CN110819714B - 一种抑癌基因及其应用 - Google Patents

一种抑癌基因及其应用 Download PDF

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CN110819714B
CN110819714B CN201911152615.5A CN201911152615A CN110819714B CN 110819714 B CN110819714 B CN 110819714B CN 201911152615 A CN201911152615 A CN 201911152615A CN 110819714 B CN110819714 B CN 110819714B
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黄仲曦
申维玺
温悦婷
章建平
潘星华
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Shenzhen Hospital of Southern Medical University
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Abstract

本发明公开了鼻咽癌的诊治标志物，所述标志物为VILL，实验证明，VILL及其蛋白在鼻咽癌组织中表达显著下调，提示可以将VILL应用于鼻咽癌的诊断或治疗中；进一步，体外细胞实验证实VILL与鼻咽癌细胞的增殖、凋亡、迁移和侵袭有关，提示VILL可以作为药物靶标应用于鼻咽癌的临床治疗。本发明还公开了与VILL基因相互作用蛋白EIF3J，发现EIF3J与VILL呈正相关，提示可通过EIF3J提高VILL基因的表达水平，从达到治疗鼻咽癌或鼻咽癌转移的目的，提供了一种新的治疗思路。

Description

一种抑癌基因及其应用
技术领域
本发明属于生物技术领域，具体涉及一种与鼻咽癌相关的抑癌基因及其应用。
背景技术
鼻咽癌起源于鼻咽腔顶部和侧壁粘膜，其发病率已成为我国头颈部肿瘤之首。鼻咽癌在我国南部地区高发，其发病率向北递减，且男性发病率高于女性。有趣的是，有研究表明两广地区因其普遍讲粤语，故而鼻咽癌发病率较高。值得注意的是，南方人群即使移民到其他地区后，鼻咽癌发病率仍居高不下。这提示：家族遗传、生活习俗、易感基因和地域分布等于鼻咽癌发生发展密切相关。
目前，血浆EBV DNA检测是鼻咽癌早期筛查非常有效的方法之一，具有高敏感性、高特异性，但在对鼻咽癌疗效评估和患者群体选择依然存在争议。另一方面，确诊鼻咽癌也离不开病理活检。随着科技发展，精准医学越发得到认可、提倡并推广。因此寻求一种无创精准有效来预测并评估鼻咽癌发生发展的标志物得到了广泛关注。现在的研究结果表明游离DNA(cell free DNA，cfDNA) 存在于多种恶性肿瘤外周血中，如肺癌、肝癌、食管癌、乳腺癌、结直肠癌等，并且与肿瘤发生发展呈正相关，但对鼻咽癌应用价值的研究较少。
发明内容
针对现有技术中的不足，本发明人通过检测鼻咽癌患者外周血中的cfDNA，利用cfDNA来开发与鼻咽癌发生发展密切相关的新的标志物，并将其应用于临床，为诊治鼻咽癌提供新的靶点。
本发明第一个的目的在于提供一种检测生物标志物的试剂在制备用于诊断鼻咽癌的产品中的应用，所述生物标志为VILL基因其核苷酸序列如SEQ ID NO：1所示，该基因编码的蛋白氨基酸序列如SEQ ID NO：2所示。
进一步地，所述样品为组织。
进一步地，与参照相比，所述生物标志物表达水平下调。
进一步地，所述试剂选自：
特异性扩增VILL基因的引物；或
特异性识别VILL基因的探针；或
特异性结合VILL基因编码的蛋白的抗体或配体。
进一步地，所述引物序列如SEQ ID NO：3～4所示。
本发明的第二个目的在于提供如下任一项所述的应用
(1)产品在制备诊断鼻咽癌的工具中的应用，所述产品包括检测VILL基因表达水平的试剂；
(2)VILL基因在制备治疗鼻咽癌的药物组合物中的应用；
(3)VILL基因在制备治疗鼻咽癌迁移的药物组合物中的应用；
(3)VILL基因过表达在制备治疗鼻咽癌的药物组合物中的应用。
进一步地，所述(1)中的试剂选自：
特异性扩增VILL基因的引物；或
特异性识别VILL基因的探针；或
特异性结合VILL基因编码的蛋白的抗体或配体。
进一步地，所述特异性扩增引物序列如SEQ ID NO：3～4所示。
本发明的第三个目的在于提供EIF3J在使用以VILL作为靶点治疗鼻咽癌药物组合物中的应用。
本发明通过对4例正常人与6例鼻咽癌患者的外周血cfDNA进行甲基化差异分析以及10例鼻咽癌患者免疫组化，发现VILL基因在鼻咽癌患者中显著低表达，并且在鼻咽癌细胞株中进行了验证，发现VILL在EBV阳性的鼻咽癌细胞中几乎不表达，而临床数据表明鼻咽癌患者几乎都携带EBV病毒。因此，VILL 基因第一内含子的MHB5区域高度甲基化以及EBV病毒可能是导致VILL在鼻咽癌患者中低表达的原因之一。
VILL(villin like，人绒毛膜样蛋白)，位于3号染色体短臂22区2带(3p22.2)，其蛋白一级结构具有6个凝胶样氨基酸重复序列与一个帽子区域，与 VIL1(villin，绒毛膜蛋白)高度相似而得名(图7)。但我们也发现二者预测的三维结构显著不同，这说明VILL与VIL1在细胞中发挥的功能可能存在差异。目前对VIL1的研究较为深入，研究表明VIL1在胃肠道肿瘤中发挥促瘤作用。但对VILL的研究很少。根据其基因位点、蛋白结构等信息，推测VILL可能是抑瘤基因。
另外，通过利用GenCLiP3分析VILL相互作用蛋白，预测与VILL可能相互作用蛋白有：HSPA8、ASAP2、TIMM13、HSPA6和EIF3J。鼻咽癌细胞HONE1 EBV过表达VILL后，RT-PCR检测结果显示VILL与EIF3J正相关，差异有统计学意义。
eIF3(eukaryotic initiation factor 3，真核翻译起始因子3)是最大的真核起始因子，在哺乳动物细胞中，其分子量超过了600KDa。eIF3是真核翻译起始进程中起着核心作用。例如，eIF3可以使eIF2·GTP·Met-tRNAi三联体复合物稳定地结合到40S核糖体亚基上，并促使43S前起始复合物的形成；它能够通过促进mRNA与40S亚基结合；而且，它还可以与游离的40S亚基结合，影响40S 亚基与60S亚基及其他eIF的结合或解离等等。此外，eIF3还被发现参与翻译过程以及细胞周期的调控。通过调节不同类型的mRNA的翻译起始，eIF3就可能选择性地调控蛋白的合成，从而对细胞的生长进行调控。而研究表明，EIF3J 使eIF3与40S核糖体亚基的结合更稳定。
本发明的有益效果：本发明经过广泛而深入的研究，通过正常人与鼻咽癌患者的外周血cfDNA甲基化差异分析以及鼻咽癌患者免疫组化分析，发现差异表达的基因，探讨其与鼻咽癌患的发生之间的关系，从而为鼻咽癌的检测及靶向治疗寻找更好的途径和方法。通过筛选，本发明首次发现了鼻咽癌中VILL基因显著性下调。实验证明，通过提高VILL的表达水平，能够有效地抑制鼻咽癌细胞的生长和侵袭，提示检测VILL基因的表达水平可成为鼻咽癌诊断的辅助诊断指标之一，提高VILL基因表达可成为预防或治疗鼻咽癌或鼻咽癌转移的新途径。
另外，本发明通过筛选与VILL基因相互作用蛋白，发现EIF3J与VILL正相关，提示可通过EIF3J提高VILL基因的表达水平，从达到治疗鼻咽癌或鼻咽癌转移的目的。为此，提供了一种新的治疗思路。
附图说明
图1为VILL基因第一内含子的MHB5区域在UCSC基因组浏览器中的详细信息示意图。
图2为VILL在鼻咽癌组织及鼻咽癌细胞及其去甲基化后表达情况示意图。 A为鼻咽癌组织标本中VILL表达对比图(20μm)，其中normal代表覆盖的正常黏膜细胞和周边正常组织细胞中VILL的表达；N+Ca代表周边正常组织和癌巢细胞中VILL的表达对比；Ca代表癌巢细胞中VILL的表达。B为RT-PCR检测VILL在各鼻咽癌细胞株表达情况。C为WB检测VILL在各鼻咽癌细胞株表达情况。D为RT-PCR检测5株鼻咽癌细胞去甲基化后(blank表示0uM；12表示12uM 5-氮杂-2'-脱氧胞苷)表达情况。E为WB检测5株鼻咽癌细胞去甲基化后(12uM 5-氮杂-2'-脱氧胞苷)表达情况。*p<0.05，**p<0.01，***p<0.001， #p<0.0001，ns p>0.05。
图3为RT-PCR和WB检测VILL过表达鼻咽癌细胞HONE1 EBV的mRNA 和蛋白表达水平示意图。A为WB检测VILL过表达鼻咽癌细胞HONE1 EBV蛋白表达水平；B为RT-PCR检测VILL过表达鼻咽癌细胞HONE1 EBV的mRNA 表达水平。*p<0.05，**p<0.01，***p<0.001，#p<0.0001，ns p>0.05。
图4为过表达VILL抑制了鼻咽癌细胞HONE1 EBV增殖示意图。A为EdU 检测VILL过表达对鼻咽癌细胞HONE1 EBV细胞周期S期的影响(200×)；B 为EdU占总细胞比例量化统计图，来自15个随机视野。C为CCK8检测VILL 过表达对鼻咽癌细胞HONE1 EBV增殖速度的影响。D为平板克隆检测VILL过表达对鼻咽癌细胞HONE1 EBV克隆形成能力的影响。E为平板克隆量化统计图。F为流式细胞周期检测VILL过表达对鼻咽癌细胞HONE1 EBV各细胞周期的影响。G为各细胞周期分布量化统计图。*p<0.05，**p<0.01，***p<0.001， #p<0.0001，ns p>0.05。
图5为过表达VILL抑制了鼻咽癌细胞HONE1 EBV迁移示意图。A为细胞划痕实验检测VILL过表达对鼻咽癌细胞HONE1 EBV愈合能力的影响(40×)； B为划痕实验愈合比例量化统计图；C为Transwell小室实验检测VILL过表达对鼻咽癌细胞HONE1 EBV迁移能力的影响(100x)；D为Transwell小室迁移细胞数量化统计图；*p<0.05，**p<0.01，***p<0.001，#p<0.0001，ns p>0.05。
图6为VILL可能相互作用的蛋白示意图。A为GenCLiP3预测与VILL相互作用的蛋白；B为RT-PCR检测鼻咽癌细胞HONE1 EBV过表达VILL后，可能相互作用蛋白mRNA水平变化；*p<0.05，**p<0.01，***p<0.001，#p<0.0001， ns p>0.05。
图7为利用SWISS预测VILL和villin蛋白结构示意图。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点，下面结合具体实施例及其附图对本发明做进一步的详细描述。
1.材料与方法
1.1 主要材料
4例正常人与6例鼻咽癌患者的外周血、10例人鼻咽癌组织由南方医院提供(cfDNA甲基化检测交由深圳市艾斯基因科技有限公司执行；免疫组化交由南方医科大学广州华银医学检验中心执行)。人肾胚细胞293T来自南方医科大学肿瘤研究所，人鼻咽癌细胞株HONE1 EBV由香港大学S.-W.Tsao教授提供； pTSB02-EGFP-FLAG-VILL和pTSB02-EGFP-FLAG购自上海权阳生物科技有限公司。LipofectamineTM2000购自美国Invitrogen公司，慢病毒载体质粒psPAX2 (PS)、穿梭质粒pMD2.G(PM)由瑞士日内瓦大学DidierTrono博士惠赠，质粒提取试剂盒购自广州东盛生物科技有限公司，Polybren购自Millipore公司，嘌呤霉素购自上海碧云天生物技术有限公司。TRIzol、逆转录试剂盒，RT-PCR 试剂盒购自TAKARA公司。1640培养基、Opti-MEN购自Gibco公司。CCK8 试剂盒购自Glpbio公司，细胞周期试剂盒购自Abpbio公司，EdU试剂盒购自锐博公司。兔抗人VILL抗体购自美国Abcam公司(ab111242，1:500稀释)，免疫组化兔抗人VILL抗体、鼠抗人Flag抗体购自sigma公司(HPA035675，1:200稀释；F1804，1:1000)，兔抗人GAPDH、兔抗人β-actin购自proteintech公司，兔二抗、鼠二抗购自abbkine公司。
1.2 实验方法
1.2.1 细胞培养及去甲基化处理
取生长状态良好的CNE1、5-8F、HOEN1、HONE1 EBV和HK1 EBV细胞用胰蛋白酶消化上述细胞，并接种于6孔板中，待细胞密度达70％左右时，分别给予0、12uM(uMol/L)5-Aza-CdR(DNA甲基化转移酶抑制剂)，每24h更换一次含药培养基，培养3天后收取RNA和蛋白。
1.2.2 慢病毒包装
在25cm培养瓶接种293T细胞，37℃，5％CO2培养箱中培养；待细胞密度达70％-80％时开始转染过表达慢病毒载体质粒(包括对照质粒oe-con： pTSB02-EGFP-FLAG；过表达质粒oe-VILL：pTSB02-EGFP-FLAG-VILL)。使用脂质体转染法，将转染的DNA溶液与磷脂混合，形成脂质体结构，将脂质体悬液加入到细胞培养液中，与受体细胞膜发生融合，DNA片段随即进入细胞质或细胞核内。转染前1h更换培养基为预热的Opti-MEN培养基(1.7ml)；按照包装系统(内含对照质粒oe-con、过表达慢病毒质粒oe-VILL、包装质粒与穿梭质粒)配置转染液，混匀后室温静置20min，轻柔加入培养瓶中(加液时细胞朝上，以免冲落细胞)，然后置于培养箱中培养；6h-8h后改换为新鲜培养基， 48h后，慢病毒载体将在293T内合成并包装成慢病毒，分泌到培养基上清中。48h与72h分别收集上清，并将两次上清混合后1,000rpm离心5min，0.45μ m PVDF滤器过滤至50mL离心管中，分装，-80℃冰箱保存。
1.2.3 HONE1 EBV稳转细胞株的筛选
在6孔板中接种HONE1 EBV细胞，完全培养基培养；待细胞密度达60％-70％时，弃旧培，加入上述含慢病毒上清1ml和促转染液polybrene(终浓度为10ug/ml)，6h-8h加全培1ml；48h后，慢病毒携带的外源DNA既可以整合到HONE1 EBV细胞染色体中并稳定表达，此时即可消化并传代；待细胞贴壁后按2μg/mL的浓度加入嘌呤霉素做筛选，并每2d-3d换液进行稳转株的筛选；一星期后换为含浓度为1μg/mL嘌呤霉素的完全培养基；等细胞长满后消化、扩大培养。
1.2.4 RT-PCR法检测细胞mRNA水平
将稳转细胞HONE1 EBV接种于6孔板中，待其长满后，利用TRIzol法提取总RNA，用反转录试剂盒将RNA反转录成cDNA，利用罗氏Lightcycler96 荧光定量PCR仪进行扩增。反应条件为：95℃预变性2min；95℃变性5s，60℃退火30s，共45个循环；95℃5s、65℃1min、95℃1s，1个循环溶解；37℃30s， 1个循环冷却。以GAPDH作为内参，对RT-PCR结果进行定量分析。利用 Primer-BLAST在线设计引物。实验重复3次，取均值。引物序列如表1所示：
表1：引物序列
Figure BDA0002283954380000071
Figure BDA0002283954380000081
1.2.5 Western blot法检测细胞蛋白水平
细胞蛋白提取试剂盒提取总蛋白，BCA法进行总蛋白浓度和定量检测。制胶，每孔加入50μg的蛋白样品，80V恒压电泳30min后，100V恒压电泳1.5 h。待电泳结束后，湿转法将蛋白电转移到PVDF膜上，5％脱脂奶粉封闭1.5h 后，加入相应的一抗，4℃孵育过夜。随后二抗室温孵育1.5h，利用BIO RAD 激光成像系统成像以及image J分析实验结果，以β-actin或GAPDH为内参，分析目的蛋白表达水平。实验重复3次，取均值。
1.2.6 CCK8法检测细胞增殖活性
取对数生长期细胞接种到96孔细胞培养板中，调整细胞密度为103个细胞每孔。每孔中加入10μl的CCK8溶液后37℃孵育2h，酶标仪检测450nm波长处每孔的吸光度值(OD值)。实验重复3次，取均值。
1.2.7 平板克隆形成实验
取对数生长的细胞，将细胞消化，用PBS洗两次，计数。将细胞稀释至 104/ml，6孔板里每孔加入2ml完全培养基，取50ul的细胞悬液加至6孔板，500 个细胞/孔。将细胞铺匀，培养10-14天左右。用PBS洗两遍，加甲醇固定15min，再用PBS洗两遍，加0.1％结晶紫染色15min，用清水洗去结晶紫，晾干。拍照，计数。
1.2.8 EdU实验
取对数生长的细胞，将细胞消化，用PBS洗两次，计数；将细胞稀释至 105/ml，加入100ul的细胞悬液加至96孔，10000个细胞/孔，5个复孔，培养过夜；按照EdU说明书操作；倒置荧光显微镜镜检，拍照，计数。
1.2.9 流式细胞术检测细胞周期
收集对数生长期细胞，计数，以105/孔接种于6孔板，置37℃，5％CO2条件下培养过夜，使细胞贴壁，次日更换培养液；48h后，中止培养，胰酶消化后，收集细胞，1000r/min离心5min，弃上清，冷PBS洗涤细胞1-2次，离心去上清；一边涡旋一边向细胞沉淀中加入70％预冷乙醇混匀，4℃固定18h以上；离心，弃去乙醇上清，加入预冷的PBS洗沉淀2-3次，离心去上清；加入200ul细胞周期染色液，震荡混匀，室温、避光反应30min；流式细胞仪检测。独立重复实验三次。
1.2.10 细胞transwell小室实验
取对数生长期细胞，饥饿培养过夜，0.25％胰酶消化，调整细胞密度为 106/mL，取100μL细胞悬液加至24孔transwell小室的上室，下室预先加入600 μL含有10％FBS的1640培养基，37℃、5％CO2培养箱培养28h；取出小室，甲醇固定15min；PBS洗2遍，结晶紫染色15min，显微镜下观察计数。
1.2.11 细胞划痕实验
先用marker笔在6孔板背后用直尺划至少5条线。每孔加入约5×105个细胞。第二天用枪头比着直尺，尽量垂至于背后的横线划痕。用PBS洗细胞2次，去处划下的细胞，加入无血清培养基。放入37℃，5％CO2培养箱培养。按0， 6，12，24小时拍照。
1.3 统计学方法
采用SPSS 20.0软件进行统计学处理。计量数据以均数±标准误表示，实验结果采用T检验进行分析。以P<0.05为差异有统计学意义。
2.实验结果
2.1 从cfDNA甲基化结果分析发现MHB5区域的高度甲基化(表2，表3)，落在了VILL第一内含子上。如图1所示，MHB5区域有显著的H3K27AC信号，提示其是转录调控区域；Chip-Seq数据显示转录因子Txn与其结合；多基因组比对显示该区域序列高度保守。这可能是VILL在鼻咽癌患者中表达下调的原因之一。
表2：10份cfDNA甲基化测序样品的CpG位点统计结果
Figure BDA0002283954380000101
表3 ：MHB5在10份样品中的详细检测信息
Figure BDA0002283954380000111
2.2 VILL在鼻咽癌组织及鼻咽癌细胞株的表达情况：本发明人收集了10例鼻咽癌组织进行VILL免疫组化染色，发现VILL在10例鼻咽癌组织的癌巢中几乎不表达(图2A)。并且也在鼻咽癌细胞株进行了验证(图2B)。经过WB 及qPCR实验，我们发现VILL在EBV阳性的鼻咽癌细胞株中表达极少，而临床数据显示鼻咽癌患者99％以上含EBV病毒，因此EBV病毒也可能是导致VILL 低表达的原因之一。接着，我们对其中5株鼻咽癌细胞株进行去甲基化处理(5- 氮杂-2'-脱氧胞苷，5-Aza-CdR)检测VILL的表达。结果显示，去甲基化后鼻咽癌细胞株VILL表达增高(图2)。
2.3 VILL过表达鼻咽癌细胞HONE1 EBV的鉴定RT-PCR和WB结果显示，转染了oe-con和oe-VILL后，鼻咽癌细胞HONE1 EBV表达VILL mRNA和蛋白明显高于对照组(oe-con)，且差异具有统计学意义(图3)。
2.4 VILL过表达对鼻咽癌细胞HONE1 EBV增殖的影响CCK8、平板克隆、 EdU和细胞周期实验结果显示，鼻咽癌细胞HONE1 EBV过表达VILL后，细胞增殖能力明显低于对照组，差异有统计学意义(图4)。
2.5.VILL过表达对鼻咽癌细胞HONE1 EBV迁移的影响细胞迁移-Transwell 小室实验、划痕实验结果显示，鼻咽癌细胞HONE1 EBV过表达VILL后，细胞迁移能力明显低于对照组，差异有统计学意义(图5)。
2.6 VILL相互作用蛋白预测利用GenCLiP3分析与VILL可能相互作用蛋白有：HSPA8、ASAP2、TIMM13、HSPA6和EIF3J。鼻咽癌细胞HONE1 EBV 过表达VILL后，RT-PCR检测结果显示VILL与EIF3J和ASAP6正相关(但 ASAP6 Ct值较大，暂不考虑)。差异有统计学意义(图6)。
由上述结果可知，VILL基因在鼻咽癌患者中显著低表达，在EBV阳性的鼻咽癌中几乎不表达，而临床数据表明鼻咽癌患者几乎都携带EBV病毒。因此， VILL基因第一内含子的MHB5区域高度甲基化以及EBV病毒可能是导致VILL 在鼻咽癌患者中低表达的原因之一。鼻咽癌细胞HONE1 EBV过表达VILL后，细胞的增殖和迁移能力均受到了抑制，因此VILL可以作为诊断和治疗鼻咽癌的重要靶点。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
SEQUENCE LISTING
<110> 一种抑癌基因及其应用
<120> 南方医科大学深圳医院、广州序科码生物技术有限责任公司
<130> 10.2
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 19130
<212> DNA
<213> human
<400> 1
gagggcggga agggccctct gtggggctgc cattttggct gggacctaaa tgcagtaaag 60
gagcagctac gggaatatag agagtggggc ttccaggcag agaagcctgc agtgcaaagg 120
tctgcagaca acgacctggg cgtcttcaag ggacacaagg aatcatattg ccagaacaca 180
gtaagccatg gggagagtga tgggagaggc agccgggaac ggccattgag agtttcatcc 240
cagttttagt ggaagccact ggaaggtttg gggagggggg tgatgtgagc gggtttatgg 300
ttaaattctg aggttgaatt ttccctctag ttagtcaaaa tcatagaaaa tagaatggtg 360
gttgcaaggg gctgggggca atggggagtc tatgtttaat gggtgcagag ttgcagagtt 420
tccgttttgt aagatgaaag agttctggaa atggtggcag tggttgcaca acaatatgag 480
tgtccttaac accacagaac cgtatactta aaagtggata caggccgggc gtggtggctt 540
acgcctgtaa tcccaacact ttgggaggct gaggccggtg gatcgctgga ggtcaggagt 600
tcgcaaccag cctgaccaac atagtgaaac cccgtctcta ccaaaaatac aaaattagcc 660
gggtgtggtg gtgcatgcct gtaatcccag ctactcaggg gactgaggca ggagaatcac 720
ttgaacccgg gaggcggagg ctgcagtgag ccaagatcat gccattgcac tccagcctcg 780
gcaacaagag caaaactctg tctcaaataa atacataaat aaaaagggga tacaatggta 840
aattttaagt atattttact acaatttttt aaatcttgtg aaaaaaagat tttttatatt 900
ttaccacaat ttttttaaat cttaggaaaa aagatccctc tggctactgt ggtaggatgg 960
tcgtagcggc caggcagacc cagagacaca gagggaggct ggctccggag caggcaggag 1020
gcgtgagagg gagggccagg cccaggatcc accatgaacc agctgccgac gggccagtgg 1080
cttgcgggag agagaaatca gggcaactcc aggtttgggg cctaagcacc ctctggatgt 1140
tcacactgaa cgtgttcctg gggctcagag agggtagatc tgggaggggc ctgaggcgat 1200
gttaaatgtg agaccaaacg tgctgggaga gatgtgacag acggaagatg gagacttagg 1260
ccggggcaag gttcggggga gttgggtcag ggagacactg gcgtttccat gctatttgca 1320
gccttcagac tggaactggg aaggaaactg gaaaacaaac aggagtgaag gcccaagctc 1380
tgctcctgct gccatttaga ggcctgaagg agacaggtgg agtgagaggg gaaggaacaa 1440
tggtgactga actgactgcc gcttggttga cagcgggcat tggtgactta ggaaagagcc 1500
attgccaggg aattcaagaa caaacagagc gggtgggctg aggggagatg ggggaagagt 1560
cggtgggaag aagtgaggac acagagtctt cctgggcaca gagacaaaaa gggacaggga 1620
aacgggtatg cacaggtaca gtggacacac tgccactgtc acagacaggg tccctgtccc 1680
aagtggggat cagacagaca tgccaggata caggcacaca catgaggtcg gacaggcaat 1740
ggcacacatg gggacgagcc aaatgcctgt gacacaggca tcccttcaca cacagggacc 1800
ctgcattgtg tctgttaaac acagtcacac actagctgta ggtcttgctg gacacagact 1860
gcccccgagt ccccaagtcc ccagacccct gtaacagaga cctttgtggg gtgaagactg 1920
aacacaggga gagggtcttg ggcagtgggc aggctttagc agcgtggctt tgatgggatg 1980
gcccacgctg gctgggcctt ggctgggact gaggccctaa gccgccaaga ggaggttgtg 2040
cccctggagt ttctggaagg ctctgtttgc agcccctcca ccccacagtg agagagagag 2100
agtggcagtg gagtggcagc tgaggtgagg gagaggtgtc ttcgctgcaa gagctggact 2160
ggcctctccc atgatgggta tcagggttcc ctgaggaggg ctgtgccagg cagtgagctc 2220
acatttgggt agggacagca gaaatatcta gagggagccc tgaccacctt cccctcctga 2280
agtgagctgg gtctcacaga gcctgacccc acagccccca gccttggtcc tacttcctcc 2340
taaagtttgc tggagtggcc tggccctctt ggtccaggcg gagcccacca ctccgggtga 2400
gggatcaggg gcccatctag tactccagag aaggaaatcc cagtatcttc cacaccccac 2460
ctcttagccc agtccaggcc tccatcccct gctctctctg gccactggta cccaggcagt 2520
gagacctggg agacttaacc atagccctgg ggagggcgct ggtcccgacg gcttgctcga 2580
tatttgttga ctggctgact aagtgaatgc ctagcccgct gagcactgtg tggtggggtg 2640
tgtcctgcag ggcaggagct aggattgagt tacagtggtg gggaccagga gtagggaggc 2700
agagactggc cccgtgccag gcctagcact gcccacaggc agggcgaagg cgcgggagca 2760
gtttccagca ggtaggcctg ggcagagagt tggggggcgg gggtttccag ggaaccagtg 2820
gggcagagtg ggctattcaa gtcaagggca tagagcatag ggtcccaggg ctcagggcca 2880
cagagcctcc gcggcagaat tgggaggcgg cagcagccca gcaggtgaaa acactgatgg 2940
ggcagggcag ctgtggccgg cagcccaagg ggaagaaagt ggcagatttt gtagatgggc 3000
agggaagtcc gtggcccaca aattcagtgt gcctgccccc aaccctggcc ccatcctcct 3060
ttcccctcac tctagagggc ggcaccactc cctgctccat ggaaactgag caaggtaatc 3120
tcgggctccc ttcaaaggta cccacaaaag aaggaagggc tcctgtaggg agagaaagag 3180
gcactggagg agggagaaag cgaggagaga ggaggcaccc agggacacga ggggcagggg 3240
gagagaagag ggggtgtgtg aagcgggtag ggagtgcccc gggaaagaga ggggagcctg 3300
gggaagagaa ggaaagacct ggggaggagg gtggggaggg agagagggag gggtgtaggg 3360
gaagacaggg aaccaggaaa ggaggaggtg cctggggagg aaaaatggga gaaaaaggga 3420
aggatcccag gggagagggg aaatcagagg ggagaaagaa gaaccagggg gaggcagggg 3480
cacccagtgg ggacagaggg aaaccctgga aaagcggagg cgtggggcat ggggagagaa 3540
agggaaacca gggaaggaat tgggcatccg gaagagaaag gggccctgag tggaggaagt 3600
gcacccccag ggaagagaag gagggcttct aggagaaggg agaggaccag ggataggagg 3660
gttcccactt gggaaggtgg tgaaagggag ttccccagag gaaagagggc aggagggttt 3720
gggaggctgg agggggcact gtggggggtt cccctgagga aagagggcag gagggcttgg 3780
gaggctggag gtggcactgt ggggagggag gaggccccca atctttgagc acccattcct 3840
ccctggagtc caggtgaaag agaatgaaca caggtgccat gcgagtgcga cctcagccct 3900
ggcagctgag ggcttggctc tacgggctgg ggctggatat cagacgggct cccggacccc 3960
tgggggcgat gctgcctctc ccagtgacac cagccgagtc ccaacttttt ggctgccagt 4020
ttgactttcc agtcccccat gtgccctggt tgacactgtt ctgaggccat ccagtagtct 4080
ttctggcact ctgtcaccat gttgtactct tccatgagct acagctcaca gtttctgtct 4140
gcacctctgg tcactgcctg cctgggcctc tggtaactgt cactggtcac tatttctgcc 4200
tattactcag tgtccttcta cggggccacc actggtggac atcctgcttg tcacccaatg 4260
tcctcacgtt ttctgttgct tgagatcacc ttttcatggc cactatgtct gcctgtcact 4320
catgtcattg tcacaccaac aggccatcaa ctgtggcaac agttcttggg gcaattaggg 4380
tgcacggacg gctcccttct ctattcattt tcactcagtc cagtcgcagg gcagcacact 4440
tcactcattc aacaagtact tactgagtac ctgctggggg ccgggcagct gggatcccac 4500
agtaaacaac aagacaaacc cctgccttca ggaatctgct gctctggtga gggagaccat 4560
agccaacaac catcctatgg tttccatagg gtgtcgggag gagctgtgag ctgtgacgga 4620
cccagctcta accccctaga aggacagccc actctgcccc tgctcccctg ccccacagcc 4680
tcctccgctc ccacatttct ttggctacat gttgggtgtt gccacagtgt actgtgaact 4740
caccagggcc aaaccagggg ccctgacaaa ggtgtaagga atgtgagcca ctctatgaca 4800
cctattgtgc cacacctgat tctgtaactc tgtcccctca ccttgttctg cctggagcca 4860
agggtgttcc tgctattgat aagacacagt gggagtggag ggggaggagg gaaggactaa 4920
cacaccaggg tcaccctgag tgagagattc agaaagaagt gacttttgga ccaaacctca 4980
cacacaggtt tctgaaggag tggtgcaggg aagcctcagg taattgaata tgaagtgcca 5040
agacataaca aaatcgacag aagtgagggt cccttccttc ccccaggtac ttctcattcc 5100
atgtcactgt cattcactgc cctctcattc tctccctctc tgcacgcgca gaaccacaga 5160
gcacagactc gagcaagcta aatcgctatt tgcagagcct gtggctgttt gcagagcctg 5220
tgtttataca gagttcagct ctgtttcaag aatttgtcag ctgaagcggg aagatcactt 5280
gaacccagaa ggtggaggtt gcagtgaagt gtgttcgaat catgcactgc aggctagggt 5340
ggcagagcaa gaccctgtct cgaaaaaact ggaaaaaaaa attgtcaagt gtttatgcct 5400
aatctgccca acccactcat atcagctgag cctgtcactt gctgcagaat ctgagtcttc 5460
atagtctgtg tgctgacatt tgtgtgttta cagagccctc ctaataaaag tcatgttcta 5520
taatgaagtt gtacaggtag ccaggtgtcg gtctccagcc tgagaactct ggctgttgtt 5580
ccttgtgtcg tcccatattc ctgcctggcc tgcgatggac atcagcaagg gcctcccagg 5640
catgcaggga ggcctccaca tatggatctc tgaggtgaga ggcacgacca aataggagag 5700
ttggtgacat ggaagagcgt ggggttttcc acactaggct tctcccgccc ccaactcaga 5760
gcgtcctctt ccaccccacc ccagcgggtg tcatgggtag aggcctcctg agttgttaca 5820
gggaactctc ctctcccaga accggaagat ggtgccggta cccgaggggg cttacgggaa 5880
cttttttgag gaacactgct atgtcatcct ccacgtgagt cgcttgggga agtctgcctg 5940
agaggggtgg catggcccgg cactggggag actgaggcac aggcataaac tctgccctgg 6000
gaagcggcaa gttgagagcc ggagaatcac atcccacaag ggggcggtta ccgttgaggc 6060
ttcttagtcc ttccctccca cttcctgatc tccgcggaag cccctgccta gcgtctcccc 6120
atggcccttg gtacatcctc cccttctcca cccgcacctc cgtcttcccc gcaacacata 6180
tacacaaaca cccggaccct aggtccccca gagcccgaag gccacgcagg gggcgtccag 6240
cgacctgcac tactgggtcg ggaagcaggc gggtgcggaa gcgcagggcg ctgcggaggc 6300
cttccagcag cgcctacagg acgagctggg gggccagacc gtgctgcacc gcgaggcgca 6360
gggccacgag tccgactgct tctgcagcta cttccgcccg ggaatcatgt gagtgcgggg 6420
gcgaccgggg caggagggag ccgtggaggc ggtgccttgg ctggggcagt cttgggatgg 6480
ccatcgtcca catccctgaa tggggcaagc cgggaggggt tgggtaacac ctttattgag 6540
atacaactca tacgccatac gattcaccca tttaaagtac atatacatgt cagtgatttt 6600
taacagttgt gtgcaaccat caccacaatt ttagaacatt ttcatcaccc taaaaagaag 6660
ccccataccc ctttgccacc actccccatt tccctccaac acccccatcc cctgccctag 6720
gcaaccactc atctgctttg tctatacatt tgcctcttcc gggcatgtca tagaaataga 6780
atcattcaac aaatggtctt ccctgacggg atctttttgt tttaacacac tgttttcatg 6840
gtttatccat gttgtagcat gtgttattac ttctttttat taccaagtaa tattgcattt 6900
tatgaatata ctacatttta tttatccatt catcacttaa tagacatttg agttgtttcc 6960
actttttggc tgctatgaat ctgtttttat gaacatttgt atacaagttt ttgtgtggac 7020
aaatgttttc atttcccttg gtatatacct aggagcagac accaatgttg ttttgagttt 7080
gcttttatgt tactgtgctg gtaggaatat atctttcgct gtaaattgct tcacaggtgt 7140
tttaaattaa cactagcaat atatttggaa atataggaaa ttgctatttg atcatttcca 7200
tttcatacag ttgaacctat atatgcatat agcatataca tgaacacatg cacacaaaca 7260
tatgcgcaga tgcagaaaca cacaccaccc tcatgtatgt ccacctatag atggacatgt 7320
gtgaatatcc atgttcacct acacacatat atgctgttgc ccaccttcat gcactcacca 7380
tctagactta cagggataaa caaatacaca gtgtgccccc aaatgtatgt acatgtacac 7440
atatatgcat gtgtccacat gactacgcat ggaagtgcac tacacatgga aatgaatgca 7500
cactcatgcc ctatgtgtgc acccatgtac ttatacatac atgtgttcac acagactcca 7560
gtgcatgtat gaagcaggca catacgtgca cgtgctcaca tctgcagtca ttcatgcaca 7620
tggcagtctg tgttcttata tacacagaat gtatataccc tcctgctatt cccacctcag 7680
ctacaggaag ggaggcctag catctgacct caagcatgtg gagaccaact tgttcaacat 7740
ccagcgactg ctgcacatca aagggaggaa gcacgtgtct gccactgagg tgaggctgcc 7800
aggggagcct cttgacctct gaactcggct cagactgggt gtactgaggt ataaggaggt 7860
tggaaattgg ctggggttgt ggggaacaag gagctgtcag cattcccaga tggtcttgaa 7920
tgaggagaac agagaggaca ccaggcttct agccccactt ctgccactgc tccccggacc 7980
ttttgtttat gcgtctatct cagggacata ttgagaacat gggcacagct acttgggagg 8040
ctgaggcagg aggatcgcct gagcccagga gtgattcatg atgacaccac tgtactccag 8100
cagcttgggt gacagagaaa gaccctgtct ctgaaacaaa aaaagaactt gggcagatgg 8160
ggcagatgat atggcgcctg tgaatggtcc ttgttagcag cacagtgaga ggtggtgcat 8220
aacctcgcac tctctcacac acattgtcat tgtcacctat atgcacacac actgtctccc 8280
actgttatcc ccgggtgctc ctctggtcca tagagtgccc ttgtgcaaat cagaacagtg 8340
cccctttctt ccatctgtca ggaaattaca ggaaaatact ggttttgtta gaacaaaaca 8400
ttttgctgcc atctggccag aatttggtgt aatgaacatt caggtgtaca acgtctacag 8460
tgtgaatggt aacccttgag gtatgcaagt gagtctgcat aggcagcctg tgacacagag 8520
acaatcccac ataggctctg catgtcaccc acagacccag tgaccataca tctctggtca 8580
ttcagggaca ccatgaagcc ccatgtttct cctattatcc tggcataatt atctaaagta 8640
ctccctttta ctctcaaaag tgtgtccgtt tggagactat cttcatccat aggtattcac 8700
tgtcaccata agcacacttc ttcacatgtg atgtccccat atacatacat gtgtatatgc 8760
acacacacaa aagataaaca gatacacccc acctgtatac atgtgtacat gtatacatgt 8820
acagcaacag acatgcactg agtgagaagt gtcataagtc atgtacaccc tgtggaatgc 8880
agcattatct acagtcacac atgcctacgt acacccagtt tgcatacagc ttcatgcaca 8940
cgtgacacat cccagctatg ctcccctcta gcggatgctg gtggtatgac actctgtctc 9000
tctccctggc tctggcaggt ggagctctcc tggaacagct ttaataaggg tgacatcttc 9060
ctgctggacc taggcaagat gatgattcag tggaatgggc ccaagaccag catttctgag 9120
aaggctcggg tcagtgtctg cccaaggaac tggggagtac ggggcttggg cggggaatga 9180
tcctccagtt gaccatcctc cggccaccca aagagttggg cttggctctg ctacaaccca 9240
agaaacgcct atgagttaca agctgagttc agaccctgca ttgttggtgt ccccctggat 9300
gccaggatga ggggacatca gcccttctcc ccatcagcct ctgggagctg agcagtgaca 9360
ggagaagtct ctgctgtgag agggcacact ggtgacaccc tgactcccag gtcctctccc 9420
gcaggggctg gctttgacct acagcctccg ggacagggaa cgtggtggtg gtcgtgcaca 9480
gattggtgtg gtggatgatg aggccaaagc cccggacctc atgcagatca tggaggctgt 9540
gctgggccgc agggtgggca gcctgcgtgc cgccacgccc agcaaggata tcaaccagct 9600
gcagaaggcc aatgttcgcc tgtaccagtg agtacccctg gggtgggcag gggtgggtgg 9660
gacagtccag gactctgtgt ccatcactac tgcaataaca cggcatctct agaaggccct 9720
ccagcaaagg ctgctgggtt tctgggacag gtcatgtgga ccgtgggtcc agcctgtact 9780
cttccatggc atgtggctgc cttgctgaga aacctattgt acatacaggg ggactgtggc 9840
gccctgccag ggccagggag ctgcctaaag ctccacagca aggctgcagt aaaagtgaag 9900
actacaactc ggggccccag cccccatgcc ttctgtctct gagggactgg ggtggggtcc 9960
taaatggggc tggagtggag acagatcagg gaggggctgg gctggccact cctgggttcc 10020
tgtcccccca gtgtctatga gaagggcaaa gacctggtgg tcctggagtt ggcgaccccc 10080
ccactgaccc aggacctgct gcaggaggag gtgaggaagg cctggcccca gctacttgca 10140
tccttcccca tccacaaccc cagcccagtc tggaccacct actgaccagc cccacccttg 10200
ctcccaggac ttctacatcc tggaccaggg tggcttcaag atctatgtgt ggcagggacg 10260
catgtctagc ctccaggaga gaaaggctgc cttcagccgg gctgtggtga gccctggggc 10320
tctgtctgag aggaacagag cactgccctg gggtctgagt ggggaggcag ctccgcccca 10380
gggtctaagg gaggaatcag ccctccccta gagtttgagt tgggaaatcc tatgctggag 10440
tcttaaaggg gaggtagccc tgccctggga gccctgagag taaagaccag gtggcctctg 10500
agaaaccatg tgtaaaaggg gcacgtggcc ctgccttcag gtctggggcc ctactgactg 10560
ggcggtcccg ctttctgagg gtggggctgg ggaggagaca gggctctctc tgtctggggt 10620
ccgtgagggt tgacatggcc tgtcctgcac gaggcctaga ctaagggggc cctgcctgcc 10680
ctcagggacc tcagagaagt cggtggtgac agagtcaatt cgctaagtgg gtcatgagct 10740
cggttaatta acgcttcttg gagctcggct gtcccagagc ggtaggtccc caggagcggc 10800
agtggggcag tggactctca gggtcgcagg actgacgatg ccttccgccc cagggcttca 10860
tccaggccaa gggctacccg acctacacca acgtggaggt ggtgaacgac ggcgccgagt 10920
cggccgcgtt caagcagctc ttccggactt ggtctgagaa gcggcgcagg aaccagaagc 10980
tcggcgggag gggtgagcgg gcggggcggg gctgacgggg gcggggcggg actggcgggg 11040
gcggggcctg gcaggaatcg gcaactcgtt gggatgggtg agcgggcggg gcggggccgg 11100
agggggcggg gcctggtctg cacggggcgg aacagagcct caggatgagg aaaggggcgt 11160
gggctgcgga ggacgcggcg ggacctggaa tcttggaggg atggttgagg agtgggcggg 11220
gcggagatgg tgttgggggg gggcagaggg cgccactgac gcctactgtc cccccttcag 11280
ataaatcgat tcatgtaaag ctggacgtgg gcaagctgca cacccagcct aagttagcgg 11340
cccagctcag gatggtggac gacggctctg ggaaggtgga ggtgaggggt actgggttag 11400
ctgggggaag atgggcacac ggaggtaagc tttgcctaca ggtaaacggt atgactgcta 11460
tctaaagtga cccaggtgca catccacaca caatcaggga cacaaaggga cacgtgtaca 11520
tttacatact ctcctagcag gcagttcata catagtcacg cctaatcata cacagccacg 11580
ggcactcaca caacacagac acacagtaca gtaacacttg gacacttaaa ttctccctgt 11640
agtcacatgt aggacaaaat caaacatatg cttatacaga cacacatact aacaccttgt 11700
aattgcacat aatcacagcc aagtacacaa ccacaaatat gaagctgcaa aaactctaca 11760
tcagacatga ccacggtcac acacaaccat caatccgcag agacacacca acacagacac 11820
acgatcacac atcatcaatt acagccccac acacccaacc ccaattaaca gccatacact 11880
gtcaaccact catctggtct cagcccagtg tcctgcgaga cgcaggggtt gagggctact 11940
tccaggctct gtagagggtg tggttgtatc ttgacttgtt ggctgacccc ctaggcagag 12000
ccctgaattt tttaggccct agtttttaaa taaagtgggg tcagaaactg tggtcccatc 12060
tggccccaag gctgatgtgg ggatcctgtg gtcttgtggg aacagaactg ctgggcctat 12120
gcgaggggtc ctagttctcg aggtaggtgg aggtcagaga aaaggcaaga tttccttctt 12180
cctttgccct aatatggatg gacagaagtg atgagtggga agagactgag agaggaggag 12240
atggggcaag ggggaagaag ggggagacag ggaaagagga agacagggaa ggagcacaaa 12300
gagagacaga gaagaaaaga ctgggaagga caggggatga ggaggaggca gagggaaggg 12360
aggaggaggt ggggtggagg aagagcaggg agagaggggg tacagggaga agagagggag 12420
ggtcagtgat agatgggata cacagagaga gacagagagc cagacagtgg gagagggaga 12480
caggaaagca gaaggaaagg cgagggacgg agggaaagag agccctggga ctcaggtgcc 12540
aagcacaaag tcagggacag gatccctcct ggtaggcagt ggacaggcag gcagagacag 12600
ggcctgagcc aggggtcctg gctgcccggt aattgccact gttaggtggc ctgtagtcaa 12660
gtcagccact tttgtgaaag acagggctct ggctgctctg gtctggaggt cggctcccca 12720
gggacaactg tcagggtcag tgtgggaaag gacacccagg cattgtctcc tgccatgcag 12780
aggtgaggta agggaggcgt catcatcccc aatatacagg tgggaaaggc ccgaagctta 12840
gaacaatgtc actgccaaca cagggcctac ctgggatttg accagagtcc gcctggctcc 12900
aggctctgcc acccacagga agaagaaact acactgacag atgtgagaca gtgtttcccc 12960
ttcagtcttt gaacaggctt tgtgttttct aaatgacact ggataaaagg gaattcattc 13020
aagagctcca aggcttccct ttccgcccgg cttctgttgc cctggcctga gcagcgagca 13080
gctgggaggg gactgaactg cccctaacca gggttgtggc tggtggggtt gggactaggg 13140
ctgggcatgt ggctgggatt gggcccatct cccaagtgtg gtgggtttca ggggtccgtg 13200
gggaaagggt cacctgcaga tccttgtaca aagcccagca caaggcccag aatgggcctg 13260
gagctgaggg tgggcccctg gcctgcgtgg atgaggaagg cctcagaggc tggggagagc 13320
tttccggttg ggtacaggat gggtgggctg gttcaggaag agcagccacc tgtgcccatt 13380
ggtcccttat tccccaggtg tggtgcatcc aggacttaca caggcagccc gtggacccca 13440
agcgtcatgg acagctgtgt gcaggcaact gctaccttgt gctctacaca taccagaggc 13500
tgggccgtgt ccagtacatc ctgtacctat ggcaggtgtg ccagcctgag ggaggcagca 13560
ctcaccttaa agcccaaggg ctgggctctg ggagtgaaat gtgagagctg ggccaggccc 13620
tcactcactg cccccacctg cagggccacc aggccactgc ggatgagatt gaggccctga 13680
acagcaacgc tgaggaacta gatgtcatgt atggtggcgt cctagtacag gagcatgtga 13740
ccatgggcag cgagcccccc cacttcctcg ccatcttcca gggccagctg gtgatcttcc 13800
aggtaggtct caccttgcca ctctggccac agcctgccca gttctgcatg ggccatggcc 13860
cccgcacaca cttctaagca ccttctcttt gggcctgggg cctgcttatc atcccctagt 13920
ttcccagagc ttgtccaagg cccaggagct ccaaggttgg ccccctgcct gagtttccct 13980
ccccagtccc agagccagat gaggaatttg agttgaaagg gagccagggg cttgcctggg 14040
gctgcacact agcaaacctg gtccccactt tccacctcca aggcctgagc acccatgccc 14100
caaccactca gagatggggg gaaaggtcca tttaaaaagg gtctgtccct gtcccagact 14160
gatgggattc ctgcactcca cacttagagg gtcctgtact cctcatgtga aatcaccagt 14220
gagggccttg atggcttcaa gcaagggtcc ctgagctctg aggcaatatg tcccacactg 14280
ggacaggaag gcgcccctgg gagcccttgc ccagcctgag gccttgctct cctataggag 14340
agagctgggc accatggaaa ggggcagtca gcatccacca caaggctttt ccaagtgcaa 14400
ggcactgaca gccacaacac caggaccatg gaggtgccag cccgtgcctc atccctcaac 14460
tccagtgaca tcttcttgct ggtcacagcc agcgtctgct acctctggtt tgggaaggta 14520
cccacagcac tgaccacttg attcatgccc agatgtagtg gtgccaggct gggggtgggt 14580
cctctccaca gggcatgcag actttgagtc cagcctcaga taggccgatg gggggaatgg 14640
ggaggggaag aagccacagg gtcccctcta gaacttgggg aaagttacaa agtgacaggt 14700
ggacctgaga gtccgcaggt cagagccagg cccaggccgc ctcccctcct tactgcctgg 14760
agcacagggc atgtcccttc ccaggtctct tgggaaaagg ggaggcctat gccagtttgc 14820
tggagttttc tctaatattc agcataaact atgactgacc ctctaagcct gggggcctcc 14880
tatcccatgc tctggaccct gcgagggtcc cagaccctgc gatccagatc aactggggac 14940
acaggcctgt agggtgagga agatgccttg ttcactggtg tcctgctgtc agcttaggcc 15000
tcctgtgact tgggcccagc cccaccccat acaccctgtg aacgggacgt ggggccggag 15060
gtgaaggccc ctcctcatgc agggcctgag ccatctcttt gcctgctagg gctgtaatgg 15120
tgatcagcgt gagatggcac gggtggtggt cactgtcatt tccaggaaga atgaggaaac 15180
ggtgctggag ggtcaggagc ctccccactt ctgggaggcc ctgggaggcc gggcccccta 15240
ccccagcaac aagaggtaac agggttggga ggagagtgtt cttacccaga ggaggaattg 15300
aggcccagag aggagggtgg gtgactggga ttcacacaga gagctggcaa gacgagacta 15360
gaaccaagga ctctcagttt cccactcaca gaggctccca caagtacagc ccacgcttcg 15420
ctcccaggcc tgggtaactt gcgtctctca gatgctgggg agagtggggg cagtggcgac 15480
ctctgctggc agcatcccct ccctgcattg tcctggtcct gggccagggg acaaagcacc 15540
cttccccacc ctacagtcct cagggatcac ggtgcccagt gtgtgtgtat acagtgtcat 15600
atgatgtgtg ggtatacagt gtgtgtggat gtgcaagaac aggtgtattc agggtgttgt 15660
gtgtggtgtg taagtggaca gggtgtgtgt aggaggtagg agtgaggcct caaggggagg 15720
agggagctga gtcctggtcg ctcttggtct gcctgtgggg cttggtttat gtgctgcact 15780
ccccacaatt ctcctgagct cagtactccc gtctccttct cccagcctgg cttgcctcct 15840
tccactttgg gccggagacc tactctctgc caggggtggg ctgaggagct cctcccatcc 15900
cagggagggc gagactccag ctcaggccct cccctacccc tgcagccctc tcctagggaa 15960
attcatgggg caccttgcgt cacagctggg tggggcgaga gctttctcca tgagcagaga 16020
gggaccctgt acccagagca gagcggagtg ctcggggaga aaccacgggg ctcagaggag 16080
ggcccagggc tcccactcct gagaagtggc ttcccaggcc ctggggtggg gcacttgtgc 16140
catgggctgg ggtgcccctc tgggtggctc acagccttgc tgtccgtgct ggccaggctc 16200
cctgaggagg tccccagctt ccagccacga ctgtttgagt gctccagcca catgggctgc 16260
ctggtcctcg cagaagtggg gttcttcagc caggaggacc tggacaagta tgacatcatg 16320
ttactggaca cctggcagga ggtaaggtgg ccatccctgc ctggtggggc tgtgaacggg 16380
ggtgtgtttc tgtttgtgta actgggtgtg tgtgtatcta gccatctgcc tctgtacaca 16440
gggctgtggg tgagtctgac cctgtcactg agcaattgca ttgcggtgca tatgagacca 16500
tggatgagtg tgcatcacca ggtggcagca gtgagactcc gggagattag gagtgtgttg 16560
gaactcaggg gtaactgaga ccatgtttgc agttgtgtgt ggccctgtgt gccttatgat 16620
actatgtgtg gctccagcga cagtgcctgc ggccgagcat gtgtcggtgc tgcatgagtg 16680
actgtgattg gaatgcccaa tcgcatcatt catgtgtgtg gcttggtgtg agtgtttgcc 16740
ttcgtgcacc tgtgtgtgac actttgtcag tgactgtgag gtcacgtgat gctagaactg 16800
gccagaccag tggcaactgg tgaaaatgtg tctgtgagca agactatgac tggctgtggg 16860
cgaatgatgg atgggtgtgt ttgtgggtct gtgtttctgc gcacagtggt ataactggcc 16920
gggtgtgcgt ggctgtgtaa acaagtgctg ttgtgtttgc atgcagctct gtgtctgaca 16980
acagtggatg acagtgtgtg gctatggttt gctgtctggc tgtgtggcga tggatgactg 17040
tgtgtctgtg taagctgcaa taatgtgtgt tctctgcatc tctgtggggc cgtgtctaca 17100
gcactgtttg aattggtgat gaagctgtgt gctcccatct actcagaaca aacccagagc 17160
tcttaccaca gccctacgtg acctggccag ccccaactct ttgacccaat ttccccctgc 17220
tcccccctca ctgcttctgc tgcagccaca atggcctctt tgtgttccct gaacatgccg 17280
ggcacacggc cacctcgggg cttttacacc ggttgtttct tctggttagg acacttttcc 17340
tccagacata catgtgactt gctctctcat gaaagctcta taagggcagg gatttttgtc 17400
cattttcttt cattgttgac tccccaaagc ctagaccaat gcccggcaca cagtaggtgc 17460
tcaataagta tttgtggatt gatctccagg taaatcatgc aagaaaaaaa aaatgtgtgg 17520
attgaacgtg tgtgtgcatg tatgcatgca ttcctgtgac cttctcgtgt gttcacgggt 17580
gtgtgggtac agccgcttgc tgggtgtgtc tgcttggcca gggatgtgta tgtggggttc 17640
tgtctgggac aactggacag ggagtggaca ggaaggggtc agctctgggc agcccctcca 17700
cctgcccaag gccaggtccc ctctgtggct cagatcttcc tgtggcttgg ggaagctgca 17760
agtgagtgga aggaggcggt ggcctggggc caggagtacc tgaagactca cccagcaggg 17820
aggagcccgg ccacacccat cgtgctggtc aagcagggcc atgagcctcc caccttcatt 17880
ggatggttct tcacttggga cccctacaag tggactgtga gtgaggcctg aaacccccag 17940
ccctacccta atttgtgggg agaggaacct gacctgggcc aatggaatga ggaggcactg 18000
ccctggctgg ggattgccag tgtgtgcgag agacctggtc ttgtcctcag gcccctcatg 18060
tgtcccattg gtgccccctt ctcctgccct ggccctgata cttgccccga ttcttgctcc 18120
agagccaccc gtcccacaag gaagtggtgg atggcagccc ggcagcagca tcaaccatct 18180
ctgagataac agcagtgagt cctggggccc ctagccttcc ccacagtggc ctgggctccg 18240
acagcatggt cctgatgggc aggggaagtg ccaggccctt gtaagtggga ggggctgcag 18300
ttggaggtaa gaggcctgtg ggttcagtgc agcctccctg tgggctactg attccccatc 18360
ttccccagcc tgaggctcct ctctggacag gaagtcaaca acttgcggct atccagatgg 18420
ccgggcaatg gcagggcagg tgccgtggcc ctgcaggccc tcaagggctc ccaggacagc 18480
tcagagaatg atctggtgcg aagccccaag tcggctggca gcagaaccag cagctccgtc 18540
agcagcacca gcgccacgat caacgggggc ctgcgccggg aacaactgat gcaccaggct 18600
gttgaggacc tgccagaggg cgtggaccct gcccgcaggg aggtgggcac cccctcactg 18660
ccccagcact agtgcatctg acactgagct ggaggagccc aaggcaggat ccactggtgg 18720
cgggcagtgg gcaactgccc cgggagatgt gtcttctagc tggggtggtg ggcaaacaga 18780
tgcgggagtc ccaagccctg gatgactgac actactgagt ggggcaggat tctgggctca 18840
gatgacaccc taccctgtac ctccccctct ctcccctgcc cagttctatc tctcagactc 18900
tgacttccaa gatatctttg ggaaatccaa ggaggaattc tacagcatgg ccacgtggag 18960
gcagcggcag gagaaaaagc agctgggctt cttctgaacc caagccctct cgactgcccc 19020
tatcccctgg accccaacat acctacaatg ctggggaggc cctgcttcca ctcccctcag 19080
aggcttttgg tcatcctctg cgtgtcagta aaagcaggca gcccatacga 19130
<210> 2
<211> 2704
<212> PRT
<213> human
<220>
<221> misc_feature
<222> (1748)..(1748)
<223> Xaa can be any naturally occurring amino acid
<400> 2
Met Asp Ile Ser Lys Gly Leu Pro Gly Met Gln Gly Gly Leu His Ile
1 5 10 15
Trp Ile Ser Glu Asn Arg Lys Met Val Pro Val Pro Glu Gly Ala Tyr
20 25 30
Gly Asn Phe Phe Glu Glu His Cys Tyr Val Ile Leu His Val Pro Gln
35 40 45
Ser Pro Lys Ala Thr Gln Gly Ala Ser Ser Asp Leu His Tyr Trp Val
50 55 60
Gly Lys Gln Ala Gly Ala Glu Ala Gln Gly Ala Ala Glu Ala Phe Gln
65 70 75 80
Gln Arg Leu Gln Asp Glu Leu Gly Gly Gln Thr Val Leu His Arg Glu
85 90 95
Ala Gln Gly His Glu Ser Asp Cys Phe Cys Ser Tyr Phe Arg Pro Gly
100 105 110
Ile Ile Tyr Arg Lys Gly Gly Leu Ala Ser Asp Leu Lys His Val Glu
115 120 125
Thr Asn Leu Phe Asn Ile Gln Arg Leu Leu His Ile Lys Gly Arg Lys
130 135 140
His Val Ser Ala Thr Glu Val Glu Leu Ser Trp Asn Ser Phe Asn Lys
145 150 155 160
Gly Asp Ile Phe Leu Leu Asp Leu Gly Lys Met Met Ile Gln Trp Asn
165 170 175
Gly Pro Lys Thr Ser Ile Ser Glu Lys Ala Arg Gly Leu Ala Leu Thr
180 185 190
Tyr Ser Leu Arg Asp Arg Glu Arg Gly Gly Gly Arg Ala Gln Ile Gly
195 200 205
Val Val Asp Asp Glu Ala Lys Ala Pro Asp Leu Met Gln Ile Met Glu
210 215 220
Ala Val Leu Gly Arg Arg Val Gly Ser Leu Arg Ala Ala Thr Pro Ser
225 230 235 240
Lys Asp Ile Asn Gln Leu Gln Lys Ala Asn Val Arg Leu Tyr His Val
245 250 255
Tyr Glu Lys Gly Lys Asp Leu Val Val Leu Glu Leu Ala Thr Pro Pro
260 265 270
Leu Thr Gln Asp Leu Leu Gln Glu Glu Asp Phe Tyr Ile Leu Asp Gln
275 280 285
Gly Gly Phe Lys Ile Tyr Val Trp Gln Gly Arg Met Ser Ser Leu Gln
290 295 300
Glu Arg Lys Ala Ala Phe Ser Arg Ala Val Gly Phe Ile Gln Ala Lys
305 310 315 320
Gly Tyr Pro Thr Tyr Thr Asn Val Glu Val Val Asn Asp Gly Ala Glu
325 330 335
Ser Ala Ala Phe Lys Gln Leu Phe Arg Thr Trp Ser Glu Lys Arg Arg
340 345 350
Arg Asn Gln Lys Leu Gly Gly Arg Asp Lys Ser Ile His Val Lys Leu
355 360 365
Asp Val Gly Lys Leu His Thr Gln Pro Lys Leu Ala Ala Gln Leu Arg
370 375 380
Met Val Asp Asp Gly Ser Gly Lys Val Glu Val Trp Cys Ile Gln Asp
385 390 395 400
Leu His Arg Gln Pro Val Asp Pro Lys Arg His Gly Gln Leu Cys Ala
405 410 415
Gly Asn Cys Tyr Leu Val Leu Tyr Thr Tyr Gln Arg Leu Gly Arg Val
420 425 430
Gln Tyr Ile Leu Tyr Leu Trp Gln Gly His Gln Ala Thr Ala Asp Glu
435 440 445
Ile Glu Ala Leu Asn Ser Asn Ala Glu Glu Leu Asp Val Met Tyr Gly
450 455 460
Gly Val Leu Val Gln Glu His Val Thr Met Gly Ser Glu Pro Pro His
465 470 475 480
Phe Leu Ala Ile Phe Gln Gly Gln Leu Val Ile Phe Gln Glu Arg Ala
485 490 495
Gly His His Gly Lys Gly Gln Ser Ala Ser Thr Thr Arg Leu Phe Gln
500 505 510
Val Gln Gly Thr Asp Ser His Asn Thr Arg Thr Met Glu Val Pro Ala
515 520 525
Arg Ala Ser Ser Leu Asn Ser Ser Asp Ile Phe Leu Leu Val Thr Ala
530 535 540
Ser Val Cys Tyr Leu Trp Phe Gly Lys Gly Cys Asn Gly Asp Gln Arg
545 550 555 560
Glu Met Ala Arg Val Val Val Thr Val Ile Ser Arg Lys Asn Glu Glu
565 570 575
Thr Val Leu Glu Gly Gln Glu Pro Pro His Phe Trp Glu Ala Leu Gly
580 585 590
Gly Arg Ala Pro Tyr Pro Ser Asn Lys Arg Leu Pro Glu Glu Val Pro
595 600 605
Ser Phe Gln Pro Arg Leu Phe Glu Cys Ser Ser His Met Gly Cys Leu
610 615 620
Val Leu Ala Glu Val Gly Phe Phe Ser Gln Glu Asp Leu Asp Lys Tyr
625 630 635 640
Asp Ile Met Leu Leu Asp Thr Trp Gln Glu Ile Phe Leu Trp Leu Gly
645 650 655
Glu Ala Ala Ser Glu Trp Lys Glu Ala Val Ala Trp Gly Gln Glu Tyr
660 665 670
Leu Lys Thr His Pro Ala Gly Arg Ser Pro Ala Thr Pro Ile Val Leu
675 680 685
Val Lys Gln Gly His Glu Pro Pro Thr Phe Ile Gly Trp Phe Phe Thr
690 695 700
Trp Asp Pro Tyr Lys Trp Thr Ser His Pro Ser His Lys Glu Val Val
705 710 715 720
Asp Gly Ser Pro Ala Ala Ala Ser Thr Ile Ser Glu Ile Thr Ala Glu
725 730 735
Val Asn Asn Leu Arg Leu Ser Arg Trp Pro Gly Asn Gly Arg Ala Gly
740 745 750
Ala Val Ala Leu Gln Ala Leu Lys Gly Ser Gln Asp Ser Ser Glu Asn
755 760 765
Asp Leu Val Arg Ser Pro Lys Ser Ala Gly Ser Arg Thr Ser Ser Ser
770 775 780
Val Ser Ser Thr Ser Ala Thr Ile Asn Gly Gly Leu Arg Arg Glu Gln
785 790 795 800
Leu Met His Gln Ala Val Glu Asp Leu Pro Glu Gly Val Asp Pro Ala
805 810 815
Arg Arg Glu Phe Tyr Leu Ser Asp Ser Asp Phe Gln Asp Ile Phe Gly
820 825 830
Lys Ser Lys Glu Glu Phe Tyr Ser Met Ala Thr Trp Arg Gln Arg Gln
835 840 845
Glu Lys Lys Gln Leu Gly Phe Phe Met Asp Ile Ser Lys Gly Leu Pro
850 855 860
Gly Met Gln Gly Gly Leu His Ile Trp Ile Ser Glu Asn Arg Lys Met
865 870 875 880
Val Pro Val Pro Glu Gly Ala Tyr Gly Asn Phe Phe Glu Glu His Cys
885 890 895
Tyr Val Ile Leu His Val Pro Gln Ser Pro Lys Ala Thr Gln Gly Ala
900 905 910
Ser Ser Asp Leu His Tyr Trp Val Gly Lys Gln Ala Gly Ala Glu Ala
915 920 925
Gln Gly Ala Ala Glu Ala Phe Gln Gln Arg Leu Gln Asp Glu Leu Gly
930 935 940
Gly Gln Thr Val Leu His Arg Glu Ala Gln Gly His Glu Ser Asp Cys
945 950 955 960
Phe Cys Ser Tyr Phe Arg Pro Gly Ile Ile Tyr Arg Lys Gly Gly Leu
965 970 975
Ala Ser Asp Leu Lys His Val Glu Thr Asn Met Asp Ile Ser Lys Gly
980 985 990
Leu Pro Gly Met Gln Gly Gly Leu His Ile Trp Ile Ser Glu Asn Arg
995 1000 1005
Lys Met Val Pro Val Pro Glu Gly Ala Tyr Gly Asn Phe Phe Glu
1010 1015 1020
Glu His Cys Tyr Val Ile Leu His Val Pro Gln Ser Pro Lys Ala
1025 1030 1035
Thr Gln Gly Ala Ser Ser Asp Leu His Tyr Trp Val Gly Lys Gln
1040 1045 1050
Ala Gly Ala Glu Ala Gln Gly Ala Ala Glu Ala Phe Gln Gln Arg
1055 1060 1065
Leu Gln Asp Glu Leu Gly Gly Gln Thr Val Leu His Arg Glu Ala
1070 1075 1080
Gln Gly His Glu Ser Asp Cys Phe Cys Ser Tyr Phe Arg Pro Gly
1085 1090 1095
Ile Ile Tyr Arg Lys Gly Gly Leu Ala Ser Asp Leu Lys His Val
1100 1105 1110
Glu Thr Asn Leu Phe Asn Ile Gln Arg Leu Leu His Ile Lys Gly
1115 1120 1125
Arg Lys His Val Ser Ala Thr Glu Val Glu Leu Ser Trp Asn Ser
1130 1135 1140
Phe Asn Lys Gly Asp Ile Phe Leu Leu Asp Leu Gly Lys Met Met
1145 1150 1155
Ile Gln Trp Asn Gly Pro Lys Thr Ser Ile Ser Glu Lys Ala Arg
1160 1165 1170
Met Ala Leu Gly Thr Ser Ser Pro Ser Pro Pro Ala Pro Pro Ser
1175 1180 1185
Ser Pro Gln His Ile Tyr Thr Asn Thr Arg Thr Leu Gly Pro Pro
1190 1195 1200
Glu Pro Glu Gly His Ala Gly Gly Gly Tyr Pro Thr Tyr Thr Asn
1205 1210 1215
Val Glu Val Val Asn Asp Gly Ala Glu Ser Ala Ala Phe Lys Gln
1220 1225 1230
Leu Phe Arg Thr Trp Ser Glu Lys Arg Arg Arg Asn Gln Lys Leu
1235 1240 1245
Gly Gly Arg Asp Lys Ser Ile His Val Lys Leu Asp Val Gly Lys
1250 1255 1260
Leu His Thr Gln Pro Lys Leu Ala Ala Gln Leu Arg Met Val Asp
1265 1270 1275
Asp Gly Ser Gly Lys Val Glu Val Trp Cys Ile Gln Asp Leu His
1280 1285 1290
Arg Gln Pro Val Asp Pro Lys Arg His Gly Gln Leu Cys Ala Gly
1295 1300 1305
Asn Cys Tyr Leu Val Leu Tyr Thr Tyr Gln Arg Leu Gly Arg Val
1310 1315 1320
Gln Tyr Ile Leu Tyr Leu Trp Gln Gly His Gln Ala Thr Ala Asp
1325 1330 1335
Glu Ile Glu Ala Leu Asn Ser Asn Ala Glu Glu Leu Asp Val Met
1340 1345 1350
Tyr Gly Gly Val Leu Val Gln Glu His Val Thr Met Gly Ser Glu
1355 1360 1365
Pro Pro His Phe Leu Ala Ile Phe Gln Gly Gln Leu Val Ile Phe
1370 1375 1380
Gln Glu Arg Ala Gly His His Gly Lys Gly Gln Ser Ala Ser Thr
1385 1390 1395
Thr Arg Leu Phe Gln Val Gln Gly Thr Asp Ser His Asn Thr Arg
1400 1405 1410
Thr Met Glu Val Pro Ala Arg Ala Ser Ser Leu Asn Ser Ser Asp
1415 1420 1425
Ile Phe Leu Leu Val Thr Ala Ser Val Cys Tyr Leu Trp Phe Gly
1430 1435 1440
Lys Gly Cys Asn Gly Asp Gln Arg Glu Met Ala Arg Val Val Val
1445 1450 1455
Thr Val Ile Ser Arg Lys Asn Glu Glu Thr Val Leu Glu Gly Gln
1460 1465 1470
Glu Pro Pro His Phe Trp Glu Ala Leu Gly Gly Arg Ala Pro Tyr
1475 1480 1485
Pro Ser Asn Lys Arg Leu Pro Glu Glu Val Pro Ser Phe Gln Pro
1490 1495 1500
Arg Leu Phe Glu Cys Ser Ser His Met Gly Cys Leu Val Leu Ala
1505 1510 1515
Glu Val Gly Phe Phe Ser Gln Glu Asp Leu Asp Lys Tyr Asp Ile
1520 1525 1530
Met Leu Leu Asp Thr Trp Gln Glu Ile Phe Leu Trp Leu Gly Glu
1535 1540 1545
Ala Ala Ser Glu Trp Lys Glu Ala Val Ala Trp Gly Gln Glu Tyr
1550 1555 1560
Leu Lys Thr His Pro Ala Gly Arg Ser Pro Ala Thr Pro Ile Val
1565 1570 1575
Leu Val Lys Gln Gly His Glu Pro Pro Thr Phe Ile Gly Trp Phe
1580 1585 1590
Phe Thr Trp Asp Pro Tyr Lys Trp Thr Ser His Pro Ser His Lys
1595 1600 1605
Glu Val Val Asp Gly Ser Pro Ala Ala Ala Ser Thr Ile Ser Glu
1610 1615 1620
Ile Thr Ala Glu Val Asn Asn Leu Arg Leu Ser Arg Trp Pro Gly
1625 1630 1635
Asn Gly Arg Ala Gly Ala Val Ala Leu Gln Ala Leu Lys Gly Ser
1640 1645 1650
Gln Asp Ser Ser Glu Asn Asp Leu Val Arg Ser Pro Lys Ser Ala
1655 1660 1665
Gly Ser Arg Thr Ser Ser Ser Val Ser Ser Thr Ser Ala Thr Ile
1670 1675 1680
Asn Gly Gly Leu Arg Arg Glu Gln Leu Met His Gln Ala Val Glu
1685 1690 1695
Asp Leu Pro Glu Gly Val Asp Pro Ala Arg Arg Glu Phe Tyr Leu
1700 1705 1710
Ser Asp Ser Asp Phe Gln Asp Ile Phe Gly Lys Ser Lys Glu Glu
1715 1720 1725
Phe Tyr Ser Met Ala Thr Trp Arg Gln Arg Gln Glu Lys Lys Gln
1730 1735 1740
Leu Gly Phe Phe Xaa Met Ser Ser Leu Gln Glu Arg Lys Ala Ala
1745 1750 1755
Phe Ser Arg Ala Val Gly Phe Ile Gln Ala Lys Gly Tyr Pro Thr
1760 1765 1770
Tyr Thr Asn Val Glu Val Val Asn Asp Gly Ala Glu Ser Ala Ala
1775 1780 1785
Phe Lys Gln Leu Phe Arg Thr Trp Ser Glu Lys Arg Arg Arg Asn
1790 1795 1800
Gln Lys Leu Gly Gly Arg Asp Lys Ser Ile His Val Lys Leu Asp
1805 1810 1815
Val Gly Lys Leu His Thr Gln Pro Lys Leu Ala Ala Gln Leu Arg
1820 1825 1830
Met Val Asp Asp Gly Ser Gly Lys Val Glu Gly Leu Pro Gly Ile
1835 1840 1845
Met Asp Ile Ser Lys Gly Leu Pro Gly Met Gln Gly Gly Leu His
1850 1855 1860
Ile Trp Ile Ser Glu Asn Arg Lys Met Val Pro Val Pro Glu Gly
1865 1870 1875
Ala Tyr Gly Asn Phe Phe Glu Glu His Cys Tyr Val Ile Leu His
1880 1885 1890
Val Pro Gln Ser Pro Lys Ala Thr Gln Gly Ala Ser Ser Asp Leu
1895 1900 1905
His Tyr Trp Val Gly Lys Gln Ala Gly Ala Glu Ala Gln Gly Ala
1910 1915 1920
Ala Glu Ala Phe Gln Gln Arg Leu Gln Asp Glu Leu Gly Gly Gln
1925 1930 1935
Thr Val Leu His Arg Glu Ala Gln Gly His Glu Ser Asp Cys Phe
1940 1945 1950
Cys Ser Tyr Phe Arg Pro Gly Ile Ile Tyr Arg Lys Gly Gly Leu
1955 1960 1965
Ala Ser Asp Leu Lys His Val Glu Thr Asn Leu Phe Asn Ile Gln
1970 1975 1980
Arg Leu Leu His Ile Lys Gly Arg Lys His Val Ser Ala Thr Glu
1985 1990 1995
Val Glu Leu Ser Trp Asn Ser Phe Asn Lys Gly Asp Ile Phe Leu
2000 2005 2010
Leu Asp Leu Gly Lys Met Met Ile Gln Trp Asn Gly Pro Lys Thr
2015 2020 2025
Ser Ile Ser Glu Lys Ala Arg Gly Leu Ala Leu Thr Tyr Ser Leu
2030 2035 2040
Arg Asp Arg Glu Arg Gly Gly Gly Arg Ala Gln Ile Gly Val Val
2045 2050 2055
Asp Asp Glu Ala Lys Ala Pro Asp Leu Met Gln Ile Met Glu Ala
2060 2065 2070
Val Leu Gly Arg Arg Val Gly Ser Leu Arg Ala Ala Thr Pro Ser
2075 2080 2085
Lys Asp Ile Asn Gln Leu Gln Lys Ala Asn Val Arg Leu Tyr His
2090 2095 2100
Val Tyr Glu Lys Gly Lys Asp Leu Val Val Leu Glu Leu Ala Thr
2105 2110 2115
Pro Pro Leu Thr Gln Asp Leu Leu Gln Glu Glu Asp Phe Tyr Ile
2120 2125 2130
Leu Asp Gln Gly Gly Phe Lys Ile Tyr Val Trp Gln Gly Arg Met
2135 2140 2145
Ser Ser Leu Gln Glu Arg Lys Ala Ala Phe Ser Arg Ala Val Gly
2150 2155 2160
Phe Ile Gln Ala Lys Gly Tyr Pro Thr Tyr Thr Asn Val Glu Val
2165 2170 2175
Val Asn Asp Gly Ala Glu Ser Ala Ala Phe Lys Gln Leu Phe Arg
2180 2185 2190
Thr Trp Ser Glu Lys Arg Arg Arg Asn Gln Lys Leu Gly Gly Arg
2195 2200 2205
Asp Lys Ser Ile His Val Lys Leu Asp Val Gly Lys Leu His Thr
2210 2215 2220
Gln Pro Lys Leu Ala Ala Gln Leu Arg Met Val Asp Asp Gly Ser
2225 2230 2235
Gly Lys Val Glu Val Trp Cys Ile Gln Asp Leu His Arg Gln Pro
2240 2245 2250
Val Asp Pro Lys Arg His Gly Gln Leu Cys Ala Gly Asn Cys Tyr
2255 2260 2265
Leu Val Leu Tyr Thr Tyr Gln Arg Leu Gly Arg Val Gln Tyr Ile
2270 2275 2280
Leu Tyr Leu Trp Gln Gly His Gln Ala Thr Ala Asp Glu Ile Glu
2285 2290 2295
Ala Leu Asn Ser Asn Ala Glu Glu Leu Asp Val Met Tyr Gly Gly
2300 2305 2310
Val Leu Val Gln Glu His Val Thr Met Gly Ser Glu Pro Pro His
2315 2320 2325
Phe Leu Ala Ile Phe Gln Gly Gln Leu Val Ile Phe Gln Glu Arg
2330 2335 2340
Ala Gly His His Gly Lys Gly Gln Ser Ala Ser Thr Thr Arg Leu
2345 2350 2355
Phe Gln Val Gln Gly Thr Asp Ser His Asn Thr Arg Thr Met Glu
2360 2365 2370
Val Pro Ala Arg Ala Ser Ser Leu Asn Ser Ser Asp Ile Phe Leu
2375 2380 2385
Leu Val Thr Ala Ser Val Cys Tyr Leu Trp Phe Gly Lys Gly Cys
2390 2395 2400
Asn Gly Asp Gln Arg Glu Met Ala Arg Val Val Val Thr Val Ile
2405 2410 2415
Ser Arg Lys Asn Glu Glu Thr Val Leu Glu Gly Gln Glu Pro Pro
2420 2425 2430
His Phe Trp Glu Ala Leu Gly Gly Arg Ala Pro Tyr Pro Ser Asn
2435 2440 2445
Lys Arg Leu Pro Glu Glu Val Pro Ser Phe Gln Pro Arg Leu Phe
2450 2455 2460
Glu Cys Ser Ser His Met Gly Cys Leu Val Leu Ala Glu Val Gly
2465 2470 2475
Phe Phe Ser Gln Glu Asp Leu Asp Lys Tyr Asp Ile Met Leu Leu
2480 2485 2490
Asp Thr Trp Gln Glu Ile Phe Leu Trp Leu Gly Glu Ala Ala Ser
2495 2500 2505
Glu Trp Lys Glu Ala Val Ala Trp Gly Gln Glu Tyr Leu Lys Thr
2510 2515 2520
His Pro Ala Gly Arg Ser Pro Ala Thr Pro Ile Val Leu Val Lys
2525 2530 2535
Gln Gly His Glu Pro Pro Thr Phe Ile Gly Trp Phe Phe Thr Trp
2540 2545 2550
Asp Pro Tyr Lys Trp Thr Ser His Pro Ser His Lys Glu Val Val
2555 2560 2565
Asp Gly Ser Pro Ala Ala Ala Ser Thr Ile Ser Glu Ile Thr Ala
2570 2575 2580
Glu Val Asn Asn Leu Arg Leu Ser Arg Trp Pro Gly Asn Gly Arg
2585 2590 2595
Ala Gly Ala Val Ala Leu Gln Ala Leu Lys Gly Ser Gln Asp Ser
2600 2605 2610
Ser Glu Asn Asp Leu Val Arg Ser Pro Lys Ser Ala Gly Ser Arg
2615 2620 2625
Thr Ser Ser Ser Val Ser Ser Thr Ser Ala Thr Ile Asn Gly Gly
2630 2635 2640
Leu Arg Arg Glu Gln Leu Met His Gln Ala Val Glu Asp Leu Pro
2645 2650 2655
Glu Gly Val Asp Pro Ala Arg Arg Glu Phe Tyr Leu Ser Asp Ser
2660 2665 2670
Asp Phe Gln Asp Ile Phe Gly Lys Ser Lys Glu Glu Phe Tyr Ser
2675 2680 2685
Met Ala Thr Trp Arg Gln Arg Gln Glu Lys Lys Gln Leu Gly Phe
2690 2695 2700
Phe
<210> 3
<211> 19
<212> DNA
<213> 人工合成
<400> 3
agggacgcat gtctagcct 19
<210> 4
<211> 20
<212> DNA
<213> 人工合成
<400> 4
acgttggtgt aggtcgggta 20

Claims (4)

1.检测肿瘤组织中VILL基因表达水平的试剂在制备用于辅助诊断携带EBV病毒的鼻咽癌试剂中的应用，其特征在于，所述VILL基因的核苷酸序列如SEQ ID NO：1所示；与健康参照相比，所述VILL基因表达水平下调。
2.如权利要求1所述的应用，其特征在于，所述检测肿瘤组织中VILL基因表达水平的试剂选自：
特异性扩增所述VILL基因的引物；或
特异性识别所述VILL基因的探针；或
特异性结合所述VILL基因编码的蛋白的抗体。
3.如权利要求2所述的应用，其特征在于，所述引物序列如SEQ ID NO：3～4所示。
4.如下任一项所述的应用
(1)过表达如权利要求1所述VILL基因的试剂在制备抑制携带EBV病毒的鼻咽癌进展的药物组合物中的应用；
(2)过表达如权利要求1所述VILL基因的试剂在制备抑制携带EBV病毒的鼻咽癌迁移的药物组合物中的应用。
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