CN113702635A - Birc6作为三阴性乳腺癌诊断和治疗靶标 - Google Patents

Abstract

本发明涉及一种可用作三阴性乳腺癌诊断靶标和治疗靶标的凋亡抑制蛋白(BIRC6)。特别地，本发明公开了BIRC6在三阴性乳腺癌细胞系及组织中均显著高表达，该过表达能够促进三阴性乳腺癌细胞增殖，并且与三阴性乳腺癌病人短的预后生存时间有关。降低BIRC6表达能够触发凋亡并导致三阴性乳腺癌细胞生长抑制，因此抑制BIRC6表达代表了三阴性乳腺癌治疗的一个新策略。

Description

BIRC6作为三阴性乳腺癌诊断和治疗靶标

技术领域

本发明属于生物医药技术领域，具体涉及BIRC6作为三阴性乳腺癌诊断和治疗靶标。

背景技术

三阴性乳腺癌(Triple-negative breast cancer,TNBC)特指ER、PgR表达缺失以及HER2过表达或基因扩增缺失的乳腺癌类型，占全部乳腺癌的12％-17％。与其他亚型相比，TNBC生物学上更具侵袭性，患者的早期远处复发率更高，5年预后更差。尽管PARP抑制剂，PD-L1抗体atezolizumab目前已被分别批准用于BRCA突变、PD-L1表达阳性的TNBC患者，但针对TNBC的靶向治疗尚处于早期，化疗仍是标准治疗方法。TNBC患者尽管对化疗具有一定的反应率，但是仍有60％-70％的病人对化疗不敏感。因此，急需鉴定更多可用于TNBC治疗的分子靶点并研发相应的靶向治疗手段。

BIRC6又称Apollon或BRUCE，是凋亡抑制蛋白(IAP)家族中分子量极大(接近530kDa)和研究较少的成员。BIRC6在其N端和C端分别包含BIR和UBC结构域，使其同时具有泛素结合活性与凋亡抑制活性。由于UBC结构域的存在，BIRC6具有E2/E3泛素连接酶的双重功能，即其仅需要E1就能够泛素化下游底物。研究表明，BIRC6会导致促凋亡蛋白SMAC、caspase-9、HtrA2和p53的泛素化和蛋白酶体降解。

大量证据表明BIRC6在多种癌症类型中高表达，包括新生儿急性髓系白血病、前列腺癌、非小细胞肺癌、卵巢癌、肝细胞癌、食道鳞状细胞癌和结直肠癌。然而，BIRC6在三阴性乳腺癌发生发展过程中的表达模式和生物学功能尚不清楚。更重要的是，BIRC6在癌症中过表达的分子机制也知之甚少。

发明内容

有鉴于此，本发明涉及一种可用作三阴性乳腺癌诊断靶标和治疗靶标的凋亡抑制蛋白BIRC6，该过表达能够促进三阴性乳腺癌细胞增殖，并且与三阴性乳腺癌病人短的预后生存时间有关，因此可以作为三阴性乳腺癌标志物及诊断标记。降低BIRC6表达能够触发凋亡并导致三阴性乳腺癌细胞生长抑制，因此抑制BIRC6表达代表了三阴性乳腺癌治疗的一个新策略。本发明还对BIRC6的上游调控机制进行了研究，发现EGF-JNK通路能够增加BIRC6的蛋白稳定性。

根据本发明的一个方面，提供了检测BIRC6蛋白表达水平的试剂在制备三阴性乳腺癌的诊断剂或诊断试剂盒中的应用。

在一个实施方案中，本发明提供了诊断三阴性乳腺癌的方法，所述方法包括检测受试者的样品中BIRC6的蛋白表达水平，其中相对于对照(健康或者非三阴性乳腺癌样品)，BIRC6在受试者的样品中的过表达指示受试者患有三阴性乳腺癌的(高)风险。

在一个实施方案中，本发明提供了检测BIRC6蛋白表达水平的试剂，其用于诊断三阴性乳腺癌。

在一个实施方案中，所述试剂为BIRC6特异性抗体。

在另一个优选实施方案中，所述BIRC6的蛋白序列如SEQ ID NO.1(UniProtKB/Swiss-Prot登录号Q9NR09)所示。

根据本发明的另一个方面，提供了降低或抑制BIRC6表达的试剂在制备治疗三阴性乳腺癌的药物中的应用。

在一个实施方案中，本发明提供降低或抑制BIRC6表达的试剂，其用作药物，特别是用作治疗三阴性乳腺癌的药物。

在另一个实施方案中，本发明提供治疗三阴性乳腺癌的方法，所述方法包括向有需要的受试者施用有效量的降低或抑制BIRC6表达的试剂。

降低或抑制BIRC6表达的试剂的本质对于本发明来说并不重要，只要它降低或抑制BIRC6的表达即可。

根据一个优选实验方案，降低或抑制BIRC6表达的试剂选自由以下组成的组：gapmer、反义RNA、siRNA、esiRNA、shRNA、miRNA、RNA适体、TALEN、CRISPR和锌指核酸酶。在特别优选的实施方案中，用于shRNA和CRISPR的具体序列是本申请说明书实施例中使用的那些序列。

根据一个优选实验方案，所述BIRC6的核苷酸序列如SEQ ID NO.2所示(Ensembl登录号ENSG00000115760)。

在另一个实施方案中，所述药物还包含另外的抗癌剂例如化疗药，例如用于降低或抑制BIRC6表达的试剂或抑制BIRC6与JNK结合的试剂。或者所述药物与用于降低或抑制BIRC6表达的方法或者抑制BIRC6与JNK的结合的方法组合使用。即使BIRC6的抑制足以实现治疗三阴性乳腺癌的效果，但是预期将降低或抑制BIRC6表达的试剂与其他抗癌药物如化疗剂组合时，可以获得更强甚至协同的抗癌效果。

根据本发明的另一个方面，提供了一种筛选用于治疗三阴性乳腺癌的候选药物的方法，所述办法包括下述步骤：

1)确定过表达BIRC6的癌细胞中BIRC6的表达水平；

2)将候选化合物与步骤1)中确定的癌细胞接触；

3)确定步骤2)得到的癌细胞中BIRC6的表达水平；

4)比较步骤1)和步骤3)中确定的BIRC6的表达水平，其中BIRC6的表达水平降低指示所述候选化合物具有抗癌潜力。

与现有技术相比，本发明首次证实了一个新的能够降低或拟制BIRC6表达的试剂的存在，并且通过检测三阴性乳腺癌患者使用该试剂的基因表达情况，发现该试剂能够使BIRC6的表达情况明显降低，从而为肿瘤精准诊断提供新的思路，同时也为降低三阴性乳腺癌的复发率奠定了坚实的基础。

附图说明

从下面结合附图的详细描述中，本发明的上述特征和特点将更明显，其中：

图1中A至D所示的结果显示BIRC6在三阴性乳腺癌细胞系及组织中高表达；其中：

图1A所示为通过免疫印迹实验检测BIRC6在细胞系中的蛋白表达水平并进行定量分析；

图1B显示BIRC6在三阴性乳腺癌病人的肿瘤样本中典型的免疫组化染色案例。病例J07A2470代表BIRC6阳性的病人，病例J07A2313代表BIRC6阴性的病人；

图1C为BIRC6在90例三阴性乳腺癌肿瘤样本中的表达情况；

图1D为基于BIRC6表达绘制的三阴性乳腺癌患者的Kaplan-Meier生存曲线；

图2为BIRC6促进三阴性乳腺癌的发生，其中：

图2A显示三条不同shRNA在MDA-MB-468和Hs578T细胞中对BIRC6都具有很好的敲低效率；

图2B所示为通过MTT实验检测细胞的增殖速率；

图2C显示通过克隆形成实验检测细胞的克隆形成能力；

图2D显示在Hs578T细胞中对BIRC6进行CRISPR-Cas9敲除，并在BIRC6敲除的细胞克隆中重新表达编码BIRC6编码区的质粒；

图2E显示通过MTT实验检测细胞的增殖速率；

图2F显示通过克隆形成实验检测细胞的克隆形成能力；

图2G显示通过流式细胞术检测细胞的凋亡情况；

图2H显示MDA-MB-468和Hs578T细胞的皮下移植瘤的肿瘤生长曲线；

图2I显示通过免疫组化检测肿瘤组织中凋亡标志物(cleaved CASP3)的表达水平；

图3中所示的结果显示EGF-JNK通路能够调控BIRC6的蛋白稳定性，其中：

图3A显示MDA-MB-468和Hs578T细胞血清饥饿48h后再用EGF刺激30min，随后通过免疫印迹检测BIRC6的蛋白表达；

图3B显示用JNK抑制剂SP600125处理Hs578T细胞1.5h后再用EGF刺激30min；随后通过免疫印迹检测BIRC6的蛋白表达；

图3C所示为通过免疫共沉淀实验检测BIRC6和JNK的相互作用；

图3D显示用EGF处理Hs578T细胞再用MG132(10μM)处理6h，随后实施免疫共沉淀检测BIRC6的泛素化水平；

图3E显示用SP600125处理Hs578T细胞再用环己胺(CHX,100μg/ml)处理指定时间，随后通过免疫印迹检测BIRC6的蛋白表达；

图3F显示用EGF处理Hs578T细胞再用CHX处理指定时间，随后通过免疫印迹检测BIRC6的蛋白表达。

具体实施方式

为了使本发明的目的、技术方案及优点更加清楚明白，以下结合具体实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。

本发明中所述的BIRC6是蛋白编码基因，其蛋白序列可以由SEQ ID NO:1表示，其核酸序列可以由SEQ ID NO:2表示。

术语“降低或抑制BIRC6表达”是指将BIRC6的表达水平降低至原来的表达水平的80％以下、70％以下、60％以下、50％以下、40％以下、30％以下、20％以下、15％以下或10％以下，例如5％以下、2％以下、1％以下或甚至0％。在一个实施方案中，通过基因敲除或敲减可以降低或抑制BIRC6的表达。

基因敲除或敲减的技术是本领域公知的，包括但不限于，逆转录病毒基因转移，造成突变，例如点突变、插入、删除、移码、或错义突变。可以将基因敲除的另一方式是，使用锌指核酸酶。锌指核酸酶(ZFN)是通过将锌指DNA结合结构域与DNA切割结构域融合而产生的人工限制性酶。可以对锌指结构域进行改造以靶向目的DNA序列，这可使锌指核酸酶靶向复杂基因组中的独特序列。其它可用来敲除基因的基因组定制技术有TAL效应物核酸酶(TALENs)。另一种技术是基因组编辑技术CRISPR/Cas9系统，其可以被用来实现RNA引导的基因组改造。

实现“降低或抑制BIRC6表达”的技术还可以包括使用gapmer、反义RNA、siRNA、esiRNA、shRNA、miRNA或RNA适体。

“反义RNA”是指与mRNA互补的RNA分子，也包括与其它RNA互补的RNA分子。由于核糖体不能翻译双链的RNA，所以反义RNA与mRNA特异性的互补结合，即抑制了该mRNA的翻译。例如可以作为表达质粒来递送反义构建体，其中当所述表达质粒在细胞中表达时，产生与细胞中BIRC6的至少一个独特部分互补的RNA。

反义RNA策略的另一特殊形式是gapmer。Gapmer是嵌合的反义寡核苷酸，其包含长度足以诱导RNase H切割的脱氧核苷酸单体的中心区段(central block)。Gapmer的设计和合成是本领域技术人员公知的并可以由商业公司完成。

“小干扰RNA(siRNA)”，有时称为短干扰RNA或沉默RNA，是一类双链RNA分子，长度约为20-25个碱基对，其通过RNA干扰(RNAi)途径发挥作用。它干扰了表达与互补的核苷酸序列的特定基因的转录后降解的mRNA，从而防止翻译。用于选择siRNA的靶序列的技术在本领域公知的。

“短发夹RNA”(short hairpin RNA，缩写shRNA)是包括两个短反向重复序列的RNA序列，可以经由RNA干扰(RNAi)使基因表现沉默化。

“esiRNA”的英文全名是Endoribonuclease-prepared siRNAs，是通过大肠杆菌的RNase III(一种核糖核酸酶)切割长双链RNA(dsRNA)而生成的siRNA混合物，长度在18-25bp之间，可以用于高效敲除靶标基因的表达水平。

本发明是基于以下发现：即BIRC6可用作三阴性乳腺癌的生物标志物及治疗靶点。因此，本发明提供了检测BIRC6蛋白表达水平的试剂在制备三阴性乳腺癌的诊断剂或诊断试剂盒中的应用。本发明还提供了降低或抑制BIRC6表达的试剂在制备治疗三阴性乳腺癌的药物中的应用。

另外，本发明还提供了一种筛选抗三阴性乳腺癌药物的方法，所述方法包括确定候选化合物是否能够降低或抑制BIRC6表达的步骤。

本发明在以下的实施例中进一步说明。这些实施例只是为了举例说明的目的，而不是用来限制本发明的范围。以下反应中所用的化学物质都是可商购的产品，除非另外指明。

在本发明中使用未配对的学生t检验作为统计分析。使用Microsoft Excel进行统计学计算。当P<0.05时，P值显著；**表示P<0.01。

实施例1

本发明实施例1提供了BIRC6在三阴性乳腺癌细胞系和组织中的表达水平研究

选取8种三阴性乳腺癌细胞系，7种非三阴性乳腺癌细胞系及2种正常的乳腺上皮组织细胞系，所有细胞系源自ATCC。将上述细胞用RIPA裂解液裂解获得蛋白上清，蛋白上清通过BCA法定量后与上样缓冲液混匀，随后通过SDS-PAGE凝胶(分离胶浓度6％)电泳分离蛋白。将凝胶上的蛋白转移到PVDF膜上，膜用5％脱脂牛奶在室温封闭1小时，然后用识别BIRC6的抗体(Abcam,ab19609)在摇床上4℃孵育过夜。第二天，用TBST缓冲液清洗3次，每次10分钟；接着，用相应的HRP标记的二抗在摇床上室温孵育1小时。孵育后，再次用TBST缓冲液清洗3次，每次10分钟；清洗完毕后，用ECL显影液检测化学发光信号并进行灰度定量分析。

结果发现，与正常乳腺上皮细胞系和非TNBC细胞系相比，BIRC6的表达在TNBC细胞系中趋于升高(如图1A所示)。

进一步，选取包含90例三阴性乳腺癌肿瘤组织样本的组织芯片(上海芯超)。将组织芯片与识别BIRC6(Abcam，ab19609)的一抗孵育，随后与生物素偶联的二抗和链霉亲和素-HRP孵育；通过DAB检测免疫反应信号，苏木精复染后对载玻片成像。

结果发现，BIRC6在TNBC病人的肿瘤样本中高表达，其比例高达66/90例(73.3％，图1B-C)。

此外，KM-plotter数据库的分析结果表明，相对BIRC6低表达的人群，BIRC6高表达的三阴性乳腺癌患者的无复发生存率明显更差(P＝0.0074，图1D)。

实施例2

本发明实施例2提供了BIRC6的细胞水平研究

选取Hs578T和MDA-MB-468两种TNBC细胞系，利用病毒包装的shRNA构建BIRC6敲低的稳筛细胞株(shRNA序列如下所示)。

首先进行包装病毒，将293T细胞(ATCC)接种于100mm平皿，第二天进行转染，在灭菌水中加入第三代慢病毒包装载体pMDLg/pRRE、pRSV-Rev和pMD2.G(等摩尔比混合，总量9μg)和shRNA表达载体(总量9μg)，再加入61μl 2M CaCl₂，使三者总体积达到500μl，混匀；将等体积2×HBS盐溶液逐滴加入，同时轻弹管壁，使每滴加入后及时混匀；静置2min后，立即将这1ml的磷酸钙-DNA悬液逐滴加入上述单层细胞的细胞培养基中，轻轻摇动平皿混匀。8h后吸去培养基与DNA沉淀，24h和48h后收集含有病毒的上清，并通过PEG8000浓缩病毒。

具体步骤如下：首先两次病毒上清在4℃、2000pm条件下离心5min去除细胞等杂物，再经低蛋白吸附的0.45μm滤膜过滤；其次过滤后的上清中添加50％PEG8000至终浓度为10％，混匀后再添加5M NaC1至终浓度为0.15M，混匀后至于4℃过夜；最后在4℃、2000g条件下离心10min，从而得到浓缩后的病毒沉淀，病毒沉淀用培养基重悬后分装放置-80℃备用。对于构建BIRC6敲低的稳筛细胞株，首先将细胞接种于100mm培养皿中，第二天在10ml培养基中加入病毒液和10μl聚凝胺(10mg/ml)，48h后加入嘌呤霉素筛选去除没有表达病毒的细胞，三天加一次嘌呤霉素，如此就得到所需的稳转细胞系。通过免疫印迹检测BIRC6敲低水平，敲低情况见图2A。

结果发现BIRC6敲低能够显著抑制细胞的增殖和克隆形成(如图2B-C所示)，而且，BIRC6敲低能够明显触发细胞凋亡(如图2G所示)。

此外，我们还通过CRISPR-Cas9技术进行敲除BIRC6。将表达Cas9、sgRNA和嘌呤霉素的质粒使用脂质体Lipofectamine3000瞬时转染Hs578T细胞。转染后加入嘌呤霉素筛选3天去除没有表达质粒的细胞，将单细胞悬液以0.5个细胞/100μl的密度接种到96孔板中(培养基中不含嘌呤霉素)；免疫印迹分析筛选出BIRC6敲除的细胞克隆(如图2D所示)，并通过测序进一步证实。

结果发现，类似BIRC6敲低，BIRC6敲除也能明显抑制细胞的增殖和克隆形成。

sgBIRC6 Sense 5'-3'GGGACCATCAAAGTCATCGA(SEQ ID NO:11)

sgCtrl Antisense 5'-3'GGGCGAGGAGCTGTTCACCG(SEQ ID NO:12)

进一步，我们将表达BIRC6编码区和新霉素的质粒pcDNA3-myc-BIRC6使用脂质体Lipofectamine3000转染上述BIRC6敲除的细胞中。转染后加入G418筛选去除没有表达质粒的细胞，将单细胞悬液以0.5个细胞/100μl的密度接种到96孔板中(培养基中不含G418)；免疫印迹分析筛选出重新表达BIRC6的细胞克隆(如图2E所示)。

结果发现，BIRC6的重新表达有效地挽救了BIRC6敲除诱导的生存抑制作用，能够促进三阴性乳腺癌细胞的增殖和克隆形成(图2E-F)。

实施例3

本发明实施例3提供了BIRC6的动物水平研究

我们研究BIRC6对三阴性乳腺癌细胞成瘤能力的影响。将BIRC6敲低的MDA-MB-468细胞系注射到无胸腺裸鼠(BALB/c裸鼠，雌性，上海斯莱克)的皮下，类似的，将BIRC6敲低的Hs578T细胞系注射到NOD/SCID小鼠(雌性，集萃药康)的皮下，待肿瘤大小肉眼可见时，每隔4-5天用游标卡尺测量肿瘤的长度和宽度。肿瘤大小计算公式为：V＝0.5×L×W²(V：体积，L：长度，W：宽度)。

结果发现，BIRC6敲低的MDA-MB-468细胞几乎不能形成肿瘤，BIRC6敲低的Hs578T细胞形成的肿瘤块明显小于对照组(如图2H所示)，而且免疫组化显示BIRC6敲低能够在体内触发凋亡(如图2I所示)。

总之，通过细胞增殖、克隆形成以及皮下成瘤等多种指标进行详细的实验验证，我们认为BIRC6具有明显的抑制三阴性乳腺癌发生发展的功能，是一个潜在的治疗靶点。

实施例4

本发明实施例4提供了BIRC6的上游调控机制研究

选取Hs578T和MDA-MB-468两种TNBC细胞系，血清饥饿48h后用EGF处理30min并检测BIRC6的蛋白表达，结果显示EGF刺激能在较短的时间内上调BIRC6的蛋白表达(如图3A所示)，揭示EGF是在翻译后而不是转录或翻译过程中调控BIRC6表达。Hs578T细胞被血清饥饿48h后用激酶JNK抑制剂SP600125(10μM)处理1.5小时，并用EGF处理30min，免疫印迹显示SP600125处理后JNK磷酸化和BIRC6蛋白表达的上调作用都被阻断(如图3B所示)，揭示JNK通路是EGF调控BIRC6蛋白表达的关键通路。用识别BIRC6的抗体对Hs578T细胞裂解液实施免疫共沉淀，结果显示JNK可以与BIRC6相互结合(如图3C所示)。此外，用识别BIRC6的抗体对EGF处理的Hs578T细胞裂解液实施免疫共沉淀，结果显示EGF处理激活JNK的同时显著降低了BIRC6的泛素化水平(如图3D所示)。随后，用SP600125处理Hs578T细胞后再用蛋白合成抑制剂CHX(100μg/ml)处理指定时间，结果显示SP600125降低了BIRC6蛋白的稳定性(如图3E所示)。

用EGF处理血清饥饿后的Hs578T细胞，并用CHX(100μg/ml)处理指定时间，结果显示EGF激活JNK的同时增加了BIRC6蛋白的稳定性(如图3F所示)。综上所述，这些结果表明，EGF-JNK信号通路有助于BIRC6的蛋白稳定。

以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应该以权利要求的保护范围为准。

序列表

<110> 深圳市罗湖区人民医院

<120> BIRC6作为三阴性乳腺癌诊断和治疗靶标

<130> 20210720

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4857

<212> PRT

<213> 浜虹被(Homo sapiens)

<400> 1

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Leu Pro Ser Val Ile Val Leu Ser Ala Gly Arg Lys Met Ala Ala Ala

20 25 30

Ala Ala Ala Ala Ser Gly Pro Gly Cys Ser Ser Ala Ala Gly Ala Gly

35 40 45

Ala Ala Gly Val Ser Glu Trp Leu Val Leu Arg Asp Gly Cys Met His

50 55 60

Cys Asp Ala Asp Gly Leu His Ser Leu Ser Tyr His Pro Ala Leu Asn

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

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

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Gly Gln Val Lys Cys Gln Tyr Ile Ser Ala Val Asp Lys Val Ile Phe

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Val Asp Asp Tyr Ala Val Gly Cys Arg Lys Asp Leu Asn Gly Ile Leu

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

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

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Cys Leu Glu Lys Val Asp Ile Ser Ser Thr Glu Gly Tyr Asp Leu Phe

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Arg Arg Glu Thr Phe Thr Ser Trp Pro His Val Gly Tyr Arg Trp Ala

290 295 300

Gln Pro Asp Pro Met Ala Gln Ala Gly Phe Tyr His Gln Pro Ala Ser

305 310 315 320

Ser Gly Asp Asp Arg Ala Met Cys Phe Thr Cys Ser Val Cys Leu Val

325 330 335

Cys Trp Glu Pro Thr Asp Glu Pro Trp Ser Glu His Glu Arg His Ser

340 345 350

Pro Asn Cys Pro Phe Val Lys Gly Glu His Thr Gln Asn Val Pro Leu

355 360 365

Ser Val Thr Leu Ala Thr Ser Pro Ala Gln Phe Pro Cys Thr Asp Gly

370 375 380

Thr Asp Arg Ile Ser Cys Phe Gly Ser Gly Ser Cys Pro His Phe Leu

385 390 395 400

Ala Ala Ala Thr Lys Arg Gly Lys Ile Cys Ile Trp Asp Val Ser Lys

405 410 415

Leu Met Lys Val His Leu Lys Phe Glu Ile Asn Ala Tyr Asp Pro Ala

420 425 430

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

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

450 455 460

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

465 470 475 480

Asp Ser Glu Glu His Ser Arg Ser Asp Ser Val Thr Gly His Thr Ser

485 490 495

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

500 505 510

Leu Gln Gln Pro Glu Lys Leu Gln Trp Glu Ile Val Ala Asn Val Leu

515 520 525

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

530 535 540

Thr Asn Ser Lys Ser Glu Lys Thr Lys Glu Lys His Gln Glu Gln His

545 550 555 560

Asn Ile Pro Phe Pro Cys Leu Leu Ala Gly Gly Leu Leu Thr Tyr Lys

565 570 575

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

580 585 590

Gly Leu Ser Arg Thr Gln Gly Glu Ser Ile Ser Glu Gln Gly Ser Thr

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

Leu His Asp Asp Gly Phe Thr Val Pro Gln Ile Ile Glu Met Glu Leu

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

Arg Leu Pro Lys Phe Ala Glu Glu Glu Asn Leu Cys Ile Asp Ser Ile

725 730 735

Thr Pro Cys Ala Asp Gly Ile His Leu Leu Val Gly Leu Arg Thr Cys

740 745 750

Pro Val Glu Ser Leu Ser Ala Ile Asn Gln Val Glu Ala Leu Asn Asn

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Leu Asn Lys Leu Asn Ser Ala Leu Cys Asn Arg Arg Lys Gly Glu Leu

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Glu Ser Asn Leu Ala Val Val Asn Gly Ala Asn Ile Ser Val Ile Gln

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

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Gln Arg Asn Val Ser Gly Gly Tyr Leu Val Leu Tyr Lys Met Asn Tyr

820 825 830

Ala Thr Arg Ile Val Thr Leu Glu Glu Glu Pro Ile Lys Ile Gln His

835 840 845

Ile Lys Asp Pro Gln Asp Thr Ile Thr Ser Leu Ile Leu Leu Pro Pro

850 855 860

Asp Ile Leu Asp Asn Arg Glu Asp Asp Cys Glu Glu Pro Ile Glu Asp

865 870 875 880

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

885 890 895

Ile Ser Thr Leu Gly His Leu Val Ile Thr Thr Gln Gly Gly Tyr Val

900 905 910

Lys Ile Leu Asp Leu Ser Asn Phe Glu Ile Leu Ala Lys Val Glu Pro

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Pro Lys Lys Glu Gly Thr Glu Glu Gln Asp Thr Phe Val Ser Val Ile

930 935 940

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

945 950 955 960

Leu His Phe Leu Gln Ile Gly Gly Thr Cys Asp Asp Ile Asp Glu Ala

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

Thr Val Pro Pro Cys Trp Val Glu Val Gln Gln Glu Gln Gln Gln Arg

1045 1050 1055

Arg His Pro Gln His Leu His Gln Gln His His Gly Asp Ala Ala Gln

1060 1065 1070

His Thr Arg Thr Trp Lys Leu Gln Thr Asp Ser Asn Ser Trp Asp Glu

1075 1080 1085

His Val Phe Glu Leu Val Leu Pro Lys Ala Cys Met Val Gly His Val

1090 1095 1100

Asp Phe Lys Phe Val Leu Asn Ser Asn Ile Thr Asn Ile Pro Gln Ile

1105 1110 1115 1120

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

1125 1130 1135

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

1140 1145 1150

Leu Asn Glu Glu Met Gln His Met Asp Val Glu Glu Ser Gln Cys Leu

1155 1160 1165

Arg Leu Cys Pro Phe Leu Glu Asp His Lys Glu Asp Ile Leu Cys Gly

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Pro Val Trp Leu Ala Ser Gly Leu Asp Leu Ser Gly His Ala Gly Met

1185 1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

Glu Glu Ile Cys Asn Gly Gly Met Arg Pro Val Val Arg Leu Pro Ser

1235 1240 1245

Leu Lys His Gln Ser Asn Lys Gly Tyr Ser Leu Ala Ser Leu Leu Ala

1250 1255 1260

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

1265 1270 1275 1280

Thr Ser Gly Thr Arg Lys Ser Glu Asn Leu Arg Gly Cys Asp Leu Leu

1285 1290 1295

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

1300 1305 1310

Lys Glu Arg Val Gln Arg Cys Ala Met Leu Gln Phe Ser Glu Phe His

1315 1320 1325

Glu Lys Leu Val Asn Thr Leu Cys Arg Lys Thr Asp Asp Gly Gln Ile

1330 1335 1340

Thr Glu His Ala Gln Ser Leu Val Leu Asp Thr Leu Cys Trp Leu Ala

1345 1350 1355 1360

Gly Val His Ser Asn Gly Pro Gly Ser Ser Lys Glu Gly Asn Glu Asn

1365 1370 1375

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

1380 1385 1390

Phe Phe Glu Ala Gly Arg Ser Ile Ala His Lys Cys Ala Arg Phe Leu

1395 1400 1405

Ala Leu Cys Ile Ser Asn Gly Lys Cys Asp Pro Cys Gln Pro Ala Phe

1410 1415 1420

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

1425 1430 1435 1440

Ala Ala Thr Thr Gly Gly Ser Val Tyr Trp Tyr Phe Val Leu Leu Asn

1445 1450 1455

Tyr Val Lys Asp Glu Asp Leu Ala Gly Cys Ser Thr Ala Cys Ala Ser

1460 1465 1470

Leu Leu Thr Ala Val Ser Arg Gln Leu Gln Asp Arg Leu Thr Pro Met

1475 1480 1485

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

1490 1495 1500

Pro Val Leu Phe Asp Leu Glu Met Ser Gly Ser Ser Cys Lys Asn Val

1505 1510 1515 1520

Tyr Asn Ser Ser Ile Gly Val Gln Ser Asp Glu Ile Asp Leu Ser Asp

1525 1530 1535

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

1540 1545 1550

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

1555 1560 1565

Thr Cys Val Ser Thr Ser Asp Gly Thr Arg Ile Glu Arg Asp Asp Ala

1570 1575 1580

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

1585 1590 1595 1600

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

1605 1610 1615

Leu Gln Ser Leu Ser His Ala Met Ala Ser Ala Glu Gln Gln Leu Gln

1620 1625 1630

Val Leu Gln Glu Lys Gln Gln Gln Leu Leu Lys Leu Gln Gln Gln Lys

1635 1640 1645

Ala Lys Leu Glu Ala Lys Leu His Gln Thr Thr Ala Ala Ala Ala Ala

1650 1655 1660

Ala Ala Ser Ala Val Gly Pro Val His Asn Ser Val Pro Ser Asn Pro

1665 1670 1675 1680

Val Ala Ala Pro Gly Phe Phe Ile His Pro Ser Asp Val Ile Pro Pro

1685 1690 1695

Thr Pro Lys Thr Thr Pro Leu Phe Met Thr Pro Pro Leu Thr Pro Pro

1700 1705 1710

Asn Glu Ala Val Ser Val Val Ile Asn Ala Glu Leu Ala Gln Leu Phe

1715 1720 1725

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

1730 1735 1740

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

1745 1750 1755 1760

Leu Ala Ile Ser His Ala Ser His Phe Leu Gln Pro Pro Pro His Gln

1765 1770 1775

Ser Ile Ile Ile Glu Arg Met His Ser Gly Ala Arg Arg Phe Val Thr

1780 1785 1790

Leu Asp Phe Gly Arg Pro Ile Leu Leu Thr Asp Val Leu Ile Pro Thr

1795 1800 1805

Cys Gly Asp Leu Ala Ser Leu Ser Ile Asp Ile Trp Thr Leu Gly Glu

1810 1815 1820

Glu Val Asp Gly Arg Arg Leu Val Val Ala Thr Asp Ile Ser Thr His

1825 1830 1835 1840

Ser Leu Ile Leu His Asp Leu Ile Pro Pro Pro Val Cys Arg Phe Met

1845 1850 1855

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

1860 1865 1870

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

1875 1880 1885

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

1890 1895 1900

Ala Asn Gln Pro Glu Ile Asp Gln His Leu Ala Met Met Val Ala Leu

1905 1910 1915 1920

Gln Glu Asp Ile Gln Cys Arg Tyr Asn Leu Ala Cys His Arg Leu Glu

1925 1930 1935

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

1940 1945 1950

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

1955 1960 1965

Arg Val Phe Ser Ala Tyr Gln Asp Cys Ile Gln Leu Gln Leu Gln Leu

1970 1975 1980

Asn Leu Ala His Asn Ala Val Gln Arg Leu Lys Val Ala Leu Gly Ala

1985 1990 1995 2000

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

2005 2010 2015

Thr Ser Ser Thr Glu Gln Leu Arg Thr Ile Ile Arg Tyr Leu Leu Asp

2020 2025 2030

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

2035 2040 2045

Val Leu Gln Ser Thr Phe His Ala Gln Ala Cys Glu Glu Leu Phe Lys

2050 2055 2060

His Leu Cys Ile Ser Gly Thr Pro Lys Ile Arg Leu His Thr Gly Leu

2065 2070 2075 2080

Leu Leu Val Gln Leu Cys Gly Gly Glu Arg Trp Trp Gly Gln Phe Leu

2085 2090 2095

Ser Asn Val Leu Gln Glu Leu Tyr Asn Ser Glu Gln Leu Leu Ile Phe

2100 2105 2110

Pro Gln Asp Arg Val Phe Met Leu Leu Ser Cys Ile Gly Gln Arg Ser

2115 2120 2125

Leu Ser Asn Ser Gly Val Leu Glu Ser Leu Leu Asn Leu Leu Asp Asn

2130 2135 2140

Leu Leu Ser Pro Leu Gln Pro Gln Leu Pro Met His Arg Arg Thr Glu

2145 2150 2155 2160

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

2165 2170 2175

Arg Leu Leu Asp Tyr Val Ala Thr Val Glu Asp Glu Ala Ala Ala Ala

2180 2185 2190

Lys Lys Pro Leu Asn Gly Asn Gln Trp Ser Phe Ile Asn Asn Asn Leu

2195 2200 2205

His Thr Gln Ser Leu Asn Arg Ser Ser Lys Gly Ser Ser Ser Leu Asp

2210 2215 2220

Arg Leu Tyr Ser Arg Lys Ile Arg Lys Gln Leu Val His His Lys Gln

2225 2230 2235 2240

Gln Leu Asn Leu Leu Lys Ala Lys Gln Lys Ala Leu Val Glu Gln Met

2245 2250 2255

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

2260 2265 2270

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

2275 2280 2285

Leu Ile Arg Leu Arg Arg Thr Ala Glu Trp Ser Arg Ser Asn Leu Asp

2290 2295 2300

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

2305 2310 2315 2320

Phe Thr Leu Ala His Glu Arg Cys Ile Ser Val Val Gln Lys Leu Val

2325 2330 2335

Leu Phe Leu Leu Ser Met Asp Phe Thr Cys His Ala Asp Leu Leu Leu

2340 2345 2350

Phe Val Cys Lys Val Leu Ala Arg Ile Ala Asn Ala Thr Arg Pro Thr

2355 2360 2365

Ile His Leu Cys Glu Ile Val Asn Glu Pro Gln Leu Glu Arg Leu Leu

2370 2375 2380

Leu Leu Leu Val Gly Thr Asp Phe Asn Arg Gly Asp Ile Ser Trp Gly

2385 2390 2395 2400

Gly Ala Trp Ala Gln Tyr Ser Leu Thr Cys Met Leu Gln Asp Ile Leu

2405 2410 2415

Ala Gly Glu Leu Leu Ala Pro Val Ala Ala Glu Ala Met Glu Glu Gly

2420 2425 2430

Thr Val Gly Asp Asp Val Gly Ala Thr Ala Gly Asp Ser Asp Asp Ser

2435 2440 2445

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

2450 2455 2460

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

2465 2470 2475 2480

Lys Glu Ile Asp Leu Glu Leu Leu Gln Asp Leu Met Glu Val Asp Ile

2485 2490 2495

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

2500 2505 2510

Phe Lys Pro Ile Ser Ser Thr Trp Tyr Asp Tyr Trp Gly Ala Asp Tyr

2515 2520 2525

Gly Thr Tyr Asn Tyr Asn Pro Tyr Ile Gly Gly Leu Gly Ile Pro Val

2530 2535 2540

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

2545 2550 2555 2560

Val Ser Val Ser Gln Ala Leu Asp Ala Arg Leu Glu Val Gly Leu Glu

2565 2570 2575

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

2580 2585 2590

Ser Ile Leu Gln Ala Leu Thr Asn Thr Ser Pro Thr Leu Ser Gln Ser

2595 2600 2605

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

2610 2615 2620

Gln Thr Ser Gln Leu Ile Ile Gln Leu Ser Ser Val Pro Met Leu Asn

2625 2630 2635 2640

Val Cys Phe Asn Lys Leu Phe Ser Met Leu Gln Val His His Val Gln

2645 2650 2655

Leu Glu Ser Leu Leu Gln Leu Trp Leu Thr Leu Ser Leu Asn Ser Ser

2660 2665 2670

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

2675 2680 2685

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

2690 2695 2700

Leu Thr Val Leu Ala Trp Tyr Pro Asn Thr Leu Leu Arg Thr Trp Cys

2705 2710 2715 2720

Leu Val Leu His Ser Leu Thr Leu Met Thr Asn Met Gln Leu Asn Ser

2725 2730 2735

Gly Ser Ser Ser Ala Ile Gly Thr Gln Glu Ser Thr Ala His Leu Leu

2740 2745 2750

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

2755 2760 2765

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

2770 2775 2780

Ala Thr Gln Ala Met Gln Glu Phe Leu Thr Arg Leu Gln Val His Leu

2785 2790 2795 2800

Ser Ser Thr Cys Pro Gln Ile Phe Ser Glu Phe Leu Leu Lys Leu Ile

2805 2810 2815

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

2820 2825 2830

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

2835 2840 2845

Glu Val Val Ser Val Ser Thr Ile Ser Ala Val Ile Glu Ser Val Thr

2850 2855 2860

Phe Leu Val His His Tyr Ile Thr Cys Ser Asp Lys Val Met Ser Arg

2865 2870 2875 2880

Ser Gly Ser Asp Ser Ser Val Gly Ala Arg Ala Cys Phe Gly Gly Leu

2885 2890 2895

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

2900 2905 2910

Met Thr Arg Asp Gln Leu Met Phe Asp Leu Leu Lys Leu Val Asn Ile

2915 2920 2925

Leu Val Gln Leu Pro Leu Ser Gly Asn Arg Glu Tyr Ser Ala Arg Val

2930 2935 2940

Ser Val Thr Thr Asn Thr Thr Asp Ser Val Ser Asp Glu Glu Lys Val

2945 2950 2955 2960

Ser Gly Gly Lys Asp Gly Asn Gly Ser Ser Thr Ser Val Gln Gly Ser

2965 2970 2975

Pro Ala Tyr Val Ala Asp Leu Val Leu Ala Asn Gln Gln Ile Met Ser

2980 2985 2990

Gln Ile Leu Ser Ala Leu Gly Leu Cys Asn Ser Ser Ala Met Ala Met

2995 3000 3005

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

3010 3015 3020

Gly Gly Leu Asp Ala Ile Ser Val Gly Asp Gly Leu Phe Thr Ile Leu

3025 3030 3035 3040

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

3045 3050 3055

Ile Leu Thr Tyr Met Ala Cys Gly Tyr Met Gly Arg Gln Gly Ser Leu

3060 3065 3070

Ala Thr Cys Gln Leu Ser Glu Pro Leu Leu Trp Phe Ile Leu Arg Val

3075 3080 3085

Leu Asp Thr Ser Asp Ala Leu Lys Ala Phe His Asp Met Gly Gly Val

3090 3095 3100

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

3105 3110 3115 3120

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

3125 3130 3135

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

3140 3145 3150

Glu Pro Asp Asn Ala Glu Gly Ile His Asn Phe Ala Pro Leu Gly Thr

3155 3160 3165

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

3170 3175 3180

Ala Thr Pro Pro His Arg Arg Ala Arg Ser Ala Ala Trp Ser Tyr Ile

3185 3190 3195 3200

Phe Leu Pro Glu Glu Ala Trp Cys Asp Leu Thr Ile His Leu Pro Ala

3205 3210 3215

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

3220 3225 3230

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

3235 3240 3245

Asn Met Leu Pro Leu Ser Thr Pro Val Val Thr Ser Gly Leu Thr Tyr

3250 3255 3260

Ile Lys Ile Gln Leu Val Lys Ala Glu Val Ala Ser Ala Val Cys Leu

3265 3270 3275 3280

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

3285 3290 3295

Lys Leu Leu Gly Leu Thr Ala Phe Gly Thr Thr Ser Ser Ala Thr Val

3300 3305 3310

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

3315 3320 3325

Gly Trp Leu Arg Leu Leu His His Cys Leu Thr His Ile Ser Asp Leu

3330 3335 3340

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

3345 3350 3355 3360

Thr Cys Ala Ala Leu Leu Met Ser Pro Tyr Cys Gly Met His Ser Pro

3365 3370 3375

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

3380 3385 3390

Gly Leu Lys Leu Ile Asp Ile Leu Leu Arg Asn Cys Ala Ala Ser Gly

3395 3400 3405

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

3410 3415 3420

Gly Leu Ser Ser Asp Ser Thr Ile Asp Ile Leu Tyr Gln Leu Gly Thr

3425 3430 3435 3440

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

3445 3450 3455

Val Ser Asp Ser Ala Arg Val Ala Ala Met Lys Arg Ser Gly Arg Met

3460 3465 3470

Asn Tyr Met Cys Pro Asn Ser Ser Thr Val Glu Tyr Gly Leu Leu Met

3475 3480 3485

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

3490 3495 3500

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

3505 3510 3515 3520

Leu Phe Glu Leu Leu Phe Asn Trp Ser Met Ser Leu Pro Cys Asn Met

3525 3530 3535

Val Leu Lys Lys Ala Val Asp Ser Leu Leu Cys Ser Met Cys His Val

3540 3545 3550

His Pro Asn Tyr Phe Ser Leu Leu Met Gly Trp Met Gly Ile Thr Pro

3555 3560 3565

Pro Pro Val Gln Cys His His Arg Leu Ser Met Thr Asp Asp Ser Lys

3570 3575 3580

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

3585 3590 3595 3600

Ala Pro Leu Ala Leu Thr Glu Ser His Leu Ala Thr Leu Ala Ser Ser

3605 3610 3615

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

3620 3625 3630

Ser Leu Leu Val Arg Ser Leu Ala Ser Phe Cys Phe Ser His Ile Ser

3635 3640 3645

Ser Ser Glu Ser Ile Ala Gln Ser Ile Asp Ile Ser Gln Asp Lys Leu

3650 3655 3660

Arg Arg His His Val Pro Gln Gln Cys Asn Lys Met Pro Ile Thr Ala

3665 3670 3675 3680

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

3685 3690 3695

His Ile Met Lys Asp Trp Leu Gly Gly Ser Glu Val Asn Pro Leu Trp

3700 3705 3710

Thr Ala Leu Leu Phe Leu Leu Cys His Ser Gly Ser Thr Ser Gly Ser

3715 3720 3725

His Asn Leu Gly Ala Gln Gln Thr Ser Ala Arg Ser Ala Ser Leu Ser

3730 3735 3740

Ser Ala Ala Thr Thr Gly Leu Thr Thr Gln Gln Arg Thr Ala Ile Glu

3745 3750 3755 3760

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

3765 3770 3775

Asn Gln Lys Leu Met Ala Gln Val Leu Cys Glu Leu Phe Gln Thr Ser

3780 3785 3790

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

3795 3800 3805

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

3810 3815 3820

Phe Leu Gln Ser Pro Cys Pro Leu Tyr Lys Gly Arg Ile Asn Ala Thr

3825 3830 3835 3840

Ser His Val Ile Gln His Pro Met Tyr Gly Ala Gly His Lys Phe Arg

3845 3850 3855

Thr Leu His Leu Pro Val Ser Thr Thr Leu Ser Asp Val Leu Asp Arg

3860 3865 3870

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

3875 3880 3885

Asp Asp Lys Glu Lys Lys Asn His Glu Glu Lys Glu Lys Val Lys Ala

3890 3895 3900

Glu Asn Gly Phe Gln Asp Asn Tyr Ser Val Val Val Ala Ser Gly Leu

3905 3910 3915 3920

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

3925 3930 3935

Ser Arg Arg Gly Arg Thr Ile Pro Asp Lys Ile Gly Ser Thr Ser Gly

3940 3945 3950

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

3955 3960 3965

His Lys Leu Leu Ala Gly Gln Pro Leu Pro Ala Glu Met Thr Leu Ala

3970 3975 3980

Gln Leu Leu Thr Leu Leu Tyr Asp Arg Lys Leu Pro Gln Gly Tyr Arg

3985 3990 3995 4000

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

4005 4010 4015

Ser Leu Ser Lys Thr Asp Ser Tyr Lys Arg Leu His Pro Glu Lys Asp

4020 4025 4030

His Gly Asp Leu Leu Ala Ser Cys Pro Glu Asp Glu Ala Leu Thr Pro

4035 4040 4045

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

4050 4055 4060

Cys Pro Ile Gln Ser Pro Leu Gln Val Phe Ala Gly Met Gly Gly Leu

4065 4070 4075 4080

Ala Leu Ile Ala Glu Arg Leu Pro Met Leu Tyr Pro Glu Val Ile Gln

4085 4090 4095

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

4100 4105 4110

Asp Ser Asp Gln Phe Glu Trp Val Thr Ile Glu Gln Ser Gly Glu Leu

4115 4120 4125

Val Tyr Glu Ala Pro Glu Thr Val Ala Ala Glu Pro Pro Pro Ile Lys

4130 4135 4140

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

4145 4150 4155 4160

Phe Gly Leu Phe Leu Arg Leu Pro Gly Tyr Ala Glu Val Leu Leu Lys

4165 4170 4175

Glu Arg Lys His Ala Gln Cys Leu Leu Arg Leu Val Leu Gly Val Thr

4180 4185 4190

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

4195 4200 4205

Leu Pro Thr Leu Pro Phe His Val Leu Arg Ser Leu Phe Ser Thr Thr

4210 4215 4220

Pro Leu Thr Thr Asp Asp Gly Val Leu Leu Arg Arg Met Ala Leu Glu

4225 4230 4235 4240

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

4245 4250 4255

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

4260 4265 4270

Val Ser Ser Ser His Asn Pro Thr Ser Thr Glu Glu Gln Gln Leu Tyr

4275 4280 4285

Trp Ala Lys Gly Thr Gly Phe Gly Thr Gly Ser Thr Ala Ser Gly Trp

4290 4295 4300

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

4305 4310 4315 4320

Thr Cys Leu Leu Gln Val Leu Ala Ser Tyr Ile Asn Pro Val Ser Ser

4325 4330 4335

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

4340 4345 4350

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

4355 4360 4365

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

4370 4375 4380

Met Ala Arg His Val Pro Leu Tyr Arg Ala Leu Leu Glu Leu Leu Arg

4385 4390 4395 4400

Ala Ile Ala Ser Cys Ala Ala Met Val Pro Leu Leu Leu Pro Leu Ser

4405 4410 4415

Thr Glu Asn Gly Glu Glu Glu Glu Glu Gln Ser Glu Cys Gln Thr Ser

4420 4425 4430

Val Gly Thr Leu Leu Ala Lys Met Lys Thr Cys Val Asp Thr Tyr Thr

4435 4440 4445

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

4450 4455 4460

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

4465 4470 4475 4480

Ile Gln Lys Thr Ala Glu Ile Val Tyr Ala Ala Thr Thr Ser Leu Arg

4485 4490 4495

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

4500 4505 4510

Met Lys Pro Lys Pro Leu Ser Val Leu Lys Ser Leu Glu Glu Lys Tyr

4515 4520 4525

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

4530 4535 4540

Glu Asp Glu Asp Gly Lys Leu Gly Phe Lys Val Asn Tyr His Tyr Met

4545 4550 4555 4560

Ser Gln Val Lys Asn Ala Asn Asp Ala Asn Ser Ala Ala Arg Ala Arg

4565 4570 4575

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

4580 4585 4590

Ser Ser Ser Ser Val Phe Val Arg Cys Asp Glu Glu Arg Leu Asp Ile

4595 4600 4605

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

4610 4615 4620

Cys Phe Glu Phe Asp Val Tyr Phe Pro Gln Asp Tyr Pro Ser Ser Pro

4625 4630 4635 4640

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

4645 4650 4655

Pro Asn Leu Tyr Asn Asp Gly Lys Val Cys Leu Ser Ile Leu Asn Thr

4660 4665 4670

Trp His Gly Arg Pro Glu Glu Lys Trp Asn Pro Gln Thr Ser Ser Phe

4675 4680 4685

Leu Gln Val Leu Val Ser Val Gln Ser Leu Ile Leu Val Ala Glu Pro

4690 4695 4700

Tyr Phe Asn Glu Pro Gly Tyr Glu Arg Ser Arg Gly Thr Pro Ser Gly

4705 4710 4715 4720

Thr Gln Ser Ser Arg Glu Tyr Asp Gly Asn Ile Arg Gln Ala Thr Val

4725 4730 4735

Lys Trp Ala Met Leu Glu Gln Ile Arg Asn Pro Ser Pro Cys Phe Lys

4740 4745 4750

Glu Val Ile His Lys His Phe Tyr Leu Lys Arg Val Glu Ile Met Ala

4755 4760 4765

Gln Cys Glu Glu Trp Ile Ala Asp Ile Gln Gln Tyr Ser Ser Asp Lys

4770 4775 4780

Arg Val Gly Arg Thr Met Ser His His Ala Ala Ala Leu Lys Arg His

4785 4790 4795 4800

Thr Ala Gln Leu Arg Glu Glu Leu Leu Lys Leu Pro Cys Pro Glu Gly

4805 4810 4815

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

4820 4825 4830

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

4835 4840 4845

Lys Glu Leu Pro Ser Asp Phe Gln Leu

4850 4855

<210> 2

<211> 14574

<212> DNA

<213> 浜虹被(Homo sapiens)

<400> 2

atggtgactg gtggtggtgc tgcacctccc gggactgtca ctgagccgct tcccagtgtg 60

attgtgctga gcgcaggccg gaagatggcg gctgcggctg cggcggcctc gggccccggc 120

tgctcctcgg cggcgggggc gggggcggcc ggggtctcag agtggctggt gctgcgggac 180

ggctgcatgc actgcgacgc cgacgggctg cacagcctgt cctaccaccc tgcgctcaac 240

gccatcctgg ccgtcactag ccgcgggacc atcaaagtca tcgacggcac ctcgggggcc 300

acactgcagg cctccgcgct cagtgctaaa ccaggtggac aggtgaaatg tcagtatatc 360

tctgctgtgg ataaagttat atttgtggat gattatgcag tagggtgtag gaaggacctt 420

aatggaatct tgttgttaga cactgctctg caaactccag tttcaaagca ggatgatgtg 480

gttcagcttg aattacccgt tacagaggca cagcagctct tatcagcatg tttagaaaag 540

gtagatattt ctagtacaga gggttatgat ttgttcatca cacagctcaa agatggttta 600

aaaaatacat ctcatgagac tgcagcaaac cacaaagttg ctaagtgggc cacagttaca 660

tttcatcttc ctcatcatgt gttgaagtcc attgccagtg ccattgtaaa tgaactcaag 720

aaaataaatc aaaatgttgc tgccttacct gtggcgtcct cagtgatgga cagattgtct 780

tacctcttac ctagtgcacg tccagaactc ggagtggggc caggccgttc tgtagacaga 840

tcactgatgt atagtgaagc taacagacgg gagacattta cctcatggcc tcatgtaggc 900

tataggtggg cacaaccaga tcccatggct caagctggat tttatcatca gcctgcctca 960

tctggagatg atagagccat gtgttttact tgtagtgtat gcctcgtttg ttgggaacct 1020

actgatgaac cttggtctga acacgaaaga cattccccaa actgcccatt tgtgaaaggt 1080

gagcacacac agaatgtgcc attgtcagtc actcttgcaa caagtcctgc acagtttcct 1140

tgtacggatg gaactgacag aatatcttgc tttgggtcgg ggagctgccc tcattttcta 1200

gctgctgcaa ctaaacgagg aaagatctgc atatgggatg tttccaaact tatgaaggtg 1260

cacttaaagt ttgaaattaa tgcctatgat ccagcaattg tacaacagct tattctatca 1320

ggagacccaa gctcaggagt tgattcaagg agaccaactt tggcgtggct ggaggactcc 1380

tctagttgct cagatatacc aaaattggaa ggagatagtg atgatttact ggaggattca 1440

gacagtgaag agcattccag atcagattct gtgacagggc atacatcaca gaaggaagcc 1500

atggaagtaa gccttgatat aacagcactc agcattctcc aacagccaga aaaacttcag 1560

tgggagattg ttgcaaatgt gcttgaagat actgttaagg atcttgaaga acttggggca 1620

aatccttgtt taacaaactc taagagtgaa aagacaaagg aaaagcacca ggagcaacac 1680

aacattcctt ttccatgttt attagctgga ggtttattaa catataaatc tcctgctacc 1740

tcacccatta gtagtaattc tcacaggtca ctggatggtt taagcagaac tcagggtgaa 1800

agtatatcag aacaagggtc aactgacaat gaatcctgca ctaattcaga actaaattct 1860

cctctggtaa ggaggacttt accggttttg cttctttata gcatcaagga atctgatgag 1920

aaagcaggaa agatcttttc acagatgaac aatattatga gtaaaagttt gcatgatgat 1980

ggttttactg ttccacagat tattgaaatg gagctggata gtcaggagca gttgttattg 2040

caggatcctc ctgtgactta cattcagcaa tttgcagatg cagcagccaa ccttacctct 2100

ccggattctg agaagtggaa ctctgtgttt cccaagcctg ggactttggt tcagtgcttg 2160

aggctgccaa agtttgcaga ggaggagaat ctttgtatag actcaataac tccttgtgct 2220

gacggaattc atttgttggt aggactgcgg acatgccctg ttgaatcctt gagtgcaata 2280

aatcaagtag aggccttgaa taatttaaat aaattaaact ctgcactatg taatagacgg 2340

aaaggtgagc tggaatcaaa tcttgctgta gtgaatggtg caaatattag tgtaatccaa 2400

catgaatcac cagcagatgt acagactcct ttaataattc agcctgagca gaggaatgtt 2460

agtggtggat atttagtgct ttataaaatg aattatgcca ctcggatagt gactttagaa 2520

gaggagccaa taaaaataca acatatcaaa gatccccagg acacaattac ctcgctcatt 2580

ttgcttccac ccgatatatt ggataatcga gaggatgact gtgaggaacc tattgaggac 2640

atgcagttaa cctcaaagaa tggttttgag agagaaaaaa cgtctgacat ttctactctt 2700

ggacacctgg taataaccac tcagggagga tatgtaaaaa tactagatct ttcaaacttt 2760

gaaattttgg ccaaagtgga gcctcccaaa aaggagggca ctgaggaaca ggacacattt 2820

gtttctgtga tttactgttc tggcacagac aggctgtgtg catgcaccaa aggtggtgag 2880

cttcattttc tccaaattgg aggaacctgt gatgatattg atgaagctga tatactagtg 2940

gatggatctc tttctaaagg aatagaacca tcttcagaag gttccaaacc tttatcaaat 3000

ccttcaagtc ctggcatttc aggagttgat ttattggtgg accagccatt cacccttgaa 3060

atcttgacat ccctagtgga gctaacccgc tttgagactt tgactccaag gttttcagcg 3120

actgttcctc catgctgggt agaagttcaa caagaacagc agcaaaggag gcatcctcaa 3180

catttgcatc agcaacacca tggtgatgct gctcagcata ctcgaacttg gaaactacag 3240

accgacagca acagctggga tgaacatgta tttgaattag tactacctaa agcttgtatg 3300

gttggacatg tggacttcaa attcgttttg aactcaaaca tcaccaatat tccacagata 3360

caagtgacac tgctgaaaaa taaagctcca ggattaggga aagtcaatgc tcttaacatt 3420

gaagtggaac aaaatgggaa accgtccctg gttgatttga atgaagaaat gcagcacatg 3480

gatgtagagg aatcacagtg tcttagatta tgtccatttt tggaggatca taaagaagac 3540

attctatgtg ggccagtatg gcttgctagt ggccttgatc tatcagggca tgctggaatg 3600

ttgacgttaa caagccccaa acttgttaaa ggtatggcag gaggaaaata tcgttcgttt 3660

ttaatccatg tcaaggcagt gaatgaaaga ggaacagaag agatttgtaa tggtggtatg 3720

cgtcctgtag taaggcttcc atccctaaaa caccagagta acaagggtta ttcacttgct 3780

tcacttttgg ctaaagttgc agcaggcaag gaaaaatcat ctaatgttaa gaatgaaaat 3840

acaagtggca cccgtaaatc tgaaaacctc cggggctgtg atttacttca agaggtctca 3900

gtcaccattc gaagatttaa gaaaacctca atttctaagg aaagagtgca acgatgtgcc 3960

atgttacagt tttcagaatt tcatgagaag cttcttaata ctctttgcag aaaaacagat 4020

gatggccaga tcacagaaca tgcccagagc cttgtgttgg atactctctg ttggttagct 4080

ggagttcatt caaatggacc cggaagctca aaggaaggaa atgagaacct actttcaaaa 4140

acacgaaaat ttctgtcaga catcgtacgt gtttgcttct ttgaggcagg acgaagtata 4200

gcccataagt gtgcccgatt tctagccttg tgcattagta atggcaaatg tgacccatgt 4260

caaccagcat ttggacctgt tctgttgaag gctttacttg ataatatgtc atttttacct 4320

gcagcaacaa ctggtggttc tgtctattgg tattttgtct tactgaatta tgtgaaagat 4380

gaagatctgg ctggatgcag tacagcttgt gcatctttgc ttactgcagt gtccagacag 4440

ttacaggaca ggctaacacc aatggaggct ttacttcaga caagatatgg attatatagc 4500

tcaccatttg atccagtcct ctttgatttg gagatgagtg gctcttcttg taaaaatgtt 4560

tataacagca gcattggtgt ccagtcagat gaaattgatt tatcagatgt cctttcagga 4620

aatggaaagg tcagtagttg cacagctgct gagggtagtt tcacatctct cactggactt 4680

ttggaagttg aacctctgca ctttacttgt gtgtcaacta gtgatggaac cagaatagaa 4740

agggatgatg caatgagttc cttcggggtt actcctgcag taggtggact atcatctggg 4800

acagttgggg aagcctcgac agccctgagt tcagcagccc aggtagcttt gcagtctctc 4860

tctcatgcaa tggcttcagc cgagcaacag ctacaggtgc tgcaagagaa acagcagcag 4920

cttttgaagc ttcagcaaca gaaagcaaag ctggaagcca agttacatca gacaacagct 4980

gcagcagctg cagcagcatc agcagtaggt cctgttcaca actctgtgcc ttccaaccca 5040

gtggctgccc ctggattctt cattcatcca tctgatgtta ttccacccac tccaaaaaca 5100

acacctcttt ttatgactcc accactcact ccacccaatg aagcagtttc cgttgtgatt 5160

aatgccgaac ttgcacagct tttcccaggc tcagtcattg atcccccagc agtcaatctt 5220

gctgcacata acaaaaattc caacaagtcc agaatgaatc cacttggttc tggtctagcc 5280

cttgcaattt ctcatgcttc acattttctt caacctccgc ctcaccagtc cattattata 5340

gagcgaatgc attcaggagc aagaagattt gtgaccttgg attttgggag gcctatattg 5400

ttgactgatg tattgattcc cacttgtgga gacttggcct ctttgtcaat tgacatttgg 5460

acattaggag aagaggtgga tggaaggcgg ttggtagtgg caactgatat aagcactcat 5520

tcactaattc ttcatgactt aataccacct cccgtgtgca gattcatgaa gatcactgtt 5580

attggacgtt acgggagtac aaatgccaga gccaaaatcc cattaggatt ttactatggt 5640

catacctaca tcttgccttg ggaaagtgaa ctgaagttaa tgcatgatcc tctaaaggga 5700

gagggagaat ctgcaaacca gccagaaatt gaccagcatt tagcaatgat ggttgctttg 5760

caggaggata tacagtgcag gtataacttg gcttgtcatc gtctggaaac ccttttgcaa 5820

agtattgatc ttcctcctct aaacagtgct aacaatgcac agtacttttt acgaaaacca 5880

gataaggcag ttgaggaaga cagtagggtt ttttctgctt atcaagattg tattcagcta 5940

cagcttcaac taaatttggc tcataatgca gtgcagaggc tcaaagtggc gctaggtgca 6000

agccggaaga tgttgagtga aacatcaaat ccagaagatt taattcagac atcttccaca 6060

gagcagttac gtactatcat cagatattta ctggacactt tgctcagcct gcttcatgct 6120

tctaatggac actctgttcc tgcagttttg cagagcacat ttcatgccca ggcctgtgaa 6180

gagctcttta aacacttgtg catcagtgga accccaaaga tacggttaca tactggtctt 6240

cttcttgttc aactgtgtgg tggtgaaagg tggtggggtc aatttctttc taatgtcctt 6300

caggaattgt acaattcgga acagcttctc atctttccac aggatagggt cttcatgtta 6360

ctttcctgca ttggtcaaag atcacttagt aatagtggag tattagaaag cttacttaat 6420

ctcttggata atttattgtc acctcttcag ccacagttac ccatgcatag gaggacagaa 6480

ggagtactag atattcccat gatcagttgg gttgttatgc tggtgtccag gttgctggat 6540

tatgtggcaa ctgttgaaga tgaagcagca gctgcaaaga aacctttgaa tggtaatcag 6600

tggagtttta ttaacaataa tctacacact cagagcttaa atagatcttc taaaggcagc 6660

agtagccttg atagattata ttccagaaaa atcagaaagc agcttgttca tcataaacag 6720

caacttaacc tactaaaagc aaagcagaag gcattggtag aacagatgga aaaagaaaaa 6780

atacaaagta acaaaggatc atcatataaa ctcctggtag aacaagcaaa actaaagcag 6840

gccacttcaa agcactttaa ggatttaatt cgtttacgtc ggacagcaga atggtcccgt 6900

tctaatttag acacagaagt tacaacagca aaagaaagtc ctgagataga accacttcca 6960

tttactctgg cccatgagcg ttgtatctca gtagtccaga aacttgttct gtttcttctc 7020

tccatggact ttacatgtca tgcagatctc ttattgtttg tttgtaaggt tcttgcacgc 7080

attgcaaatg ccacgaggcc aactattcat ctgtgtgaga ttgtgaacga accccagctg 7140

gaaagactgc tgttactttt ggttggaact gacttcaata gaggagatat atcttggggt 7200

ggtgcttggg ctcagtattc cttaacttgc atgctacaag atattttagc aggagaatta 7260

ctggctccag tagccgcaga agccatggag gaaggaacag tgggtgatga tgtaggtgcg 7320

acagctggtg actctgatga ctcccttcaa cagtcctcag ttcagttgct ggaaactata 7380

gatgaacctt tgacacatga cataacaggt gcacctcctc tgtcctcttt ggaaaaagat 7440

aaagaaattg accttgagtt acttcaggat ctaatggaag ttgacattga tcctttagat 7500

attgatttgg aaaaggaccc tcttgcagcc aaggttttta agccaataag cagtacatgg 7560

tatgattatt ggggtgctga ttatgggacc tacaattaca acccttacat tggaggtctg 7620

ggaattcctg tagcaaagcc accagcaaac acggagaaga acggatcaca gacagttagc 7680

gtttcagtct ctcaggccct ggatgctcgc ctagaagttg gacttgaaca gcaagcagaa 7740

ctgatgttga aaatgatgtc tactctggag gcagattcca ttttacaggc attaacaaat 7800

acatctccta cattatcaca gtctcccact ggaacagatg attcacttct agggggttta 7860

caagcagcaa accaaaccag ccagcttatt atacagttat catctgtccc aatgttaaat 7920

gtttgtttca acaaactttt ttccatgctt caagtccatc atgttcagtt ggagtcactt 7980

ctccaattgt ggctcacact gagcctgaat tctagttcaa ctggaaacaa agaaaatgga 8040

gcagacatat ttttatataa tgctaatagg atacctgtta tttcattaaa tcaagcatca 8100

ataactagct ttctcacagt gttagcttgg tatcccaata ctttgctccg gacatggtgc 8160

cttgtgcttc atagcctaac actcatgaca aacatgcagc ttaattctgg ttccagcagt 8220

gccattggaa ctcaggagag tactgctcat ttgttggttt cagatccaaa cctaattcat 8280

gtattagtga aatttctttc tggcaccagt ccacatggaa caaatcaaca cagtccacag 8340

gttggtccta cagctacaca agctatgcaa gaatttctta ctcgattaca agtgcatctt 8400

tcttcaacat gtcctcagat attcagtgaa tttttgctca agctaattca tatactttca 8460

actgaaaggg gtgctttcca gacaggccaa ggacctctcg atgcccaagt gaagctctta 8520

gaattcactc tggagcagaa ttttgaagtc gtttcagtta gtactatttc tgccgtgata 8580

gaatcggtta catttttagt gcaccactat atcacttgct cagacaaagt aatgtcaaga 8640

agtggatcag atagctccgt gggtgctcga gcatgctttg ggggactctt tgccaatctt 8700

attcgtccgg gtgatgcaaa agcagtttgt ggcgaaatga caagagatca actcatgttt 8760

gatttgttaa aacttgttaa cattttagtg cagctgcctc tttcaggcaa tagggaatac 8820

agtgcaagag tgtctgtgac cacaaataca acagatagtg tttcagatga agaaaaagtc 8880

tcaggaggca aagatggcaa tggaagcagt accagtgttc aaggatcgcc tgcatatgtt 8940

gctgacttag tcttagccaa ccaacaaatt atgagccaga ttttgtctgc tctgggcctg 9000

tgtaatagca gtgccatggc aatgataatt ggagcaagtg gattacatct cactaaacat 9060

gaaaactttc atggtgggtt ggatgccata tcagttgggg atggattatt taccatactg 9120

acaaccctta gtaaaaaagc ttctacagtc cacatgatgc tgcagccaat tttaacatac 9180

atggcctgtg gatatatggg cagacaaggc tctcttgcta cttgccagtt atctgagcca 9240

ttattgtggt tcattttgag agtattggat actagtgatg ccttgaaagc atttcatgat 9300

atgggtggtg ttcagctcat atgcaataat atggttacta gtacaagggc tattgtgaac 9360

actgcaagaa gtatggtatc aactattatg aaatttcttg actctggtcc aaataaagct 9420

gttgacagca cattgaaaac aagaatacta gcttctgagc ctgacaatgc tgaagggatt 9480

cataactttg cacccctcgg tacaatcaca tctagcagtc ctactgccca accagctgaa 9540

gtgctattgc aggccacacc tcctcacaga agagctcgct ctgctgcttg gtcctacatc 9600

tttcttccag aggaggcttg gtgtgacctt accattcacc ttcctgcagc agtgctgctt 9660

aaggagatac atatccagcc tcatcttgca tctcttgcaa cctgcccttc ctcagtgtct 9720

gttgaagtaa gtgcagatgg ggtaaatatg ctacctttgt ccactcctgt tgtcacaagt 9780

ggcctcacct acataaaaat tcagcttgta aaagccgaag tagcttctgc tgtctgcctt 9840

agactacatc gtccacggga tgccagcaca ttaggccttt cacaaattaa attattgggg 9900

ctcactgctt ttggtaccac ctcttctgca acagttaata atccattcct tccatctgaa 9960

gatcaggtat ccaaaacaag tattggatgg ttacggttat tacatcattg ccttactcac 10020

ataagtgatc tagaaggaat gatggcaagt gcagctgcac ctactgctaa tctgctgcag 10080

acttgtgcgg ccttattgat gtcaccttac tgtggaatgc attcacccaa catcgaggtt 10140

gtgcttgtaa agataggact gcagtctact agaattggcc tgaagctcat agacattctc 10200

ctgagaaatt gtgcagcatc aggcagtgat cctacagatt tgaatagtcc tttacttttt 10260

ggaagactaa atggactctc ttctgactct acgatagata ttctttacca gcttggaaca 10320

actcaggatc ctggtacaaa agacagaatt caggccttgt taaaatgggt tagtgattct 10380

gcaagagtgg ctgctatgaa gagaagtggc aggatgaact acatgtgtcc taactcctca 10440

acagtagagt atggtcttct gatgccatct ccttctcatt tgcactgtgt agcagccatt 10500

ctgtggcata gttatgagct gcttgtagaa tatgacttac cagcactcct ggaccaagag 10560

ctctttgagt tactttttaa ttggtccatg tctcttccct gcaatatggt tttgaagaaa 10620

gctgttgaca gtctactttg ctcaatgtgt cacgtacacc caaactattt ttctttgctc 10680

atgggctgga tgggaattac ccctcctcca gtgcaatgtc atcatagact gtccatgaca 10740

gatgatagca aaaagcagga tcttagttca tctttaacag atgactctaa aaatgcacaa 10800

gcacctctcg cattaactga atcacatttg gctacccttg cttcctcttc tcaatctcct 10860

gaagctatta aacaattact agactcaggt ttgccttctc ttcttgtgag gagtctggct 10920

agtttctgct ttagccacat ttctagctca gaaagcattg cccagtcaat agatatttcc 10980

caggacaaac tcaggcgcca tcatgtccca caacaatgta ataagatgcc tatcacagcc 11040

gacctagttg ctcctattct taggtttttg acagaagttg gcaatagcca tattatgaaa 11100

gattggcttg gtggttctga agtcaatcca ctatggacag cacttctgtt tttattgtgt 11160

cactctgggt ccacttctgg aagccataat ttaggtgcac aacagaccag tgcaagatca 11220

gcttctcttt cttcagctgc tacaacagga ctgactactc aacagcgcac agcaattgag 11280

aatgcaactg ttgcgttctt tctacagtgc atttcatgcc atcctaataa tcaaaagctg 11340

atggcacagg ttctttgtga actatttcag acatctcctc aaagagggaa ccttccaaca 11400

tctgggaaca tttcagggtt tatacgaaga ttatttttac agttgatgct ggaagatgag 11460

aaagtgacaa tgtttcttca gtctccatgt ccactgtaca aaggtagaat taatgctact 11520

agccacgtca tccagcatcc aatgtatgga gcaggccaca aattccgtac tcttcatttg 11580

ccagtctcaa caacattatc agatgttctt gacagagtgt cagatactcc aagtatcaca 11640

gctaaattaa ttagtgaaca aaaagatgac aaagaaaaga aaaaccatga agagaaagaa 11700

aaagttaaag cggaaaatgg atttcaagac aattacagtg ttgttgttgc ctctgggctg 11760

aagtctcaat ctaaacgtgc tgtgtcagct acaccacctc gcccaccatc caggaggggg 11820

aggacaatac ctgataaaat aggaagtact tcaggagcag aggctgccaa caaaataatt 11880

actgtcccag tgtttcacct gtttcacaaa ctcttggcag gccagccatt gccagctgaa 11940

atgacacttg cccagctttt aactctccta tatgaccgaa aacttcctca gggttaccgc 12000

tcaatagatc tgactgttaa attgggatca agagttataa cagaccccag tctatcaaaa 12060

acagattctt ataaaagact acaccctgaa aaagatcatg gagacttact tgctagctgt 12120

ccagaagatg aggctctcac tccaggtgat gaatgcatgg atgggatact ggatgaatct 12180

ttgcttgaaa cctgtccaat tcagtcacca ttacaagttt ttgcaggaat gggtggactg 12240

gctcttattg ctgaaagact acccatgcta tatccagaag taattcaaca ggtgagtgct 12300

ccagttgtaa catctaccac tcaggaaaag ccgaaggata gcgatcagtt tgaatgggtg 12360

accattgaac agtcagggga gttagtttat gaagcaccag aaactgttgc ggctgaacct 12420

ccacctatca agtcagcagt acagaccatg tctcccatac ctgcccattc tttggctgct 12480

tttggattat ttcttcgtct tccgggctat gcggaagtgc tactgaaaga gagaaaacat 12540

gcccagtgcc ttcttcgatt ggtattggga gtgacagatg atggagaagg aagtcatatt 12600

cttcaatctc catcagccaa tgtgcttcca acccttcctt tccacgtcct tcgtagcttg 12660

tttagcacta cacctttgac aactgatgat ggtgtacttc taaggcggat ggcattggaa 12720

attggagcct tacacctcat tcttgtctgt ctctctgctt tgagccacca ttccccacga 12780

gttccaaact ctagcgtgaa tcaaactgag ccacaggtgt caagctctca taaccctaca 12840

tcaacagaag aacaacagtt atattgggcc aaagggactg gctttggaac aggctctaca 12900

gcttctgggt gggatgtgga acaagcctta actaagcaaa ggctggaaga ggaacatgtt 12960

acctgccttc tgcaggttct tgccagttac ataaatcccg tcagtagtgc ggtaaatgga 13020

gaagctcagt catctcatga gactagaggg cagaacagta atgcccttcc ttctgtactt 13080

ctcgagcttc tcagtcagtc ctgcctcatc ccagccatgt catcttatct acgaaatgat 13140

tcagttctgg acatggcaag acatgtgcca ctctatcggg cactgctgga attgcttcgg 13200

gccattgctt cttgtgctgc catggtgccc ctattgttgc ccctttctac agagaacggt 13260

gaagaggaag aagaacagtc agaatgtcaa acttctgttg gtacattgtt agccaaaatg 13320

aagacctgtg ttgataccta taccaaccgt ttaagatcta aaagggaaaa tgttaaaaca 13380

ggagtaaaac cagatgcgtc tgatcaagaa ccagaaggac ttactctttt ggtaccagac 13440

atccaaaaga ctgctgagat agtttatgca gccaccacca gtttgcggca agcaaatcag 13500

gaaaaaaaac tgggtgaata ctccaagaag gcggctatga aacccaaacc tttgtcagta 13560

ttaaagtcac ttgaagaaaa atatgtggct gttatgaaga aattacagtt tgatacgttt 13620

gaaatggttt ctgaagatga agatgggaaa ttgggattta aagtaaatta ccactacatg 13680

tctcaggtga aaaatgctaa tgatgcgaac agtgctgcca gagctcgccg ccttgcccag 13740

gaagctgtga cgctttcaac ctcactgcct ctgtcttcat cctctagtgt gtttgtacgc 13800

tgtgatgagg agcgacttga tatcatgaag gttctaataa ctggtccagc ggacacccct 13860

tatgcaaatg gctgctttga gtttgatgtg tattttcctc aagattatcc cagttcaccc 13920

cctcttgtga atctagagac aactggtggt catagcgtgc gattcaatcc aaacctttat 13980

aatgatggca aggtttgttt aagcatctta aacacgtggc atggaagacc agaagagaag 14040

tggaatcctc agacctcaag ctttttgcaa gtgttggtgt ctgtccagtc ccttatatta 14100

gtagctgagc cttattttaa tgaaccggga tatgaacggt ctagaggcac tcccagtggc 14160

acacagagtt ctcgagaata tgatggaaac attcgacaag caacagttaa gtgggcaatg 14220

ctagaacaaa tcagaaaccc ttcaccatgt tttaaagagg taatacacaa acatttttac 14280

ttgaaaagag ttgagataat ggcccaatgt gaggagtgga ttgcggatat ccagcagtac 14340

agcagtgata agcgggtagg caggactatg tctcaccatg cagcagctct caagcgtcac 14400

actgctcagc tccgcgaaga gttgctgaaa cttccctgcc ctgaaggctt ggatcctgac 14460

actgacgatg ccccagaggt gtgcagagcc acaacaggtg ctgaggagac tctaatgcat 14520

gatcaggtta aacccagcag cagcaaagaa ctccccagtg acttccagtt atga 14574

Claims (8)

1.一种检测BIRC6蛋白表达水平的试剂在制备三阴性乳腺癌的诊断试剂或诊断试剂盒中的应用。

2.根据权利1所述的应用，其特征在于，所述试剂为BIRC6特异性抗体。

3.根据权利要求1或2所述的应用，其特征在于，所述BIRC6的蛋白序列如SEQ ID NO.1所示。

4.一种降低或抑制BIRC6表达的试剂在制备治疗三阴性乳腺癌的药物中的应用。

5.根据权利要求4所述的应用，其特征在于，所述降低或抑制BIRC6表达的试剂选自由以下组成的组：gapmer、反义RNA、siRNA、esiRNA、shRNA、miRNA、RNA适体、TALEN、CRISPR和锌指核酸酶。

6.根据权利要求5所述的应用，其特征在于，所述BIRC6的核苷酸序列如SEQ ID NO.2所示。

7.根据权利要求4-6任意一项所述的应用，其特征在于，所述药物还包括另外的抗癌剂，所述抗癌药为用于降低或抑制BIRC6表达的试剂或抑制BIRC6与JNK结合的试剂。

8.一种筛选用于治疗三阴性乳腺癌的候选药物的方法，其特征在于，该方法包括下述步骤：

1)确定过表达BIRC6的癌细胞中BIRC6的表达水平；

2)将候选化合物与步骤1)中确定的癌细胞接触；

3)确定步骤2)得到的癌细胞中BIRC6的表达水平；

4)比较步骤1)和步骤3)中确定的BIRC6的表达水平，其中BIRC6的表达水平降低指示所述候选化合物具有抗癌潜力。

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