgaca tcctaccagt atttgtccta ccgggttttt gttgttttct gttctgtata 16740

atagtatcta atgttggcaa aaattgaatt ttttgaagta tacagagtgt tatgggtttt 16800

ggaatttgtg gacacagatt tagaagatca ccatttacaa ataaaatatt ttacatctat 16860

aaaaaaaaaa aa 16872

<210> 2

<211> 4911

<212> PRT

<213> 人类

<400> 2

Met Ser Ser Glu Glu Asp Lys Ser Val Glu Gln Pro Gln Pro Pro Pro

1 5 10 15

Pro Pro Pro Glu Glu Pro Gly Ala Pro Ala Pro Ser Pro Ala Ala Ala

20 25 30

Asp Lys Arg Pro Arg Gly Arg Pro Arg Lys Asp Gly Ala Ser Pro Phe

35 40 45

Gln Arg Ala Arg Lys Lys Pro Arg Ser Arg Gly Lys Thr Ala Val Glu

50 55 60

Asp Glu Asp Ser Met Asp Gly Leu Glu Thr Thr Glu Thr Glu Thr Ile

65 70 75 80

Val Glu Thr Glu Ile Lys Glu Gln Ser Ala Glu Glu Asp Ala Glu Ala

85 90 95

Glu Val Asp Asn Ser Lys Gln Leu Ile Pro Thr Leu Gln Arg Ser Val

100 105 110

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

115 120 125

Ile Ser Glu Gln Leu Cys Ala Phe Cys Tyr Cys Gly Glu Lys Ser Ser

130 135 140

Leu Gly Gln Gly Asp Leu Lys Gln Phe Arg Ile Thr Pro Gly Phe Ile

145 150 155 160

Leu Pro Trp Arg Asn Gln Pro Ser Asn Lys Lys Asp Ile Asp Asp Asn

165 170 175

Ser Asn Gly Thr Tyr Glu Lys Met Gln Asn Ser Ala Pro Arg Lys Gln

180 185 190

Arg Gly Gln Arg Lys Glu Arg Ser Pro Gln Gln Asn Ile Val Ser Cys

195 200 205

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

210 215 220

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

225 230 235 240

Ala Leu Phe Asp Ser Thr Gly Thr Cys Trp Ala His His Arg Cys Val

245 250 255

Glu Trp Ser Leu Gly Val Cys Gln Met Glu Glu Pro Leu Leu Val Asn

260 265 270

Val Asp Lys Ala Val Val Ser Gly Ser Thr Glu Arg Cys Ala Phe Cys

275 280 285

Lys His Leu Gly Ala Thr Ile Lys Cys Cys Glu Glu Lys Cys Thr Gln

290 295 300

Met Tyr His Tyr Pro Cys Ala Ala Gly Ala Gly Thr Phe Gln Asp Phe

305 310 315 320

Ser His Ile Phe Leu Leu Cys Pro Glu His Ile Asp Gln Ala Pro Glu

325 330 335

Arg Ser Lys Glu Asp Ala Asn Cys Ala Val Cys Asp Ser Pro Gly Asp

340 345 350

Leu Leu Asp Gln Phe Phe Cys Thr Thr Cys Gly Gln His Tyr His Gly

355 360 365

Met Cys Leu Asp Ile Ala Val Thr Pro Leu Lys Arg Ala Gly Trp Gln

370 375 380

Cys Pro Glu Cys Lys Val Cys Gln Asn Cys Lys Gln Ser Gly Glu Asp

385 390 395 400

Ser Lys Met Leu Val Cys Asp Thr Cys Asp Lys Gly Tyr His Thr Phe

405 410 415

Cys Leu Gln Pro Val Met Lys Ser Val Pro Thr Asn Gly Trp Lys Cys

420 425 430

Lys Asn Cys Arg Ile Cys Ile Glu Cys Gly Thr Arg Ser Ser Ser Gln

435 440 445

Trp His His Asn Cys Leu Ile Cys Asp Asn Cys Tyr Gln Gln Gln Asp

450 455 460

Asn Leu Cys Pro Phe Cys Gly Lys Cys Tyr His Pro Glu Leu Gln Lys

465 470 475 480

Asp Met Leu His Cys Asn Met Cys Lys Arg Trp Val His Leu Glu Cys

485 490 495

Asp Lys Pro Thr Asp His Glu Leu Asp Thr Gln Leu Lys Glu Glu Tyr

500 505 510

Ile Cys Met Tyr Cys Lys His Leu Gly Ala Glu Met Asp Arg Leu Gln

515 520 525

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

530 535 540

Glu Met Glu Val Glu Gly Pro Glu Asp Gln Met Val Phe Ser Glu Gln

545 550 555 560

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

565 570 575

Pro Asp Ala Val Gln Val His Thr Glu Glu Gln Gln Lys Ser His Pro

580 585 590

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

595 600 605

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

610 615 620

Ser Glu Asp Leu Lys Met Ser Ser Glu Val Lys His Ile Cys Gly Glu

625 630 635 640

Asp Gln Ile Glu Asp Lys Met Glu Val Thr Glu Asn Ile Glu Val Val

645 650 655

Thr His Gln Ile Thr Val Gln Gln Glu Gln Leu Gln Leu Leu Glu Glu

660 665 670

Pro Glu Thr Val Val Ser Arg Glu Glu Ser Arg Pro Pro Lys Leu Val

675 680 685

Met Glu Ser Val Thr Leu Pro Leu Glu Thr Leu Val Ser Pro His Glu

690 695 700

Glu Ser Ile Ser Leu Cys Pro Glu Glu Gln Leu Val Ile Glu Arg Leu

705 710 715 720

Gln Gly Glu Lys Glu Gln Lys Glu Asn Ser Glu Leu Ser Thr Gly Leu

725 730 735

Met Asp Ser Glu Met Thr Pro Thr Ile Glu Gly Cys Val Lys Asp Val

740 745 750

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

755 760 765

Ser Phe Ser Ser Ser Ala Asp Ile Ser Lys Ala Asp Val Ser Ser Ser

770 775 780

Pro Thr Pro Ser Ser Asp Leu Pro Ser His Asp Met Leu His Asn Tyr

785 790 795 800

Pro Ser Ala Leu Ser Ser Ser Ala Gly Asn Ile Met Pro Thr Thr Tyr

805 810 815

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

820 825 830

Arg Lys Phe Ser Pro Gly Arg Pro Arg Ser Lys Gln Gly Ala Trp Ser

835 840 845

Thr His Asn Thr Val Ser Pro Pro Ser Trp Ser Pro Asp Ile Ser Glu

850 855 860

Gly Arg Glu Ile Phe Lys Pro Arg Gln Leu Pro Gly Ser Ala Ile Trp

865 870 875 880

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

885 890 895

Arg Gly Ala Gly Leu Ser Gly Arg Gly Gly Arg Gly Arg Ser Lys Leu

900 905 910

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

915 920 925

Ile Ser Ser Asn Lys Asp Asp Glu Glu Asn Ser Met His Asn Thr Val

930 935 940

Val Leu Phe Ser Ser Ser Asp Lys Phe Thr Leu Asn Gln Asp Met Cys

945 950 955 960

Val Val Cys Gly Ser Phe Gly Gln Gly Ala Glu Gly Arg Leu Leu Ala

965 970 975

Cys Ser Gln Cys Gly Gln Cys Tyr His Pro Tyr Cys Val Ser Ile Lys

980 985 990

Ile Thr Lys Val Val Leu Ser Lys Gly Trp Arg Cys Leu Glu Cys Thr

995 1000 1005

Val Cys Glu Ala Cys Gly Lys Ala Thr Asp Pro Gly Arg Leu Leu

1010 1015 1020

Leu Cys Asp Asp Cys Asp Ile Ser Tyr His Thr Tyr Cys Leu Asp

1025 1030 1035

Pro Pro Leu Gln Thr Val Pro Lys Gly Gly Trp Lys Cys Lys Trp

1040 1045 1050

Cys Val Trp Cys Arg His Cys Gly Ala Thr Ser Ala Gly Leu Arg

1055 1060 1065

Cys Glu Trp Gln Asn Asn Tyr Thr Gln Cys Ala Pro Cys Ala Ser

1070 1075 1080

Leu Ser Ser Cys Pro Val Cys Tyr Arg Asn Tyr Arg Glu Glu Asp

1085 1090 1095

Leu Ile Leu Gln Cys Arg Gln Cys Asp Arg Trp Met His Ala Val

1100 1105 1110

Cys Gln Asn Leu Asn Thr Glu Glu Glu Val Glu Asn Val Ala Asp

1115 1120 1125

Ile Gly Phe Asp Cys Ser Met Cys Arg Pro Tyr Met Pro Ala Ser

1130 1135 1140

Asn Val Pro Ser Ser Asp Cys Cys Glu Ser Ser Leu Val Ala Gln

1145 1150 1155

Ile Val Thr Lys Val Lys Glu Leu Asp Pro Pro Lys Thr Tyr Thr

1160 1165 1170

Gln Asp Gly Val Cys Leu Thr Glu Ser Gly Met Thr Gln Leu Gln

1175 1180 1185

Ser Leu Thr Val Thr Val Pro Arg Arg Lys Arg Ser Lys Pro Lys

1190 1195 1200

Leu Lys Leu Lys Ile Ile Asn Gln Asn Ser Val Ala Val Leu Gln

1205 1210 1215

Thr Pro Pro Asp Ile Gln Ser Glu His Ser Arg Asp Gly Glu Met

1220 1225 1230

Asp Asp Ser Arg Glu Gly Glu Leu Met Asp Cys Asp Gly Lys Ser

1235 1240 1245

Glu Ser Ser Pro Glu Arg Glu Ala Val Asp Asp Glu Thr Lys Gly

1250 1255 1260

Val Glu Gly Thr Asp Gly Val Lys Lys Arg Lys Arg Lys Pro Tyr

1265 1270 1275

Arg Pro Gly Ile Gly Gly Phe Met Val Arg Gln Arg Ser Arg Thr

1280 1285 1290

Gly Gln Gly Lys Thr Lys Arg Ser Val Ile Arg Lys Asp Ser Ser

1295 1300 1305

Gly Ser Ile Ser Glu Gln Leu Pro Cys Arg Asp Asp Gly Trp Ser

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

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

1355 1360 1365

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

1370 1375 1380

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

1385 1390 1395

Met Asn Thr Gly Phe Leu Asp Pro Ser Leu Asp Pro Leu Leu Ser

1400 1405 1410

Ser Ser Ser Ala Pro Thr Lys Ser Gly Thr His Gly Pro Ala Asp

1415 1420 1425

Asp Pro Leu Ala Asp Ile Ser Glu Val Leu Asn Thr Asp Asp Asp

1430 1435 1440

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

1445 1450 1455

Ser Asp Ile Gly Pro Val Thr Asp Asp Pro Ser Ser Leu Pro Gln

1460 1465 1470

Pro Asn Val Asn Gln Ser Ser Arg Pro Leu Ser Glu Glu Gln Leu

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515

Asp Val Glu Asp Leu Phe Thr Ala Val Leu Ser Pro Ala Asn Thr

1520 1525 1530

Gln Pro Thr Pro Leu Pro Gln Pro Pro Pro Pro Thr Gln Leu Leu

1535 1540 1545

Pro Ile His Asn Gln Asp Ala Phe Ser Arg Met Pro Leu Met Asn

1550 1555 1560

Gly Leu Ile Gly Ser Ser Pro His Leu Pro His Asn Ser Leu Pro

1565 1570 1575

Pro Gly Ser Gly Leu Gly Thr Phe Ser Ala Ile Ala Gln Ser Ser

1580 1585 1590

Tyr Pro Asp Ala Arg Asp Lys Asn Ser Ala Phe Asn Pro Met Ala

1595 1600 1605

Ser Asp Pro Asn Asn Ser Trp Thr Ser Ser Ala Pro Thr Val Glu

1610 1615 1620

Gly Glu Asn Asp Thr Met Ser Asn Ala Gln Arg Ser Thr Leu Lys

1625 1630 1635

Trp Glu Lys Glu Glu Ala Leu Gly Glu Met Ala Thr Val Ala Pro

1640 1645 1650

Val Leu Tyr Thr Asn Ile Asn Phe Pro Asn Leu Lys Glu Glu Phe

1655 1660 1665

Pro Asp Trp Thr Thr Arg Val Lys Gln Ile Ala Lys Leu Trp Arg

1670 1675 1680

Lys Ala Ser Ser Gln Glu Arg Ala Pro Tyr Val Gln Lys Ala Arg

1685 1690 1695

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

1700 1705 1710

Asp Ser Met Lys Arg Gln Gln Gln Gln Asp Ser Ile Asp Pro Ser

1715 1720 1725

Ser Arg Ile Asp Ser Glu Leu Phe Lys Asp Pro Leu Lys Gln Arg

1730 1735 1740

Glu Ser Glu His Glu Gln Glu Trp Lys Phe Arg Gln Gln Met Arg

1745 1750 1755

Gln Lys Ser Lys Gln Gln Ala Lys Ile Glu Ala Thr Gln Lys Leu

1760 1765 1770

Glu Gln Val Lys Asn Glu Gln Gln Gln Gln Gln Gln Gln Gln Phe

1775 1780 1785

Gly Ser Gln His Leu Leu Val Gln Ser Gly Ser Asp Thr Pro Ser

1790 1795 1800

Ser Gly Ile Gln Ser Pro Leu Thr Pro Gln Pro Gly Asn Gly Asn

1805 1810 1815

Met Ser Pro Ala Gln Ser Phe His Lys Glu Leu Phe Thr Lys Gln

1820 1825 1830

Pro Pro Ser Thr Pro Thr Ser Thr Ser Ser Asp Asp Val Phe Val

1835 1840 1845

Lys Pro Gln Ala Pro Pro Pro Pro Pro Ala Pro Ser Arg Ile Pro

1850 1855 1860

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

1865 1870 1875

Pro Gln Val Phe Ser Pro Gly Ser Ser Asn Ser Arg Pro Pro Ser

1880 1885 1890

Pro Met Asp Pro Tyr Ala Lys Met Val Gly Thr Pro Arg Pro Pro

1895 1900 1905

Pro Val Gly His Ser Phe Ser Arg Arg Asn Ser Ala Ala Pro Val

1910 1915 1920

Glu Asn Cys Thr Pro Leu Ser Ser Val Ser Arg Pro Leu Gln Met

1925 1930 1935

Asn Glu Thr Thr Ala Asn Arg Pro Ser Pro Val Arg Asp Leu Cys

1940 1945 1950

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

1955 1960 1965

Thr Pro Arg Pro Val Met Thr Asp Gln Phe Pro Lys Ser Leu Gly

1970 1975 1980

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

1985 1990 1995

Ile Ala Ala Gly Thr Ser Asp His Phe Thr Lys Pro Ser Pro Arg

2000 2005 2010

Ala Asp Val Phe Gln Arg Gln Arg Ile Pro Asp Ser Tyr Ala Arg

2015 2020 2025

Pro Leu Leu Thr Pro Ala Pro Leu Asp Ser Gly Pro Gly Pro Phe

2030 2035 2040

Lys Thr Pro Met Gln Pro Pro Pro Ser Ser Gln Asp Pro Tyr Gly

2045 2050 2055

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

2060 2065 2070

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

2075 2080 2085

Gln Ser Asn Asp Pro Tyr Ser Gln Pro Pro Leu Thr Pro His Pro

2090 2095 2100

Ala Val Asn Glu Ser Phe Ala His Pro Ser Arg Ala Phe Ser Gln

2105 2110 2115

Pro Gly Thr Ile Ser Arg Pro Thr Ser Gln Asp Pro Tyr Ser Gln

2120 2125 2130

Pro Pro Gly Thr Pro Arg Pro Val Val Asp Ser Tyr Ser Gln Ser

2135 2140 2145

Ser Gly Thr Ala Arg Ser Asn Thr Asp Pro Tyr Ser Gln Pro Pro

2150 2155 2160

Gly Thr Pro Arg Pro Thr Thr Val Asp Pro Tyr Ser Gln Gln Pro

2165 2170 2175

Gln Thr Pro Arg Pro Ser Thr Gln Thr Asp Leu Phe Val Thr Pro

2180 2185 2190

Val Thr Asn Gln Arg His Ser Asp Pro Tyr Ala His Pro Pro Gly

2195 2200 2205

Thr Pro Arg Pro Gly Ile Ser Val Pro Tyr Ser Gln Pro Pro Ala

2210 2215 2220

Thr Pro Arg Pro Arg Ile Ser Glu Gly Phe Thr Arg Ser Ser Met

2225 2230 2235

Thr Arg Pro Val Leu Met Pro Asn Gln Asp Pro Phe Leu Gln Ala

2240 2245 2250

Ala Gln Asn Arg Gly Pro Ala Leu Pro Gly Pro Leu Val Arg Pro

2255 2260 2265

Pro Asp Thr Cys Ser Gln Thr Pro Arg Pro Pro Gly Pro Gly Leu

2270 2275 2280

Ser Asp Thr Phe Ser Arg Val Ser Pro Ser Ala Ala Arg Asp Pro

2285 2290 2295

Tyr Asp Gln Ser Pro Met Thr Pro Arg Ser Gln Ser Asp Ser Phe

2300 2305 2310

Gly Thr Ser Gln Thr Ala His Asp Val Ala Asp Gln Pro Arg Pro

2315 2320 2325

Gly Ser Glu Gly Ser Phe Cys Ala Ser Ser Asn Ser Pro Met His

2330 2335 2340

Ser Gln Gly Gln Gln Phe Ser Gly Val Ser Gln Leu Pro Gly Pro

2345 2350 2355

Val Pro Thr Ser Gly Val Thr Asp Thr Gln Asn Thr Val Asn Met

2360 2365 2370

Ala Gln Ala Asp Thr Glu Lys Leu Arg Gln Arg Gln Lys Leu Arg

2375 2380 2385

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

2390 2395 2400

Gln Glu Lys Gly Ser Gln Asp Ser Pro Ala Val Pro His Pro Gly

2405 2410 2415

Pro Leu Gln His Trp Gln Pro Glu Asn Val Asn Gln Ala Phe Thr

2420 2425 2430

Arg Pro Pro Pro Pro Tyr Pro Gly Asn Ile Arg Ser Pro Val Ala

2435 2440 2445

Pro Pro Leu Gly Pro Arg Tyr Ala Val Phe Pro Lys Asp Gln Arg

2450 2455 2460

Gly Pro Tyr Pro Pro Asp Val Ala Ser Met Gly Met Arg Pro His

2465 2470 2475

Gly Phe Arg Phe Gly Phe Pro Gly Gly Ser His Gly Thr Met Pro

2480 2485 2490

Ser Gln Glu Arg Phe Leu Val Pro Pro Gln Gln Ile Gln Gly Ser

2495 2500 2505

Gly Val Ser Pro Gln Leu Arg Arg Ser Val Ser Val Asp Met Pro

2510 2515 2520

Arg Pro Leu Asn Asn Ser Gln Met Asn Asn Pro Val Gly Leu Pro

2525 2530 2535

Gln His Phe Ser Pro Gln Ser Leu Pro Val Gln Gln His Asn Ile

2540 2545 2550

Leu Gly Gln Ala Tyr Ile Glu Leu Arg His Arg Ala Pro Asp Gly

2555 2560 2565

Arg Gln Arg Leu Pro Phe Ser Ala Pro Pro Gly Ser Val Val Glu

2570 2575 2580

Ala Ser Ser Asn Leu Arg His Gly Asn Phe Ile Pro Arg Pro Asp

2585 2590 2595

Phe Pro Gly Pro Arg His Thr Asp Pro Met Arg Arg Pro Pro Gln

2600 2605 2610

Gly Leu Pro Asn Gln Leu Pro Val His Pro Asp Leu Glu Gln Val

2615 2620 2625

Pro Pro Ser Gln Gln Glu Gln Gly His Ser Val His Ser Ser Ser

2630 2635 2640

Met Val Met Arg Thr Leu Asn His Pro Leu Gly Gly Glu Phe Ser

2645 2650 2655

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

2660 2665 2670

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

2675 2680 2685

Leu Asp Ser Asp Asp Pro Ser Val Lys Glu Leu Asp Val Lys Asp

2690 2695 2700

Leu Glu Gly Val Glu Val Lys Asp Leu Asp Asp Glu Asp Leu Glu

2705 2710 2715

Asn Leu Asn Leu Asp Thr Glu Asp Gly Lys Val Val Glu Leu Asp

2720 2725 2730

Thr Leu Asp Asn Leu Glu Thr Asn Asp Pro Asn Leu Asp Asp Leu

2735 2740 2745

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

2750 2755 2760

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

2765 2770 2775

Leu Pro Ile Asp Asp Lys Leu Asp Asn Gln Cys Val Ser Val Glu

2780 2785 2790

Pro Lys Lys Lys Glu Gln Glu Asn Lys Thr Leu Val Leu Ser Asp

2795 2800 2805

Lys His Ser Pro Gln Lys Lys Ser Thr Val Thr Asn Glu Val Lys

2810 2815 2820

Thr Glu Val Leu Ser Pro Asn Ser Lys Val Glu Ser Lys Cys Glu

2825 2830 2835

Thr Glu Lys Asn Asp Glu Asn Lys Asp Asn Val Asp Thr Pro Cys

2840 2845 2850

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

2855 2860 2865

Ser Leu His Pro Cys Asp Pro Asp Leu Phe Glu Lys Arg Thr Asn

2870 2875 2880

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

2885 2890 2895

Gln Leu Pro Ala Gln Asp Val Ile Asn Ser Cys Gly Ile Thr Gly

2900 2905 2910

Ser Thr Pro Val Leu Ser Ser Leu Leu Ala Asn Glu Lys Ser Asp

2915 2920 2925

Asn Ser Asp Ile Arg Pro Ser Gly Ser Pro Pro Pro Pro Thr Leu

2930 2935 2940

Pro Ala Ser Pro Ser Asn His Val Ser Ser Leu Pro Pro Phe Ile

2945 2950 2955

Ala Pro Pro Gly Arg Val Leu Asp Asn Ala Met Asn Ser Asn Val

2960 2965 2970

Thr Val Val Ser Arg Val Asn His Val Phe Ser Gln Gly Val Gln

2975 2980 2985

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

2990 2995 3000

Leu Gly Thr Gly Lys Pro Ala Thr Gln Thr Gly Pro Gln Thr Ser

3005 3010 3015

Gln Ser Gly Thr Ser Ser Met Ser Gly Pro Gln Gln Leu Met Ile

3020 3025 3030

Pro Gln Thr Leu Ala Gln Gln Asn Arg Glu Arg Pro Leu Leu Leu

3035 3040 3045

Glu Glu Gln Pro Leu Leu Leu Gln Asp Leu Leu Asp Gln Glu Arg

3050 3055 3060

Gln Glu Gln Gln Gln Gln Arg Gln Met Gln Ala Met Ile Arg Gln

3065 3070 3075

Arg Ser Glu Pro Phe Phe Pro Asn Ile Asp Phe Asp Ala Ile Thr

3080 3085 3090

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

3095 3100 3105

Lys Val Met Ala Gln Asn Asn Leu Gly Met Pro Pro Met Val Met

3110 3115 3120

Ser Arg Phe Pro Phe Met Gly Gln Val Val Thr Gly Thr Gln Asn

3125 3130 3135

Ser Glu Gly Gln Asn Leu Gly Pro Gln Ala Ile Pro Gln Asp Gly

3140 3145 3150

Ser Ile Thr His Gln Ile Ser Arg Pro Asn Pro Pro Asn Phe Gly

3155 3160 3165

Pro Gly Phe Val Asn Asp Ser Gln Arg Lys Gln Tyr Glu Glu Trp

3170 3175 3180

Leu Gln Glu Thr Gln Gln Leu Leu Gln Met Gln Gln Lys Tyr Leu

3185 3190 3195

Glu Glu Gln Ile Gly Ala His Arg Lys Ser Lys Lys Ala Leu Ser

3200 3205 3210

Ala Lys Gln Arg Thr Ala Lys Lys Ala Gly Arg Glu Phe Pro Glu

3215 3220 3225

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

3230 3235 3240

Val Gln Lys Gln Leu Glu Gln Ile Arg Lys Gln Gln Lys Glu His

3245 3250 3255

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

3260 3265 3270

Ala Met Ala Pro Pro Thr Met Met Pro Ser Val Gln Pro Gln Pro

3275 3280 3285

Pro Leu Ile Pro Gly Ala Thr Pro Pro Thr Met Ser Gln Pro Thr

3290 3295 3300

Phe Pro Met Val Pro Gln Gln Leu Gln His Gln Gln His Thr Thr

3305 3310 3315

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

3320 3325 3330

Gly Trp Gln Pro Asn Ser Ala Pro Ala His Leu Pro Leu Asn Pro

3335 3340 3345

Pro Arg Ile Gln Pro Pro Ile Ala Gln Leu Pro Ile Lys Thr Cys

3350 3355 3360

Thr Pro Ala Pro Gly Thr Val Ser Asn Ala Asn Pro Gln Ser Gly

3365 3370 3375

Pro Pro Pro Arg Val Glu Phe Asp Asp Asn Asn Pro Phe Ser Glu

3380 3385 3390

Ser Phe Gln Glu Arg Glu Arg Lys Glu Arg Leu Arg Glu Gln Gln

3395 3400 3405

Glu Arg Gln Arg Ile Gln Leu Met Gln Glu Val Asp Arg Gln Arg

3410 3415 3420

Ala Leu Gln Gln Arg Met Glu Met Glu Gln His Gly Met Val Gly

3425 3430 3435

Ser Glu Ile Ser Ser Ser Arg Thr Ser Val Ser Gln Ile Pro Phe

3440 3445 3450

Tyr Ser Ser Asp Leu Pro Cys Asp Phe Met Gln Pro Leu Gly Pro

3455 3460 3465

Leu Gln Gln Ser Pro Gln His Gln Gln Gln Met Gly Gln Val Leu

3470 3475 3480

Gln Gln Gln Asn Ile Gln Gln Gly Ser Ile Asn Ser Pro Ser Thr

3485 3490 3495

Gln Thr Phe Met Gln Thr Asn Glu Arg Arg Gln Val Gly Pro Pro

3500 3505 3510

Ser Phe Val Pro Asp Ser Pro Ser Ile Pro Val Gly Ser Pro Asn

3515 3520 3525

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

3530 3535 3540

Phe Gln Gln Ser Pro Val Arg Pro Ser Phe Thr Pro Ala Leu Pro

3545 3550 3555

Ala Ala Pro Pro Val Ala Asn Ser Ser Leu Pro Cys Gly Gln Asp

3560 3565 3570

Ser Thr Ile Thr His Gly His Ser Tyr Pro Gly Ser Thr Gln Ser

3575 3580 3585

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

3590 3595 3600

Lys Lys Arg Thr Arg Lys Lys Lys Arg Asp Asp Asp Ala Glu Ser

3605 3610 3615

Thr Lys Ala Pro Ser Thr Pro His Ser Asp Ile Thr Ala Pro Pro

3620 3625 3630

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

3635 3640 3645

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

3650 3655 3660

Val Gly Pro Ser Thr Pro Asn Met Ala Ala Gly Gln Leu Cys Thr

3665 3670 3675

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

3680 3685 3690

Pro Asn Gln Gln Thr Tyr Ala Asn Ser Glu Val Asp Lys Leu Ser

3695 3700 3705

Met Glu Thr Pro Ala Lys Thr Glu Glu Ile Lys Leu Glu Lys Ala

3710 3715 3720

Glu Thr Glu Ser Cys Pro Gly Gln Glu Glu Pro Lys Leu Glu Glu

3725 3730 3735

Gln Asn Gly Ser Lys Val Glu Gly Asn Ala Val Ala Cys Pro Val

3740 3745 3750

Ser Ser Ala Gln Ser Pro Pro His Ser Ala Gly Ala Pro Ala Ala

3755 3760 3765

Lys Gly Asp Ser Gly Asn Glu Leu Leu Lys His Leu Leu Lys Asn

3770 3775 3780

Lys Lys Ser Ser Ser Leu Leu Asn Gln Lys Pro Glu Gly Ser Ile

3785 3790 3795

Cys Ser Glu Asp Asp Cys Thr Lys Asp Asn Lys Leu Val Glu Lys

3800 3805 3810

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

3815 3820 3825

Gly Gly Phe Gly Cys Gly Asn Gln Leu Pro Lys Thr Asp Gly Gly

3830 3835 3840

Ser Glu Thr Lys Lys Gln Arg Ser Lys Arg Thr Gln Arg Thr Gly

3845 3850 3855

Glu Lys Ala Ala Pro Arg Ser Lys Lys Arg Lys Lys Asp Glu Glu

3860 3865 3870

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

3875 3880 3885

Lys Gln Gln Asn Asn Leu Ser Asn Pro Pro Thr Pro Pro Ala Ser

3890 3895 3900

Leu Pro Pro Thr Pro Pro Pro Met Ala Cys Gln Lys Met Ala Asn

3905 3910 3915

Gly Phe Ala Thr Thr Glu Glu Leu Ala Gly Lys Ala Gly Val Leu

3920 3925 3930

Val Ser His Glu Val Thr Lys Thr Leu Gly Pro Lys Pro Phe Gln

3935 3940 3945

Leu Pro Phe Arg Pro Gln Asp Asp Leu Leu Ala Arg Ala Leu Ala

3950 3955 3960

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

3965 3970 3975

Pro His Asn Asn Gln Glu Glu Leu Arg Ile Gln Asp His Cys Gly

3980 3985 3990

Asp Arg Asp Thr Pro Asp Ser Phe Val Pro Ser Ser Ser Pro Glu

3995 4000 4005

Ser Val Val Gly Val Glu Val Ser Arg Tyr Pro Asp Leu Ser Leu

4010 4015 4020

Val Lys Glu Glu Pro Pro Glu Pro Val Pro Ser Pro Ile Ile Pro

4025 4030 4035

Ile Leu Pro Ser Thr Ala Gly Lys Ser Ser Glu Ser Arg Arg Asn

4040 4045 4050

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

4055 4060 4065

Gly Pro Ser Pro Asn Gly Pro Arg Ser Gly Leu Ile Ser Val Ala

4070 4075 4080

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

4085 4090 4095

Ala Ala Phe Ser Asp Leu Leu His Val Arg Ile Pro Asn Ser Tyr

4100 4105 4110

Glu Val Ser Ser Ala Pro Asp Val Pro Ser Met Gly Leu Val Ser

4115 4120 4125

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

4130 4135 4140

Leu Arg Gly Pro Pro Pro Gly Ser Ala Asn Pro Pro Arg Leu Val

4145 4150 4155

Ser Ser Tyr Arg Leu Lys Gln Pro Asn Val Pro Phe Pro Pro Thr

4160 4165 4170

Ser Asn Gly Leu Ser Gly Tyr Lys Asp Ser Ser His Gly Ile Ala

4175 4180 4185

Glu Ser Ala Ala Leu Arg Pro Gln Trp Cys Cys His Cys Lys Val

4190 4195 4200

Val Ile Leu Gly Ser Gly Val Arg Lys Ser Phe Lys Asp Leu Thr

4205 4210 4215

Leu Leu Asn Lys Asp Ser Arg Glu Ser Thr Lys Arg Val Glu Lys

4220 4225 4230

Asp Ile Val Phe Cys Ser Asn Asn Cys Phe Ile Leu Tyr Ser Ser

4235 4240 4245

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

4250 4255 4260

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

4265 4270 4275

Gln Tyr Ser Asn Asn Ile Ser Thr Leu Asp Val His Cys Leu Pro

4280 4285 4290

Gln Leu Pro Glu Lys Ala Ser Pro Pro Ala Ser Pro Pro Ile Ala

4295 4300 4305

Phe Pro Pro Ala Phe Glu Ala Ala Gln Val Glu Ala Lys Pro Asp

4310 4315 4320

Glu Leu Lys Val Thr Val Lys Leu Lys Pro Arg Leu Arg Ala Val

4325 4330 4335

His Gly Gly Phe Glu Asp Cys Arg Pro Leu Asn Lys Lys Trp Arg

4340 4345 4350

Gly Met Lys Trp Lys Lys Trp Ser Ile His Ile Val Ile Pro Lys

4355 4360 4365

Gly Thr Phe Lys Pro Pro Cys Glu Asp Glu Ile Asp Glu Phe Leu

4370 4375 4380

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

4385 4390 4395

Tyr Arg Lys Cys Cys Phe Cys His Glu Glu Gly Asp Gly Leu Thr

4400 4405 4410

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

4415 4420 4425

His Leu Asn Cys Ala Leu Trp Ser Thr Glu Val Tyr Glu Thr Gln

4430 4435 4440

Ala Gly Ala Leu Ile Asn Val Glu Leu Ala Leu Arg Arg Gly Leu

4445 4450 4455

Gln Met Lys Cys Val Phe Cys His Lys Thr Gly Ala Thr Ser Gly

4460 4465 4470

Cys His Arg Phe Arg Cys Thr Asn Ile Tyr His Phe Thr Cys Ala

4475 4480 4485

Ile Lys Ala Gln Cys Met Phe Phe Lys Asp Lys Thr Met Leu Cys

4490 4495 4500

Pro Met His Lys Pro Lys Gly Ile His Glu Gln Glu Leu Ser Tyr

4505 4510 4515

Phe Ala Val Phe Arg Arg Val Tyr Val Gln Arg Asp Glu Val Arg

4520 4525 4530

Gln Ile Ala Ser Ile Val Gln Arg Gly Glu Arg Asp His Thr Phe

4535 4540 4545

Arg Val Gly Ser Leu Ile Phe His Thr Ile Gly Gln Leu Leu Pro

4550 4555 4560

Gln Gln Met Gln Ala Phe His Ser Pro Lys Ala Leu Phe Pro Val

4565 4570 4575

Gly Tyr Glu Ala Ser Arg Leu Tyr Trp Ser Thr Arg Tyr Ala Asn

4580 4585 4590

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

4595 4600 4605

Pro Val Phe Val Ile Arg Ile Val Glu Gln Gly His Glu Asp Leu

4610 4615 4620

Val Leu Ser Asp Ile Ser Pro Lys Gly Val Trp Asp Lys Ile Leu

4625 4630 4635

Glu Pro Val Ala Cys Val Arg Lys Lys Ser Glu Met Leu Gln Leu

4640 4645 4650

Phe Pro Ala Tyr Leu Lys Gly Glu Asp Leu Phe Gly Leu Thr Val

4655 4660 4665

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

4670 4675 4680

Cys Glu Asn Tyr Thr Phe Arg Tyr Gly Arg Asn Pro Leu Met Glu

4685 4690 4695

Leu Pro Leu Ala Val Asn Pro Thr Gly Cys Ala Arg Ser Glu Pro

4700 4705 4710

Lys Met Ser Ala His Val Lys Arg Phe Val Leu Arg Pro His Thr

4715 4720 4725

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

4730 4735 4740

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

4745 4750 4755

Ser Ser Gln Tyr Arg Lys Met Lys Thr Glu Trp Lys Ser Asn Val

4760 4765 4770

Tyr Leu Ala Arg Ser Arg Ile Gln Gly Leu Gly Leu Tyr Ala Ala

4775 4780 4785

Arg Asp Ile Glu Lys His Thr Met Val Ile Glu Tyr Ile Gly Thr

4790 4795 4800

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

4805 4810 4815

Ser Gln Asn Arg Gly Val Tyr Met Phe Arg Met Asp Asn Asp His

4820 4825 4830

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

4835 4840 4845

His Ser Cys Ala Pro Asn Cys Val Ala Glu Val Val Thr Phe Glu

4850 4855 4860

Arg Gly His Lys Ile Ile Ile Ser Ser Ser Arg Arg Ile Gln Lys

4865 4870 4875

Gly Glu Glu Leu Cys Tyr Asp Tyr Lys Phe Asp Phe Glu Asp Asp

4880 4885 4890

Gln His Lys Ile Pro Cys His Cys Gly Ala Val Asn Cys Arg Lys

4895 4900 4905

Trp Met Asn

4910

<210> 3

<211> 3218

<212> DNA

<213> 人类

<400> 3

acttaaatcc ctcccactgg aacagcaggc tccagtttca cccagcaagc gtgagaacag 60

gtactgcttc ctgagcaccg ccagccaccg gcaccaagac cggccacatc ccagcactgc 120

ccacctctgc tcccagccgc cagatgacgg aggctctcgc cagaccctca gcacgcagag 180

ctggcttctg atagaagtga tcgggaaaga aagcaaagcg ggaggtgcct ctttagaaac 240

cacgaagtgc acgcggcgtc gacagtgatc acgccacctg gacagccaga gtccaaggca 300

taaggaggaa aatgagtctc ctcaaagagc ggaagccaaa aaagccacat tacatcccca 360

ggcctccagg aaagcccttc aagtataaat gtttccaatg tccctttact tgcaatgaga 420

agtcacatct ttttaatcac atgaagtatg gtctttgtaa aaactcgatt actttagtat 480

cagagcagga tcgagttccc aagtgcccta aatctaactc actagacccc aagcaaacca 540

accagcccga tgccacggcg aagccagcct cttccaagtc tgtcgcaaat ggactctctg 600

ccttcgactc gaagcttcag cacagctctg ccagggaaga catcaaggaa aacctggagc 660

tgcaagcccg gggaacccac aggtgcctgg gacagaagcc agccctccac agggcatcac 720

cctgcaagag cccagctccg gaagccgccc tcggtgccca gcctgctctg gaaggcgcag 780

ctcggccttc tgcatttgtt ccagtcggcg agcacagact caaggggcca gacaacgccg 840

aggcgcccga gacactggct ttacacaacc ccactgccaa ggccgtgtct ttccacacca 900

agtcggcctt ccacactcct ggctacccct ggaaagccgg ctcacctttc cttccaccag 960

agtttccaca taaaatctca tctacaaagg ggcttggggc catttcccct tacatgcacc 1020

ccacaatccc agagtacccg cctcactttt acacagagca cgggctggcc accatctact 1080

cgccttacct gctggctggg agctcgcctg agtgtgacgc acccctgctg tcagtctacg 1140

gaacccaaga cccgagacac ttcctgcctc acccggggcc gatccctaag cacctggctc 1200

catctccagc cacatacgat cactacaggt ttttccagca atatccctct aacctgccga 1260

ttccttacgg attttacagg ccagagtctg cattttcctc ctatggtctc agactcccac 1320

ctgtcactgg cctcacccga gatcagagct ctcacctgct tgaagaagcc accctggtct 1380

atccagcctc gagtccttcc aggttaaacc cttcggaccc caacagaaaa cacgtcgagt 1440

tcgaaagtcc aattcctgag gctaaagact cctccaaggc tgggcagaga gacacggaag 1500

ggtccaaaat gagcccccgc gcagggagtg cagccacggg ctccccaggg aggccgagcc 1560

ccaccgactt catgcagacg agccagacct gcgaaggcct gtacgacctc tccaacaagg 1620

cagcctccag cgcactggga agactctacc cgccagagca aagcctcaca gccttcaggc 1680

ctgttaagaa aagcacagaa tgcctacctg cccaggctgc tgagaccaca gcagagtctc 1740

cagtaagcct caatgttgtg aacggagacc ctcctgctcc gaccggaagc gcctctctcg 1800

tctcggaggc cgcgccttcc agtccggacg acagctccgg gatgggcccc ctcaacctct 1860

ccaagaaatc agagataaac ctggcagcca cccacgaacc cacgtaccaa ggcagccccc 1920

aggcggaaac cgccagcttc tcagagctgc aggaccttcc actcaatctc tcggtgaagg 1980

acccctgtaa cacccaggct ccgaggcctg ccttccccgg tcgaccacga gctgcagaac 2040

ctgctgctgc tgttccacag aagactggga cagaaggttc tgaggatggg cccagccacc 2100

ctgagaccaa gccaggcagc ctcgacggtg acggggcccc acccacaggc cccggcgagg 2160

aggctccaga cgcatgcgcg gtggacagca gcgaggagca gaagcagacg gcagccgtgg 2220

ccctgtgcca gctggcggcc tacagcccca ggaacatccg ggtgggcgat ggggatgctg 2280

cggccccgga acctgcctgc cggcaagaca cacccacact gagctccatg gagagccaag 2340

aggcccagtg tgacctcaga cccaaaggac aaaagaggac aagtctaagg gatgctggaa 2400

aatcccagca aggagctaag aaggcgaagc tgcaggacac ggccagagtg ttcacactac 2460

gaaggagggc ccgggtgtcc taacgccggg ttcacacgtg tgttcacaga gctacggcca 2520

cacacacgcc ttccaaggtg gcaagctaca acacctctga actggcactt tcacattttt 2580

acaaatgcag ctgctgcttc tcaaaaaaac aaacaaacaa acaaacaaaa aaccctccaa 2640

ttcagttttt ataaatatta agcatgaata ttaaaaggtg cttctacttt tggttgtaaa 2700

aacacctgaa tgactctaag actgatatgt attttcaagt ctaagctgtc ttacagaaga 2760

tcttttataa atgtttcctt ataaatatct caccattaca acaaattgtt ttaactgttt 2820

ttctattagc tctagctgca tatttgatgt aaatgacaat tactgaaaaa atgtcagaaa 2880

aaacattttc agtactaaca ttaaagtgcc atatgtaaaa aagaaaaatg tgatttgtat 2940

aactaaataa cacacaaaca tcaagaggct atttatacaa ataatttatt tccactaggg 3000

aaagtgcatt actggtgaag gtattatcaa tttattctac ttgcttataa tgttacagtg 3060

aatgttctgg cttactctgc ctcactttcc attccccaaa atgatgtgta tgttgctaat 3120

tttccaataa actcatatga accataagga aacataaaat gcaaataaac ataaatctat 3180

gttatccttt aaaaaaaaaa aaaaaaaaaa aaaaaaaa 3218

<210> 4

<211> 723

<212> PRT

<213> 人类

<400> 4

Met Ser Leu Leu Lys Glu Arg Lys Pro Lys Lys Pro His Tyr Ile Pro

1 5 10 15

Arg Pro Pro Gly Lys Pro Phe Lys Tyr Lys Cys Phe Gln Cys Pro Phe

20 25 30

Thr Cys Asn Glu Lys Ser His Leu Phe Asn His Met Lys Tyr Gly Leu

35 40 45

Cys Lys Asn Ser Ile Thr Leu Val Ser Glu Gln Asp Arg Val Pro Lys

50 55 60

Cys Pro Lys Ser Asn Ser Leu Asp Pro Lys Gln Thr Asn Gln Pro Asp

65 70 75 80

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

85 90 95

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

100 105 110

Glu Asn Leu Glu Leu Gln Ala Arg Gly Thr His Arg Cys Leu Gly Gln

115 120 125

Lys Pro Ala Leu His Arg Ala Ser Pro Cys Lys Ser Pro Ala Pro Glu

130 135 140

Ala Ala Leu Gly Ala Gln Pro Ala Leu Glu Gly Ala Ala Arg Pro Ser

145 150 155 160

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

165 170 175

Glu Ala Pro Glu Thr Leu Ala Leu His Asn Pro Thr Ala Lys Ala Val

180 185 190

Ser Phe His Thr Lys Ser Ala Phe His Thr Pro Gly Tyr Pro Trp Lys

195 200 205

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

210 215 220

Thr Lys Gly Leu Gly Ala Ile Ser Pro Tyr Met His Pro Thr Ile Pro

225 230 235 240

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

245 250 255

Ser Pro Tyr Leu Leu Ala Gly Ser Ser Pro Glu Cys Asp Ala Pro Leu

260 265 270

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

275 280 285

Gly Pro Ile Pro Lys His Leu Ala Pro Ser Pro Ala Thr Tyr Asp His

290 295 300

Tyr Arg Phe Phe Gln Gln Tyr Pro Ser Asn Leu Pro Ile Pro Tyr Gly

305 310 315 320

Phe Tyr Arg Pro Glu Ser Ala Phe Ser Ser Tyr Gly Leu Arg Leu Pro

325 330 335

Pro Val Thr Gly Leu Thr Arg Asp Gln Ser Ser His Leu Leu Glu Glu

340 345 350

Ala Thr Leu Val Tyr Pro Ala Ser Ser Pro Ser Arg Leu Asn Pro Ser

355 360 365

Asp Pro Asn Arg Lys His Val Glu Phe Glu Ser Pro Ile Pro Glu Ala

370 375 380

Lys Asp Ser Ser Lys Ala Gly Gln Arg Asp Thr Glu Gly Ser Lys Met

385 390 395 400

Ser Pro Arg Ala Gly Ser Ala Ala Thr Gly Ser Pro Gly Arg Pro Ser

405 410 415

Pro Thr Asp Phe Met Gln Thr Ser Gln Thr Cys Glu Gly Leu Tyr Asp

420 425 430

Leu Ser Asn Lys Ala Ala Ser Ser Ala Leu Gly Arg Leu Tyr Pro Pro

435 440 445

Glu Gln Ser Leu Thr Ala Phe Arg Pro Val Lys Lys Ser Thr Glu Cys

450 455 460

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

465 470 475 480

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

485 490 495

Val Ser Glu Ala Ala Pro Ser Ser Pro Asp Asp Ser Ser Gly Met Gly

500 505 510

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

515 520 525

Glu Pro Thr Tyr Gln Gly Ser Pro Gln Ala Glu Thr Ala Ser Phe Ser

530 535 540

Glu Leu Gln Asp Leu Pro Leu Asn Leu Ser Val Lys Asp Pro Cys Asn

545 550 555 560

Thr Gln Ala Pro Arg Pro Ala Phe Pro Gly Arg Pro Arg Ala Ala Glu

565 570 575

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

580 585 590

Gly Pro Ser His Pro Glu Thr Lys Pro Gly Ser Leu Asp Gly Asp Gly

595 600 605

Ala Pro Pro Thr Gly Pro Gly Glu Glu Ala Pro Asp Ala Cys Ala Val

610 615 620

Asp Ser Ser Glu Glu Gln Lys Gln Thr Ala Ala Val Ala Leu Cys Gln

625 630 635 640

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

645 650 655

Ala Ala Pro Glu Pro Ala Cys Arg Gln Asp Thr Pro Thr Leu Ser Ser

660 665 670

Met Glu Ser Gln Glu Ala Gln Cys Asp Leu Arg Pro Lys Gly Gln Lys

675 680 685

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

690 695 700

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

705 710 715 720

Arg Val Ser

Claims (4)

1.检测KMT2C突变和ZNF750突变的试剂在制备用于预测食管鳞状细胞癌（ESCC）预后的试剂盒中的用途，其中所述KMT2C突变是在SEQ ID NO：2所示序列基础上发生如下所示突变中的一种或多种：V752I，A1685S，A2204T，V2790I，P3465L和P3308L，所述ZNF750突变是在SEQ ID NO：4所示序列基础上发生如下所示突变中的一种或多种：K101fs，Y25C，S70X，P9S，W207X和H13Q。

2.权利要求1所述的用途，其中所述ZNF750突变是在SEQ ID NO：4所示序列基础上发生如下所示突变中的一种或多种：Y25C和H13Q。

3.权利要求1所述的用途，其中所述KMT2C突变是在SEQ ID NO：2所示序列基础上发生A1685S突变，所述ZNF750突变是在SEQ ID NO：4所示序列基础上发生Y25C突变。

4.权利要求1-3中任一项所述的用途，其中所述ESCC为I-II期的ESCC，所述试剂包含检测所述KMT2C突变和ZNF750突变的引物。