疫苗制备方法和抗肿瘤疫苗的制作方法

专利名称：疫苗制备方法和抗肿瘤疫苗的制作方法
技术领域：
本发明涉及生物技术领域，具体而言涉及一种疫苗制备方法和制备的生物疫苗，特别是新型的抗肿瘤疫苗，如EGFR分子疫苗。分子疫苗属新生物技术药物(New Biotechnological drug)范畴，涉及到多种生物技术，如PCR技术、分子克隆技术、基因表达技术、现代疫苗技术、以及生物技术药物学和药剂学等。
背景技术：
疫苗(vaccine)是一种能刺激机体免疫系统产生抗特异性靶物质(如病毒、细菌等)的免疫反应的物质。经典的疫苗概念来源于抗感染免疫，它主要是使用处理过的病原微生物(如病毒、细菌等)及其衍生物免疫机体产生体液免疫反应以预防感染性疾病。比如，灭活疫苗(inactivated vaccine)是利用化学方法杀死实际感染成分的衍生物。弱毒疫苗(attenuated vaccine)是改变过的活病毒或细菌使其在接种的生物体内不能增殖。这两类疫苗都是通过表面蛋白(抗原)作用于B和T淋病细胞来进行免疫的。当病原体生物感染人体时，B和T淋病细胞就会迅速作出反应，在病原微生物产生破坏之前将其消灭。然而，这两类疫苗潜藏着危险，因为它们能被传染性病菌所污染。例如，每年总有一小部分儿童因为接种脊髓灰质炎疫苗而感染此病。因此，一个发展方向是亚单位疫苗(subunit vaccine)。亚单位疫苗是利用DNA重组技术生产的单纯由表面蛋白组成、能引起免疫系统反应的疫苗，也称为基因工程疫苗或者DNA重组疫苗。参见，基因工程学原理，马建岗，主编，西安交通大学出版社，p240，2001.11。除了上述采用病原体本身、其具有免疫原性的构成成分、以及基因表达产物外，也有人研究使用“裸”DNA作为疫苗的基因疫苗，是由来源于病原体的一个抗原编码基因及作为其载体的质粒DNA组成。通过注射或粒子轰击等途径将基因疫苗导入人体后，这段基因可以在活体细胞中合成抗原蛋白，从而引起机体免疫反应。目前，基因表达文库免疫技术是发现免疫活性基因的最系统和客观的手段。参见，第四次浪潮生物经济，封展旗，杨同卫，编著，经济管理出版社，p169-171，2000。
疫苗的主要作用在于预防疾病。现在也有应用疫苗来治疗疾病。所以，针对正常的人体和动物体而言，疫苗的作用在于增强机体的防病和抗病能力，起到预防疾病的目的；对于患有某种疾病或者病患的人体和动物体而言，疫苗的作用在于诱导机体产生针对特定致病因素的反应，达到消除病灶，治疗疾病或者病患的目的。可以认为疫苗就是通过影响机体的免疫系统达到预防，或者治疗，或者预防和治疗疾病或者病患的物质的总称。
动物体内细胞分裂调节失控而无限增殖的细胞称为肿瘤细胞(tumor cell)。具有转移能力的肿瘤称为恶性肿瘤(malignancy)，上皮组织的恶性肿瘤称为癌。参见，细胞生物学，翟中和等，主编，高等教育出版社，p423-427，2000(2001重印)。对于肿瘤的起源和宿主对于肿瘤的应答已经进行了大量的基础研究和临床应用研究。关于起源，已经显示大量的环境因素对于动物具有致癌性和致突变性。有些肿瘤事实上与暴露于某种特定的物质有关(例如石棉与造船厂工人的间皮瘤)。已知病毒也可以诱发动物肿瘤，例如乙型肝炎病毒与肝癌有关。宿主免疫应答在许多情况下产生以抵抗肿瘤，而在某些情况下可能是保护性的。
肿瘤细胞和正常细胞可以根据其抗原的质和量的差异区分开来。肿瘤细胞的抗原，特别是肿瘤特异抗原为肿瘤细胞特有，成为肿瘤预防和治疗中的研究重点。
用于肿瘤治疗的肿瘤疫苗(cancer vaccine)尤其具有重要的意义。这种治疗性疫苗不同于传统的预防性疫苗，主要用于治疗已罹患癌症的病人，其目的以激发患者机体对肿瘤的特异性免疫应答，最终达到有效的排斥。研制和开发新型肿瘤疫苗已成为近年国际上肿瘤免疫治疗的热点，主要包括肿瘤细胞疫苗、基因修饰疫苗、多肽瘤苗和基因/DNA疫苗等。
肿瘤细胞疫苗是将病人或者患病动物的肿瘤细胞通过物理或者化学方法处理后的具有治疗或者辅助治疗作用的完整的死细胞。处理方法可以是X光照射，有机溶剂处理等。这种疫苗被通过注射等方法施用于机体后，可以诱导或者增强病人对其肿瘤的免疫力。基因修饰疫苗、多肽瘤苗和基因/DNA疫苗也均是通过利用肿瘤抗原及其片段，或者编码肿瘤抗原及其片段的多核苷酸，以及含有这些多核苷酸的载体或者细胞对于肿瘤进行治疗或者辅助治疗的疫苗。
如上所述，疫苗在疾病预防和/或治疗中具有重要作用。肿瘤作为一种严重的动物体细胞异常生长现象，随着环境污染的加剧，发生频率有所增强。因此开发新的疫苗，特别是肿瘤疫苗是必须的。本发明提供了一种制备疫苗的新方法，提供了一种新型肿瘤疫苗-EGFR分子疫苗(EGFR molecular vaccine)，满足了上述需要。
发明概述第一个方面，本发明提供了一种制备疫苗的方法。该方法包括1)分析特定病原体的特异抗原；2)获得编码特异抗原的多核苷酸序列；3)获得与该多核苷酸序列有足够差异的多核苷酸序列；4)利用步骤3)中所得的多核苷酸序列制备疫苗。
第二个发面，本发明提供了一种制备肿瘤疫苗的方法。该方法包括
1)分析动物体内的肿瘤细胞的特异抗原；2)获得编码特异抗原的多核苷酸序列；3)获得与该多核苷酸序列有足够差异的多核苷酸序列；4)利用步骤3)中所得的多核苷酸序列制备疫苗。
第三个方面，本发明提供了一种制备EGFR疫苗的方法。该方法包括1)分析动物体内的肿瘤细胞的EGFR抗原；2)获得编码特异抗原的多核苷酸序列；3)获得与该多核苷酸序列有足够差异的多核苷酸序列；4)利用步骤3)中所得的多核苷酸序列制备疫苗。
第四个方面，本发明提供了一种核酸疫苗，其中含有与编码一种病原体上的一种特定抗原的多核苷酸序列有一定的差异，而且其同源性需要达到一定数值的多核苷酸序列。疫苗中所含多核苷酸序列与病原体中该特定抗原的多核苷酸序列所编码的多肽的同源性为30-95％。
第五个方面，本发明提供了一种核酸疫苗，其中含有与编码一种病原体上的两种或者两种以上特定抗原的多核苷酸序列有一定的差异，而且其同源性分别要达到一定数值的两种或者两种以上多核苷酸序列。疫苗中所含多核苷酸序列与病原体中相应的特定抗原的多核苷酸序列所编码的多肽的同源性为30-95％。
第六个方面，本发明提供了一种蛋白质或者多肽疫苗，其中含有与一种病原体上的一种特定抗原的蛋白质部分中的氨基酸序列有一定的差异，而且其同源性为30-95％的蛋白质或者多肽分子。
第七个方面，本发明提供了一种蛋白质或者多肽疫苗，其中含有与一种病原体上的两种或者两种以上特定抗原的蛋白质部分中的氨基酸序列有一定的差异，而且其同源性为30-95％的两种或者两种以上蛋白质或者多肽分子。
第八个方面，本发明提供了一种表皮生长因子受体(EGFR)核酸疫苗，其中含有与编码一种生物体内的EGFR分子的基因中的核苷酸序列有一定的差异，而且其同源性需要达到一定数值的一种多核苷酸序列。疫苗中所含多核苷酸序列编码的表皮生长因子受体与该生物体内的表皮生长因子受体中的氨基酸序列的同源性为30-95％。
第九个方面，本发明提供了一种表皮生长因子受体(EGFR)核酸疫苗，其中含有与编码一种生物体内的EGFR分子的基因中的核苷酸序列有一定的差异，而且其同源性需要达到一定数值的两种或者两种以上的多核苷酸序列。疫苗中所含多核苷酸序列编码的表皮生长因子受体与该生物体内的表皮生长因子受体中的氨基酸序列的同源性为30-95％。疫苗中所含的两种或者两种以上的多核苷酸序列可以具有不同的来源，其与该生物体内的EGFR分子的基因中的核苷酸序列的同源性可以不同。
第十个方面，本发明提供了一种表皮生长因子受体(EGFR)蛋白质或者多肽疫苗，其中含有与一种生物体内的EGFR分子的变体。疫苗中所含表皮生长因子受体的氨基酸序列与该生物体内的表皮生长因子受体中的氨基酸序列的同源性为30-95％。
第十个方面，本发明提供了一种表皮生长因子受体(EGFR)蛋白质或者多肽疫苗，其中含有与两种或者两种以上的与生物体内EGFR分子不同的变体。疫苗中所含表皮生长因子受体的氨基酸序列与该生物体内的表皮生长因子受体中的氨基酸序列的同源性为30-95％。
第十一个方面，本发明提供了的表皮生长因子受体核酸疫苗和表皮生长因子受体蛋白质或者多肽疫苗用于肿瘤预防和治疗中的用途。
发明祥述在此所用，“病原体”是指侵入生物体的外来生物体，包括细菌、病毒、真菌、原虫、蠕虫等，它们可以侵入另外的生物体中，导致一定的疾病或者疾病状态。
在此所用，“肿瘤细胞”是指生物体内发生了生长和分裂失控，脱离了衰老和死亡的正常途径的细胞。“肿瘤细胞”群构成肿瘤组织。在本发明中，术语“肿瘤”是以其广义而言，包括任何类型的细胞不正常的增殖，也指肿瘤组织或者肿瘤细胞，需要对这种增殖予以遏制或者将这些细胞以及组织予以抑制或者消除。
在此所用，“生物体”是指动物体，尤其指“人体”。对于存在有免疫系统的动物，本发明的方法和疫苗均可以适用。
可以利用本技术领域的基础研究结果或者已知的方法确定病原体或者肿瘤细胞中的特定抗原作为制备本发明所述疫苗的靶标。由于特定病原体或者体内的异常细胞具有特定的标记分子，比如它们的特定抗原，所以，以这些分子作为靶标，可以设计疫苗来激发生物体针对这些有害物质、生物或者细胞的免疫反应，达到防病和治病的目的。
从GenBank等公开的数据库，或者众多的网站上可以找到相应的有用抗原的基因序列，或者其氨基酸序列。根据中心法则，利用氨基酸序列或者基因序列进行推导所得的序列也可以应用于本发明中。有用的网站包括，但不限于，http//www.sanger.ac.uk/Proiects/Microbes.http//bioweb.pasteur.fr/Genoist/TubercuList.
在本发明中，使用的针对靶抗原的核酸序列或者多肽的氨基酸序列不同于编码该靶抗原的蛋白质或者多肽部分的核酸序列或者其氨基酸序列。其同源性以编码产物而言为30-95％。
同源性通常用序列分析软件来测定。在上下文中对于两种多肽序列，术语“同源性”指相同或具有特定百分比的氨基酸残基的两个或多个序列，当在同一比较窗口或指定区域内尽可能一致地比较或对齐时，所述氨基酸残基是相同的，这正如用大量序列比较算法或通过人工对齐和视觉观察所测定那样。
对于序列比较，通常一个序列作为参考序列，测试序列与其进行比较。当使用序列比较算法时，将测试序列和参考序列输入计算机，指定序列算法程序参数。然后用序列比较算法基于程序参数计算测试序列相对于参考序列的序列同源性百分比。在本发明中，某种选定的抗原中的氨基酸序列作为参考序列，疫苗中核苷酸序列的编码序列或者疫苗中的蛋白质或者多肽中的氨基酸序列就是测试序列，该测试序列与参考序列的同源性是本发明的一个方面。
如果将靶抗原的核酸序列或者多肽的氨基酸序列看作“母序列或者参考序列”，那么本发明所用的核酸序列或者多肽的氨基酸序列可以看作“子序列或者衍生序列”。
“母序列或者参考序列”的来源可以是现有技术，比如上面提到的数据库，网站，公开的发明和论文等。它们也可以来源于对特定抗原，不论微生物抗原还是体内肿瘤的抗原的研究。可以是对基因组，染色体，蛋白质或者多肽，mRNA，DNA分子进行研究和分析获得母序列或者参考序列或者其序列信息。
“子序列或者衍生序列”是由“母序列或者参考序列”演变而来的序列，它们与“母序列或者参考序列”有一定的差异，但是又不是完全不同。“子序列或者衍生序列”可以是自然界已经存在的与“母序列或者参考序列”具有结构上关联的序列，也可以是通过人工方法得到的序列。前者是指不同生物或者生物个体内存在的编码同源蛋白的序列。比如，人表皮生长因子受体的编码序列和小鼠表皮生长因子受体的编码序列，可以将它们看作本发明所称“母序列”与“子序列”的关系，或者“参考序列”与“衍生序列”的关系。这种称呼可以相互交换。比如将小鼠表皮生长因子受体的编码序列看作“母序列”，则人表皮生长因子受体的编码序列被看作“子序列”；或者人表皮生长因子受体的编码序列被看作“母序列”，小鼠表皮生长因子受体的编码序列看作“子序列”。也可以以“母序列或者参考序列”为起始物质，经过突变或者修饰得到“子序列或者衍生序列”。还可以通过人工合成方法得到与“母序列或者参考序列”有点差异的“子序列或者衍生序列”。通过检索有关数据库，在计算机上运行有关程序得到“母序列或者参考序列”的“子序列或者衍生序列”，然后通过人工合成得到具有“子序列或者衍生序列”的核酸、蛋白质以及其片段的物质。通过筛选有关分子文库，也可以得到本发明所称“子序列或者衍生序列”。具有“子序列或者衍生序列”的核酸，核酸片段，寡核苷酸，多核苷酸以及具有“子序列或者衍生序列”的蛋白质，多肽，及其片段可以用于本发明的实施中。在本发明实施中，对这些具有“子序列或者衍生序列”的核酸，核酸片段，寡核苷酸，多核苷酸以及具有“子序列或者衍生序列”的蛋白质，多肽，及其片段可以进行各种修饰。也可以将它们包括在适当的载体或者细胞或者病毒中，达到本发明的发明目的。上述用于本发明实施中的各种物质可以是分离的，可以是纯化的；它们之间也可以是混合的，还可以混合进一定物质，比如佐剂。
此处所用，短语“核酸”或“核酸序列”指寡核苷酸、核苷酸、多核苷酸，或指寡核苷酸、核苷酸、多核苷酸中任一种的片段，指基因组或合成来源的DNA或RNA，指肽核酸(PNA)，或者指任何天然或合成来源的类似DNA或类似RNA的物质。
特定多肽或蛋白质的“编码序列”或“编码特定多肽或蛋白质的核苷酸序列”是当在置于适当调节序列控制下时被转录或翻译到多肽或蛋白质中的核酸序列。
术语“基因”指涉及产生多肽链的DNA片段；包括编码区之前和之后的区(前导区和非转录尾区)，以及适当的时候，包括个体编码片段(外显子)之间的间插序列(内含子)。
正如此处所用，“氨基酸”或“氨基酸序列”指寡肽、肽、多肽或蛋白序列，或指寡肽、肽、多肽或蛋白序列中任意一种的片段、部分或亚单位，指天然分子或合成分子。
正如此处所用，术语“多肽”指通过肽键或修饰的肽键即肽等排物(peptideisostere)彼此连接在一起的氨基酸，并且可以含有除了20个由基因编码的氨基酸以外的修饰氨基酸。多肽可以通过任一种天然方法修饰，如翻译后加工，或通过本技术领域已知的化学修饰技术修饰。修饰可以发生在多肽中的任何地方，包括肽骨架、氨基酸侧链和氨基末端或羧基末端。应该意识到，在给定的多肽中，相同类型的修饰可以在数个位点以相同或变化的程度存在。而且，给定的多肽可以具有许多类型的修饰。这些修饰包括乙酰化作用、酰化作用、ADP-核糖基化作用、酰胺化作用、黄素的共价附着、亚铁血红素部分的共价附着、核苷酸或核苷酸衍生物的共价附着、脂质或脂质衍生物的共价附着、交联环化、二硫键形成、脱甲基作用、共价交联的形成、半胱氨酸的形成、焦谷氨酸的形成、甲酰化作用、γ-羧化作用、糖基化作用、GPI锚形成、羟基化作用、碘化作用、甲基化作用、豆蔻酰化、氧化、蛋白水解加工、磷酸化作用、异戊二烯化、外消旋作用、硒化作用、硫酸化作用、t-RNA介导的将氨基酸加入到蛋白质的过程如精氨酰化。(参考Creighton，T.E.，Proteins-structure and Molecular Properties第二版，W.H.Freeman andCompany，New York(1993)；Posttranslational Covalent Modification of Proteins，B.C.Johnson编著，Academic Press，NewYork，1-12页(1983))。
正如此处所用，术语“分离的”指物质离开其原始环境(例如，如果是天然存在的，则离开其天然环境)。例如，存在于活的动物体内的天然存在的多核苷酸或多肽不是分离的，但从天然系统中的一些或全部共存物质中分离出的相同多核苷酸或多肽是分离的。这样的多核苷酸可以是载体的一部分，和/或这样的多核苷酸或多肽可以是组合物的一部分，它们仍然是分离的，原因在于这些载体或组合物不是天然环境的一部分。
正如此处所用，术语“纯化的”不要求完全纯化；更确切地说，该术语是一个相对定义。
正如此处所用，术语“重组的”指核酸与“骨架”核酸相邻，而在其天然环境中与“骨架”核酸不是相邻的。根据本发明的骨架分子包括核酸，如表达载体、自我复制核酸、病毒、整合核酸和其它用于维持或操纵相关核酸插入物的载体或核酸。
“重组的”多肽或蛋白质指通过重组DNA技术产生的多肽或蛋白质；即，通过编码期望多肽或蛋白质的外源DNA构建物转化的细胞产生的多肽或蛋白质。“合成的”多肽或蛋白质是那些通过化学合成制备的多肽或蛋白质。也可以固相化学肽合成方法来合成本发明的多肽或片段。这些方法自从20世纪60年代早期在本技术领域就已经已知(Merrifield，R.B.，J.Am.Chem.Soc.，852149-2154，1963)(也可以参见Stewart，J.M.和Young，J.D.，Solid Phase Peptide Synthesis，第二版，PierceChemical Co.，Rockford，I11，11-12页))，并且最近已经被用于可通过商业途径获得的实验室肽设计和合成试剂盒(Cambridge Research Biochemicals)。这些可通过商业途径获得的实验室试剂盒通常使用了H.M.Geysen等人，Proc.Natl.Acad.Sci.，USA，813998(1984)的教导，在大量“棒”或“针”的尖端上提供合成肽，所有棒或针与单个板子相连接。当使用这样的系统时，一个具有棒或针的板子被翻转，且插入到具有相应孔或容器的第二个板子中，所述孔或容器含有将适当氨基酸附着或固定到针或棒尖端的溶液。通过重复这一处理步骤，即将棒或针的尖端翻转且插入到适当溶液中，氨基酸就被构建到期望肽中。此外，可以得到数量众多的FMOC肽合成系统。例如，多肽或片段的装配可以使用Applied Biosystems，Inc.提供的Model 431A自动肽合成仪在固体载体上进行。该肽合成仪提供了现成获取本发明所述肽，或者通过直接合成或者通过合成一系列片段，这些片段可以使用其它已知技术偶联。
当起动启动子转录的RNA聚合酶将编码序列转录到mRNA中时，启动子序列与编码序列是“可操纵连接”的。
“质粒”由前面的小写字母“p”，和/或其后的大写字母和/或数字标明。此处的起始质粒或者通过商业途径获得，是不受限制地公开获得，或者可以用与公开方法一致的方法从可获得的质粒构建而来。另外，与此处所描述的质粒等效的质粒在本领域是已知的，对于普通技术人员而言是明了的。在本发明中，构建了一些典型的质粒，参考附图2。这些质粒是实施本发明的一种形式。
“寡核苷酸”或者指一个单链多脱氧核苷酸，或者指两个互补的多脱氧核苷酸链，它们可以化学合成。这样化学合成的寡核苷酸没有5’磷酸，因此在没有用ATP添加磷酸和存在激酶的情况下，不会与另一个寡核苷酸连接。合成寡核苷酸将与没有发生去磷酸化作用的片段连接。
正如此处所用，“片段”是天然存在的蛋白质的一部分，其可以以至少两种不同的构象存在。片段可以具有与天然蛋白质相同或基本上相同的氨基酸序列。“基本上相同”指氨基酸序列大部分是相同的，但不是完全相同。
术语“变体”指在一个或多个碱基对、密码子、内含子、外显子或氨基酸残基(分别地)被修饰而不同于本发明的“母序列或者参考序列”。变体可以通过很多方法产生，这些方法包括，例如易错PCR、改组、寡核苷酸介导的诱变、装配PCR、有性PCR诱变、体内诱变、盒式诱变、递归集团诱变(recursive ensemblemutagenesis)、指数集团诱变(exponential ensemble mutagenesis)、位点特异性诱变、基因再装配、GSSM及其任意组合。
从又一个方面来说，本发明所述的“子序列或者衍生序列”可以看作“母序列或者参考序列”的“变体”。因为它们在结构上与抗原中的“母序列或者参考序列”具有一定的相似性，而且又具有一定的差异。所以含有“子序列或者衍生序列”的物质可以诱发机体针对该抗原的免疫反应，尤其可以打破机体对该抗原业已产生的耐受性。其实，真是由于本发明所述疫苗中所用物质与抗原结构有一定相同性，机体发动的针对本发明疫苗中的物质的免疫反应就会交叉作用于该抗原。同时因为本发明所述疫苗中所用物质与抗原结构有一定差异，所以本发明的疫苗特别适用于诱发机体针对产生耐受性的抗原进行反应。
以下以EGFR分子疫苗为例来说明本发明的方法。事实上，本发明并不限于EGFR分子疫苗，本技术领域的技术人员根据本发明的教导可以方便地将本发明的方法用于其他疫苗的制备中。本发明更重要的价值在于提出了一种制备疫苗，特别是打破机体耐受性的方法；尤其在于肿瘤治疗上有重要价值。
EGFR分子疫苗就是利用经现代生物工程技术改造进化的或在生物进化过程中形成的来源于人或其他异种生物的同源EGFR分子作为抗原而构建的各种新生物技术疫苗，包括重组蛋白疫苗、重组基因疫苗、重组病毒疫苗、基因修饰疫苗和稳定转化共生菌。这是一种新型概念的肿瘤疫苗。
同源EGFR分子就是指与人EGFR分子在氨基酸或核苷酸水平上存在不同程度的相同程度的其它物种来源(如小鼠、大鼠、鸡、鲐、果蝇等，见SEQ ID NO 1-14)或人工进化的EGFR分子的总称。
人表皮生长因子受体(epidermal growth factor receptor，EGFR)是一种分子量为170KD的跨膜单链糖蛋白，由1186个氨基酸组成，具有酪氨酸蛋白激酶(PTK)活性。EGFR由胞外配体结合区、跨膜疏水区、近膜区、PTK活性区和受体C端尾肽五个部分组成。EGFR有多种不同形式的转录方式(见SEQ ID NO 1-5)，还有多种三种突变体，其中主要的突变体是第III类EGFRvIII(又称ΔEGFR或de2-7EGFR，见SEQ ID NO 6)，是恶性肿瘤细胞膜上所特有的，具有高度的肿瘤特异性。人EGFR分子是一类基因家族，除EGFR(亦称c-erbB1/HER1)外其它家族成员还包括c-erbB2/HER2、c-erbB3/HER3和c-erbB4/HER4，它们分别编码膜蛋白p185erbB2、p160 erbB3、p180 erbB4。虽然这些成员具有高度同源的氨基酸序列和相似的结构特征，但配体各不相同。本发明所涉及的肿瘤疫苗靶分子是EGFR(c-erbB1/HER1)，不包括c-erbB2/HER2、c-erbB3/HER3和c-erbB4/HER4。
EGFR分子的配体EGF或TGF-可通过自分泌(autocrine)或旁分泌(paracrine)等途径作用于EGFR，使其PTK活化，经过一系列的信号传递，继而导致细胞的分裂增殖。
EGFR广泛分布于正常的哺乳动物上皮细胞表面，平均每个细胞的受体数为5-10万。多种肿瘤细胞如肺癌、乳腺癌、卵巢癌、结肠癌、前列腺癌、胃癌、膀胱癌、头颈部鳞癌和胶质瘤等，都过量表达EGFR，数量可达1-3×106/细胞。以肺癌为例，EGFR的过量表达还与肺癌的浸润、转移和预后密切相关。因此，EGFR被认为是肿瘤治疗的一个理想的靶分子。目前，以EGFR为靶分子的治疗，主要表现为EGFR单克隆抗体和小分子化合物。此外，以EGFRvIII为靶分子的肿瘤肽疫苗、以及利用反义核酸技术进行基因治疗等取得了一些有益的进展。以EGFR分子为靶分子设计抗肿瘤分子疫苗，目前尚无任何文献报道和专利报告。
肿瘤细胞在其恶性转化、增殖的过程中要产生一种或多种肿瘤抗原。然而，在大多数情况下，肿瘤抗原的免疫原性较弱，并不足以引起机体的主动免疫反应。从免疫学的角度看，肿瘤细胞就是一种能不断表达“正常”抗原(基因过度表达)和/或“异常”抗原(基因修饰、突变或缺失等)的宿主体内“自身”组织细胞，因此肿瘤抗原可看作是自体抗原。在正常情况下，机体对自体抗原不产生免疫应答，即呈现出免疫耐受。事实上，自体抗原是宿主免疫系统必须耐受的最相关和最丰富的抗原。自体抗原耐受的诱导和维持是由多种机制介导的，它们能防止对正常组织的不适当破坏。然而，当机体隐蔽的抗原释放、或生物、物理与化学等因素使自体抗原发生改变等情况下，却可导致自体免疫反应，造成靶抗原所在的细胞、组织或器官的病理性损伤和功能障碍。这样，如果能使肿瘤细胞隐蔽的自体抗原释放或者使其发生某种改变的话，则可能因此诱导出机体对自身肿瘤细胞的特异性自体免疫反应，从而使肿瘤缩小乃至消退。如前文所述，EGFR分子在多种肿瘤中过量表达，因此，EGFR分子可看作是一种肿瘤抗原，也是一种自体抗原。因此，可对人EGFR抗原分子(见SEQ ID NO 1-6)进行各种生物技术改造，制备成分子疫苗，从而诱导抗肿瘤免疫反应，达到抗肿瘤作用的目的。
生物在进化过程中，不同物种之间存在着具有不同程度同源性的大量分子，如EGFR分子就广泛存在于人、鼠、果蝇、鱼类、鸟类等生物，它们相互之间存在不同的同源性程度，人的EGFR分子在氨基酸水平上与鼠、鸡和果蝇的EGFR分子的同源性分别为90％、64％和32％。我们可以利用异种同源分子在进化过程中所形成的细微差别来打破免疫耐受、增强免疫原性、诱导肿瘤细胞的自体免疫反应进而达到抗肿瘤的目的。这其中的机制很可能是由于在进化过程中同源基因的中性突变虽不导致其功能的丧失或改变，但却可能影响到或改变了其免疫应答的方式。当异种同源基因导入到受试对象体内表达出相应的异种同源蛋白，受试者将之识别为外来抗原，一方面产生相应的抗体或细胞毒性淋巴细胞来清除它，另一方面由于这些表达出的异种同源蛋白与受试者体内相应的蛋白分子存在某种程度上的差异而与之产生非特异性的交叉反应，从而诱导自身免疫反应，打破机体对自身的这种蛋白的免疫耐受(见说明书附图1)。
如前文所述，人的EGFR与其他物种(如鼠、鸡、果蝇等)的EGFR在氨基酸水平上表现出不同程度的同源性，通过BLAST分析发现人的EGFR在氨基酸水平上与鼠、鸡和果蝇的同源性分别90％、64％和32％。我们利用异种同源EGFR分子产生的种与种之间的免疫交叉反应来诱导机体的主动免疫反应，克服自身肿瘤细胞的免疫耐受，通过其产生的抗体竞争性抑制EGFR与配体EGF或TGF-的结合，从而抑制肿瘤细胞的生长，此外还通过其所诱导的CTL来抑制肿瘤细胞的生长。
此外，现代生物工程技术的发展，如易错PCR技术、DNA“洗牌术”(DNAshuffling)、噬菌体展示技术等，为EGFR分子的人工基因直接进化(gene directedevolution)提供了技术支持。
本发明涉及抗肿瘤EGFR分子疫苗，包括自体EGFR分子疫苗(autologousEGFR vaccine)、异种EGFR分子疫苗(xenogeneic EGFR vaccine)和基因直接进化EGFR分子疫苗(directed evolution EGFR Vaccine)。
本发明主要涉及一种新型的肿瘤疫苗——EGFR分子疫苗，它是利用经现代生物工程技术改造的或在生物进化过程中形成的来源于人或其他异种生物的同源EGFR分子作为抗原而构建的各种新生物技术疫苗，包括蛋白疫苗、基因疫苗、病毒疫苗、基因修饰疫苗和稳定转化共生菌。EGFR分子疫苗对各种过表达EGFR分子的实体瘤都具有抗肿瘤作用效应，其抗肿瘤作用机制在于EGFR分子作为免疫交叉抗原，能打破机体对自身EGFR分子的免疫耐受，诱导机体产生针对EGFR分子自身免疫样交叉反应。抗肿瘤EGFR分子疫苗也可以是利用纳米生物技术制备而成的纳米粒制剂。
一、EGFR分子的来源、选择与改造本发明涉及的EGFR分子包括自体EGFR分子(autologous EGFR molecule)和异种EGFR分子(xenogeneic EGFR molecule)和基因直接进化EGFR分子(directedevolution EGFR molecule)。
利用在生物进化过程中形成的或经现代生物工程技术改造的同源EGFR分子的差异性进行抗肿瘤免疫治疗是本发明的最大特征。
EGFR在自然界中分布广泛，从哺乳动物(如人、鼠等)、鸟类(如鸡等)、鱼类(如鲐等)到低等生物(如线虫、果蝇等)都有EGFR的表达，这些生物来源的EGFR分子之间都存在一定的差异，其同源性程度在30-100％之间。本发明所涉及的异种EGFR分子的同源性在30-95％之间，主要涉及的生物物种是人、鼠、鸡和果蝇，在氨基酸水平上，人的EGFR分子与鼠、鸡和果蝇的EGFR分子的同源性分别为90％、64％和32％，人、鼠、鸡和果蝇的EGFR分子分别代表了本发明中所涉及的同源性程度的监测点。
自体EGFR分子由于存在免疫耐受，故免疫原性较弱。利用现代生物工程技术可对自体EGFR分子进行人工直接进化(gene directed evolution)和改造，增强EGFR分子作为抗原的免疫原性。通常是利用易错PCR、随机引物延伸技术和DNA“洗牌”术(DNA shuffling)等对自体EGFR分子进行人工突变，建立基因突变库，然后使用噬菌体展示技术(phage display)、核糖体展示技术(ribosome display)等进行筛选，从而得到免疫原性强的EGFR分子。
此外，利用细菌、病毒等不同生物的表达修饰系统的差异，也可以在蛋白质水平上对自体EGFR分子进行人工改造，增强自体EGFR的免疫原性。
二、EGFR重组基因疫苗基因疫苗是利用现代分子生物学技术构建制备的以核酸为基础的新型疫苗，主要是DNA疫苗。
本发明的EGFR分子疫苗之一就是DNA疫苗。依据GenBank等各公开的数据库所收藏的各种生物来源(如人、小鼠、大鼠、鸡、鲐、果蝇等，见SEQ ID NO1-14)的EGFR分子的序列(包括基因、cDNA、mRNA和氨基酸序列)按常规方法设计引物或探针，用PCR、RT-PCR、杂交等技术，从各种商品化的基因文库、cDNA文库或各种细胞系、组织等中克隆分离出不同种属生物来源的EGFR分子的胞外区段的cDNA(发明人发现EGFR胞外区段才是引起免疫反应的反应域)，或进一步用基因直接进化技术筛选到的具有强免疫原性的EGFR分子。各种来源的EGFR cDNA的胞外段序列经测序鉴定后，用分子克隆技术构建其真核生物表达质粒系统，然后转染入CHO细胞系，观察并检测其表达EGFR的情况和水平。各重组EGFR分子的真核生物表达质粒可经过限制性酶切分析、SDS-PAGE和Western Blot等进行鉴定、确证。用碱法抽提经过鉴定的重组EGFR分子表达质粒，用超离心、超滤等方法去除大肠杆菌内毒素，即得到纯净的质重组粒DNA，这些质粒DNA即可作为DNA疫苗进行免疫。
DNA分子疫苗的代表性质粒图谱如图2A所示，其具体构建过程简要叙述如下依据GenBank等各公开的数据库所收藏的人、小鼠、鸡、的EGFR分子的cDNA序列(分别对应于SEQ ID NO 1-5，7-9，19)设计PCR引物(人的引物为5’GACCATGGAGGAAAAGAAAGTTTGC 3’，5’ACGAATTCTTAGGACGGGATCTTAGGCCCA3’；小鼠的引物为5’GACCATGGAGGAAAAGAAAGTCTGC 3’，5’ACGAATTCTTAATAGATGGTATCTTTGGC 3’；鸡的引物为5’GACCATGGAGGAGAAGAAAGTTTGTC 3’，5’ACGAATTCTTAAGATGGAGTTTTGGAGCC 3’)，分别以人肺癌细胞株A431、小鼠肺癌细胞株LL2和鸡胚的总RNA为模板进行RT-PCR扩增，电泳收集纯化扩增的EGFR片段(均为1.9kb)，然后进行PCR产物亚克隆。把PCR亚克隆测序确证后，用NcoI和EcoRI酶切，收集1.9kb片段并纯化，插入到用NcoI和EcoRI双酶切的pORF-MCS(InvivoGen公司)载体，筛选重组质粒。候选的重组质粒经限制性酶切分析及PCR扩增等双重鉴定，命名为pORF-hEGFR、pORF-mEGFR和pORF-chEGFR。至于pcDNA-hEGFR、pcDNA-mEGFR和pcDNA-chEGFR的构建过程与上述方法一致(pcDNA3.1(+)载体来源于Invitrogen公司)，只是PCR引物略有改动，分别是人的引物5’GAGCTAGCATGGAGGAAAAGAAAGTTTGC 3’，5’CACTCGAGTTAGGACGGGATCTTAGGCCCA 3’；小鼠的引物为5’GAGCTAGCATGGAGGAAAAGAAAGTCTGC 3’，5’CACTCGAGTTAATAGATGGTATCTTTGGC 3’；鸡的引物为5’GAGCTAGCATGGAGGAGAAGAAAGTTTGTC 3’，5’CACTCG AGTTAAGATGGAGTTTTGGAGCC 3’。
用人的EGFR基因胞外区段构建和制备DNA疫苗免疫Lewis肺癌的模型小鼠，发现免疫后的第8周，注射人EGFR疫苗的小鼠生存率为78％，明显高于注射过小鼠EGFR疫苗(25％)和对照实验动物(10％～15％)，同时并未发现模型小鼠的肺、肝、心、肾等有病理性改变，进一步的研究表明诱导的自体免疫反应主要依赖于CD4+T淋巴细胞，CTL活性检测没有发现靶细胞特异性的杀伤效应。免疫组化结果显示肿瘤组织有自身抗体沉着，而肺、肝等组织则没有，自身反应抗体主要为IgG。
以EGFR分子为模板设计的各种反义RNA和RNAi可看作是EGFR重组基因疫苗的特例，它主要不是通过提高EGFR分子的免疫原性，诱导产生抗EGFR抗体和特异性的CTL，进而阻断EGFR信号通路，诱导肿瘤细胞凋亡，抑制肿瘤细胞的生长和扩散，而是在DNA和RNA水平上直接抑制和阻断EGFR分子的表达。
三、EGFR重组蛋白疫苗蛋白疫苗是比较传统形式的疫苗，但蛋白质有较好的免疫原性。本发明的EGFR分子疫苗之一就是重组蛋白疫苗，包括以各种大肠杆菌重组表达载体、酵母重组表达载体、棒状病毒重组表达载体等表达系统表达出的重组蛋白所构建而成的疫苗。
利用分子克隆技术(如PCR、RT-PCR、杂交等技术)从各种商品化的基因文库、cDNA文库或各种细胞系、组织等中克隆分离出不同种属生物来源的EGFR分子的胞外区段的cDNA，或进一步用基因直接进化技术筛选到的具有强免疫原性的EGFR分子，然后用分子克隆技术构建其原核生物表达质粒系统，转化合适的大肠杆菌宿主，观察并检测其表达EGFR的情况和水平。各重组EGFR分子的表达质粒可经过限制性酶切分析、SDS-PAGE和Western Blot等进行鉴定、确证。经确证的重组EGFR分子原核表达质粒转化E.coli，大量培养重组菌落，低温离心收集菌体，菌体经PBS重悬后用超声法破碎细胞，用离子交换层析、亲和层析等方法分离纯化重组EGFR蛋白，此重组的EGFR蛋白质即可作为蛋白疫苗进行免疫。
E.coli表达的重组EGFR蛋白疫苗的代表性质粒图谱如图2B所示，其具体构建过程简要叙述如下依据GenBank等各公开的数据库所收藏的人、小鼠、鸡、的EGFR分子的cDNA序列(分别对应于SEQ ID NO 1-5，7-9，19)设计PCR引物(人的引物为5’GACCATGGAGGAAAAGAAAGTTTGC 3’，5’ACAGATCTAGGACGGGATCTTAGGCCCA 3’；小鼠的引物为5’GACCATGGAGGAAAAGAAAGTCTGC 3’，5’ACAGATCTATAGATGGTATCTTTGGC 3’；鸡的引物为5’GACCATGGAGGAGAAGAAAGTTTGTC 3’，5’ACAGATCTAGATGGAGTTTT GGAGCC 3’)，分别以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等为模板进行PCR扩增，电泳收集纯化扩增的EGFR片段(均为1.9kb)，然后进行PCR产物亚克隆。把PCR亚克隆测序确证后，用NcoI和BglII酶切，收集1.9kb片段并纯化，插入到用NcoI和BglII双酶切的pQE60(QIAGEN公司)载体，筛选重组质粒。候选的重组质粒经限制性酶切分析及PCR扩增等双重鉴定，命名为pQE-hEGFR、pQE-mEGFR和pQE-chEGFR。
除上述利用大肠杆菌重组表达系统表达各种来源的EGFR分子外，还可以利用酵母重组表达系统、棒状病毒重组表达系统等表达EGFR重组蛋白，从而制备重组蛋白疫苗。EGFR重组蛋白疫苗的构建流程图见图3。
酵母表达的重组EGFR蛋白疫苗的代表性质粒图谱如图2C所示，其具体构建过程简要叙述如下依据GenBank等各公开的数据库所收藏的人、小鼠、鸡、的EGFR分子的cDNA序列(分别对应于SEQ ID NO 1-5，7-9，19)设计PCR引物(人的引物为5’ATACTCGAGAAAAGAGAGCTGGAGGAAAAGAAAG 3’，5’GCTCTAGAATGGCACAGGTGGCACA 3’；小鼠的引物为5’ATGCTCGAGAAAAGAGAGTTGGAGGAAAAGAAAGTC 3’，5’AAGCGGCCGCCATAGATGGTATCTTTG 3’；鸡的引物为5’ATACTCGAGAAA AGAGAGGTGGAGGAGAAGAAAG3’，5’CGTCTAGAAGATGGAGTTTTGGAG 3’)，分别以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等为模板进行PCR扩增，电泳收集纯化扩增的EGFR片段(均为1.9kb)，然后进行PCR产物亚克隆。把PCR亚克隆测序确证后，用XhoI和XbaI(对小鼠的克隆为XhoI和NotI)酶切，收集1.9kb片段并纯化，插入到用XhoI和XbaI双酶切(对小鼠的克隆为XhoI和NotI双酶切)的pPICZ A(Invitrogen公司)载体，转化E.coli筛选重组质粒。候选的重组质粒经限制性酶切分析及PCR扩增等双重鉴定，命名为酵母表达质粒pYE-hEGFR、pYE-mEGFR和pYE-chEGFR。这些酵母表达质粒经PmeI酶切线性化后，用电穿孔法转化酵母菌株X33或GS115，用Zeocin抗性筛选稳定转化子，在MMH(Minimal Methanolwith histidine，MMH)和MDH(Minimal Dextrose with histidine，MDH)琼脂平板上鉴定和挑选Mut+转化子。挑选6-10个Mut+转化子进行小规模表达，用SDS-PAGE、Western Blot、ELISA等方法对所表达的重组蛋白进行鉴定。选择其中表达效率最高的Mut+转化子做大规模表达，建立各级酵母表达种子库。重组菌株大量摇瓶培养或发酵，低温离心收集菌体，菌体经PBS重悬后用超声法破碎细胞，用离子交换层析、亲和层析等方法分离纯化重组EGFR蛋白，此重组的EGFR蛋白质即可作为蛋白疫苗进行免疫。
同样地，也可用其他的酵母表达系统构建类似的酵母重组表达质粒。
重组蛋白疫苗具有比DNA疫苗更强的诱导免疫交叉反应的作用，产生高滴度的抗EGFR抗体以及特异性的CTL，从而抑制肿瘤细胞的生长和转移。
四、EGFR重组病毒疫苗重组病毒也是一种良好的分子疫苗转运系统，这些重组病毒表达系统构建的分子疫苗包括重组腺病毒疫苗、Lentivirus疫苗、腺病毒相关病毒疫苗、逆转录病毒疫苗、痘苗病毒疫苗和单纯疱疹病毒疫苗。
腺病毒载体是目前在肿瘤基因治疗中最有效的载体之一，它具有滴度高、安全性好、能感染分裂或不分裂细胞、不整合插入染色体等优点。此外，腺病毒还具有较强的免疫原性，这对于基因治疗也许是一个缺点，但在基因免疫治疗中却可能是一个很大的优点。重组腺病毒疫苗是EGFR重组病毒疫苗最重要的一种。同前文所述，首先克隆出各种同种、异种或基因直接进化的EGFR cDNA，然后用分子生物学技术构建其重组腺病毒表达载体，转染293细胞，即得到重组的腺病毒。重组的腺病毒经PCR、Western Blot等加以确证。利用293细胞大量扩增经确证的EGFR重组腺病毒疫苗，用超离心、超滤等技术分离、纯化重组腺病毒，此经纯化的EGFR重组腺病毒即可作为疫苗进行免疫。EGFR重组腺病毒疫苗的构建流程示意图见图4。
EGFR重组腺病毒是利用AdEasy系统构建而成的，其具体构建过程简要叙述如下依据GenBank等各公开的数据库所收藏的人、小鼠、鸡、的EGFR分子的cDNA序列(分别对应于SEQ ID NO 1-5，7-9，19)设计PCR引物(人的引物为5’GAAGATCTATGGAGGAAAAGAAAGTTTGC 3’，5’ACGATATCTTAAGGACGGGATCTTAGGCCCA 3’；小鼠的引物为5’GAAGATCTATGGAGGAAAAGAAAGTCTGC 3’，5’ACGATATCTTAATAGATGGTATCTTTGGC 3’；鸡的引物为5’GAAGATCTATGGAGGAGAAGAAAGTTTGTC 3’ ，5’ACGATATCTTAAGATGGA GTTTTGGAGCC 3’)，分别以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等为模板进行PCR扩增，电泳收集纯化扩增的EGFR片段(均为1.9kb)，然后进行PCR产物亚克隆。把PCR亚克隆测序确证后，用BglII和EcoRV酶切，收集1.9kb片段并纯化，插入到用BglII和EcoRV双酶切的pShuttle-CMV(Quantum Biotechnologies公司)载体，筛选重组质粒。候选的重组质粒经限制性酶切分析及PCR扩增等双重鉴定，命名为腺病毒穿梭表达质粒pShuttle-hEGFR、pShuttle-mEGFR和pShuttle-chEGFR(图2D)。分别将经PmeI酶切的腺病毒穿梭表达载体pShuttle-EGFR与包含腺病毒基因组的骨架载体pAdEasy-1或pAdEasy-2共转化E.coli BJ5183，得到重组腺病毒载体质粒pAd-hEGFR、pAd-mEGFR和pAd-chEGFR。这些重组腺病毒载体质粒经PacI酶切后，用磷酸钙-DNA共沉淀法转染入腺病毒包装细胞株293细胞中，得到相应的重组腺病毒Ad-hEGFR、Ad-mEGFR和Ad-chEGFR。用PCR、Western blot等确证EGFR基因构建入重组腺病毒载体中并能在真核细胞中得到正确有效的表达。
根据EGFR重组腺病毒疫苗中所包含腺病毒基因组的不同，又有EGFR重组I代腺病毒(腺病毒穿梭表达质粒pShuttle-EGFR与pAdEasy-1的重组体，命名为Ad-hEGFR I、Ad-mEGFR I和Ad-chEGFR I)疫苗和EGFR重组II代腺病毒(腺病毒穿梭表达载体pShuttle-EGFR与pAdEasy-2的重组体，命名为Ad-hEGFR II、Ad-mEGFR II和Ad-chEGFR II)疫苗之分。
此外，EGFR重组腺病毒疫苗还可以用甘露聚糖(Mannan)等进行靶向修饰。
EGFR重组腺病毒疫苗由于腺病毒良好的基因导入性，能有效诱导机体产生抗肿瘤免疫反应，抑制EGFR高表达肿瘤的生长。
Lentivirus病毒载体是新一代的基因治疗载体，它来源于HIV-1的复制缺陷型lentivirus，与传统的来源于的莫洛尼氏白血病毒(Moloney Leukemia Virus，MoMLV)的逆转录病毒载体不同，它对处于分裂和不分裂状态的哺乳动物细胞都能进行有效转染，且生物安全性更好。此外，与腺病毒载体不同的是，Lentivirus病毒载体能整合到细胞染色体中，从而使导入的外源基因获得稳定、长久的表达。重组Lentivirus病毒疫苗也是EGFR重组病毒疫苗较重要的一种。同前文所述，首先克隆出各种同种、异种或基因直接进化的EGFR cDNA，然后用分子生物学技术构建其重组Lentivirus表达载体，转染293FT细胞，即得到重组的Lentivirus病毒。重组的Lentivirus病毒经PCR、Western Blot等加以确证。利用293FT细胞大量扩增经确证的EGFR重组Lentivirus病毒疫苗，用超离心、超滤等技术分离、纯化重组Lentivirus病毒，此经纯化的EGFR重组Lentivirus病毒即可作为疫苗进行免疫。EGFR重组Lentivirus病毒疫苗的构建流程示意图见图4B。
EGFR重组Lentivirus病毒是利用ViraPowerTMLentiviral GatewayExpressionKit(Invitrogen公司)构建而成的，其具体构建过程简要叙述如下依据GenBank等各公开的数据库所收藏的人、小鼠、鸡、的EGFR分子的cDNA序列(分别对应于SEQ ID NO 1-5，7-9，19)设计PCR引物(人的引物为5’GACCATGGAGGAAAAGAAAGTTTGC 3’，5’ACGATATCAGGACGGGATCTTAGGCCCA 3’；小鼠的引物为5’GACCATGGAGGAAAAGAAAGTCTGC 3’，5’ACGATATCATAGATGGTATCTTTGGC 3’；鸡的引物为5’GACCATGGAGGAGAAGAAAGTTTGTC 3’，5’ACGATATCAGATGGAGTTTTGGAGCC 3’)，分别以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等为模板进行PCR扩增，电泳收集纯化扩增的EGFR片段(均为1.9kb)，然后进行PCR产物亚克隆。把PCR亚克隆测序确证后，用NcoI和EcoRV酶切，收集1.9kb片段并纯化，插入到用NcoI和EcoRV双酶切的pENTR11(Invitrogen公司)载体中，筛选重组质粒，命名为pENTR-hEGFR、pENTR-mEGFR和pENTR-chEGFR(图2E)。分别将这些载体pENTR-EGFR与包含Lentivirus病毒基因组的骨架载体pLenti6/V5-DEST共转化E.coli DH5，得到重组Lentivirus病毒载体质粒pLenti-hEGFR、pLenti-mEGFR和pLenti-chEGFR(图2E)。这些重组Lentivirus病毒载体质粒与包装混合物一起(ViraPowerTMPackagingMix)用磷酸钙-DNA共沉淀法转染入Lentivirus病毒包装细胞株293FT细胞中，得到相应的重组Lentivirus病毒Lenti-hEGFR、Lenti-mEGFR和Lenti-chEGFR。用PCR、Western blot等确证EGFR基因构建入重组Lentivirus病毒载体中并能在真核细胞中得到正确有效的表达。
五、EGFR分子疫苗的修饰和改进前文的重组EGFR分子疫苗(包括重组EGFR基因疫苗、蛋白疫苗和病毒疫苗)只是基本形式，本发明还在高效性、特异性等方面有许多修饰和改进，这些改进包括1.EGFR分子疫苗佐剂的选择合适的佐剂是有效提高疫苗的免疫效应的良好载体，不同的疫苗形式有不同的佐剂。本发明EGFR DNA疫苗的佐剂主要是福氏佐剂和脂质体，EGFR蛋白疫苗的佐剂主要是铝盐，EGFR重组病毒疫苗通常不使用佐剂。
2.EGFR基因修饰疫苗基因修饰疫苗是指转染了人或其他异种生物的EGFR分子的细胞疫苗，包括使用稳定转化的各种肿瘤细胞株、血管内皮细胞和树突状细胞制备的细胞疫苗、用各种重组病毒感染的肿瘤细胞或肿瘤组织制备的疫苗。EGFR基因修饰疫苗是EGFR分子疫苗的一种特例形式。
前文已描述多种肿瘤包括肺癌、乳腺癌、卵巢癌、结肠癌、前列腺癌、胃癌、膀胱癌、头颈部鳞癌和胶质瘤等都高表达EGFR，但这些肿瘤均对EGFR耐受，并不产生抗EGFR的免疫反应。但是，利用基因修饰疫苗可打破免疫耐受，产生抗EGFR免疫反应。用重组的各种来源的EGFR分子的真核表达质粒转染各种肿瘤细胞株(如肺癌细胞株A431、乳腺癌细胞株MCF7等)、肿瘤血管内皮细胞等，筛选稳定表达EGFR分子的转化株，收获稳定转化株，用副甲醛固定法等制备细胞疫苗。此外，也可以用各种来源的EGFR构建的各种EGFR重组病毒疫苗感染各种肿瘤细胞株、肿瘤组织和肿瘤血管内皮细胞等，用副甲醛固定法等制备细胞疫苗。
3.EGFR稳定转化共生菌EGFR分子疫苗还有活体疫苗形式，这就是EGFR稳定转化共生菌。
用各种来源的(包括自体的、异体的和基因直接进化的)EGFR构建的原核生物重组表达质粒，转化肠道共生菌，如双岐杆菌等，筛选其稳定表达菌株。这些稳定表达的肠道共生菌菌株能持续分泌外源EGFR分子，诱导机体免疫反应，是一种活体形式的EGFR疫苗，它是EGFR分子疫苗的特例。
4.EGFR分子疫苗的靶向纳米粒制剂单纯的重组EGFR分子疫苗(包括重组EGFR基因疫苗、蛋白疫苗和病毒疫苗)依然有它的缺陷，其特异性不够，免疫原性仍有待提高。本发明利用纳米生物技术对EGFR分子疫苗进行靶向修饰，制备EGFR分子疫苗的靶向纳米粒制剂。这些靶向纳米粒以EGFR分子(包括DNA和蛋白质形式)为抗原靶分子，以生物性分子如脂质体、可降解性高分子生物材料如聚丙交酯-乙交酯(polyDL-lactide-co-glycolide polymer，PLGA)等为纳米材料，以MIP-3、MIP-3等基因以及甘露聚糖(Mannan)、Flt3-L等为修饰基团，以树突状细胞为靶细胞构建而成。
本发明的EGFR分子疫苗靶向纳米粒制剂有多种形式，主要包括①甘露糖化纳米粒(包括甘露糖化腺病毒重组EGFR疫苗、甘露糖化重组EGFR蛋白疫苗、甘露糖化脂质体重组EGFR基因疫苗、甘露糖化重组EGFR蛋白疫苗等)，②基因靶向纳米粒(基因靶向纳米脂质体重组EGFR疫苗、基因靶向纳米PLGA重组EGFR疫苗、基因靶向重组EGFR腺病毒疫苗等)，它们同时表达EGFR和MIP-3等基因，③肿瘤新生血管靶向腺病毒重组EGFR疫苗(RGD修饰的腺病毒重组EGFR疫苗)。
本发明的纳米粒粒径通常在500nm以下，可分为三种规格200-500nm、100-200nm和50-100nm，以50-100nm粒径的纳米粒的效果为最佳，纳米粒峰值在80nm左右。
甘露糖化腺病毒重组EGFR疫苗的制备过程简要描述如下用常规方法扩增EGFR重组腺病毒(I代、II代均可)，层析或超离心法纯化重组腺病毒。将70mgmannan(sigma)溶于5ml 0.1M的磷酸盐缓冲液(pH6.0)中，终浓度14mg/ml，加45ml 0.01M高碘酸钠溶液，在4℃下混合氧化60分钟，加入10l乙二醇，在4℃下孵育30分钟，即得Ox-M(Oxidative Mannan，Ox-M)混合物。将Ox-M混合物倒入用重碳酸盐缓冲液(pH6.0-9.0)平衡Sephadex-G25层析柱进行层析分离，OX-M即被洗入2ml的空容器。将纯化的Ox-M与1×1014腺病毒颗粒混合，室温过夜，即获得所需Ox-M-腺病毒。在Ox-M-腺病毒中加入1mg/ml硼氢化钠，室温放置3小时即得Red-M-腺病毒(Reductive Mannan，Red-M)。Ox-M-腺病毒与Red-M-腺病毒经超滤脱盐、浓缩后，过滤细菌，小管分装，-80℃低温保存。此经纯化的甘露糖化重组EGFR腺病毒即可作为疫苗进行免疫。
本发明认为腺病毒颗粒的粒径在80nm左右，是天然的纳米粒。对腺病毒颗粒进行修饰，使腺病毒纤维蛋白(Adenoviral fiber protein)表达RGD三肽，该三肽对肿瘤血管内皮细胞有特异靶向性。用RGD修饰的腺病毒重组EGFR疫苗，可看作是天然的靶向纳米EGFR分子疫苗。
本发明利用AdEasy系统构建RGD修饰腺病毒的重组EGFR疫苗，图5显示了RGD修饰的腺病毒重组EGFR疫苗的构建流程，其具体过程是腺病毒基因组骨架质粒pAdEasy-1和pAdEasy-2经限制性内切酶SpeI(Sp)酶切后，用T4 DNA聚合酶补平(filling，f)末端，再用PacI(P)酶切，分别电泳回收6211bp和3579bp的片段，分别命名为AdFiber I/Sp/f/P和AdFiber II/Sp/f/P，该片段包含了完整的腺病毒纤维蛋白基因。把AdFiber I/Sp/f/P和AdFiber II/Sp/f/P片段插入经BamHI酶切-T4 DNA聚合酶补平-PacI酶切处理(BamHI/filling/PacI-digested)的pShuttle载体，将所得的重组质粒分别命名为pSh-AdFiber I和pSh-AdFiber II。pSh-AdFiberI用NheI酶切-T4 DNA聚合酶补平-KpnI酶切处理(NheI/filling/KpnI)，电泳回收2090bp的片段AdFiber I/Nh/f/K，将该片段插入到经SmaI/KpnI双酶切的pUC18载体中，所得的重组质粒命名为pUC-AdFiber I；而pSh-AdFiber II用AvrII酶切-T4 DNA聚合酶补平-HindIII酶切处理(AvrII/filling/HindIII)，电泳回收838bp的片段AdFiber I/A/f/H，将该片段插入到经SmaI/HindIII双酶切的pUC18载体中，所得的重组质粒命名为pUC-AdFiberII。设计一系列PCR引物以便以pUC-AdFiberI和pUC-AdFiberII为模板扩增腺病毒疣足(Adenovirus knob，Ad-knob)基因序列，引物分别是F1(5’GAAAGCTAGC CCTGCAAACATCA 3’)、R1( 5’ACTCCCGGGAGTTGTGTCTCCTGTTTCCTG3’)、F2(5’ACTCCCGGGAGTGC ATACTCTATGTCA 3’)、R2(5’TATGGTACCGGGAGGTGGTGA 3’)、F3(5’AACCTAGGGAGGTTAACCTAAGCACTG 3’)、和R3(5’CTCAAGCTTTTTGG AATTGTTTGA 3’)。以引物F1-R1、F2-R2、F3-R1和F2-R3分别进行第一轮PCR，得到产物PCR1、PCR2、PCR3和PCR4，再以F1-R2和F3-R3为引物，以第一次扩增产物PCR1与PCR2、PCR3与PCR4为模板进行第二轮PCR扩增，得到PCR产物PCR1-PCR2(PCR I)、PCR3-PCR4(PCR II)，将第二轮PCR扩增的PCR I和PCR II插入到经EcoRV酶切的pBR322载体中，所得到的重组质粒命名为pBR-PCR I和pBR-PCR II。把RGD-4C双螺旋寡聚核苷酸(RGD-4C duplex)5’TGTGACTGCCGCGGAGACTGTTTCTGC 3’3’ACACTGACGGCGCCTCTGACAAAGACG 5’插入到SmaI酶切的pBR-PCR I and pBR-PCR II载体中，将所得到的重组质粒命名为pBR-PCR/RGD I和pBR-PCR/RGD II，并对重组结构进行测序确证。用NheI/KpnI双酶切pBR-PCR/RGD I，电泳回收PCR/RGD I片段，然后插入到NheI/KpnI双酶切的pUC-AdFiber I载体中，所得的重组质粒命名为pUC-AdFiber-RGD I；用AvrII/HindIII双酶切pBR-PCR/RGD II，电泳回收PCR/RGDII片段，再插入到AvrII/HindIII双酶切的pUC-AdFiber II载体中，将所得到的重组质粒命名为pUC-AdFiber-RGD II。然后，用SpeI/PacI双酶切pUC-AdFiber-RGDI和pUC-AdFiber-RGD II载体，电泳回收AdFiber-RGD I、AdFiber-RGD II片段，插入到SpeI/PacI双酶切的pAdEasy-1、pAdEasy-2载体中，所得的重组质粒分别命名为pAdEasy-RGD.I、pAdEasy-RGD II。将经PmeI线性化的腺病毒穿梭质粒pShuttle-hEGFR、pShuttle-mEGFR和pShuttle-chEGFR分别与pAdEasy-RGD I和pAdEasy-RGD II共转化E.coli BJ5183，所得的重组质粒命名为腺病毒质粒pAd-RGD-EGFR I和pAd-RGD-EGFR II，腺病毒质粒pAd-RGD-EGFR I转染293细胞，所得的重组腺病毒命名为Ad-RGD-EGFR I，腺病毒质粒pAd-RGD-EGFR II转染293E4pIX细胞，所得的重组腺病毒命名为Ad-RGD-EGFR II。经纯化的Ad-RGD-EGFR I和Ad-RGD-EGFR II可作为疫苗进行免疫，对肿瘤血管内皮细胞具有特异的靶向性。
本发明发现纳米靶向的EGFR分子疫苗能比各种常规的EGFR分子疫苗更有效地提高EGFR的免疫原性，诱导更强烈的抗肿瘤免疫反应。纳米靶向EGFR分子疫苗抗肿瘤作用原理示意图见图6。
5.EGFR分子疫苗与其它免疫反应促进因子等的协同应用EGFR分子疫苗的抗肿瘤作用效应可在其它免疫促进因子的协同作用下得到增强，这些免疫促进因子包括各种细胞因子(cytokine，如IL-2、TNF-、IFN-、GM-CSF等)、各种趋化因子(Chemokine，如MIP3、MIP3、IP10等)、各种应急因子(如HSP70、HSP90等)、各种免疫刺激因子(如B7等)。各种免疫促进因子与EGFR分子疫苗的协同作用既可以通过基因融合在基因水平上实现，也可以通过蛋白质联合应用在蛋白质水平上实现，还可以通过共转染肿瘤细胞、树突状细胞和血管内皮细胞等在细胞水平上实现。
六、EGFR分子疫苗的抗肿瘤作用效应EGFR分子疫苗的重要生物学功能就是以同种、异种或直接进化的EGFR分子为抗原分子所构建的各种新生物技术疫苗，包括蛋白疫苗、基因疫苗、病毒疫苗和基因修饰疫苗等，都具有抗肿瘤作用，这些抗肿瘤作用表现为保护性抗肿瘤作用、治疗性抗肿瘤作用以及抗肿瘤转移作用。
为观察EGFR重组DNA疫苗的抗肿瘤效应，给随即分组的小鼠(每组15只)分别肌肉注射100g的EGFR重组DNA疫苗hEe-p、mEe-p、c-p或生理盐水，每周一次，连续四周。在第四次免疫结束后一周，给每只免疫小鼠分别皮下接种5×105个LL/2c Lewis肺癌(图7A和C)或MA782/5S乳腺癌细胞(图7B和D)。从图中可以看出，用mEe-p、c-p或生理盐水免疫小鼠的肿瘤持续生长，而hEe-p免疫小鼠的保护性免疫效应则非常明显，并且hEe-p免疫小鼠的生存率也明显高于mEe-p、c-p或生理盐水免疫小鼠，hEe-p免疫的小鼠生存期超过5个月，其在荷瘤150天后，接种LL/2c Lewis肺癌和MA782/5S乳腺癌的生存率分别有60％和66％。研究还发现，保护性免疫效应呈剂量依存效应，150g剂量的免疫效果与100g的免疫效果相当，而5-15g的剂量则几乎没有免疫效果。此外，hEe-p免疫EGFR阴性肿瘤(如H22肝癌和MMT-06052鼠乳腺癌)的荷瘤小鼠没有保护性免疫效应。
除保护性免疫效应外，EGFR重组DNA疫苗还具有治疗性免疫效应(图8)。同样地，给随即分组的小鼠(每组15只)首先皮下接种1 106个LL/2c Lewis肺癌(图8A和C)或MA782/5S乳腺癌活细胞(图8B和D)，5天后分别肌肉注射100g的hEe-p、mEe-p、c-p或生理盐水，每周一次，连续四周。从图中可以看出，用mEe-p、c-p或生理盐水免疫小鼠的肿瘤持续生长，而hEe-p免疫小鼠的治疗性免疫效应则非常明显，并且hEe-p免疫小鼠的生存率也明显高于mEe-p、c-p或生理盐水免疫小鼠，hEe-p免疫的小鼠生存期超过5个月，其在荷瘤150天后，接种LL/2c Lewis肺癌和MA782/5S乳腺癌的生存率分别有40％和53％。
重组EGFR蛋白疫苗也同样有保护性免疫效应和治疗性免疫效应(图9)。同前文所述，选择6-8周龄雌性C57BL/6或BALB/c小鼠，随机分组，建立LL/2c Lewis肺癌、MA782/5S乳腺癌和C26结肠癌荷瘤小鼠模型。荷瘤小鼠皮下注射重组蛋白疫苗5-50μg或佐剂氢氧化铝磷胶及生理盐水100μl，每周一次，连续四周。重组chEGFR蛋白疫苗免疫小鼠产生了明显的抗肿瘤保护性免疫效应和治疗性免疫效应，可抑制LL/2c Lewis肺癌和MA782/5S乳腺癌的生长，延长荷瘤小鼠的生存时间，但对EGFR阴性的C26肿瘤无明显的影响。而用重组mEGFR蛋白疫苗、铝佐剂免疫或注射生理盐水等对照组的荷瘤小鼠的肿瘤则生长迅速，小鼠生存期明显缩短。疫苗组肿瘤体积(t检验)和生存期(log-rank检验)与各对照组比较均有显著差异(P＜0.05)。图9就显示了重组EGFR蛋白疫苗对MA782/5S乳腺癌荷瘤小鼠的抗肿瘤作用效果。
转移是肿瘤进展以及放化疗失败的常见原因，肿瘤细胞在血液、淋巴循环中的存在以及微小转移灶的形成是转移的关键。本发明继续观察到重组EGFR分子疫苗还有抗肿瘤转移作用(图10)。在尾静脉注射转移模型的治疗中发现，chEGFR蛋白疫苗免疫的荷瘤小鼠较少发生肺部转移或程度远较对照组轻，而重组mEGFR蛋白疫苗、铝佐剂免疫或注射生理盐水等对照组的荷瘤小鼠则100％出现肺部转移，且转移病灶的个数明显为多，甚至无法计数。图10显示了LL/2c Lewis肺癌荷瘤小鼠经重组EGFR蛋白疫苗免疫的抗肿瘤转移效应，从图中可以看出，各组治疗结束时，chEGFR蛋白疫苗免疫小鼠的LL/2c移植瘤肺部转移灶数量和肺湿重均显著较对照组为轻。
此外，EGFR分子疫苗还能抑制体外(in vitro)肿瘤细胞的生长及具有体内(invivo)过继免疫抗肿瘤效应(图11)。EGFR分子疫苗第四次免疫7天后，收集小鼠血清，用亲和层析法纯化血清中的免疫球蛋白(immunoglobulin，Ig)。向2×105/ml的处于对数生长期的EGFR阳性肿瘤细胞(A549，LL/2c，MA782/5S)和EGFR阴性肿瘤细胞(H22和MMT-06052)中加入不同浓度的Ig(1-1000mg/ml)共培养72小时，用台盼蓝染色法鉴定活细胞，计算细胞生长抑制率。结果显示用人EGFR重组DNA疫苗hEe-p免疫小鼠来源的Ig处理的EGFR阳性肿瘤细胞呈现明显的抑制生长状态，而EGFR阴性的肿瘤细胞则没有影响(图11A)。作为对照，用未免疫的正常小鼠来源的Ig与相应的肿瘤共培养，无论对EGFR阳性肿瘤细胞还是EGFR阴性肿瘤细胞都没有抑制作用(图11B)。
这些纯化的来源于免疫小鼠的Ig还具有过继免疫作用效应。裸鼠皮下接种1×105-1×106个肿瘤细胞1天后，按10-300mg/kg静脉输注纯化的Ig，每周二次，连续三周。结果显示过继输入的来源于人EGFR重组DNA疫苗hEe-p免疫小鼠的Ig具有显著的肿瘤抑制作用。作为对照，把纯化的Ig与固定的EGFR阳性肿瘤细胞或EGFR阴性肿瘤细胞在4℃下振荡1小时以吸附Ig，共重复4次。结果发现来源于人EGFR重组DNA疫苗hEe-p免疫小鼠的Ig的抗肿瘤效应由于被EGFR阳性肿瘤细胞(LL/2c，MA782/5S)预吸附而消除，但对EGFR阴性肿瘤细胞(H22)仍然有效(图11C)。
除上述过表达EGFR的肺癌、乳腺癌外，EGFR分子疫苗对其他各种过表达EGFR的实体瘤，包括卵巢癌、结肠癌、前列腺癌、胃癌、膀胱癌、头颈部鳞癌和胶质瘤等，也有较好的抗肿瘤作用效应。
七、EGFR分子疫苗的抗肿瘤作用原理及生物安全性现代免疫学认为，抗原或抗原表位与相应受体间的相互识别除特异性外，还存在一定程度的伸展性(plasticity)、是混杂性(promiscuity)或退变性(degeneracy)，从而通过分子模拟(molecular mimicry)机制诱导针对自身抗原的免疫交叉反应，打破免疫耐受。
本发明发现EGFR分子作为一种免疫交叉抗原，能打破机体对自身EGFR分子的免疫耐受，诱导机体产生针对EGFR分子自身免疫样交叉反应，这些免疫反应包括针对EGFR分子的主动免疫反应(包括细胞免疫反应和体液免疫反应)和被动免疫反应(过继免疫反应)。
为了研究EGFR分子疫苗的抗肿瘤作用原理，我们首先对EGFR分子疫苗免疫小鼠进行体液免疫检测，包括应用流式细胞术、Western印迹、免疫沉淀等验证小鼠/兔免疫血清中抗EGFR自身抗体的存在，应用ELISA检测小鼠/兔免疫血清中抗EGFR抗体的滴度及抗体类型，应用免疫组织化学法检测免疫小鼠肿瘤组织内的自身抗体，应用肿瘤细胞成集落实验、血清过继免疫实验检测自身抗体的功能等。
每只小鼠在免疫前及免疫后每周经鼠尾静脉采血或处死小鼠时取血，收集血清备用。Western Blot检测表明重组EGFR分子疫苗免疫诱导的抗体可以特异识别相应的免疫原(重组EGFR蛋白或表达于肿瘤细胞上的EGFR)，但不能识别EGFR阴性细胞。同时，用流式细胞仪对所获得的抗体空间表位进行识别，也发现重组EGFR分子疫苗能诱导产生特异的抗体，可以识别表达于肿瘤细胞表面的EGFR。ELISA自身抗体测定结果显示，在初次免疫2周后重组EGFR分子疫苗免疫小鼠开始产生抗小鼠自身EGFR抗体，滴度可达1∶100至1∶5000，以后逐渐升高，至第4周抗体滴度可达1∶10000至1∶500000，到第8周抗体滴度仍能维持在1∶500至1∶1000，而相应的对照组则检测不到明显的抗体产生。对所产生的抗体亚型作进一步检测，发现EGFR分子疫苗产生的抗体以IgG为主。图12显示了EGFR分子疫苗(重组蛋白疫苗和重组DNA疫苗)免疫小鼠所诱导产生的抗体类型，从图中可以看出，重组EGFR分子疫苗明显诱导产生了IgG1、IgG2a和IgG2b等抗体，但IgM和IgA并没有升高，并且这些抗体可被CD4抗体阻断，但不能被抗CD8、抗NK或对照抗体所阻断，而其他对照组蛋白疫苗、佐剂和生理盐水组则不能检测到特异抗体。
EGFR分子疫苗还诱导了细胞免疫。对EGFR分子疫苗免疫的小鼠进行的细胞免疫检测主要包括应用51Cr释放法测定CTL活性，应用ELISPOT检测细胞因子水平(主要是免疫血清中IFN-γ和IL-4浓度)，应用免疫细胞去除(Depletion ofimmune cell subsets)实验检测T细胞类型(CD4+T淋巴细胞、CD8+T淋巴细胞或NK细胞等)。
重组chEGFR疫苗免疫小鼠后，取脾单个核细胞作ELISPOT检测，发现脾单个核细胞中存在大量抗原特异B细胞，而经重组mEGFR疫苗免疫后也能发现少量特异B细胞的存在，但空白组及佐剂或空载体组未能发现有统计意义的特异B细胞。重组EGFR分子疫苗免疫小鼠3周后，取免疫小鼠脾T细胞，并用健康脾单个核细胞为抗原呈递细胞加以免疫原在体外再刺激扩增可能已活化T细胞。结果显示经重组chEGFR分子疫苗免疫后均含有较多量的IFN-及IL-4产生细胞，而重组mEGFR分子疫苗在体外再刺激源于chEGFR疫苗免疫小鼠的脾T细胞也含有较多的IFN-及IL-4产生细胞，明显多于同等条件下未体外刺激的T细胞。进一步以标准的51Cr释放法测定了重组EGFR分子疫苗免疫后脾T细胞对鼠、人等相关肿瘤细胞的特异杀伤活性。结果显示重组chEGFR分子疫苗免疫后的小鼠脾T细胞在40∶1时对EGFR阳性表达LL/2c、MA781/5S肿瘤细胞的特异杀伤活性分别达40.27％、42.83％，但对重组mEGFR分子疫苗免疫的小鼠脾T细胞对MA781/5S或LLC肿瘤细胞、以及EGFR阴性C26细胞未发现杀伤活性，同时这种作用可被相应抗CD8及MHC-I单克隆抗体阻断，而不能被抗CD4及MHC-II单克隆抗体阻断，其他对照组脾T细胞则未检测到有统计意义的杀伤活性。图13显示了EGFR重组DNA疫苗免疫小鼠所表现出的CTL效应，从图中可以看出人EGFR重组DNA疫苗hEe-p免疫小鼠来源的T细胞比其他对照组来源的T细胞对EGFR阳性的肿瘤细胞有着更高的细胞毒性，并且这种细胞毒性在体外可被抗CD8或抗MHC I单抗所阻断，但不能被抗CD4单抗所阻断，显示这种细胞杀伤活性来源于MHC I依赖的CD8+T细胞。另外，激活的脾细胞对YAC-1靶细胞没有显示出NK活性的增加。此外，过继输注人EGFR重组DNA疫苗hEe-p免疫小鼠来源的CD4剔除(CD8+)或CD8剔除(CD4+)T细胞显示出对EGFR阳性肿瘤细胞的抗肿瘤作用，但对同源的EGFR阴性肿瘤细胞的抗肿瘤作用不明显，而相应的对照组也未显示出抗肿瘤作用效果。
本发明还对EGFR分子疫苗免疫小鼠的潜在长期毒性进行了观察。研究中没有观察到诸如小鼠体重减轻、皮毛皱缩、食欲减退、寿命缩短等明显的毒副作用。通过对免疫小鼠的肝、肺、脾、脑等器官的显微病理检查，没有发现病理变化，并且免疫荧光染色也未在主要脏器中发现自身抗体沉着(图14)。


图1 同源分子疫苗的作用原理示意2 重组EGFR质粒图谱A.重组EGFR真核表达质粒图谱；B.重组EGFR原核表达质粒图谱；C.重组EGFR酵母表达质粒图谱；D.EGFR重组腺病毒穿梭质粒图谱；E.EGFR重组Lentivirus病毒前体质粒图谱图3 EGFR重组蛋白疫苗的构建流程示意4 EGFR重组病毒疫苗的构建流程示意图A.EGFR重组腺病毒疫苗的构建流程示意图；B.EGFR重组Lentivirus病毒疫苗的构建流程示意5 RGD修饰的腺病毒重组EGFR疫苗构建流程示意6 纳米靶向EGFR分子疫苗抗肿瘤作用原理示意7.重组EGFR DNA疫苗的抗肿瘤保护性免疫效应A.LL/2c Lewis肺癌荷瘤的免疫小鼠的肿瘤体积变化；B.MA782/5S乳腺癌荷瘤的免疫小鼠的肿瘤体积变化；C.LL/2c Lewis肺癌荷瘤的免疫小鼠的生存率；D.MA782/5S乳腺癌荷瘤的免疫小鼠的生存率。hEe-p，人EGFR胞外段DNA疫苗；mEe-p，鼠EGFR胞外段DNA疫苗；c-p，空质粒对照；Saline，生理盐水对照。
图8.重组EGFR DNA疫苗的抗肿瘤治疗性免疫效应A.LL/2c Lewis肺癌荷瘤的免疫小鼠的肿瘤体积变化；B.MA782/5S乳腺癌荷瘤的免疫小鼠的肿瘤体积变化；C.LL/2c Lewis肺癌荷瘤的免疫小鼠的生存率；D.MA782/5S乳腺癌荷瘤的免疫小鼠的生存率。hEe-p，人EGFR胞外段DNA疫苗；mEe-p，鼠EGFR胞外段DNA疫苗；c-p，空质粒对照。
图9 重组EGFR蛋白疫苗的抗肿瘤免疫效应A.保护性免疫效应；B.治疗性免疫效应；C.荷瘤(MA782/5S乳腺癌)小鼠生存曲线。edCER，鸡EGFR胞外段重组蛋白疫苗；edMER，鼠EGFR胞外段重组蛋白疫苗；Adj，铝佐剂；NS，生理盐水。
图10 重组EGFR蛋白疫苗的抗肿瘤转移效应，A.LL/2c肿瘤肺部转移灶数量；B.LL/2c肿瘤转移后肺湿重。edCER，鸡EGFR胞外段重组蛋白疫苗；edMER，鼠EGFR胞外段重组蛋白疫苗；Adj，铝佐剂；NS，生理盐水。
图11 EGFR分子疫苗的体外(in vitro)抑制肿瘤细胞生长效应及体内(invivo)过继免疫抗肿瘤效应A.EGFR重组DNA疫苗hEe-p免疫小鼠来源的Ig对EGFR阳性肿瘤细胞(A549，LL/2c，MA782/5S)和EGFR阴性肿瘤细胞(H22 andMMT-06052)的生长抑制状况。B.用未免疫的正常小鼠来源的Ig对肿瘤细胞的生长抑制状况。C.EGFR重组DNA疫苗hEe-p免疫小鼠来源的Ig的体内(in vivo)过继免疫抗肿瘤效应。hEe-p，人EGFR胞外段DNA疫苗；mEe-p，鼠EGFR胞外段DNA疫苗；c-p，空质粒对照；Saline，生理盐水对照。
图12 EGFR分子疫苗诱导产生的抗体类型A.EGFR重组DNA疫苗诱导产生的抗体类型；B.EGFR重组蛋白疫苗诱导产生的抗体类型。hEe-p，人EGFR胞外段DNA疫苗；mEe-p，鼠EGFR胞外段DNA疫苗；c-p，空质粒对照；Saline，生理盐水对照；edCER，鸡EGFR胞外段重组蛋白疫苗；edMER，鼠EGFR胞外段重组蛋白疫苗；Adj，铝佐剂；NS，生理盐水。
图13 EGFR重组DNA疫苗免疫小鼠的CTL效应A.人EGFR重组DNA疫苗hEe-p及其对照组免疫小鼠来源的T细胞对LL/2c Lewis肺癌细胞在不同E∶T比例下的51Cr释放分析结果；B.过继输注2×107个来源于人EGFR重组DNA疫苗hEe-p免疫小鼠的CD4剔除(CD8+)T细胞对EGFR阳性的LL/2c Lewis肺癌细胞和EGFR阴性的B16黑色素瘤细胞的抗肿瘤作用；C.过继输注2×107个来源于人EGFR重组DNA疫苗hEe-p免疫小鼠的CD8剔除(CD4+)T细胞对EGFR阳性的LL/2c Lewis肺癌细胞和EGFR阴性的B16黑色素瘤细胞的的抗肿瘤作用；D.过继输注2×107个来源于人EGFR重组DNA疫苗hEe-p免疫小鼠的CD4剔除(CD8+)T细胞对EGFR阳性的MA782/5S乳腺癌细胞和EGFR阴性的MethA纤维肉瘤细胞的的抗肿瘤作用；E.过继输注2×107个来源于人EGFR重组DNA疫苗hEe-p免疫小鼠的CD8剔除(CD4+)T细胞对EGFR阳性的MA782/5S乳腺癌细胞和EGFR阴性的MethA纤维肉瘤细胞的抗肿瘤作用。 人EGFR重组DNA疫苗hEe-p免疫小鼠来源的T细胞过继免疫的荷瘤小鼠； 鼠EGFR重组DNA疫苗mEe-p免疫小鼠来源的T细胞过继免疫的荷瘤小鼠； 非免疫小鼠来源的T细胞过继免疫的荷瘤小鼠。
图14肿瘤细胞及组织自身抗体的免疫荧光显微图谱A.EGFR分子疫苗免疫的LL/2c Lewis肺癌细胞的自身抗体沉着；B.EGFR分子疫苗免疫的MA782/5S鼠乳腺癌细胞的自身抗体沉着；C.未免疫的LL/2c Lewis肺癌细胞无自身抗体沉着；D.未免疫的MA782/5S鼠乳腺癌细胞无自身抗体沉着；E.CD4+T阻断的EGFR分子疫苗免疫的LL/2c Lewis肺癌细胞无自身抗体沉着；F.CD4+T阻断的EGFR分子疫苗免疫的MA782/5S鼠乳腺癌细胞无自身抗体沉着；G CD8+T阻断的EGFR分子疫苗免疫的LL/2c Lewis肺癌细胞显示自身抗体沉着；H.CD8+T阻断的EGFR分子疫苗免疫的MA782/5S鼠乳腺癌细胞显示自身抗体沉着；I.EGFR分子疫苗免疫小鼠的肝脏无自身抗体沉着；J.EGFR分子疫苗免疫小鼠的肾脏无自身抗体沉着；K.未免疫小鼠的肝脏无自身抗体沉着；L.未免疫小鼠的肾脏无自身抗体沉着。
具体实施例方式
实施例1 EGFR重组DNA疫苗依据GenBank等各公开的数据库所收藏的人、小鼠、鸡、的EGFR分子的cDNA序列(分别对应于SEQ ID NO 1-5，7-9，19)设计PCR引物(人的引物为5’GACCATGGAGGAAAAGAAAGTTTGC 3’，5’ACGAATTCTTAGGACGGGATCTTAGGCCCA3’；小鼠的引物为5’GACCATGGAGGAAAAGAAAGTCTGC 3’，5’ACGAATTCTTAATAGATGGTATCTTTGGC 3’；鸡的引物为5’GACCATGGAGGAGAAGAAAGTTTGTC 3’，5’ACGAATTCTTAAGATGGAGTTTTGGAGCC 3’)，分别以人肺癌细胞株A431、小鼠肺癌细胞株LL2和鸡胚的总RNA为模板进行RT-PCR扩增，电泳收集纯化扩增的EGFR片段(均为1.9kb)，然后进行PCR产物亚克隆。把PCR亚克隆测序确证后，用NcoI和EcoRI酶切，收集1.9kb片段并纯化，插入到用NcoI和EcoRI双酶切的pORF-MCS(InvivoGen公司)载体，筛选重组质粒。候选的重组质粒经限制性酶切分析及PCR扩增等双重鉴定，命名为pORF-hEGFR、pORF-mEGFR和pORF-chEGFR。
同样地，设计针对于真核表达载体pcDNA3.1(+)(Invitrogen公司)的PCR引物，分别是人的引物5’GAGCTAGCATGGAGGAAA AGAAAGTTTGC 3’，5’CACTCGAGTTAGGACGGGATCTTAGGCCCA 3’；小鼠的引物为5’GAGCTAGCATGGAGGAAAAGAAAGTCTGC 3’，5’CACTCGAG TTAATAGATGGTATCTTTGGC 3’；鸡的引物为5’GAGCTAGCATGGAGGAGAA GAAAGTTTGTC 3’，5’CACTCG AGTTAAGATGGAGTTTTGGAGCC 3’，依前述RNA模板进行RT-PCR扩增。将纯化的1.9kb的PCR产物用NheI和XhoI双酶切，插入到对应的双酶切载体pcDNA3.1(+)中，筛选重组质粒，将重组质粒分别命名为pcDNA-hEGFR、pcDNA-mEGFR和pcDNA-chEGFR。
这些真核表达质粒pORF-EGFR或pcDNA-EGFR等转染入CHO、NIH3T3、Vero等细胞系，观察并用SDS-PAGE、ELISA、Western Blot等技术检测其表达EGFR的情况和水平。用碱法抽提经过鉴定的重组EGFR分子表达质粒，用超离心、超滤等方法去除大肠杆菌内毒素，即得到纯净的质重组粒DNA，这些质粒DNA即可作为DNA疫苗进行免疫。
概括地讲，EGFR重组DNA疫苗的构建过程是依据GenBank等各公开的数据库所收藏的各种生物来源(如人、小鼠、大鼠、鸡、鲐、果蝇等，见SEQ IDNO 1-14)的关于EGFR分子的序列(包括基因、cDNA、mRNA和氨基酸序列)用常规方法设计引物或探针，用PCR、RT-PCR、杂交等技术，从各种商品化的基因文库、cDNA文库(ClonTech，Strategene公司等生产的各种基因文库、cDNA文库)或各种细胞系(如人肺癌细胞株A431、鼠Lewis肺癌细胞株LL2等)、组织(如肺癌、乳腺癌组织、果蝇、鸡胚等)等中克隆分离出不同种属生物来源的EGFR分子的胞外区段的cDNA，或进一步用基因直接进化技术筛选到的具有强免疫原性的EGFR分子。各种来源的EGFR cDNA的胞外段序列经测序鉴定后，用分子克隆技术插入真核生物表达质粒(如pcDNA3.1、pORF-mcs、pBLAST-mcs、pSecTag2等)，筛选重组表达质粒，经限制性酶切分析鉴定后转染入CHO、NIH3T3、Vero等细胞系，观察并用SDS-PAGE、ELISA、Western Blot等技术检测其表达EGFR的情况和水平。用碱法抽提经过鉴定的重组EGFR分子表达质粒，用超离心、超滤等方法去除大肠杆菌内毒素，即得到纯净的质重组粒DNA，这些质粒DNA即可作为DNA疫苗进行免疫。
选择6-8周龄小鼠，用常规方法建立各种荷瘤小鼠模型，每只小鼠注射100gEGFR重组表达质粒，每周1次，连续4周。观察荷瘤小鼠的生长情况及肿瘤的发展情况，8周后处死小鼠，收集免疫小鼠血清及各种脏器，用流式细胞术、ELISA、Western blot等方法检测体液免疫反应，用Cr51、ELISpot等检测细胞免疫反应，用免疫组化方法检测免疫的毒、副作用。进一步提纯免疫小鼠血清，按常规方法建立裸鼠的肿瘤模型，进行过继免疫治疗，观察肿瘤的生长情况。
实施例2 EGFR重组蛋白疫苗(E.coli表达)同前文所述，依据GenBank等各公开的数据库所收藏的人、小鼠、鸡、的EGFR分子的cDNA序列(分别对应于SEQ ID NO 1-5，7-9，19)设计PCR引物(人的引物为5’GACCATGGAGGAAAAGAAAGTTTGC 3’，5’ACAGATCTAGGACGGGATCTTAGGCCCA 3’；小鼠的引物为5’GACCATGGAGGAAAAGAAAGTCTGC 3’，5’ACAGATCTATAGATGGTATCTTTGGC 3’；鸡的引物为5’GACCATGGA GGAGAAGAAAGTTTGTC 3’，5’ACAGATCTAGATGGAGTTTTGGAGCC 3’)，分别以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等为模板进行PCR扩增，电泳收集纯化扩增的EGFR片段(均为1.9kb)，然后进行PCR产物亚克隆。把PCR亚克隆测序确证后，用NcoI和BglII酶切，收集1.9kb片段并纯化，插入到用NcoI和BglII双酶切的pQE60(QIAGEN公司)载体，筛选重组质粒。候选的重组质粒经限制性酶切分析及PCR扩增等双重鉴定，命名为pQE-hEGFR、pQE-mEGFR和pQE-chEGFR。
同样地，可针对其他不同的原核表达载体(如pET32、pLLp、pSE420等)设计PCR引物，以构建其他的重组原核表达质粒。
经限制性酶切分析等鉴定后的各重组原核表达质粒，转化不同的大肠杆菌宿主(如E.coli TOP10F′、E.coli BL21(DE3)pLys、E.coli M15、E.coli DH5、E.coliJM109等)，观察并用SDS-PAGE、ELISA、Western Blot等技术检测其表达EGFR的情况和水平，确定最适的大肠杆菌表达宿主菌株。用经确证的重组EGFR表达质粒转化最适大肠杆菌菌株，建立稳定表达菌株和各级种子库。重组菌株大量摇瓶培养或发酵，低温离心收集菌体，菌体经PBS重悬后用超声法破碎细胞，用离子交换层析、亲和层析等方法分离纯化重组EGFR蛋白，此重组的EGFR蛋白质即可作为蛋白疫苗进行免疫。
选择6-8周龄小鼠，用常规方法建立各种荷瘤小鼠模型，每只小鼠注射5-50gEGFR重组蛋白，每周1次，连续4周。观察荷瘤小鼠的生长情况及肿瘤的发展情况，8周后处死小鼠，收集免疫小鼠血清及各种脏器，用流式细胞术、ELISA、Western blot等方法检测体液免疫反应，用Cr51、ELISpot等检测细胞免疫反应，用免疫组化方法检测免疫的毒、副作用。进一步提纯免疫小鼠血清，按常规方法建立裸鼠的肿瘤模型，进行过继免疫治疗，观察肿瘤的生长情况。
实施例3 EGFR重组蛋白疫苗(酵母Pichia pastoris表达)
同前文所述，依据GenBank等各公开的数据库所收藏的人、小鼠、鸡、的EGFR分子的cDNA序列(分别对应于SEQ ID NO 1-5，7-9，19)设计PCR引物(人的引物为5’ATACTCGAGAAAAGAGAGCTGGAGGAAAAGAAAG 3’，5’GCTCTAGAATGGCACAGGTGGCACA 3’；小鼠的引物为5’ATGCTCGAGAAAA GAGAGTTGGAGGAAAAGAAAGTC 3’，5’AAGCGGCCGCCATAGATGGTATCTTTG 3’；鸡的引物为5’ATACTCGAGAAAAGAGAGGTGGAGGAGAAGAAAG 3’，5’CGTCTAGAAGATGGAGTTTTGGAG 3’)，分别以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等为模板进行PCR扩增，电泳收集纯化扩增的EGFR片段(均为1.9kb)，然后进行PCR产物亚克隆。把PCR亚克隆测序确证后，用XhoI和XbaI(对小鼠的克隆为XhoI和NotI)酶切，收集1.9kb片段并纯化，插入到用XhoI和XbaI双酶切(对小鼠的克隆为XhoI和NotI双酶切)的pPICZ A(Invitrogen公司)载体，转化E.coli筛选重组质粒。候选的重组质粒经限制性酶切分析及PCR扩增等双重鉴定，命名为酵母表达质粒pYE-hEGFR、pYE-mEGFR和pYE-chEGFR。这些酵母表达质粒经PmeI酶切线性化后，用电穿孔法转化酵母菌株X33或GS115，用Zeocin抗性筛选稳定转化子，在MMH(Minimal Methanol with histidine，MMH)和MDH(Minimal Dextrose with histidine，MDH)琼脂平板上鉴定和挑选Mut+转化子。挑选6-10个Mut+转化子进行小规模表达，用SDS-PAGE、Western Blot、ELISA等方法对所表达的重组蛋白进行鉴定。选择其中表达效率最高的Mut+转化子做大规模表达，建立各级酵母表达种子库。重组菌株大量摇瓶培养或发酵，低温离心收集菌体，菌体经PBS重悬后用超声法破碎细胞，用离子交换层析、亲和层析等方法分离纯化重组EGFR蛋白，此重组的EGFR蛋白质即可作为蛋白疫苗进行免疫。
同样地，也可用其他的酵母表达系统构建类似的酵母重组表达质粒。
选择6-8周龄小鼠，用常规方法建立各种荷瘤小鼠模型，每只小鼠注射5-50gEGFR重组蛋白，每周1次，连续4周。观察荷瘤小鼠的生长情况及肿瘤的发展情况，8周后处死小鼠，收集免疫小鼠血清及各种脏器，用流式细胞术、ELISA、Western blot等方法检测体液免疫反应，用Cr51、ELISpot等检测细胞免疫反应，用免疫组化方法检测免疫的毒、副作用。进一步提纯免疫小鼠血清，按常规方法建立裸鼠的肿瘤模型，进行过继免疫治疗，观察肿瘤的生长情况。
实施例4 EGFR重组腺病毒疫苗同前文所述，依据GenBank等各公开的数据库所收藏的人、小鼠、鸡、的EGFR分子的cDNA序列(分别对应于SEQ ID NO 1-5，7-9，19)设计PCR引物(人的引物为5’GAAGATCTATGGAGGAAAAGAAAGTTTGC 3’，5’ACGATATCTTAAGGACGGGATCTTAGGCCCA 3’；小鼠的引物为5’GAAGATCTATGGAGGAAAAGAAAGTCTGC 3’，5’ACGATATCTTAATAGATGGTATCTTTGGC 3’；鸡的引物为5’GAAGATCTATGGAGGAGAAGAAAGTTTGTC 3’，5’ACGATATCTTAAGATGGAGTTTTGGAGCC 3’)，分别以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等为模板进行PCR扩增，电泳收集纯化扩增的EGFR片段(均为1.9kb)，然后进行PCR产物亚克隆。把PCR亚克隆测序确证后，用BglII和EcoRV酶切，收集1.9kb片段并纯化，插入到用BglII和EcoRV双酶切的pShuttle-CMV(Quantum Biotechnologies公司)载体，筛选重组质粒。候选的重组质粒经限制性酶切分析及PCR扩增等双重鉴定，命名为腺病毒穿梭表达质粒pShuttle-hEGFR、pShuttle-mEGFR和pShuttle-chEGFR(图2D)。分别将经PmeI酶切的腺病毒穿梭表达载体pShuttle-EGFR与包含腺病毒基因组的骨架载体pAdEasy-1或pAdEasy-2共转化E.coli BJ5183，得到重组腺病毒载体质粒pAd-hEGFR、pAd-mEGFR和pAd-chEGFR。这些重组腺病毒载体质粒经PacI酶切后，用磷酸钙-DNA共沉淀法转染入腺病毒包装细胞株293细胞中，得到相应的重组腺病毒Ad-hEGFR、Ad-mEGFR和Ad-chEGFR。用PCR、Western blot和酶切分析确证EGFR基因构建入重组腺病毒载体中并能在真核细胞中得到正确有效的表达，用超离心法大量制备重组腺病毒Ad-EGFR，用上层琼脂法、TCID50等法测定每批次的重组腺病毒的滴度(pfu)。利用293细胞大量扩增经确证的EGFR重组腺病毒疫苗，用超离心、超滤等技术分离、纯化重组腺病毒，此经纯化的EGFR重组腺病毒即可作为疫苗进行免疫。
同前文所述，选择6-8周龄小鼠，用常规方法建立各种荷瘤小鼠模型，每只小鼠注射1×109 PFU的EGFR重组腺病毒，每周1次，连续4周。观察荷瘤小鼠的生长情况及肿瘤的发展情况，8周后处死小鼠，收集免疫小鼠血清及各种脏器，用流式细胞术、ELISA、Western blot等方法检测体液免疫反应，用Cr51、ELISpot等检测细胞免疫反应，用免疫组化方法检测免疫的毒、副作用。进一步提纯免疫小鼠血清，按常规方法建立裸鼠的肿瘤模型，进行过继免疫治疗，观察肿瘤的生长情况。
实施例5 EGFR重组Lentivirus病毒疫苗如前文所述，本发明利用ViraPowerTMLentiviral GatewayExpression Kit(Invitrogen公司)构建EGFR重组Lentivirus病毒，其具体构建过程是依据GenBank等各公开的数据库所收藏的人、小鼠、鸡、的EGFR分子的cDNA序列(分别对应于SEQ ID NO 1-5，7-9，19)设计PCR引物(人的引物为5’GACCATGGAGGAAAAGAAAGTTTGC 3’，5’ACGATATCAGGACGGGA TCTTAGGCCCA 3’；小鼠的引物为5’GACCATGGAGGAAAAGAAAGTCTGC 3’，5’ACGATATCATAGATGGTATCTTTGGC 3’；鸡的引物为5’GACCATGGAGGAGAAGAAAGTTTGTC 3’，5’ACGATATCAGATGGAGTTTTGGAGCC 3’)，分别以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等为模板进行PCR扩增，电泳收集纯化扩增的EGFR片段(均为1.9kb)，然后进行PCR产物亚克隆。把PCR亚克隆测序确证后，用NcoI和EcoRV酶切，收集1.9kb片段并纯化，插入到用NcoI和EcoRV双酶切的pENTR11(Invitrogen公司)载体中，筛选重组质粒，命名为pENTR-hEGFR、pENTR-mEGFR和pENTR-chEGFR(图2E)。分别将这些载体pENTR-EGFR与包含Lentivirus病毒基因组的骨架载体pLenti6/V5-DEST共转化E.coli DH5，得到重组Lentivirus病毒载体质粒pLenti-hEGFR、pLenti-mEGFR和pLenti-chEGFR(图2E)。这些重组Lentivirus病毒载体质粒与包装混合物一起(ViraPowerTMPackagingMix)用磷酸钙-DNA共沉淀法转染入Lentivirus病毒包装细胞株293FT细胞中，得到相应的重组Lentivirus病毒Lenti-hEGFR、Lenti-mEGFR和Lenti-chEGFR。用PCR、Western blot等确证EGFR基因构建入重组Lentivirus病毒载体中并能在真核细胞中得到正确有效的表达。利用293FT细胞大量扩增经确证的EGFR重组Lentivirus病毒疫苗，用超离心、超滤等技术分离、纯化重组Lentivirus病毒，此经纯化的EGFR重组Lentivirus病毒即可作为疫苗进行免疫。
实施例6 甘露糖化腺病毒重组EGFR疫苗如前文所述，用常规方法扩增EGFR重组腺病毒(I代、II代均可)，层析或超离心法纯化重组腺病毒。将70mg mannan(sigma)溶于5ml 0.1M的磷酸盐缓冲液(pH6.0)中，终浓度14mg/ml，加45ml 0.01M高碘酸钠溶液，在4℃下混合氧化60分钟，加入10l乙二醇，在4℃下孵育30分钟，即得Ox-M(Oxidative Mannan)混合物。将Ox-M混合物倒入用重碳酸盐缓冲液(pH6.0-9.0)平衡Sephadex-G25层析柱进行层析分离，OX-M即被洗入2ml的空容器。将纯化的Ox-M与1×1014腺病毒颗粒混合，室温过夜，即获得所需Ox-M-腺病毒。在Ox-M-腺病毒中加入1mg/ml硼氢化钠，室温放置3小时即得Red-M-腺病毒。Ox-M-腺病毒与Red-M-腺病毒经超滤脱盐、浓缩后，过滤细菌，小管分装，-80℃低温保存。此经纯化的甘露糖化重组EGFR腺病毒即可作为疫苗进行免疫。
同前文所述，选择6-8周龄小鼠，用常规方法建立各种荷瘤小鼠模型，每只小鼠注射1×1010PFU的重组EGFR甘露糖化腺病毒疫苗，每周1次，连续4周。观察荷瘤小鼠的生长情况及肿瘤的发展情况，8周后处死小鼠，收集免疫小鼠血清及各种脏器，用流式细胞术、ELISA、Western blot等方法检测体液免疫反应，用Cr51、ELISpot等检测细胞免疫反应，用免疫组化方法检测免疫的毒、副作用。进一步提纯免疫小鼠血清，按常规方法建立裸鼠的肿瘤模型，进行过继免疫治疗，观察肿瘤的生长情况。
实施例7 RGD修饰的腺病毒重组EGFR疫苗的构建如前文所述，本发明利用AdEasy系统构建RGD修饰腺病毒的重组EGFR疫苗，其具体过程是腺病毒基因组骨架质粒pAdEasy-1和pAdEasy-2经限制性内切酶SpeI(Sp)酶切后，用T4 DNA聚合酶补平(filling，f)末端，再用PacI(P)酶切，分别电泳回收6211bp和3579bp的片段，分别命名为AdFiber I/Sp/f/P和AdFiber II/Sp/f/P，该片段包含了完整的腺病毒纤毛(Adenovirus fiber)基因。把AdFiber I/Sp/f/P和AdFiber II/Sp/f/P片段插入经BamHI酶切-T4 DNA聚合酶补平-PacI酶切处理(BamHI/filling/PacI-digested)的pShuttle载体，将所得的重组质粒分别命名为pSh-AdFiber I和pSh-AdFiber II。pSh-AdFiber I用NheI酶切-T4DNA聚合酶补平-KpnI酶切处理(NheI/filling/KpnI)，电泳回收2090bp的片段AdFiber I/Nh/f/K，将该片段插入到经SmaI/KpnI双酶切的pUC18载体中，所得的重组质粒命名为pUC-AdFiber I；而pSh-AdFiber II用AvrII酶切-T4 DNA聚合酶补平-HindIII酶切处理(AvrII/filling/HindIII)，电泳回收838bp的片段AdFiberI/A/f/H，将该片段插入到经SmaI/HindIII双酶切的pUC18载体中，所得的重组质粒命名为pUC-AdFiber II。设计一系列PCR引物以便以pUC-AdFiber I和pUC-AdFiberII为模板扩增腺病毒疣足(Adenovirus knob，Ad-knob)基因序列，引物分别是F1(5’GAAAGCTAGC CCTGCAAACATCA 3’)、R1(5’ACTCCCGGGAGTTGTGTCTCCTGTTTCCTG 3’)、F2(5’ACTCCCGGGAGTGCATACTCTATGTCA 3’)、R2(5’TATGGTAC CGGGAGGTGGTGA 3’)、F3(5’AACCTAGGGAGGTTAACCTAAGCACTG 3’)、和R3(5’CTCAAGCTTTTTGGAATTGTTTGA 3’)。以引物F1-R1、F2-R2、F3-R1和F2-R3分别进行第一轮PCR，得到产物PCR1、PCR2、PCR3和PCR4，再以F1-R2和F3-R3为引物，以第一次扩增产物PCR1与PCR2、PCR3与PCR4为模板进行第二轮PCR扩增，得到PCR产物PCR1-PCR2(PCR I)、PCR3-PCR4(PCR II)，将第二轮PCR扩增的PCR I和PCR II插入到经EcoRV酶切的pBR322载体中，所得到的重组质粒命名为pBR-PCR I和pBR-PCR II。把RGD-4C双螺旋寡聚核苷酸(RGD-4C duplex)5’TGTGACTGCCGCGGAGACTGTTTCTGC 3’3’ACACTGACGGCGCCTCTGACAAAGACG 5’插入到SmaI酶切的pBR-PCR I and pBR-PCR II载体中，将所得到的重组质粒命名为pBR-PCR/RGD I和pBR-PCR/RGD II，并对重组结构进行测序确证。用NheI/KpnI双酶切pBR-PCR/RGD I，电泳回收PCR/RGD I片段，然后插入到NheI/KpnI双酶切的pUC-AdFiber I载体中，所得的重组质粒命名为pUC-AdFiber-RGD I；用AvrII/HindIII双酶切pBR-PCR/RGD II，电泳回收PCR/RGDII片段，再插入到AvrII/HindIII双酶切的pUC-AdFiber II载体中，将所得到的重组质粒命名为pUC-AdFiber-RGD II。然后，用SpeI/PacI双酶切pUC-AdFiber-RGDI阳pUC-AdFiber-RGD II载体，电泳回收AdFiber-RGD I、AdFiber-RGD II片段，插入到SpeI/PacI双酶切的pAdEasy-1、pAdEasy-2载体中，所得的重组质粒分别命名为pAdEasy-RGD I、pAdEasy-RGD II。将经PmeI线性化的腺病毒穿梭质粒pShuttle-hEGFR、pShuttle-mEGFR和pShuttle-chEGFR分别与pAdEasy-RGD I和pAdEasy-RGD II共转化E.coli BJ5183，所得的重组质粒命名为腺病毒质粒pAd-RGD-EGFR I和pAd-RGD-EGFR II，腺病毒质粒pAd-RGD-EGFR I转染293细胞，所得的重组腺病毒命名为Ad-RGD-EGFR I，腺病毒质粒pAd-RGD-EGFR II转染293E4pIX细胞，所得的重组腺病毒命名为Ad-RGD-EGFR II。
经纯化的Ad-RGD-EGFR I和Ad-RGD-EGFR II可作为疫苗进行免疫，对肿瘤血管内皮细胞具有特异的靶向性。
实施例8 重组EGFR分子疫苗的药效学观察药效学研究是观察和验证重组EGFR分子疫苗的抗肿瘤作用的重要指标。在本发明中，药效学观察的实验包括普通荷瘤小鼠(含保护性免疫实验、治疗性免疫实验、肿瘤转移模型实验)、兔免疫试验、猴免疫试验、裸鼠过继免疫实验、剂量依存实验、体外细胞实验等。这里主要叙述以肺癌为模型的普通荷瘤小鼠实验，其它可类比。
1.荷瘤小鼠保护性免疫实验 随机分组的6-8周龄C57BL/6小鼠双侧后肢股四头肌肌肉交叉注射EGFR分子疫苗，每周一次，连续四周。第5周各组小鼠均右腋皮下接种LL/2肿瘤细胞1×106。分别在首次免疫第0、1、3、5周经鼠尾静脉或处死小鼠时采血。离心后(5000转/分，3分钟)收集血清，-20℃保存备用。
2.荷瘤小鼠治疗性免疫实验 随机分组的6-8周龄C57BL/6小鼠均右腋皮下接种LL/2肿瘤细胞1×106。接种后第4天(可触及肿瘤)，随机分组，小鼠后肢肌肉交叉注射。每周一次，连续四周。各组分别在首次免疫第0、2、4、6周经鼠尾静脉或处死小鼠时采血。离心后(5000转/分，3分钟)收集血清，-20℃保存备用。观察并记录肿瘤重量、体积及生存曲线。
3.荷瘤小鼠转移模型实验 选6-8周龄C57BL/6雌性小鼠，随机分组，小鼠后肢肌肉交叉注射疫苗及对照组。每周一次，连续四周。2周后，取对数期生长的LL/2细胞，按2×105/只接种于C57BL/6小鼠右后腿肌肉内，继续上述疫苗注射，4周后断颈处死小鼠，取肺称重，检查并计数肺部转移灶后，用10％中性缓冲福尔马林固定。
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<221>CDS<222>(1)..(3630)<223>
<400>1atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa 96Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt 144Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat 192Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560
ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag 240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg 288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac 336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat 384Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115120 125aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg 432Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130 135140cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag 480His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225230235 240aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260265 270acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320
gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325 330335tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345 350gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375380cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400atc aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405410 415ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc agg acc aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att tca 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg 1440Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485490 495aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500 505510gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515520525gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535 540gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550 555560gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565570 575
gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610 615 620acc tac gga tgc act ggg cca ggt ctt gaa ggc tgt cca acg aat ggg 1920Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly625630 635640cct aag atc ccg tcc atc gcc act ggg atg gtg ggg gcc ctc ctc ttg 1968Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu645 650 655ctg ctg gtg gtg gcc ctg ggg atc ggc ctc ttc atg cga agg cgc cac 2016Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His660 665 670atc gtt cgg aag cgc acg ctg cgg agg ctg ctg cag gag agg gag ctt 2064Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu675 680685gtg gag cct ctt aca ccc agt gga gaa gct ccc aac caa gct ctc ttg 2112Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu690695 700agg atc ttg aag gaa act gaa ttc aaa aag atc aaa gtg ctg ggc tcc 2160Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser705 710715 720ggt gcg ttc ggc acg gtg tat aag gga ctc tgg atc cca gaa ggt gag 2208Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu725 730735aaa gtt aaa att ccc gtc gct atc aag gaa tta aga gaa gca aca tct 2256Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser740 745 750ccg aaa gcc aac aag gaa atc ctc gat gaa gcc tac gtg atg gcc agc 2304Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser755760765gtg gac aac ccc cac gtg tgc cgc ctg ctg ggc atc tgc ctc acc tcc 2352Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser770775 780acc gtg cag ctc atc acg cag ctc atg ccc ttc ggc tgc ctc ctg gac 2400Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp785 790795 800tat gtc cgg gaa cac aaa gac aat att ggc tcc cag tac ctg ctc aac 2448Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn805 810815tgg tgt gtg cag atc gca aag ggc atg aac tac ttg gag gac cgt cgc 2496Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg820 825 830
ttg gtg cac cgc gac ctg gca gcc agg aac gta ctg gtg aaa aca ccg 2544Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro835 840845cag cat gtc aag atc aca gat ttt ggg ctg gcc aaa ctg ctg ggt gcg 2592Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala850 855860gaa gag aaa gaa tac cat gca gaa gga ggc aaa gtg cct atc aag tgg 2640Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp865 870 875 880atg gca ttg gaa tca att tta cac aga atc tat acc cac cag agt gat 2688Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp885 890 895gtc tgg agc tac ggg gtg acc gtt tgg gag ttg atg acc ttt gga tcc 2736Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser900 905 910aag cca tat gac gga atc cct gcc agc gag atc tcc tcc atc ctg gag 2784Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu915 920 925aaa gga gaa cgc ctc cct cag cca ccc ata tgt acc atc gat gtc tac 2832Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr930 935 940atg atc atg gtc aag tgc tgg atg ata gac gca gat agt cgc cca aag 2880Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys945 950 955 960ttc cgt gag ttg atc atc gaa ttc tcc aaa atg gcc cga gac ccc cag 2928Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln965 970975cgc tac ctt gtc att cag ggg gat gaa aga atg cat ttg cca agt cct 2976Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro980 985990aca gac tcc aac ttc tac cgt gcc ctg atg gat gaa gaa gac atg gac 3024Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp99510001005gac gtg gtg gat gcc gac gag tac ctc atc cca cag cag ggc ttc 3069Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe101010151020ttc agc agc ccc tcc acg tca cgg act ccc ctc ctg agc tct ctg 3114Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu102510301035agt gca acc agc aac aat tcc acc gtg gct tgc att gat aga aat 3159Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn104010451050ggg ctg caa agc tgt ccc atc aag gaa gac agc ttc ttg cag cga 3204Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg105510601065tac agc tca gac ccc aca ggc gcc ttg act gag gac agc ata gac 3249Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp107010751080
gac acc ttc ctc cca gtg cct gaa tac ata aac cag tcc gtt ccc 3294Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro1085 10901095aaa agg ccc gct ggc tct gtg cag aat cct gtc tat cac aat cag 3339Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln1100 11051110cct ctg aac ccc gcg ccc agc aga gac cca cac tac cag gac ccc 3384Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro1115 11201125cac agc act gca gtg ggc aac ccc gag tat ctc aac act gtc cag 3429His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln1130 11351140ccc acc tgt gtc aac agc aca ttc gac agc cct gcc cac tgg gcc 3474Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala1145 11501155cag aaa ggc agc cac caa att agc ctg gac aac cct gac tac cag 3519Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln1160 11651170cag gac ttc ttt ccc aag gaa gcc aag cca aat ggc atc ttt aag 3564Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys1175 11801185ggc tcc aca gct gaa aat gca gaa tac cta agg gtc gcg cca caa 3609Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln1190 11951200agc agt gaa ttt att gga gca tga 3633Ser Ser Glu Phe Ile Gly Ala1205 1210<210>2<211>1210<212>PRT<213>人(homo sapiens)<400>2Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 7075 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115 120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130 135140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305310 315320
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325 330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375 380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485 490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500 505510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515520525Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550555560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610 615 620Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly625630 635640Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu645 650 655Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His660 665 670Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu675680 685Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu690695 700Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser705 710715 720Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu725 730735Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser740 745 750Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser755 760 765Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser770775780Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp785 790795800Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn805 810 815Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg820 825 830
Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro835 840845Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala850 855860Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp865870 875 880Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp885 890 895Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser900 905 910Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu915 920 925Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr930 935 940Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys945 950955960Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln965 970975Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro980 985 990Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp995 10001005Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe1010 1015 1020Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu1025 1030 1035Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn1040 1045 1050Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg1055 1060 1065Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp1070 1075 1080
Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro108510901095Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln110011051110Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro111511201125His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln113011351140Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala114511501155Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln116011651170Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys117511801185Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln119011951200Ser Ser Glu Phe Ile Gly Ala12051210<210>3<211>1974<212>DNA<213>人(homo sapiens)<220>
<221>CDS<222>(1)..(1971)<223>
<400>3atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa 96Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt 144Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe354045ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat 192Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag 240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 7075 80acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg 288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac 336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat 384Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115120 125aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg 432Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130 135140cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag 480His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260265 270acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325330335tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345350gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375380cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400atc aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405410 415ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc agg acc aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att tca 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg 1440Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485 490 495aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500505 510gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515 520525gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
530 535540gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550555 560gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600605aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610615 620acc tac gga tgc act ggg cca ggt ctt gaa ggc tgt cca acg aat gga 1920Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly625630 635640agc tac ata gtg tct cac ttt cca aga tca ttc tac aag atg tca gtg 1968Ser Tyr Ile Val Ser His Phe Pro Arg Ser Phe Tyr Lys Met Ser Val645 650 655cac tga 1974His<210>4<211>657<212>PRT<213>人(homo sapiens)<400>4Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130 135140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290 295300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345 350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360 365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485 490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500505 510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515 520525Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610 615 620Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly625630 635640Ser Tyr Ile Val Ser His Phe Pro Arg Ser Phe Tyr Lys Met Ser Val645 650 655His<210>5<211>2118<212>DNA<213>人(homo sapiens)<220>
<221>CDS<222>(1)..(2115)<223>
<400>5atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa 96Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt 144Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat 192Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag 240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80
acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg 288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac 336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat 384Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115 120 125aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg 432Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag 480His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195 200205aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210215 220ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265 270acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325330335
tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345350gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375 380cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400atc aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405410 415ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc agg acc aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att tca 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg 1440Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485490 495aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500505510gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515520525gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535 540gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550 555 560gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590
aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600605aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610 615620acc tac ggg cca gga aat gag agt ctc aaa gcc atg tta ttc tgc ctt 1920Thr Tyr Gly Pro Gly Asn Glu Ser Leu Lys Ala Met Leu Phe Cys Leu625 630635640ttt aaa cta tca tcc tgt aat caa agt aat gat ggc agc gtg tcc cac 1968Phe Lys Leu Ser Ser Cys Asn Gln Ser Asn Asp Gly Ser Val Ser His645650655cag agc ggg agc cca gct gct cag gag tca tgc tta gga tgg atc cct 2016Gln Ser Gly Ser Pro Ala Ala Gln Glu Ser Cys Leu Gly Trp Ile Pro660 665670tct ctt ctg ccg tca gag ttt cag ctg ggt tgg ggt gga tgc agc cac 2064Ser Leu Leu Pro Ser Glu Phe Gln Leu Gly Trp Gly Gly Cys Ser His675680685ctc cat gcc tgg cct tct gca tct gtg atc atc acg gcc tcc tcc tgc 2112Leu His Ala Trp Pro Ser Ala Ser Val Ile Ile Thr Ala Ser Ser Cys690 695 700cac tga 2118His705<210>6<211>705<212>PRT<213>人(homo sapiens)<400>6Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp Hls Phe35 4045Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 7075 80
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val8590 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115 120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150 155 160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180185190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235 240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245 250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305310 315 320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325330335
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345 350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360 365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375 380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500 505510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515520525Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535 540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610615 620Thr Tyr Gly Pro Gly Asn Glu Ser Leu Lys Ala Met Leu Phe Cys Leu625630 635640Phe Lys Leu Ser Ser Cys Asn Gln Ser Asn Asp Gly Ser Val Ser His645650655Gln Ser Gly Ser Pro Ala Ala Gln Glu Ser Cys Leu Gly Trp Ile Pro660 665670Ser Leu Leu Pro Ser Glu Phe Gln Leu Gly Trp Gly Gly Cys Ser His675680685Leu His Ala Trp Pro Ser Ala Ser Val Ile Ile Thr Ala Ser Ser Cys690695 700His705<210>7<211>1887<212>DNA<213>人(homo sapiens)<220>
<221>CDS<222>(1)..(1884)<223>
<400>7atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa 96Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt 144Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat 192Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn505560ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag 240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg 288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac 336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat 384Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115120 125aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg 432Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag 480His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180185190agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225230235240aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245 250 255gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275280285gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295300ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310 315 320gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325330 335tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345 350gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375 380cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390 395400atc aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405 410415ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc agg acc aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att tca 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg 1440Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485 490 495aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500 505510gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val SerCys Arg Asn515520525gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535 540gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600605aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca 8ac tgc 1872Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610 615620acc tac ggg tcc taa 1887Thr Tyr Gly Ser625<210>8<211>628<212>PRT<213>人(homo sapiens)<400>8Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 7075 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130 135140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245 250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290 295300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325 330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345 350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370375380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390 395400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405 410415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450 455 460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500 505 510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515520525Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535 540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550555560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595600605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610 615620Thr Tyr Gly Ser
625<210>9<211>1218<212>DNA<213>人(homo sapiens)-<220>
<221>CDS<222>(1)..(1215)<223>
<400>9atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct48Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa96Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt144Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat192Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat384Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115120 125aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg432Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag480His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160
agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230235240aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325330 335tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345350gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375 380cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385 390395400atc aca ggt ttg agc tga 1218Ile Thr Gly Leu Ser405
<210>10<211>405<212>PRT<213>人(homo sapiens)<400>10Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 7075 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225230235240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325 330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360 365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Leu Ser405<210>11<211>2829<212>DNA<213>人(homo sapiens)<220>
<221>CDS<222>(1)..(2826)<223>
<400>11atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag cgt aat tat gtg 96Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Arg Asn Tyr Val20 25 30gtg aca gat cac ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat 144Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr3540 45gag atg gag gaa gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct 192Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro5055 60tgc cgc aaa gtg tgt aac gga ata ggt att ggt gaa ttt aaa gac tca 240Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser65 70 75 80ctc tcc ata aat gct acg aat att aaa cac ttc aaa aac tgc acc tcc 288Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser85 90 95atc agt ggc gat ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc 336Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser100105 110ttc aca cat act cct cct ctg gat cca cag gaa ctg gat att ctg aaa 384Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys115 120125acc gta aag gaa atc aca ggg ttt ttg ctg att cag gct tgg cct gaa 432Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu130 135140aac agg acg gac ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc 480Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly145150155160agg acc aag caa cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac 528Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn165 170175ata aca tcc ttg gga tta cgc tcc ctc aag gag ata agt gat gga gat 576Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp180185190gtg ata att tca gga aac aaa aat ttg tgc tat gca aat aca ata aac 624Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn195200205tgg aaa aaa ctg ttt ggg acc tcc ggt cag aaa acc aaa att ata agc 672Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser
210215 220aac aga ggt gaa aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc 720Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala225230235 240ttg tgc tcc ccc gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc 768Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val245 250 255tct tgc cgg aat gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac 816Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn260265270ctt ctg gag ggt gag cca agg gag ttt gtg gag aac tct gag tgc ata 864Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile275 280285cag tgc cac cca gag tgc ctg cct cag gcc atg aac atc acc tgc aca 912Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr290295300gga cgg gga cca gac aac tgt atc cag tgt gcc cac tac att gac ggc 960Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly305 310315 320ccc cac tgc gtc aag acc tgc ccg gca gga gtc atg gga gaa aac aac 1008Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn325 330335acc ctg gtc tgg aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc 1056Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys340 345 350cat cca aac tgc acc tac gga tgc act ggg cca ggt ctt gaa ggc tgt 1104His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys355360365cca acg aat ggg cct aag atc ccg tcc atc gcc act ggg atg gtg ggg 1152Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly370375 380gcc ctc ctc ttg ctg ctg gtg gtg gcc ctg ggg atc ggc ctc ttc atg 1200Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met385390395 400cga agg cgc cac atc gtt cgg aag cgc acg ctg cgg agg ctg ctg cag 1248Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln405 410415gag agg gag ctt gtg gag cct ctt aca ccc agt gga gaa gct ccc aac 1296Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn420 425430caa gct ctc ttg agg atc ttg aag gaa act gaa ttc aaa aag atc aaa 1344Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys435440445gtg ctg ggc tcc ggt gcg ttc ggc acg gtg tat aag gga ctc tgg atc 1392Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile450455 460cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gaa tta aga 1440Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg
465 470475480gaa gca aca tct ccg aaa gcc aac aag gaa atc ctc gat gaa gcc tac 1488Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr485490495gtg atg gcc agc gtg gac aac ccc cac gtg tgc cgc ctg ctg ggc atc 1536Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile500 505510tgc ctc acc tcc acc gtg cag ctc atc acg cag ctc atg ccc ttc ggc 1584Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly515 520 525tgc ctc ctg gac tat gtc cgg gaa cac aaa gac aat att ggc tcc cag 1632Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln530535 540tac ctg ctc aac tgg tgt gtg cag atc gca aag ggc atg aac tac ttg 1680Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu545550555 560gag gac cgt cgc ttg gtg cac cgc gac ctg gca gcc agg aac gta ctg 1728Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu565570 575gtg aaa aca ccg cag cat gtc aag atc aca gat ttt ggg ctg gcc aaa 1776Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys580 585 590ctg ctg ggt gcg gaa gag aaa gaa tac cat gca gaa gga ggc aaa gtg 1824Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val595 600605cct atc aag tgg atg gca ttg gaa tca att tta cac aga atc tat acc 1872Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr610615 620cac cag agt gat gtc tgg agc tac ggg gtg acc gtt t9g gag ttg atg 1920His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met625 630 635 640acc ttt gga tcc aag cea tat gac gga atc cct gcc agc gag atc tcc 1968Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser645650 655tcc atc ctg gag aaa gga gaa cgc ctc cct cag cca ccc ata tgt acc 2016Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr660665 670atc gat gtc tac atg atc atg gtc aag tgc tgg atg ata gac gca gat 2064Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp675 680685agt cgc cca aag ttc cgt gag ttg atc atc gaa ttc tcc aaa atg gcc 2112Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala690695 700cga gac ccc cag cgc tac ctt gtc att cag ggg gat gaa aga atg cat 2160Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His705710715 720ttg cca agt cct aca gac tcc aac ttc tac cgt gcc ctg atg gat gaa 2208Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu
725 730735gaa gac atg gac gac gtg gtg gat gcc gac gag tac ctc atc cca cag 2256Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln740745750cag ggc ttc ttc agc agc ccc tcc acg tca cgg act ccc ctc ctg agc 2304Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser755760 765tct ctg agt gca acc agc aac aat tcc acc gtg gct tgc att gat aga 2352Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg770775780aat ggg ctg caa agc tgt ccc atc aag gaa gac agc ttc ttg cag cga 2400Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg785790 795 800tac agc tca gac ccc aca ggc gcc ttg act gag gac agc ata gac gac 2448Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp805810 815acc ttc ctc cca gtg cct gaa tac ata aac cag tcc gtt ccc aaa agg 2496Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg820 825 830ccc gct ggc tct gtg cag aat cct gtc tat cac aat cag cct ctg aac 2544Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn835 840845ccc gcg ccc agc aga gac cca cac tac cag gac ccc cac agc act gca 2592Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala850855 860gtg ggc aac ccc gag tat ctc aac act gtc cag ccc acc tgt gtc aac 2640Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn865 870 875 880agc aca ttc gac agc cct gcc cac tgg gcc cag aaa ggc agc cac caa 2688Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln885890 895att agc ctg gac aac cct gac tac cag cag gac ttc ttt ccc aag gaa 2736Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu900905910gcc aag cca aat ggc atc ttt aag ggc tcc aca gct gaa aat gca gaa 2784Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu915 920925tac cta agg gtc gcg cca caa agc agt gaa ttt att gga gca tga 2829Tyr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala930935 940<210>12<211>942<212>PRT<213>人(homo sapiens)
<400>12Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Arg Asn Tyr Val20 25 30Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr35 4045Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro5055 60Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser65 70 75 80Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser85 90 95Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser100 105 110Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys115 120125Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu130 135140Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly145150155160Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn165 170175Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp180185190Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn195 200205Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser210215 220Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala225230235 240Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val
245 250 255Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn260 265 270Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile275 280285Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr290295300Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly305 310315 320Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn325 330 335Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys340 345 350His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys355360365Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly370 375380Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met385390395 400Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln405 410415Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn420 425430Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys435440 445Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile450 455460Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg465 470 475 480Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr485 490 495Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile
500 505 510Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly515520 525Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln530 535 540Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu545550555 560Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu565570 575Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys580 585 590Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val595 600 605Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr610615620His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met625 630 635640Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser645650 655Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr660665 670Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp675 680685Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala690695 700Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His705710 715 720Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu725730735Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln740 745 750Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser
755 760765Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg770775780Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg785790 795800Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp805810 815Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg820 825 830Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn835840 845Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala850 855860Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn865 870 875 880Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln885890 895Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu900905910Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu915 920925TVr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala930935 940<210>13<211>3633<212>DNA<213>小鼠(Mus musculus)<220>
<221>CDS<222>(1)..(3630)<223>
<400>13atg cga ccc tca ggg acc gcg aga acc aca ctg ctg gtg ctg ctg acc 48Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr1 5 10 15gcg ctc tgc gcc gca ggt ggg gcg ttg gag gaa aag aaa gtc tgc caa 96Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30ggc aca agt aac agg ctc acc caa ctg ggc act ttt gaa gac cac ttt 144Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045ctg agc ctg cag agg atg tac aac aac tgt gaa gtg gtc ctt ggg aac 192Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn505560ttg gaa att acc tat gtg caa agg aat tac gac ctt tcc ttc tta aag 240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80acc atc cag gag gtg gcc ggc tat gtc ctc att gcc ctc aac acc gtg 288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag aga atc cct ttg gag aac ctg cag atc atc agg gga aat gct ctt 336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu100 105 110tat gaa aac acc tat gcc tta gcc atc ctg tcc aac tat ggg aca aac 384Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn115120 125aga act ggg ctt agg gaa ctg ccc atg cgg aac tta cag gaa atc ctg 432Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140att ggt gct gtg cga ttc agc aac aac ccc atc ctc tgc aat atg gat 480Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp145 150155160act atc cag tgg agg gac atc gtc caa aac gtc ttt atg agc aac atg 528Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met165 170175tca atg gac tta cag agc cat ccg agc agt tgc ccc aaa tgt gat cca 576Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro180185 190agc tgt ccc aat gga agc tgc tgg gga gga gga gag gag aac tgc cag 624Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln195200 205aaa ttg acc aaa atc atc tgt gcc cag caa tgt tcc cat cgc tgt cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg210215 220ggc agg tcc ccc agt gac tgc tgc cac aac caa tgt gct gcg ggg tgt 720Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225230235240
aca ggg ccc cga gag agt gac tgt ctg gtc tgc caa aag ttc caa gat 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp245250255gag gcc aca tgc aaa gac acc tgc cca cca ctc atg ctg tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270acc acc tat cag atg gat gtc aac cct gaa ggg aag tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285gcc acc tgt gtg aag aag tgc ccc cga aac tac gtg gtg aca gat cat 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300ggc tca tgt gtc cga gcc tgt ggg cct gac tac tac gaa gtg gaa gaa 960Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu305 310315 320gat ggc atc cgc aag tgt aaa aaa tgt gat ggg ccc tgt cgc aaa gtt 1008Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val325330335tgt aat ggc ata ggc att ggt gaa ttt aaa gac aca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn340 345350gct aca aac atc aaa cac ttc aaa tac tgc act gcc atc agc ggg gac 1104Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp355 360365ctt cac atc ctg cca gtg gcc ttt aag ggg gat tct ttc acg cgc act 1152Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr370 375 380cct cct cta gac cca cga gaa cta gaa att cta aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu385390395 400ata aca ggc ttt ttg ctg att cag gct tgg cct gat aac tgg act gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp405410 415ctc cat gct ttc gag aac cta gaa ata ata cgt ggc aga aca aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430cat ggt cag ttt tct ttg gcg gtc gtt ggc ctg aac atc aca tca ctg 1344His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu435440 445ggg ctg cgt tcc ctc aag gag atc agt gat ggg gat gtg atc att tct 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460gga aac cga aat ttg tgc tac gca aac aca ata aac tgg aaa aaa ctc 1440Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttc ggg aca ccc aat cag aaa acc aaa atc atg aac aac aga gct gag 1488Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu485490 495
aaa gac tgc aag gcc gtg aac cac gtc tgc aat cct tta tgc tcc tcg 1536Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser500 505510gaa ggc tgc tgg ggc cct gag ccc agg gac tgt gtc tcc tgc cag aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn515520525gtg agc aga ggc agg gag tgc gtg gag aaa tgc aac atc ctg gag ggg 1632Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly530535 540gaa cca agg gag ttt gtg gaa aat tct gaa tgc atc cag tgc cat cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550 555560gaa tgt ctg ccc cag gcc atg aac atc acc tgt aca ggc agg ggg cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575gac aac tgc atc cag tgt gcc cac tac att gat ggc cca cac tgt gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590aag acc tgc cca gct ggc atc atg gga gag aac aac act ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605aag tat gca gat gcc aat aat gtc tgc cac cta tgc cac gcc aac tgt 1872Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys610 615620acc tat gga tgt gct ggg cca ggt ctt caa gga tgt gaa gtg tgg cca 1920Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro625 630635640tct ggg cca aag ata cca tct att gcc act ggg att gtg ggt ggc ctc 1968Ser Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Ile Val Gly Gly Leu645 650 655ctc ttc ata gtg gtg gtg gcc ctt ggg att ggc cta ttc atg cga aga 2016Leu Phe Ile Val Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg660 665 670cgt cac att gtt cga aag cgt aca cta cgc cgc ctg ctt caa gag aga 2064Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg675 680685gag ctc gtg gaa cct ctc aca ccc agc gga gaa gct cca aac caa gcc 2112Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala690695 700cac ttg agg ata tta aag gaa aca gaa ttc aaa aag atc aaa gtt ctg 2160His Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu705 710715 720ggt tcg gga gca ttt ggc aca gtg tat aag ggt ctc tgg atc cca gaa 2208Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu725 730 735ggt gag aaa gta aaa atc ccg gtg gcc atc aag gag tta aga gaa gcc 2256Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala740 745 750
aca tct cca aaa gcc aac aaa gaa atc ctt gac gaa gcc tat gtg atg 2304Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met755 760 765gct agt gtg gac aac cct cat gta tgc cgc ctc ctg ggc atc tgt ctg 2352Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu770775 780acc tcc act gtc cag ctc att aca cag ctc atg ccc tac ggt tgc ctc 2400Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys Leu785 790 795800ctg gac tac gtc cga gaa cac aag gac aac att ggc tcc cag tac ctc 2448Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu805 810 815ctc aac tgg tgt gtg cag att gca aag ggc atg aac tac ctg gaa gat 2496Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp820 825 830cgg cgt ttg gtg cac cgt gac ttg gca gcc agg aat gta ctg gtg aag 2544Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys835 840845aca cca cag cat gtc aag atc aca gat ttt ggg ctg gcc aaa ctg ctt 2592Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu850 855 860ggt gct gaa gag aaa gaa tat cat gcc gag ggg ggc aaa gtg cct atc 2640Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile865 870 875 880aag tgg atg gct ttg gaa tca att tta cac cga att tat aca cac caa 2688Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln885890 895agt gat gtc tgg agc tat ggt gtc act gtg tgg gaa ctg atg acc ttt 2736Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe900 905 910ggg tcc aag cct tat gat gga atc cca gca agt gac atc tca tcc atc 2784Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Asp Ile Ser Ser Ile915 920 925cta gag aaa gga gaa cgc ctt cca cag cca cct atc tgc acc atc gat 2832Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp930935940gtc tac atg atc atg gtc aag tgc tgg atg ata gat gct gat agc cgc 2880Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg945 950 955960cca aag ttc cga gag ttg att ctt gaa ttc tcc aaa atg gcc cga gac 2928Pro Lys Phe Arg Glu Leu Ile Leu Glu Phe Ser Lys Met Ala Arg Asp965970975cca cag cgc tac ctt gtt atc cag ggg gat gaa aga atg cat ttg cca 2976Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro980 985 990agc cct aca gac tcc aac ttt tac cga gcc ctg atg gat gaa gag gac 3024Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp9951000 1005
atg gag gat gta gtt gat gct gat gag tat ctt acc cca cag caa3069Met Glu Asp Val Val Asp Ala Asp Glu Tyr Leu Thr Pro Gln Gln101010151020ggc ttc ttc aac agc ccg tcc acg tcg agg act ccc ctc ttg agt3114Gly Phe Phe Asn Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser1025 10301035tct ctg agt gca act agc aac aat tcc act gtg gct tgc att aat3159Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asn1040 10451050aga aat ggg agc tgc cgt gtc aaa gaa gac gcc ttc ttg cag cgg3204Arg Asn Gly Ser Cys Arg Val Lys Glu Asp Ala Phe Leu Gln Arg1055 10601065tac agc tcc gac ccc aca ggt gct gta aca gag gac aac ata gat3249Tyr Ser Ser Asp Pro Thr Gly Ala Val Thr Glu Asp Asn Ile Asp1070 10751080gac gca ttc ctc cct gta cct gaa tat gta aac caa tct gtt ccc3294Asp Ala Phe Leu Pro Val Pro Glu Tyr Val Asn Gln Ser Val Pro1085 10901095aag agg cca gca ggc tct gtg cag aac cct gtc tat cac aat cag3339Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln1100 11051110ccc ctg cat cca gct cct gga aga gac ctg cat tat caa aat ccc3384Pro Leu His Pro Ala Pro Gly Arg Asp Leu His Tyr Gln Asn Pro1115 11201125cac agc aat gca gtg ggc aac cct gag tat ctc aac act gcc cag3429His Ser Asn Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Ala Gln1130 11351140cct acc tgt ctc agt agt ggg ttt aac agc cct gca ctc tgg atc3474Pro Thr Cys Leu Ser Ser Gly Phe Asn Ser Pro Ala Leu Trp Ile1145 11501155cag aaa ggc agt cac caa atg agc cta gac aac cct gac tac cag3519Gln Lys Gly Ser His Gln Met Ser Leu Asp Asn Pro Asp Tyr Gln1160 11651170cag gac ttc ttc ccc aag gaa acc aag cca aat ggc ata ttt aag3564Gln Asp Phe Phe Pro Lys Glu Thr Lys Pro Asn Gly Ile Phe Lys1175 11801185ggc ccc aca gct gaa aat gca gag tac cta cgg gtg gca cct cca3609Gly Pro Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Pro1190 11951200agc agc gag ttt att gga gca tga 3633Ser Ser Glu Phe Ile Gly Ala1205 1210<210>14<211>1210<212>PRT<213>小鼠(Mus musculus)
<400>14Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr1 5 1015Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn50 5560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu100 105 110Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn115120 125Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp145 150155160Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met165 170175Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro180185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln195 200205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg210 215 220Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp245 250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290 295300Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu305310 315320Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val325330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn340 345350Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp355 360365Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr370 375 380Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp405410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu485490 495
Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser500 505510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn515520525Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly530535 540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550 555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp595600 605Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys610 615 620Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro625 630 635 640Ser Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Ile Val Gly Gly Leu645 650 655Leu Phe Ile Val Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg660 665 670Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg675 680685Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala690695 700His Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu705 710715 720Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu725730 735Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala740 745 750
Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met755 760 765Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu770775 780Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys Leu785 790 795800Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu805 810 815Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp820 825 830Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys835 840845Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu850 855 860Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile865 870 875 880Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln885890 895Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe900 905 910Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Asp Ile Ser Ser Ile915 920 925Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp930935940Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg945 950955 960Pro Lys Phe Arg Glu Leu Ile Leu Glu Phe Ser Lys Met Ala Arg Asp965 970 975Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro980 985 990Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp9951000 1005
Met Glu Asp Val Val Asp Ala Asp Glu Tyr Leu Thr Pro Gln Gln101010151020Gly Phe Phe Asn Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser102510301035Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asn104010451050Arg Asn Gly Ser Cys Arg Val Lys Glu Asp Ala Phe Leu Gln Arg105510601065Tyr Ser Ser Asp Pro Thr Gly Ala Val Thr Glu Asp Asn Ile Asp107010751080Asp Ala Phe Leu Pro Val Pro Glu Tyr Val Asn Gln Ser Val Pro108510901095Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln110011051110Pro Leu His Pro Ala Pro Gly Arg Asp Leu His Tyr Gln Asn Pro111511201125His Ser Asn Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Ala Gln113011351140Pro Thr Cys Leu Ser Ser Gly Phe Asn Ser Pro Ala Leu Trp Ile114511501155Gln Lys Gly Ser His Gln Met Ser Leu Asp Asn Pro Asp Tyr Gln116011651170Gln Asp Phe Phe Pro Lys Glu Thr Lys Pro Asn Gly Ile Phe Lys117511801185Gly Pro Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Pro119011951200Ser Ser Glu Phe Ile Gly Ala1205 1210<210>15<211>1932<212>DNA<213>小鼠(Mus musculus)
<220>
<221>CDS<222>(1)..(1929)<223>
-<400>15atg cga ccc tca ggg acc gcg aga acc aca ctg ctg gtg ctg ctg acc48Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr1 5 10 15gcg ctc tgc gcc gca ggt ggg gcg ttg gag gaa aag aaa gtc tgc caa96Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30ggc aca agt aac agg ctc acc caa ctg ggc act ttt gaa gac cac ttt144Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe354045ctg agc ctg cag agg atg tac aac aac tgt gaa gtg gtc ctt ggg aac192Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn505560ttg gaa att acc tat gtg caa agg aat tac gac ctt tcc ttc tta aag240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 7580acc atc cag gag gtg gcc ggc tat gtc ctc att gcc ctc aac acc gtg288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag aga atc cct ttg gag aac ctg cag atc atc agg gga aat gct ctt336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu100 105 110tat gaa aac acc tat gcc tta gcc atc ctg tcc aac tat ggg aca aac384Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn115120 125aga act ggg ctt agg gaa ctg ccc atg cgg aac tta cag gaa atc ctg432Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140att ggt gct gtg cga ttc agc aac aac ccc atc ctc tgc aat atg gat480Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp145 150155160act atc cag tgg agg gac atc gtc caa aac gtc ttt atg agc aac atg528Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met165 170175tca atg gac tta cag agc cat ccg agc agt tgc ccc aaa tgt gat cca576Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro180185 190agc tgt ccc aat gga agc tgc tgg gga gga gga gag gag aac tgc cag624Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln
195 200205aaa ttg acc aaa atc atc tgt gcc cag caa tgt tcc cat cgc tgt cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg210 215 220ggc agg tcc ccc agt gac tgc tgc cac aac caa tgt gct gcg ggg tgt 720Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225230235240aca ggg ccc cga gag agt gac tgt ctg gtc tgc caa aag ttc caa gat 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp245250255gag gcc aca tgc aaa gac acc tgc cca cca ctc atg ctg tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260265 270acc acc tat cag atg gat gtc aac cct gaa ggg aag tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285gcc acc tgt gtg aag aag tgc ccc cga aac tac gtg gtg aca gat cat 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300ggc tca tgt gtc cga gcc tgt ggg cct gac tac tac gaa gtg gaa gaa 960Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu305310 315320gat ggc atc cgc aag tgt aaa aaa tgt gat ggg ccc tgt cgc aaa gtt 1008Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val325330335tgt aat ggc ata ggc att ggt gaa ttt aaa gac aca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn340 345350gct aca aac atc aaa cac ttc aaa tac tgc act gcc atc agc ggg gac 1104Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp355 360365ctt cac atc ctg cca gtg gcc ttt aag ggg gat tct ttc acg cgc act 1152Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr370 375380cct cct cta gac cca cga gaa cta gaa att cta aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu385 390395400ata aca ggc ttt ttg ctg att cag gct tgg cct gat aac tgg act gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp405410 415ctc cat gct ttc gag aac cta gaa ata ata cgt ggc aga aca aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430cat ggt cag ttt tct ttg gcg gtc gtt ggc ctg aac atc aca tca ctg 1344His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu435440 445ggg ctg cgt tcc ctc aag gag atc agt gat ggg gat gtg atc att tct 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455460gga aac cga aat ttg tgc tac gca aac aca ata aac tgg aaa aaa ctc 1440Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttc ggg aca ccc aat cag aaa acc aaa atc atg aac aac aga gct gag 1488Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu485490 495aaa gac tgc aag gcc gtg aac cac gtc tgc aat cct tta tgc tcc tcg 1536Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser500 505510gaa ggc tgc tgg ggc cct gag ccc agg gac tgt gtc tcc tgc cag aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn515520525gtg agc aga ggc agg gag tgc gtg gag aaa tgc aac atc ctg gag ggg 1632Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly530535 540gaa cca agg gag ttt gtg gaa aat tct gaa tgc atc cag tgc cat cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550555560gaa tgt ctg ccc cag gcc atg aac atc acc tgt aca ggc agg ggg cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565570 575gac aac tgc atc cag tgt gcc cac tac att gat ggc cca cac tgt gtc 1776Asp Asn Cys Ile Gln Cys Ala HisTyr Ile Asp Gly Pro His Cys Val580 585590aag acc tgc cca gct ggc atc atg gga gag aac aac act ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp595600 605aag tat gca gat gcc aat aat gtc tgc cac cta tgc cac gcc aac tgt 1872Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys610 615 620acc tat gga gta atg gtt cct gaa atg ttg ctc caa agt atc att tta 1920Thr Tyr Gly Val Met Val Pro Glu Met Leu Leu Gln Ser Ile Ile Leu625 630635640aaa cct att tga 1932Lys Pro Ile<210>16<211>643<212>PRT<213>小鼠(Mus musculus)<400>16Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr
1 5 10 15Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn505560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 7580Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu100 105 110Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn115120 125Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp145 150155160Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met165 170175Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro180185190Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln195 200205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg210 215 220Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp245250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290 295300Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu305 310315320Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val325330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn340 345350Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp355 360365Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr370 375 380Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp405 410415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465 470475 480Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu485 490 495Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser500 505510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn
515 520 525Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly530535 540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys610 615 620Thr Tyr Gly Val Met Val Pro Glu Met Leu Leu Gln Ser Ile Ile Leu625 630635640Lys Pro Ile<210>17<211>1968<212>DNA<213>小鼠(Mus musculus)<220>
<221>CDS<222>(1)..(1965)<223>
<400>17atg cga ccc tca ggg acc gcg aga acc aca ctg ctg gtg ctg ctg acc48Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr1 5 10 15gcg ctc tgc gcc gca ggt ggg gcg ttg gag gaa aag aaa gtc tgc caa96Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30
ggc aca agt aac agg ctc acc caa ctg ggc act ttt gaa gac cac ttt144Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045ctg agc ctg cag agg atg tac aac aac tgt gaa gtg gtc ctt ggg aac192Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn505560ttg gaa att acc tat gtg caa agg aat tac gac ctt tcc ttc tta aag240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 7075 80acc atc cag gag gtg gcc ggc tat gtc ctc att gcc ctc aac acc gtg288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag aga atc cct ttg gag aac ctg cag atc atc agg gga aat gct ctt336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu100 105 110tat gaa aac acc tat gcc tta gcc atc ctg tcc aac tat ggg aca aac384Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn115120 125aga act ggg ctt agg gaa ctg ccc atg cgg aac tta cag gaa atc ctg432Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140att ggt gct gtg cga ttc agc aac aac ccc atc ctc tgc aat atg gat480Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp145 150155160act atc cag tgg agg gac atc gtc caa aac gtc ttt atg agc aac atg528Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met165 170175tca atg gac tta cag agc cat ccg agc agt tgc ccc aaa tgt gat cca576Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro180185 190agc tgt ccc aat gga agc tgc tgg gga gga gga gag gag aac tgc cag624Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln195200 205aaa ttg acc aaa atc atc tgt gcc cag caa tgt tcc cat cgc tgt cgt672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg210 215 220ggc agg tcc ccc agt gac tgc tgc cac aac caa tgt gct gcg ggg tgt720Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240aca ggg ccc cga gag agt gac tgt ctg gtc tgc caa aag ttc caa gat768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp245250255gag gcc aca tgc aaa gac acc tgc cca cca ctc atg ctg tac aac ccc816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270acc acc tat cag atg gat gtc aac cct gaa ggg aag tac agc ttt ggt864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275280285
gcc acc tgt gtg aag aag tgc ccc cga aac tac gtg gtg aca gat cat 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290 295300ggc tca tgt gtc cga gcc tgt ggg cct gac tac tac gaa gtg gaa gaa 960Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu305310 315320gat ggc atc cgc aag tgt aaa aaa tgt gat ggg ccc tgt cgc aaa gtt 1008Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val325330335tgt aat ggc ata ggc att ggt gaa ttt aaa gac aca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Set Ile Asn340 345350gct aca aac atc aaa cac ttc aaa tac tgc act gcc atc agc ggg gac 1104Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp355 360365ctt cac atc ctg cca gtg gcc ttt aag ggg gat tct ttc acg cgc act 1152Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr370 375 380cct cct cta gac cca cga gaa cta gaa att cta aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu385390395 400ata aca ggc ttt ttg ctg att cag gct tgg cct gat aac tgg act gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp405 410 415ctc cat gct ttc gag aac cta gaa ata ata cgt ggc aga aca aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430cat ggt cag ttt tct ttg gcg gtc gtt ggc ctg aac atc aca tca ctg 1344His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu435440 445ggg ctg cgt tcc ctc aag gag atc agt gat ggg gat gtg atc att tct 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460gga aac cga aat ttg tgc tac gca aac aca ata aac tgg aaa aaa ctc 1440Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttc ggg aca ccc aat cag aaa acc aaa atc atg aac aac aga gct gag 1488Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu485490 495aaa gac tgc aag gcc gtg aac cac gtc tgc aat cct tta tgc tcc tcg 1536Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser500 505510gaa ggc tgc tgg ggc cct gag ccc agg gac tgt gtc tcc tgc cag aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn515520525gtg agc aga ggc agg gag tgc gtg gag aaa tgc aac atc ctg gag ggg 1632Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly530 535540
gaa cca agg gag ttt gtg gaa aat tct gaa tgc atc cag tgc cat cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550555 560gaa tgt ctg ccc cag gcc atg aac atc acc tgt aca ggc agg ggg cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575gac aac tgc atc cag tgt gcc cac tac att gat ggc cca cac tgt gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590aag acc tgc cca gct ggc atc atg gga gag aac aac act ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605aag tat gca gat gcc aat aat gtc tgc cac cta tgc cac gcc aac tgt 1872Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys610615620acc tat gga tgt gct ggg cca ggt ctt caa gga tgt gaa gtg tgg cca 1920Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro625630 635640tct ggg tac gtt caa tgg cag tgg atc tta aag acc ttt tgg atc taa 1968Ser Gly Tyr Val Gln Trp Gln Trp Ile Leu Lys Thr Phe Trp Ile645 650 655<210>18<211>655<212>PRT<213>小鼠(Mus musculus)<400>18Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr1 5 10 15Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe354045Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn505560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu100 105 110Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn115120 125Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130 135140Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp145 150 155 160Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met165 170175Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro180185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln195 200205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg210 215 220Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp245250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290 295300Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu305 310315320Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val325330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn340 345350
Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp355 360365Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr370 375 380Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp405410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu485490 495Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser500 505510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn515520525Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly530535 540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605
Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys610 615620Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro625 630635640Ser Gly Tyr Val Gln Trp Gln Trp Ile Leu Lys Thr Phe Trp Ile645 650 655<210>19<211>1962<212>DNA<213>鸡<220>
<221>CDS<222>(1)..(1962)<223>
<400>19atg ggt gta cgc agc ccc ctg tcc gcc tct ggg cct cgc ggg gcc gct 48Met Gly Val Arg Ser Pro Leu Ser Ala Ser Gly Pro Arg Gly Ala Ala1 5 10 15gtc ctg gtg cta ctg ctg ctg ggc gtc gcg ctg tgt tcc gcc gtg gag 96Val Leu Val Leu Leu Leu Leu Gly Val Ala Leu Cys Ser Ala Val Glu20 25 30gag aag aaa gtt tgt caa ggg aca aat aac aag ttg acc cag ctg ggg 144Glu Lys Lys Val Cys Gln Gly Thr Asn Asn Lys Leu Thr Gln Leu Gly3540 45cac gtg gaa gac cat ttc acc agc ctg cag aga atg tac aac aac tgc 192His Val Glu Asp His Phe Thr Ser Leu Gln Arg Met Tyr Asn Asn Cys50 55 60gaa gtg gta ctg a9t aac ctg gag att acc tac gtg gag cat aat cgc 240Glu Val Val Leu Ser Asn Leu Glu Ile Thr Tyr Val Glu His Asn Arg65 70 75 80gat ctc acc ttc ctt aag acc ata cag gag gtt gca ggc tat gtg ctc 288Asp Leu Thr Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr Val Leu85 90 95att gcg ctt aac atg gtg gac gtc att ccc tta gaa aac ctc cag att 336Ile Ala Leu Asn Met Val Asp Val Ile Pro Leu Glu Asn Leu Gln Ile100105 110atc cga ggg aat gtg ctt tat gac aac tct ttt gcc ctg gca gtt tta 384Ile Arg Gly Asn Val Leu Tyr Asp Asn Ser Phe Ala Leu Ala Val Leu
115 120125tcc aat tac cac atg aat aaa acc cag gga ctt cga gag ctg cca atg432Ser Asn Tyr His Met Asn Lys Thr Gln Gly Leu Arg Glu Leu Pro Met130135140aaa cgg cta tca gaa att ctc aat gga ggt gtt aaa atc agc aac aac480Lys Arg Leu Ser Glu Ile Leu Asn Gly Gly Val Lys Ile Ser Asn Asn145150 155 160ccc aaa ctg tgc aac atg gac act gtt ctc tgg aat gac atc att gat528Pro Lys Leu Cys Asn Met Asp Thr Val Leu Trp Asn Asp Ile Ile Asp165 170 175aca agc agg aag cct ctc aca gta ctt gac ttt gca agc aat ctt tct576Thr Ser Arg Lys Pro Leu Thr Val Leu Asp Phe Ala Ser Asn Leu Ser180185190tct tgt cca aaa tgc cat ccg aac tgc aca gaa gac cac tgc tgg ggt624Ser Cys Pro Lys Cys His Pro Asn Cys Thr Glu Asp His Cys Trp Gly195200205gct ggt gaa cag aac tgc cag act tta aca aaa gtc atc tgt gcc cag672Ala Gly Glu Gln Asn Cys Gln Thr Leu Thr Lys Val Ile Cys Ala Gln210215220caa tgc tct ggc cgg tgc aga gga aag gtg ccc agt gac tgc tgc cac720Gln Cys Ser Gly Arg Cys Arg Gly Lys Val Pro Ser Asp Cys Cys His225230 235240aat cag tgt gct gca ggg tgc aca gga cct cgg gag agt gac tgc ctg768Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys Leu245 250 255gca tgc cgc aag ttt cgg gat gat gct acc tgc aag gac aca tgt ccc816Ala Cys Arg Lys Phe Arg Asp Asp Ala Thr Cys Lys Asp Thr Cys Pro260265270cca ctg gtc ctc tat aac ccc acc acc tat caa atg gat gtc aac cct864Pro Leu Val Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn Pro275 280285gag gga aaa tac agc ttt gga gcc act tgt gtg agg gaa tgt cca cac912Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Arg Glu Cys Pro His290 295300aac tat gtg gtg aca gat cat ggc tcc tgc gtt cgc tcg tgt aat act960Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ser Cys Asn Thr305 310315320gat act tac gaa gtg gaa gaa aat ggt gtt cgg aag tgt aaa aaa tgt 1008Asp Thr Tyr Glu Val Glu Glu Asn Gly Val Arg Lys Cys Lys Lys Cys325 330335gat ggg cta tgt agc aaa gtg tgc aat ggc att gga ata ggt gaa ctt 1056Asp Gly Leu Cys Ser Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Leu340345350aaa ggg atc cta tcc ata aat gcc aca aac atc gac tcc ttc aaa aac 1104Lys Gly Ile Leu Ser Ile Asn Ala Thr Asn Ile Asp Ser Phe Lys Asn355 360365tgt acg aag atc aat ggg gat gtc agc att ctt cct gtt gca ttt cta 1152Cys Thr Lys Ile Asn Gly Asp Val Ser Ile Leu Pro Val Ala Phe Leu
370 375 380ggg gat gcc ttc aca aag aca cta ccc ctt gac cct aag aag ctg gat 1200Gly Asp Ala Phe Thr Lys Thr Leu Pro Leu Asp Pro Lys Lys Leu Asp385390 395400gtc ttt aga aca gtc aaa gaa ata tca gga ttt ttg ttg att cag gcc 1248Val Phe Arg Thr Val Lys Glu Ile Ser Gly Phe Leu Leu Ile Gln Ala405 410 415tgg cct gat aat gct act gat ctc tat gct ttt gaa aat ctg gag att 1296Trp Pro Asp Asn Ala Thr Asp Leu Tyr Ala Phe Glu Asn Leu Glu Ile420 425430atc cga ggc cga acc aag cag cac ggc cag tat tcc ctt gct gtt gtt 1344Ile Arg Gly Arg Thr Lys Gln His Gly Gln Tyr Ser Leu Ala Val Val435 440445aac ttg aaa ata cag tcg ttg ggg ctg cgc tcc ctc aag gaa ata agt 1392Asn Leu Lys Ile Gln Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser450 455460gat gga gac att gcc att atg aag aac aag aac ctc tgc tat gct gac 1440Asp Gly Asp Ile Ala Ile Met Lys Asn Lys Asn Leu Cys Tyr Ala Asp465 470 475480acc atg aac tgg cgc agc ttg ttt gct act cag agt cag aaa aca aaa 1488Thr Met Asn Trp Arg Ser Leu Phe Ala Thr Gln Ser Gln Lys Thr Lys485490495att ata cag aac aga aat aaa aat gat tgt act gct gac agg cat gtg 1536Ile Ile Gln Asn Arg Asn Lys Asn Asp Cys Thr Ala Asp Arg His Val500505 510tgt gac ccg ctg tgc tcg gac gtg ggc tgc tgg ggc cca ggg ccc ttc 1584Cys Asp Pro Leu Cys Ser Asp Val Gly Cys Trp Gly Pro Gly Pro Phe515520525cac tgc ttt tcc tgc agg ttt ttc agt cgc cag aag gag tgt gta aaa 1632His Cys Phe Ser Cys Arg Phe Phe Ser Arg Gln Lys Glu Cys Val Lys530 535540cag tgc aac atc ctg caa ggg gag cca cgt gag ttt gaa aga gac tcc 1680Gln Cys Asn Ile Leu Gln Gly Glu Pro Arg Glu Phe Glu Arg Asp Ser545 550555560aaa tgc ctc ccc tgc cac tca gag tgt ctg gta cag aac tcc act gca 1728Lys Cys Leu Pro Cys His Ser Glu Cys Leu Val Gln Asn Ser Thr Ala565 570575tac aac aca acc tgc tct gga ccg ggc cca gac cac tgc atg aag tgt 1776Tyr Asn Thr Thr Cys Ser Gly Pro Gly Pro Asp His Cys Met Lys Cys580585 590gcc cat ttt ata gat ggt ccc cac tgt gtg aag gcc tgc ccc gct ggg 1824Ala His Phe Ile Asp Gly Pro His Cys Val Lys Ala Cys Pro Ala Gly595 600605gtc ctg ggt gag aat gat acc ctg gtc tgg aag tat gca gat gcc aat 1872Val Leu Gly Glu Asn Asp Thr Leu Val Trp Lys Tyr Ala Asp Ala Asn610615620gct gtt tgc cag ctc tgc cat cca aac tgt aca cga ggg tgc aaa ggg 1920Ala Val Cys Gln Leu Cys His Pro Asn Cys Thr Arg Gly Cys Lys Gly
625 630 635 640cca ggt ctt gaa gga tgt cca aat ggc tcc aaa act cca tct1962Pro Gly Leu Glu Gly Cys Pro Asn Gly Ser Lys Thr Pro Ser645 650<210>20<211>654<212>PRT<213>鸡<400>20Met Gly Val Arg Ser Pro Leu Ser Ala Ser Gly Pro Arg Gly Ala Ala1 5 1015Val Leu Val Leu Leu Leu Leu Gly Val Ala Leu Cys Ser Ala Val Glu20 25 30Glu Lys Lys Val Cys Gln Gly Thr Asn Asn Lys Leu Thr Gln Leu Gly3540 45His Val Glu Asp His Phe Thr Ser Leu Gln Arg Met Tyr Asn Asn Cys50 5560Glu Val Val Leu Ser Asn Leu Glu Ile Thr Tyr Val Glu His Asn Arg65 7075 80Asp Leu Thr Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr Val Leu85 90 95Ile Ala Leu Asn Met Val Asp Val Ile Pro Leu Glu Asn Leu Gln Ile100105 110Ile Arg Gly Asn Val Leu Tyr Asp Asn Ser Phe Ala Leu Ala Val Leu115120125Ser Asn Tyr His Met Asn Lys Thr Gln Gly Leu Arg Glu Leu Pro Met130135140Lys Arg Leu Ser Glu Ile Leu Asn Gly Gly Val Lys Ile Ser Asn Asn145150 155 160Pro Lys Leu Cys Asn Met Asp Thr Val Leu Trp Asn Asp Ile Ile Asp165 170 175Thr Ser Arg Lys Pro Leu Thr Val Leu Asp Phe Ala Ser Asn Leu Ser
180 185 190Ser Cys Pro Lys Cys His Pro Asn Cys Thr Glu Asp His Cys Trp Gly195200 205Ala Gly Glu Gln Asn Cys Gln Thr Leu Thr Lys Val Ile Cys Ala Gln210215220Gln Cys Ser Gly Arg Cys Arg Gly Lys Val Pro Ser Asp Cys Cys His225230235 240Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys Leu245 250 255Ala Cys Arg Lys Phe Arg Asp Asp Ala Thr Cys Lys Asp Thr Cys Pro260265270Pro Leu Val Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn Pro275 280285Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Arg Glu Cys Pro His290 295300Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ser Cys Asn Thr305 310315320Asp Thr Tyr Glu Val Glu Glu Asn Gly Val Arg Lys Cys Lys Lys Cys325330 335Asp Gly Leu Cys Ser Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Leu340345350Lys Gly Ile Leu Ser Ile Asn Ala Thr Asn Ile Asp Ser Phe Lys Asn355 360365Cys Thr Lys Ile Asn Gly Asp Val Ser Ile Leu Pro Val Ala Phe Leu370 375380Gly Asp Ala Phe Thr Lys Thr Leu Pro Leu Asp Pro Lys Lys Leu Asp385390 395400Val Phe Arg Thr Val Lys Glu Ile Ser Gly Phe Leu Leu Ile Gln Ala405 410415Trp Pro Asp Asn Ala Thr Asp Leu Tyr Ala Phe Glu Asn Leu Glu Ile420 425430Ile Arg Gly Arg Thr Lys Gln His Gly Gln Tyr Ser Leu Ala Val Val
435 440445Asn Leu Lys Ile Gln Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser450455 460Asp Gly Asp Ile Ala Ile Met Lys Asn Lys Asn Leu Cys Tyr Ala Asp465 470 475 480Thr Met Asn Trp Arg Ser Leu Phe Ala Thr Gln Ser Gln Lys Thr Lys485 490 495Ile Ile Gln Asn Arg Asn Lys Asn Asp Cys Thr Ala Asp Arg His Val500 505510Cys Asp Pro Leu Cys Ser Asp Val Gly Cys Trp Gly Pro Gly Pro Phe515520525His Cys Phe Ser Cys Arg Phe Phe Ser Arg Gln Lys Glu Cys Val Lys530535 540Gln Cys Asn Ile Leu Gln Gly Glu Pro Arg Glu Phe Glu Arg Asp Ser545 550555560Lys Cys Leu Pro Cys His Ser Glu Cys Leu Val Gln Asn Ser Thr Ala565570575Tyr Asn Thr Thr Cys Ser Gly Pro Gly Pro Asp His Cys Met Lys Cys580 585590Ala His Phe Ile Asp Gly Pro His Cys Val Lys Ala Cys Pro Ala Gly595 600605Val Leu Gly Glu Asn Asp Thr Leu Val Trp Lys Tyr Ala Asp Ala Asn610615620Ala Val Cys Gln Leu Cys His Pro Asn Cys Thr Arg Gly Cys Lys Gly625 630635640Pro Gly Leu Glu Gly Cys Pro Asn Gly Ser Lys Thr Pro Ser645 650<210>21<211>4134<212>DNA<213>果蝇(Drosophila melanogaster)
<220>
<221>CDS<222>(1)..(3630)<223>
<400>21atg atg att atc agc atg tgg atg agc ata tcg cga gga ttg tgg gac48Met Met Ile Ile Ser Met Trp Met Ser Ile Ser Arg Gly Leu Trp Asp1 5 10 15agc agc tcc atc ttg tcg gtg ctg ctg atc ctc gcc tgc atg gca tcc96Ser Ser Ser Ile Leu Ser Val Leu Leu Ile Leu Ala Cys Met Ala Ser20 25 30atc acc aca agc tca tcg gtc agc aat gcc ggc tat gtg gat aat ggc 144Ile Thr Thr Ser Ser Ser Val Ser Asn Ala Gly Tyr Val Asp Asn Gly35 40 45aat atg aaa gtc tgc atc ggc act aaa tct cgg ctc tcc gtg ccc tcc 192Asn Met Lys Val Cys Ile Gly Thr Lys Ser Arg Leu Ser Val Pro Ser5055 60aac aag gaa cat cat tac cga aac ctc aga gat cgg tac acg aac tgt 240Asn Lys Glu His His Tyr Arg Asn Leu Arg Asp Arg Tyr Thr Asn Cys65 70 7580acg tat gtg gat ggc aac ttg aaa ctg acc tgg cta ccc aac gag aat 288Thr Tyr Val Asp Gly Asn Leu Lys Leu Thr Trp Leu Pro Asn Glu Asn85 9095ttg gac ctc agc ttc cta gac aac ata cgg gag gtc acc ggc tat att 336Leu Asp Leu Ser Phe Leu Asp Asn Ile Arg Glu Val Thr Gly Tyr Ile100105110ctg atc agt cat gtg gac gtt aag aaa gtg gtg ttt ccc aaa cta caa 384Leu Ile Ser His Val Asp Val Lys Lys Val Val Phe Pro Lys Leu Gln115 120125atc att cgc gga cgc acg ctg ttc agc tta tcc gtg gag gag gag aag 432Ile Ile Arg Gly Arg Thr Leu Phe Ser Leu Ser Val Glu Glu Glu Lys130 135 140tat gcc ttg ttc gtc act tat tcc aaa atg tac acg ctg gag att ccc 480Tyr Ala Leu Phe Val Thr Tyr Ser Lys Met Tyr Thr Leu Glu Ile Pro145 150 155 160gat cta cgc gat gtc tta aat ggc caa gtg ggc ttc cac aac aac tac 528Asp Leu Arg Asp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn Tyr165170 175aat ctc tgc cac atg cga acg atc cag tgg tcg gag att gta tcc aac 576Asn Leu Cys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser Asn180185 190ggc acg gat gca tac tac aac tac gac ttt act gct ccg gag cgc gag 624Gly Thr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg Glu195 200205
tgt ccc aag tgc cac gag agc tgc acg cac gga tgt tgg ggc gag ggt 672Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu Gly210215220ccc aag aat tgc cag aag ttc agc aag ctc acc tgc tcg cca cag tgt 720Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser Pro Gln Cys225230235240gcc gga ggt cgt tgc tat gga cca aag ccg cgg gag tgt tgt cac ctc 768Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu Cys Cys His Leu245250255ttc tgc gcc gga gga tgc act ggt ccc acg caa aag gat tgc atc gcc 816Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln Lys Asp Cys Ile Ala260 265270tgc aag aac ttc ttc gac gag gca gta tca aag gag gaa tgc ccg ccc 864Cys Lys Asn Phe Phe Asp Glu Ala Val Ser Lys Glu Glu Cys Pro Pro275280 285atg cgc aag tac aat ccc acc acc tat gtt ctt gaa acg aat cct gag 912Met Arg Lys Tyr Asn Pro Thr Thr Tyr Val Leu Glu Thr Asn Pro Glu290295300gga aag tat gcc tat ggt gcc acc tgc gtc aag gag tgt ccc ggt cat 960Gly Lys Tyr Ala Tyr Gly Ala Thr Cys Val Lys Glu Cys Pro Gly His305 310 315320ctg ttg cgg gat aat ggc gcc tgc gtg cgc agc tgt ccc cag gac aag 1008Leu Leu Arg Asp Asn Gly Ala Cys Val Arg Ser Cys Pro Gln Asp Lys325 330335atg gac aag ggg ggc gag tgt gtg ccc tgc aat gga ccg tgc ccc aaa 1056Met Asp Lys Gly Gly Glu Cys Val Pro Cys Asn Gly Pro Cys Pro Lys340345350acc tgc ccg ggc gtt act gtc ctg cat gcc ggc aac att gac tcg ttc 1104Thr Cys Pro Gly Val Thr Val Leu His Ala Gly Asn Ile Asp Ser Phe355 360 365cgg aat tgt acg gtg atc gat ggc aac att cgc att ttg gat cag acc 1152Arg Asn Cys Thr Val Ile Asp Gly Asn Ile Arg Ile Leu Asp Gln Thr370 375 380ttc tcg ggc ttc cag gat gtc tat gcc aac tac acg atg gga cca cga 1200Phe Ser Gly Phe Gln Asp Val Tyr Ala Asn Tyr Thr Met Gly Pro Arg385390395 400tac ata ccg ctg gat ccc gag cga cgg gag gtg ttc tcc acg gtg aag 1248Tyr Ile Pro Leu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val Lys405410415gag atc acc ggg tat ctg aat atc gag gga acc cac ccg cag ttc cgg 1296Glu Ile Thr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe Arg420425 430aat ctg tcg tac ttt cgc aat ctg gaa aca att cat ggc cgc cag ctg 1344Asn Leu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln Leu435440445atg gag agc atg ttt gcc gct ttg gcg atc gtt aag tca tcc ctg tac 1392Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu Tyr450 455 460
agc ctg gag atg cgc aat ctg aag cag att agt tcc ggc agt gtg gtc 1440Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val Val465470475 480atc cag cat aat aga gac ctc tgc tac gta agc aat atc cgt tgg ccg 1488Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp Pro485490495gcc att cag aag gag ccc gaa cag aag gtg tgg gtc aac gag aat ctc 1536Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp Val Asn Glu Asn Leu500 505510agg gcg gat cta tgc gag aaa aat gga acc att tgc tcg gat cag tgc 1584Arg Ala Asp Leu Cys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln Cys515 520 525aac gag gac ggc tgc tgg gga gct ggc acg gat cag tgc ctt acc tgc 1632Asn Glu Asp Gly Cys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr Cys530 535 540aag aac ttc aat ttc aat ggc acc tgc atc gcc gac tgt ggt tat ata 1680Lys Asn Phe Asn Phe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr Ile545550555 560tcc aat gcc tac aag ttt gac aat aga acg tgc aag ata tgc cat cca 1728Ser Asn Ala Tyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His Pro565570575gag tgc cgg act tgc aat gga gct gga gca gat cac tgc cag gag tgc 1776Glu Cys Arg Thr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu Cys580585 590gtc cat gtg agg gac ggt cag cac tgt gtg tcc gag tgc ccg aag aac 1824Val His Val Arg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys Asn595600 605aag tac aac gat cgt ggt gtc tgc cga gag tgc cac gcc acc tgc gat 1872Lys Tyr Asn Asp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys Asp610615620gga tgc act ggg ccc aag gac acc atc ggc att gga gcg tgt acg acg 1920Gly Cys Thr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr Thr625 630 635 640tgc aat ttg gcc att atc aac aat gac gcc aca gta aaa cgc tgc ctg 1968Cys Asn Leu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys Leu645 650655ctg aag gac gac aag tgc ccc gat ggg tat ttc tgg gag tat gtg cat 2016Leu Lys Asp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val His660 665 670ccg caa gag cag gga tcg ctg aag cca ttg gcc ggc aga gca gtt tgc 2064Pro Gln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val Cys675 680685cga aag tgc cat ccc ctt tgc gag ctg tgc acg aac tac gga tac cat 2112Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr His690695700gaa cag gtg tgc tcc aag tgc acc cac tac aag cga cgg gag cag tgc 2160Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln Cys705 710715720
gag acc gag tgt ccg gcc gat cac tac acg gat gag gag cag cgc gag 2208Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg Glu725730 735tgc ttc cag cgc cac ccg gaa tgc aat ggt tgc acg ggt ccg ggt gcc 2256Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly Ala740 745 750gac gat tgc aag tct tgc cgc aac ttt aag ttg ttc gac gcg aat gag 2304Asp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn Glu755760765acg ggt ccc tat gtg aac tcc acg atg ttc aat tgc acc tcg aag tgt 2352Thr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys Cys770 775 780ccc ttg gag atg cga cat gtg aac tat cag tac acg gcc att gga ccc 2400Pro Leu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly Pro785790 795800tac tgc gca gct agt ccg ccg agg agc agc aag ata act gcc aat ctg 2448Tyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn Leu805 810815gat gtg aac atg atc ttc att atc act ggt gct gtt ctg gtg ccg acg 2496Asp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro Thr820 825 830atc tgc atc ctc tgc gtg gtc aca tac att tgt cgg caa aag caa aag 2544Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln Lys835 840 845gcc aag aag gaa aca gtc aag atg acc atg gct ctg tct ggc tgc gag 2592Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys Glu850855 860gat tcc gag ccg ctg cgt ccc tcg aac att gga gcc aac cta tgc aag 2640Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys Lys865870875 880ttg cgc att gtc aag gac gcc gag ttg cgc aag ggc gga gtc ctt gga 2688Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu Gly885 890895atg gga gcc ttt gga cga gtg tac aag ggc gtt tgg gtg ccg gag ggt 2736Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu Gly900 905 910gag aac gtc aag att cca gtg gcc att aag gag ctg ctc aag tcc aca 2784Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu LVs Ser Thr915 920 925ggc gcc gag tca agc gaa gag ttc ctc cgc gaa gcc tac atc atg gcc 2832Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met Ala930 935 940tct gag gag cac gtt aat ctg ctg aag ctc ctg gcc gtg tgc atg tcc 2880Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met Ser945 950 955 960tca caa atg atg cta atc acg caa ctg atg ccg ctt ggc tgc ctg ttg 2928Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys Leu Leu965 970975
gac tat gtg cga aat aac cgg gac aag atc ggc tct aag gct ctg ctc 2976Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys Ala Leu Leu980985 990aac tgg agc acg caa atc gcc aag ggc atg tcg tat ctg gag gag aag 3024Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr Leu Glu Glu Lys995 10001005cga ctg gtc cac aga gac ttg gct gcc cgc aat gtc ctg gtg cag 3069Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Gln101010151020act ccc tcg ctg gtg aag atcacc gac ttt ggg ctg gcc aag ttg 3114Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu1025 10301035ctg agc agc gat tcc aat gag tac aag gct gct ggc ggc aag atg 3159Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala Gly Gly Lys Met1040 10451050ccc atc aag tgg ttg gca ctg gag tgc atc cgc aat cgt gta ttc 3204Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg Asn Arg Val Phe1055 10601065acc agc aag tcc gat gtc tgg gcc ttt ggt gtg acc att tgg gaa 3249Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val Thr Ile Trp Glu1070 10751080ctg ctg acc ttt ggc cag cgt cca cac gag aac atc cca gct aag 3294Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn Ile Pro Ala Lys1085 10901095gat att ccc gat ctt att gaa gtc ggt ctg aag ctg gag cag ccg 3339Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys Leu Glu Gln Pro1100 11051110gag att tgt tcg ctg gac att tac tgt aca ctg ctc tcg tgc tgg 3384Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu Leu Ser Cys Trp1115 11201125cac ttg gat gcc gcc atg cgt cca acc ttc aag cag ctg act acg 3429His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys Gln Leu Thr Thr1130 11351140gtc ttt gct gag ttc gcc aga gat ccg ggt cgc tat ctg gcc att 3474Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg Tyr Leu Ala Ile1145 11501155ccc ggg gat aag ttc acc cgg ctg ccg gct tac acg agt cag gat 3519Pro Gly Asp Lys Phe Thr Arg Leu Pro Ala Tyr Thr Ser Gln Asp1160 11651170gag aag gat ctc atc cga aaa ttg gct ccc acc acc gat ggg tcc 3564Glu Lys Asp Leu Ile Arg Lys Leu Ala Pro Thr Thr Asp Gly Ser117511801185gaa gcc att gcg aaa ccc gat gac tac ctg caa ccc aag gca gca 3609Glu Ala Ile Ala Lys Pro Asp Asp Tyr Leu Gln Pro Lys Ala Ala1190 11951200cct ggt cct agt cac aga acc gactgcacgg atgagatgcc caagctgaac 3660Pro Gly Pro Ser His Arg Thr12051210
cgctactgca aggatcctag taacaagaat tcgagtaccg gagacgatga gagggattcg 3720agtgcccggg aagtgggcgt gggtaatctg cgcctcgatc taccagtcga tgaggatgat 3780tatctgatgc ccacttgcca accgggtccc aacaacaaca acaacatgaa taatcccaat 3840caaaacaata tggcagctgt gggcgtggct gccggctaca tggatctcat cggagtgccc 3900gttagtgtgg acaatccgga gtatctgcta aacgcgcaga cactgggagt tggggagtcg 3960ccgataccca cccagaccat cgggataccg gtgatgggag gcccgggcac catggaggtc 4020aaggtgccaa tgccaggcag tgagccaacc agctccgatc acgagtacta caatgatacc 4080caacgggagt tgcagccact gcatcgaaac cgcaacacgg agacgagggt gtag4134<210>22<211>1210<212>PRT<213>果蝇(Drosophila melanogaster)<400>22Met Met Ile Ile Ser Met Trp Met Ser Ile Ser Arg Gly Leu Trp Asp1 5 10 15Ser Ser Ser Ile Leu Ser Val Leu Leu Ile Leu Ala Cys Met Ala Ser20 25 30Ile Thr Thr Ser Ser Ser Val Ser Asn Ala Gly Tyr Val Asp Asn Gly35 40 45Asn Met Lys Val Cys Ile Gly Thr Lys Ser Arg Leu Ser Val Pro Ser5055 60Asn Lys Glu His His Tyr Arg Asn Leu Arg Asp Arg Tyr Thr Asn Cys65 70 7580Thr Tyr Val Asp Gly Asn Leu Lys Leu Thr Trp Leu Pro Asn Glu Asn85 9095Leu Asp Leu Ser Phe Leu Asp Asn Ile Arg Glu Val Thr Gly Tyr Ile100 105 110Leu Ile Ser His Val Asp Val Lys Lys Val Val Phe Pro Lys Leu Gln115120 125Ile Ile Arg Gly Arg Thr Leu Phe Ser Leu Ser Val Glu Glu Glu Lys130 135 140
Tyr Ala Leu Phe Val Thr Tyr Ser Lys Met Tyr Thr Leu Glu Ile Pro145 150155160Asp Leu Arg Asp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn Tyr165170 175Asn Leu Cys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser Asn180185 190Gly Thr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg Glu195 200205Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu Gly210 215220Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser Pro Gln Cys225230235240Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu Cys Cys His Leu245 250255Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln Lys Asp Cys Ile Ala260 265270Cys Lys Asn Phe Phe Asp Glu Ala Val Ser Lys Glu Glu Cys Pro Pro275280 285Met Arg Lys Tyr Asn Pro Thr Thr Tyr Val Leu Glu Thr Asn Pro Glu290295300Gly Lys Tyr Ala Tyr Gly Ala Thr Cys Val Lys Glu Cys Pro Gly His305 310 315320Leu Leu Arg Asp Asn Gly Ala Cys Val Arg Ser Cys Pro Gln Asp Lys325 330335Met Asp Lys Gly Gly Glu Cys Val Pro Cys Asn Gly Pro Cys Pro Lys340345350Thr Cys Pro Gly Val Thr Val Leu His Ala Gly Asn Ile Asp Ser Phe355 360 365Arg Asn Cys Thr Val Ile Asp Gly Asn Ile Arg Ile Leu Asp Gln Thr370 375 380Phe Ser Gly Phe Gln Asp Val Tyr Ala Asn Tyr Thr Met Gly Pro Arg385390395 400
Tyr Ile Pro Leu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val Lys405410415Glu Ile Thr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe Arg420 425 430Asn Leu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln Leu435440445Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu Tyr450 455 460Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val Val465470475480Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp Pro485 490 495Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp Val Asn Glu Asn Leu500 505 510Arg Ala Asp Leu Cys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln Cys515520 525Asn Glu Asp Gly Cys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr Cys530 535 540Lys Asn Phe Asn Phe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr Ile545550555 560Ser Asn Ala Tyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His Pro565570575Glu Cys Arg Thr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu Cys580585 590Val His Val Arg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys Asn595 600605Lys Tyr Asn Asp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys Asp610 615 620Gly Cys Thr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr Thr625630 635 640Cys Asn Leu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys Leu645 650 655
Leu Lys Asp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val His660665670Pro Gln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val Cys675 680685Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr His690695700Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln Cys705 710715 720Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg Glu725730 735Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly Ala740 745750Asp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn Glu755760765Thr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys Cys770775780Pro Leu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly Pro785790 795800Tyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn Leu805 810815Asp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro Thr820 825 830Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln Lys835840 845Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys Glu850 855 860Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys Lys865870 875 880Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu Gly885 890895Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu Gly900905 910
Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser Thr915 920 925Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met Ala930935940Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met Ser945 950955960Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys Leu Leu965970975Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys Ala Leu Leu980985 990Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr Leu Glu Glu Lys995 10001005Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Gln101010151020Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu102510301035Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala Gly Gly Lys Met104010451050Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg Asn Arg Val Phe105510601065Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val Thr Ile Trp Glu107010751080Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn Ile Pro Ala Lys108510901095Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys Leu Glu Gln Pro110011051110Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu Leu Ser Cys Trp111511201125His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys Gln Leu Thr Thr113011351140Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg Tyr Leu Ala Ile114511501155
Pro Gly Asp Lys Phe Thr Arg Leu Pro Ala Tyr Thr Ser Gln Asp116011651170Glu Lys Asp Leu Ile Arg Lys Leu Ala Pro Thr Thr Asp Gly Ser117511801185Glu Ala Ile Ala Lys Pro Asp Asp Tyr Leu Gln Pro Lys Ala Ala119011951200Pro Gly Pro Ser His Arg Thr12051210<210>23<211>4281<212>DNA<213>果蝇(Drosophila melanogaster)<400>23atgctgctgc gacggcgcaa cggcccctgc cccttccccc tgctgcttct gctcctggcc 60cactgcattt gcatttggcc cgcgtcggcg gcccgcgatc gctacgcccg ccagaacaat 120cgccagcgcc atcaggatat agatcgcgat cgggatcgag atcgattcct ataccgcagc 180agttcggccc aaaatcgaca gaggggcggg gccaacttcg ccctgggact gggagccaac 240ggagtcacca ttcccaccag tctggaggat aagaacaaga acgagttcgt caaggggaaa 300atctgcatcg gcactaaatc tcggctctcc gtgccctcca acaaggaaca tcattaccga 360aacctcagag atcggtacac gaactgtacg tatgtggatg gcaacttgaa actgacctgg 420ctacccaacg agaatttgga cctcagcttc ctagacaaca tacgggaggt caccggctat 480attctgatca gtcatgtgga cgttaagaaa gtggtgtttc ccaaactaca aatcattcgc 540ggacgcacgc tgttcagctt atccgtggag gaggagaagt atgccttgtt cgtcacttat 600tccaaaatgt acacgctgga gattcccgat ctacgcgatg tcttaaatgg ccaagtgggc 660ttccacaaca actacaatct ctgccacatg cgaacgatcc agtg9tcgga gattgtatcc 720aacggcacgg atgcatacta caactacgac tttactgctc cggagcgcga gtgtcccaag 780tgccacgaga gctgcacgca cggatgttgg ggcgagggtc ccaagaattg ccagaagttc 840agcaagctca cctgctcgcc acagtgtgcc ggaggtcgtt gctatggacc aaagccgcgg 900gagtgttgtc acctcttctg cgccggagga tgcactggtc ccacgcaaaa ggattgcatc 960gcctgcaaga acttcttcga cgaggcagta tcaaaggagg aatgcccgcc catgcgcaag 1020tacaatccca ccacctatgt tcttgaaacg aatcctgagg gaaagtatgc ctatggtgcc 1080
acctgcgtca aggagtgtcc cggtcatctg ttgcgggata atggcgcctg cgtgcgcagc 1140tgtccccagg acaagatgga caaggggggc gagtgtgtgc cctgcaatgg accgtgcccc 1200aaaacctgcc cgggcgttac tgtcctgcat gccggcaaca ttgactcgtt ccggaattgt 1260acggtgatcg atggcaacat tcgcattttg gatcagacct tctcgggctt ccaggatgtc 1320tatgccaact acacgatggg accacgatac ataccgctgg atcccgagcg acgggaggtg 1380ttctccacgg tgaaggagat caccgggtat ctgaatatcg agggaaccca cccgcagttc 1440cggaatctgt cgtactttcg caatctggaa acaattcatg gccgccagct gatggagagc 1500atgtttgccg ctttggcgat cgttaagtca tccctgtaca gcctggagat gcgcaatctg 1560aagcagatta gttccg9cag tgtggtcatc cagcataata gagacctctg ctacgtaagc 1620aatatccgtt ggccggccat tcagaaggag cccgaacaga aggtgtgggt caacgagaat 1680ctcagggcgg atctatgcga gaaaaatgga accatttgct cggatcagtg caacgaggac 1740ggctgctggg gagctggcac ggatcagtgc cttacctgca agaacttcaa tttcaatggc 1800acctgcatcg ccgactgtgg ttatatatcc aatgcctaca agtttgacaa tagaacgtgc 1860aagatatgcc atccagagtg ccggacttgc aatggagctg gagcagatca ctgccaggag 1920tgcgtccatg tgagggacgg tcagcactgt gtgtccgagt gcccgaagaa caagtacaac 1980gatcgtggtg tctgccgaga gtgccacgcc acctgcgatg gatgcactgg gcccaaggac 2040accatcggca ttggagcgtg tacgacgtgc aatttggcca ttatcaacaa tgacgccaca 2100gtaaaacgct gcctgctgaa ggacgacaag tgccccgatg ggtatttctg ggagtatgtg 2160catccgcaag agcagggatc gctgaagcca ttggccggca gagcagtttg ccgaaagtgc 2220catccccttt gcgagctgtg cacgaactac ggataccatg aacaggtgtg ctccaagtgc 2280acccactaca agcgacggga gcagtgcgag accgagtgtc cggccgatca ctacacggat 2340gaggagcagc gcgagtgctt ccagcgccac ccggaatgca atggttgcac gggtccgggt 2400gccgacgatt gcaagtcttg ccgcaacttt aagttgttcg acgcgaatga gacgggtccc 2460tatgtgaact ccacgatgtt caattgcacc tcgaagtgtc ccttggagat gcgacatgtg 2520aactatcagt acacggccat tggaccctac tgcgcagcta gtccgccgag gagcagcaag 2580ataactgcca atctggatgt gaacatgatc ttcattatca ctggtgctgt tctggtgccg 2640acgatctgca tcctctgcgt ggtcacatac atttgtcggc aaaagcaaaa ggccaagaag 2700gaaacagtca agatgaccat ggctctgtct ggctgcgagg attccgagcc gctgcgtccc 2760tcgaacattg gagccaacct atgcaagttg cgcattgtca aggacgccga gttgcgcaag 2820ggcggagtcc ttggaatggg agcctttgga cgagtgtaca agggcgtttg ggtgccggag 2880ggtgagaacg tcaagattcc agtggccatt aaggagctgc tcaagtccac aggcgccgag 2940tcaagcgaag agttcctccg cgaagcctac atcatggcct ctgaggagca cgttaatctg 3000
ctgaagctcc tggccgtgtg catgtcctca caaatgatgc taatcacgca actgatgccg 3060cttggctgcc tgttggacta tgtgcgaaat aaccgggaca agatcggctc taaggctctg 3120ctcaactgga gcacgcaaat cgccaagggc atgtcgtatc tggaggagaa gcgactggtc 3180cacagagact tggctgcccg caatgtcctg gtgcagactc cctcgctggt gaagatcacc 3240gactttgggc tggccaagtt gctgagcagc gattccaatg agtacaaggc tgctggcggc 3300aagatgccca tcaagtggtt ggcactggag tgcatccgca atcgtgtatt caccagcaag 3360tccgatgtct gggcctttgg tgtgaccatt tgggaactgc tgacctttgg ccagcgtcca 3420cacgagaaca tcccagctaa ggatattccc gatcttattg aagtcggtct gaagctggag 3480cagccggaga tttgttcgct ggacatttac tgtacactgc tctcgtgctg gcacttggat 3540gccgccatgc gtccaacctt caagcagctg actacggtct ttgctgagtt cgccagagat 3600ccgggtcgct atctggccat tcccggggat aagttcaccc ggctgccggc ttacacgagt 3660caggatgaga aggatctcat ccgaaaattg gctcccacca ccgatgggtc cgaagccatt 3720gcgaaacccg atgactacct gcaacccaag gcagcacctg gtcctagtca cagaaccgac 3780tgcacggatg agatgcccaa gctgaaccgc tactgcaagg atcctagtaa caagaattcg 3840agtaccggag acgatgagag ggattcgagt gcccgggaag tgggcgtggg taatctgcgc 3900ctcgatctac cagtcgatga ggatgattat ctgatgccca cttgccaacc gggtcccaac 3960aacaacaaca acatgaataa tcccaatcaa aacaatatgg cagctgtggg cgtggctgcc 4020ggctacatgg atctcatcgg agtgcccgtt agtgtggaca atccggagta tctgctaaac 4080gcgcagacac tgggagttgg ggagtcgccg atacccaccc agaccatcgg gataccggtg 4140atgggaggcc cgggcaccat ggaggtcaag gtgccaatgc caggcagtga gccaaccagc 4200tccgatcacg agtactacaa tgatacccaa cgggagttgc agccactgca tcgaaaccgc 4260aacacggaga cgagggtgta g 4281<210>24<211>4281<212>DNA<213>大鼠(Rattus norvegicus)<220>
<221>CDS<222>(1)..(3627)<223>
<400>24atg ctg ctg cga cgg cgc aac ggc ccc tgc ccc ttc ccc ctg ctg ctt 48Met Leu Leu Arg Arg Arg Asn Gly Pro Cys Pro Phe Pro Leu Leu Leu1 5 1015ctg ctc ctg gcc cac tgc att tgc att tgg ccc gcg tcg gcg gcc cgc 96Leu Leu Leu Ala His Cys Ile Cys Ile Trp Pro Ala Ser Ala Ala Arg20 25 30gat cgc tac gcc cgc cag aac aat cgc cag cgc cat cag gat ata gat 144Asp Arg Tyr Ala Arg Gln Asn Asn Arg Gln Arg His Gln Asp Ile Asp35 4045cgc gat cgg gat cga gat cga ttc cta tac cgc agc agt tcg gcc caa 192Arg Asp Arg Asp Arg Asp Arg Phe Leu Tyr Arg Ser Ser Ser Ala Gln50 5560aat cga cag agg ggc ggg gcc aac ttc gcc ctg gga ctg gga gcc aac 240Asn Arg Gln Arg Gly Gly Ala Asn Phe Ala Leu Gly Leu Gly Ala Asn6570 75 80gga gtc acc att ccc acc agt ctg gag gat aag aac aag aac gag ttc 288Gly Val Thr Ile Pro Thr Ser Leu Glu Asp Lys Asn Lys Asn Glu Phe85 9095gtc aag ggg aaa atc tgc atc ggc act aaa tct cgg ctc tcc gtg ccc 336Val Lys Gly Lys Ile Cys Ile Gly Thr Lys Ser Arg Leu Ser Val Pro100 105 110tcc aac aag gaa cat cat tac cga aac ctc aga gat cgg tac acg aac 384Ser Asn Lys Glu His His Tyr Arg Asn Leu Arg Asp Arg Tyr Thr Asn115 120125tgt acg tat gtg gat ggc aac ttg aaa ctg acc tgg cta ccc aac gag 432Cys Thr Tyr Val Asp Gly Asn Leu Lys Leu Thr Trp Leu Pro Asn Glu130135140aat ttg gac ctc agc ttc cta gac aac ata cgg gag gtc acc ggc tat 480Asn Leu Asp Leu Ser Phe Leu Asp Asn Ile Arg Glu Val Thr Gly Tyr145 150155 160att ctg atc agt cat gtg gac gtt aag aaa gtg gtg ttt ccc aaa cta 528Ile Leu Ile Ser His Val Asp Val Lys Lys Val Val Phe Pro Lys Leu165 170 175caa atc att cgc gga cgc acg ctg ttc agc tta tcc gtg gag gag gag 576Gln Ile Ile Arg Gly Arg Thr Leu Phe Ser Leu Ser Val Glu Glu Glu180 185190aag tat gcc ttg ttc gtc act tat tcc aaa atg tac acg ctg gag att 624Lys Tyr Ala Leu Phe Val Thr Tyr Ser Lys Met Tyr Thr Leu Glu Ile195 200 205ccc gat cta cgc gat gtc tta aat ggc caa gtg ggc ttc cac aac aac 672Pro Asp Leu Arg Asp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn210 215220tac aat ctc tgc cac atg cga acg atc cag tgg tcg gag att gta tcc 720Tyr Asn Leu Cys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser225230235 240aac ggc acg gat gca tac tac aac tac gac ttt act gct ccg gag cgc 768Asn Gly Thr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg
245 250255gag tgt ccc aag tgc cac gag agc tgc acg cac gga tgt tgg ggc gag 816Glu Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu260 265270ggt ccc aag aat tgc cag aag ttc agc aag ctc acc tgc tcg cca cag 864Gly Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser Pro Gln275280285tgt gcc gga ggt cgt tgc tat gga cca aag ccg cgg gag tgt tgt cag 912Cys Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu Cys Cys His290295 300ctc ttc tgc gcc gga gga tgc act ggt ccc acg caa aag gat tgc atc 960Leu Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln Lys Asp Cys Ile305310315 320gcc tgc aag aac ttc ttc gac gag gca gta tca aag gag gaa tgc ccg 1008Ala Cys Lys Asn Phe Phe Asp Glu Ala Val Ser Lys Glu Glu Cys Pro325 330 335ccc atg cgc aag tac aat ccc acc acc tat gtt ctt gaa acg aat cct 1056Pro Met Arg Lys Tyr Asn Pro Thr Thr Tyr Val Leu Glu Thr Asn Pro340345 350gag gga aag tat gcc tat ggt gcc acc tgc gtc aag gag tgt ccc ggt 1104Glu Gly Lys Tyr Ala Tyr Gly Ala Thr Cys Val Lys Glu Cys Pro Gly355 360 365cat ctg ttg cgg gat aat ggc gcc tgc gtg cgc agc tgt ccc cag gac 1152His Leu Leu Arg Asp Asn Gly Ala Cys Val Arg Ser Cys Pro Gln Asp370375380aag atg gac aag ggg ggc gag tgt gtg ccc tgc aat gga ccg tgc ccc 1200Lys Met Asp Lys Gly Gly Glu Cys Val Pro Cys Asn Gly Pro Cys Pro385390395400aaa acc tgc ccg ggc gtt act gtc ctg cat gcc ggc aac att gac tcg 1248Lys Thr Cys Pro Gly Val Thr Val Leu His Ala Gly Asn Ile Asp Ser405 410415ttc cgg aat tgt acg gtg atc gat ggc aac att cgc att ttg gat cag 1296Phe Arg Asn Cys Thr Val Ile Asp Gly Asn Ile Arg Ile Leu Asp Gln420425430acc ttc tcg ggc ttc cag gat gtc tat gcc aac tac acg atg gga cca 1344Thr Phe Ser Gly Phe Gln Asp Val Tyr Ala Asn Tyr Thr Met Gly Pro435440 445cga tac ata ccg ctg gat ccc gag cga cgg gag gtg ttc tcc acg gtg 1392Arg Tyr Ile Pro Leu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val450 455460aag gag atc acc ggg tat ctg aat atc gag gga acc cac ccg cag ttc 1440Lys Glu Ile Thr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe465 470 475 480cgg aat ctg tcg tac ttt cgc aat ctg gaa aca att cat ggc cgc cag 1488Arg Asn Leu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln485 490 495ctg atg gag agc atg ttt gcc gct ttg gcg atc gtt aag tca tcc ctg 1536Leu Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu
500 505 510tac agc ctg gag atg cgc aat ctg aag cag att agt tcc ggc agt gtg 1584Tyr Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val515520525gtc atc cag cat aat aga gac ctc tgc tac gta agc aat atc cgt tgg 1632Val Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp530 535540ccg gcc att cag aag gag ccc gaa cag aag gtg tgg gtc aac gag aat 1680Pro Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp Val Asn Glu Asn545 550555 560ctc agg gcg gat cta tgc gag aaa aat gga acc att tgc tcg gat cag 1728Leu Arg Ala Asp Leu Cys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln565570575tgc aac gag gac ggc tgc tgg gga gct ggc acg gat cag tgc ctt acc 1776Cys Asn Glu Asp Gly Cys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr580585 590tgc aag aac ttc aat ttc aat ggc acc tgc atc gcc gac tgt ggt tat 1824Cys Lys Asn Phe Asn Phe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr595600605ata tcc aat gcc tac aag ttt gac aat aga acg tgc aag ata tgc cat 1872Ile Ser Asn Ala Tyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His610 615620cca gag tgc cgg act tgc aat gga gct gga gca gat cac tgc cag gag 1920Pro Glu Cys Arg Thr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu625630 635640tgc gtc cat gtg agg gac ggt cag cac tgt gtg tcc gag tgc ccg aag 1968Cys Val His Val Arg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys645 650655aac aag tac aac gat cgt ggt gtc tgc cga gag tgc cac gcc acc tgc 2016Asn Lys Tyr Asn Asp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys660665670gat gga tgc act ggg ccc aag gac acc atc ggc att gga gcg tgt acg 2064Asp Gly Cys Thr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr675 680685acg tgc aat ttg gcc att atc aac aat gac gcc aca gta aaa cgc tgc 2112Thr Cys Asn Leu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys690695 700ctg ctg aag gac gac aag tgc ccc gat ggg tat ttc tgg gag tat gtg 2160Leu Leu Lys Asp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val705710715720cat ccg caa gag cag gga tcg ctg aag cca ttg gcc ggc aga gca gtt 2208His Pro Gln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val725 730735tgc cga aag tgc cat ccc ctt tgc gag ctg tgc acg aac tac gga tac 2256Cys Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr740 745 750cat gaa cag gtg tgc tcc aag tgc acc cac tac aag cga cgg gag cag 2304His Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln
755 760 765tgc gag acc gag tgt ccg gcc gat cac tac acg gat gag gag cag cgc 2352Cys Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg770775 780gag tgc ttc cag cgc cac ccg gaa tgc aat ggt tgc acg ggt ccg ggt 2400Glu Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly785790 795 800gcc gac gat tgc aag tct tgc cgc aac ttt aag ttg ttc gac gcg aat 2448Ala Asp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn805810 815gag acg ggt ccc tat gtg aac tcc acg atg ttc aat tgc acc tcg aag 2496Glu Thr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys820 825830tgt ccc ttg gag atg cga cat gtg aac tat cag tac acg gcc att gga 2544Cys Pro Leu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly835840 845ccc tac tgc gca gct agt ccg ccg agg agc agc aag ata act gcc aat 2592Pro Tyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn850855 860ctg gat gtg aac atg atc ttc att atc act ggt gct gtt ctg gtg ccg 2640Leu Asp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro865870 875 880acg atc tgc atc ctc tgc gtg gtc aca tac att tgt cgg caa aag caa 2688Thr Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln885 890 895aag gcc aag aag gaa aca gtc aag atg acc atg gct ctg tct ggc tgc 2736Lys Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys900 905 910gag gat tcc gag ccg ctg cgt ccc tcg aac att gga gcc aac cta tgc 2784Glu Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys915 920925aag ttg cgc att gtc aag gac gcc gag ttg cgc aag ggc gga gtc ctt 2832Lys Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu930 935 940gga atg gga gcc ttt gga cga gtg tac aag ggc gtt tgg gtg ccg gag 2880Gly Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu945 950955 960ggt gag aac gtc aag att cca gtg gcc att aag gag ctg ctc aag tcc 2928Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser965 970 975aca ggc gcc gag tca agc gaa gag ttc ctc cgc gaa gcc tac atc atg 2976Thr Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met980 985990gcc tct gag gag cac gtt aat ctg ctg aag ctc ctg gcc gtg tgc atg 3024Ala Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met995 1000 1005tcc tca caa atg atg cta atc acg caa ctg atg ccg ctt ggc tgc 3069Ser Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys
101010151020ctg ttg gac tat gtg cga aat aac cgg gac aag atc ggc tct aag 3114Leu Leu Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys102510301035gct ctg ctc aac tgg agc acg caa atc gcc aag ggc atg tcg tat 3159Ala Leu Leu Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr104010451050ctg gag gag aag cga ctg gtc cac aga gac ttg gct gcc cgc aat 3204Leu Glu Glu Lys Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn105510601065gtc ctg gtg cag act ccc tcg ctg gtg aag atc acc gac ttt ggg 3249Val Leu Val Gln Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly107010751080ctg gcc aag ttg ctg agc agc gat tcc aat gag tac aag gct gct 3294Leu Ala Lys Leu Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala108510901095ggc ggc aag atg ccc atc aag tgg ttg gca ctg gag tgc atc cgc 3339Gly Gly Lys Met Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg110011051110aat cgt gta ttc acc agc aag tcc gat gtc tgg gcc ttt ggt gtg 3384Asn Arg Val Phe Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val111511201125acc att tgg gaa ctg ctg acc ttt ggc cag cgt cca cac gag aac 3429Thr Ile Trp Glu Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn113011351140atc cca gct aag gat att ccc gat ctt att gaa gtc ggt ctg aag 3474Ile Pro Ala Lys Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys114511501155ctg gag cag ccg gag att tgt tcg ctg gac att tac tgt aca ctg 3519Leu Glu Gln Pro Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu116011651170ctc tcg tgc tgg cac ttg gat gcc gcc atg cgt cca acc ttc aag 3564Leu Ser Cys Trp His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys117511801185cag ctg act acg gtc ttt gct gag ttc gcc aga gat ccg ggt cgc 3609Gln Leu Thr Thr Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg119011951200tat ctg gcc att ccc ggg gataagttca cccggctgcc ggcttacacg 3657Tyr Leu Ala Ile Pro Gly1205agtcaggatg agaaggatct catccgaaaa ttggctccca ccaccgatgg gtccgaagcc 3717attgcgaaac ccgatgacta cctgcaaccc aaggcagcac ctggtcctag tcacagaacc 3777gactgcacgg atgagatgcc caagctgaac cgctactgca aggatcctag taacaagaat 3837tcgagtaccg gagacgatga gagggattcg agtgcccggg aagtgggcgt gggtaatctg 3897cgcctcgatc taccagtcga tgaggatgat tatctgatgc ccacttgcca accgggtccc 3957
aacaacaaca acaacatgaa taatcccaat caaaacaata tggcagctgt gggcgtggct 4017gccggctaca tggatctcat cggagtgccc gttagtgtgg acaatccgga gtatctgcta 4077aacgcgcaga cactgggagt tggggagtcg ccgataccca cccagaccat cgggataccg 4137gtgatgggag gcccgggcac catggaggtc aaggtgccaa tgccaggcag tgagccaacc 4197agctccgatc acgagtacta caatgatacc caacgggagt tgcagccact gcatcgaaac 4257cgcaacacgg agacgagggt gtag 4281<210>25<211>1209<212>PRT<213>大鼠(Rattus norvegicus)<400>25Met Leu Leu Arg Arg Arg Asn Gly Pro Cys Pro Phe Pro Leu Leu Leu1 5 10 15Leu Leu Leu Ala His Cys Ile Cys Ile Trp Pro Ala Ser Ala Ala Arg20 25 30Asp Arg Tyr Ala Arg Gln Asn Asn Arg Gln Arg His Gln Asp Ile Asp35 4045Arg Asp Arg Asp Arg Asp Arg Phe Leu Tyr Arg Ser Ser Ser Ala Gln505560Asn Arg Gln Arg Gly Gly Ala Asn Phe Ala Leu Gly Leu Gly Ala Asn6570 75 80Gly Val Thr Ile Pro Thr Ser Leu Glu Asp Lys Asn Lys Asn Glu Phe85 9095Val Lys Gly Lys Ile Cys Ile Gly Thr Lys Ser Arg Leu Ser Val Pro100 105 110Ser Asn Lys Glu His His Tyr Arg Asn Leu Arg Asp Arg Tyr Thr Asn115 120125Cys Thr Tyr Val Asp Gly Asn Leu Lys Leu Thr Trp Leu Pro Asn Glu130135140Asn Leu Asp Leu Ser Phe Leu Asp Asn Ile Arg Glu Val Thr Gly Tyr145 150155 160
Ile Leu Ile Ser His Val Asp Val Lys Lys Val Val Phe Pro Lys Leu165 170175Gln Ile Ile Arg Gly Arg Thr Leu Phe Ser Leu Ser Val Glu Glu Glu180 185190Lys Tyr Ala Leu Phe Val Thr Tyr Ser Lys Met Tyr Thr Leu Glu Ile195 200 205Pro Asp Leu Arg Asp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn210215 220Tyr Asn Leu Cys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser225230235 240Asn Gly Thr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg245250255Glu Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu260 265270Gly Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser Pro Gln275280 285Cys Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu Cys Cys His290295 300Leu Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln Lys Asp Cys Ile305310315 320Ala Cys Lys Asn Phe Phe Asp Glu Ala Val Ser Lys Glu Glu Cys Pro325 330 335Pro Met Arg Lys Tyr Asn Pro Thr Thr Tyr Val Leu Glu Thr Asn Pro340345 350Glu Gly Lys Tyr Ala Tyr Gly Ala Thr Cys Val Lys Glu Cys Pro Gly355 360 365His Leu Leu Arg Asp Asn Gly Ala Cys Val Arg Ser Cys Pro Gln Asp370375380Lys Met Asp Lys Gly Gly Glu Cys Val Pro Cys Asn Gly Pro Cys Pro385390395400Lys Thr Cys Pro Gly Val Thr Val Leu His Ala Gly Asn Ile Asp Ser405410 415
Phe Arg Asn Cys Thr Val Ile Asp Gly Asn Ile Arg Ile Leu Asp Gln420 425 430Thr Phe Ser Gly Phe Gln Asp Val Tyr Ala Asn Tyr Thr Met Gly Pro435440 445Arg Tyr Ile Pro Leu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val450 455460Lys Glu Ile Thr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe465 470 475 480Arg Asn Leu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln485 490 495Leu Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu500505510Tyr Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val515520525Val Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp530 535540Pro Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp Val Asn Glu Asn545 550 555 560Leu Arg Ala Asp Leu Cys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln565570 575Cys Asn Glu Asp Gly Cys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr580585 590Cys Lys Asn Phe Asn Phe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr595600605Ile Ser Asn Ala Tyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His610 615620Pro Glu Cys Arg Thr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu625630 635640Cys Val His Val Arg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys645650655Asn Lys Tyr Asn Asp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys660665670
Asp Gly Cys Thr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr675 680685Thr Cys Asn Leu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys690695 700Leu Leu Lys Asp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val705710715720His Pro Gln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val725 730735Cys Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr740745750His Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln755760765Cys Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg770775780Glu Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly785790 795 800Ala Asp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn805810 815Glu Thr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys820 825830Cys Pro Leu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly835840 845Pro Tyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn850 855860Leu Asp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro865870 875 880Thr Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln885 890 895Lys Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys900 905 910Glu Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys915 920925
Lys Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu930 935 940Gly Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu945 950955 960Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser965 970 975Thr Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met980 985990Ala Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met995 10001005Ser Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys1010 1015 1020Leu Leu Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys1025 1030 1035Ala Leu Leu Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr1040 1045 1050Leu Glu Glu Lys Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn1055 1060 1065Val Leu Val Gln Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly1070 1075 1080Leu Ala Lys Leu Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala1085 1090 1095Gly Gly Lys Met Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg1100 1105 1110Asn Arg Val Phe Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val1115 1120 1125Thr Ile Trp Glu Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn1130 1135 1140Ile Pro Ala Lys Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys1145 1150 1155Leu Glu Gln Pro Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu1160 1165 1170
Leu Ser Cys Trp His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys117511801185Gln Leu Thr Thr Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg119011951200Tyr Leu Ala Ile Pro Gly1205<210>26<211>3576<212>DNA<213>斑马鱼(Danio rerio)<220>
<221>CDS<222>(1)..(3573)<223>
<400>26atg gca gga cca act gaa atc gga ttg ttt ttt acc tta ttg ctc tcc 48Met Ala Gly Pro Thr Glu Ile Gly Leu Phe Phe Thr Leu Leu Leu Ser1 5 1015ggg agc ttc tgc gcg acg ccg gaa aag aaa gtg tgt cag gga gca aac 96Gly Ser Phe Cys Ala Thr Pro Glu Lys Lys Val Cys Gln Gly Ala Asn20 25 30aac aaa ctg act ctt ctg gga acg gtg gaa gac cat tat cag gtt ctg 144Asn Lys Leu Thr Leu Leu Gly Thr Val Glu Asp His Tyr Gln Val Leu35 40 45ctc aga atg tac aga aac tgc act gtg gtt ctg gag aac ctg gaa att 192Leu Arg Met Tyr Arg Asn Cys Thr Val Val Leu Glu Asn Leu Glu Ile505560aca cat ata aca gag aaa tat gac ctg tcc ttc ctc aag agc atc cag 240Thr His Ile Thr Glu Lys Tyr Asp Leu Ser Phe Leu Lys Ser Ile Gln65 70 7580gaa gtt ggt ggc tat gtt ctt atc gcg gtc aat acg gtt tcc aaa atc 288Glu Val Gly Gly Tyr Val Leu Ile Ala Val Asn Thr Val Ser Lys Ile85 90 95cct ctg gag aac ctg cgc atc att cgc gga cac tca ctt tat gaa gac 336Pro Leu Glu Asn Leu Arg Ile Ile Arg Gly His Ser Leu Tyr Glu Asp100 105 110aaa ttt gcc ttg gcc gtc ctg gtg aac ttc aac aac agc atc gaa caa 384Lys Phe Ala Leu Ala Val Leu Val Asn Phe Asn Asn Ser Ile Glu Gln
115 120 125ggc gta aaa gag ttg ccg ctg act agt tta act gaa ata ctt aag ggt 432Gly Val Lys Glu Leu Pro Leu Thr Ser Leu Thr Glu Ile Leu Lys Gly130 135140gga gtc aag ttt tgc agg aac gat tat tta tgt aat gtg ggg acc atc 480Gly Val Lys Phe Cys Arg Asn Asp Tyr Leu Cys Asn Val Gly Thr Ile145 150 155160gag tgg gcc gac atc ctg aac atg aag agc ctg cct aca atc gtg agc 528Glu Trp Ala Asp Ile Leu Asn Met Lys Ser Leu Pro Thr Ile Val Ser165170175cat aat ata agc tat gga aaa aac tgt gga aag tgc gat cca agc tgt 576His Asn Ile Ser Tyr Gly Lys Asn Cys Gly Lys Cys Asp Pro Ser Cys180 185190ttc aat ggc tcc tgc tgg ggc acc gga ccc gac aag tgc cag aga atg 624Phe Asn Gly Ser Cys Trp Gly Thr Gly Pro Asp Lys Cys Gln Arg Met195200 205acg aaa gtg atc tgt gcg gag cag tgt tca ggg agg tgt aaa gga ccc 672Thr Lys Val Ile Cys Ala Glu Gln Cys Ser Gly Arg Cys Lys Gly Pro210 215220aga ccc att gac tgc tgt aat gaa cac tgt gct gct gga tgc act gga 720Arg Pro Ile Asp Cys Cys Asn Glu His Cys Ala Ala Gly Cys Thr Gly225 230 235 240ccc aga cct aca gac tgt ctg gcc tgt aag gac ttc cag gat gaa ggg 768Pro Arg Pro Thr Asp Cys Leu Ala Cys Lys Asp Phe Gln Asp Glu Gly245250255aca tgt aag gac gca tgt ccg cgg ctc atg ctc tac gac cca aac aca 816Thr Cys Lys Asp Ala Cys Pro Arg Leu Met Leu Tyr Asp Pro Asn Thr260 265 270cac cag ctc gcg cca aac cca tat ggg aag tac agc ttt ggg gca acg 864His Gln Leu Ala Pro Asn Pro Tyr Gly Lys Tyr Ser Phe Gly Ala Thr275280 285tgc atc aag aca tgc cca cac aac tat gtg gtg acg gat cac ggg gcc 912Cys Ile Lys Thr Cys Pro His Asn Tyr Val Val Thr Asp His Gly Ala290 295 300tgt gtg aga aca tgc agc cct ggc acc tat gaa gtg gat gag ggt gga 960Cys Val Arg Thr Cys Ser Pro Gly Thr Tyr Glu Val Asp Glu Gly Gly305 310315320gtt cgc aaa tgt aag agg tgt gaa ggc ctg tgt cca aaa gtg tgc aat 1008Val Arg Lys Cys Lys Arg Cys Glu Gly Leu Cys Pro Lys Val Cys Asn325330335ggg ttg gga atg ggg cct tta gcc aat gtc ctg tca atc aat gcc acc 1056Gly Leu Gly Met Gly Pro Leu Ala Asn Val Leu Ser Ile Asn Ala Thr340 345350aac atc gac tcc ttt gag aac tgc act aaa atc agc ggc aat gtt gcc 1104Asn Ile Asp Ser Phe Glu Asn Cys Thr Lys Ile Ser Gly Asn Val Ala355360365atc ctc agc acc aca ttc aga ggt gac cca cat act aac act tca gga 1152Ile Leu Ser Thr Thr Phe Arg Gly Asp Pro His Thr Asn Thr Ser Gly
370375 380ctg gat cca gca aag ctc agt gta ttg agt act gtc aaa gaa atc act1200Leu Asp Pro Ala Lys Leu Ser Val Leu Ser Thr Val Lys Glu Ile Thr385 390395400ggt tac ctg atg att cag ctg tgg ccg gag agc atg cag tcc ctt agt1248Gly Tyr Leu Met Ile Gln Leu Trp Pro Glu Ser Met Gln Ser Leu Ser405410 415gcc ttc gaa aac ctt gag gtc atc cga gga cgg aca aaa aca caa gga1296Ala Phe Glu Asn Leu Glu Val Ile Arg Gly Arg Thr Lys Thr Gln Gly420425 430acg tac agc ttt gct gtc acc aag acg gcc atc act cat tta ggc atg1344Thr Tyr Ser Phe Ala Val Thr Lys Thr Ala Ile Thr His Leu Gly Met435440 445cgt tct ctg agg gag atc agt gac ggg gac gtg tcc atc gtt aag aat1392Arg Ser Leu Arg Glu Ile Ser Asp Gly Asp Val Ser Ile Val Lys Asn450455 460aag aat ctc tgc tac agc agc cct gaa cac tgg aaa cgc ctc ttc aag1440Lys Asn Leu Cys Tyr Ser Ser Pro Glu His Trp Lys Arg Leu Phe Lys465470 475480tcc aaa caa cag tcg gtc aaa atg att gaa aat atg gat gct gcc acc1488Ser Lys Gln Gln Ser Val Lys Met Ile Glu Asn Met Asp Ala Ala Thr485 490495tgc gcc aat cag aac agc aca tgt aat gag atg tgc acg gct gac ggc1536Cys Ala Asn Gln Asn Ser Thr Cys Asn Glu Met Cys Thr Ala Asp Gly500505510tgc tgg ggt ccc ggc ccc acc atg tgc ttc ggc tgt gag cat tac agc1584Cys Trp Gly Pro Gly Pro Thr Met Cys Phe Gly Cys Glu His Tyr Ser515 520525cgc gga aaa cac tgc gtg gct tcc tgc aac ctg ctg aat ggt gag ccg1632Arg Gly Lys His Cys Val Ala Ser Cys Asn Leu Leu Asn Gly Glu Pro530535 540cgt gaa tat gag gtc aat aaa aca tgc atg gaa tgc gat cct gaa tgt1680Arg Glu Tyr Glu Val Asn Lys Thr Cys Met Glu Cys Asp Pro Glu Cys545 550555560ctg ctc atg aat gaa acc cag acc tgc aac ggc cct gga ccc gac aaa1728Leu Leu Met Asn Glu Thr Gln Thr Cys Asn Gly Pro Gly Pro Asp Lys565 570 575tgt aca gtg tgt gca aac tat aaa gac gga ccg cac tgt gtg cat cgc1776Cys Thr Val Cys Ala Asn Tyr Lys Asp Gly Pro His Cys Val His Arg580 585590tgc ccg caa ggt gta cca gga gag aaa gac aca ctc atc tgg aaa tac1824Cys Pro Gln Gly Val Pro Gly Glu Lys Asp Thr Leu Ile Trp Lys Tyr595 600 605gct gac gtg aca cac gtt tgc cag ccc tgt cat gaa aac tgc acc cag1872Ala Asp Val Thr His Val Cys Gln Pro Cys His Glu Asn Cys Thr Gln610 615620gga tgt acg ggg cct gat cta aag gac tgc aaa gat ttc aaa agc tct1920Gly Cys Thr Gly Pro Asp Leu Lys Asp Cys Lys Asp Phe Lys Ser Ser
625 630 635 640ggt ttg ccg atg atc gct gct ggc gtt gtc gga ggt cta ctg gcg ttt 1968Gly Leu Pro Met Ile Ala Ala Gly Val Val Gly Gly Leu Leu Ala Phe645 650 655gtt att ctg gct ctt gga gtg gcc gtt ctc ctg cgc aga cgc cac atc 2016Val Ile Leu Ala Leu Gly Val Ala Val Leu Leu Arg Arg Arg His Ile660665 670cgg agg aag agg act ctg aga cga ctc ctg caa gag aga gag ctt gtg 2064Arg Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu Val675680685gag cct ctg acc ccc agc ggc gaa gcc ccc aat cag gcc tta ctg cgc 2112Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu Arg690695 700atc ctc aaa gag acg gag ttc aag aag atc aaa gtg ctg ggc tcc ggg 2160Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser Gly705 710 715 720gct ttc ggc act gtg cac aag ggc ctt tgg gtt cca gaa gga gag aat 2208Ala Phe Gly Thr Val His Lys Gly Leu Trp Val Pro Glu Gly Glu Asn725 730 735gtg aag atc cct gtc gcc atc aag gtc tta aga gaa gcc acg tct ccc 2256Val Lys Ile Pro Val Ala Ile Lys Val Leu Arg Glu Ala Thr Ser Pro740 745 750aaa gct aac aag gag ata atg gat gag gcg tac gtc atg gcc agt gtt 2304Lys Ala Asn Lys Glu Ile Met Asp Glu Ala Tyr Val Met Ala Ser Val755 760765gag cat ccc cat gtg tgt cgt ctg ctg ggc atc tgc ttg acc tcc aca 2352Glu His Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr770 775780gtg cag ctc atc act cag ctg atg ccc tac ggc tgc ttg ctg gac tat 2400Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp Tyr785 790795 800gtc aga gaa aac aag gac cgc atc ggc tcc cag cac ctg ctc aac tgg 2448Val Arg Glu Asn Lys Asp Arg Ile Gly Ser Gln His Leu Leu Asn Trp805810 815tgc gtg cag atc gct aaa ggt atg aat tat cta gaa gag cgc cat ctt 2496Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Glu Arg His Leu820 825 830gtg cac cga gac ctg gca gca cgt aat gtg ttg gta aag acg cct cag 2544Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro Gln835840845cat gtc aag att acc gat ttc ggc ctc gcc aag ctg tta aac gcg gac 2592His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Asn Ala Asp850 855 860gag aag gag tat cac gca gat gga gga aag gtt cca att aaa tgg atg 2640Glu Lys Glu Tyr His Ala Asp Gly Gly Lys Val Pro Ile Lys Trp Met865 870 875 880gcg ctg gag tcc atc cag cac agg act tac acc cac cag agt gac gtc 2688Ala Leu Glu Ser Ile Gln His Arg Thr Tyr Thr His Gln Ser Asp Val
885 890 895tgg agc tac ggt gtg acc gtc tgg gag ttg atg acg ttt ggg acg aaa 2736Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Thr Lys900 905 910cct tat gat ggg att cct gcc agt gaa atc gcc gga gtt ctg gaa aaa 2784Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ala Gly Val Leu Glu Lys915 920 925gga gaa aga ctt cct caa ccc ccc atc tgc acc att gat gtg tac atg 2832Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met930935 940atc atg gtc aaa tgt tgg atg att gat gct gag agc aga ccc cgc ttc 2880Ile Met Val Lys Cys Trp Met Ile Asp Ala Glu Ser Arg Pro Arg Phe945 950 955 960agg gag ctc atc gca gag ttc act aaa atg gct cgt gac ccg tcc cgc 2928Arg Glu Leu Ile Ala Glu Phe Thr Lys Met Ala Arg Asp Pro Ser Arg965970 975tat ctg gtc att cag ggg gac gac cgc atg cat tta ccc agt cct tct 2976Tyr Leu Val Ile Gln Gly Asp Asp Arg Met His Leu Pro Ser Pro Ser980 985990gac tcc aag ttc tac cgc agc ctg atg agc gga gag ctg gac gag gcc 3024Asp Ser Lys Phe Tyr Arg Ser Leu Met Ser Gly Glu Leu Asp Glu Ala995 1000 1005gtg gac gca gac gag tat tta gtg ccc aat cac agc ttc ttc agc 3069Val Asp Ala Asp Glu Tyr Leu Val Pro Asn His Ser Phe Phe Ser101010151020agc ccg agc acg tcc cgc aca caa ctg ctg cac tct gtg agc ctg 3114Ser Pro Ser Thr Ser Arg Thr Gln Leu Leu His Ser Val Ser Leu102510301035aac agc agc ttt gga aac tgt aat agt aga aac ggg aat ggt tat 3159Asn Ser Ser Phe Gly Asn Cys Asn Ser Arg Asn Gly Asn Gly Tyr1040 10451050cca gtg agg gag aac agc atg gtc ctg cgc tac atc cca gac ccc 3204Pro Val Arg Glu Asn Ser Met Val Leu Arg Tyr Ile Pro Asp Pro1055 10601065aca gag cgc ttt cag gag gga gac ttt cag cct gcg ccg ggt tat 3249Thr Glu Arg Phe Gln Glu Gly Asp Phe Gln Pro Ala Pro Gly Tyr1070 10751080aac gaa tat atg aac cag aat gag tcc agc atg atc aac cca gtg 3294Asn Glu Tyr Met Asn Gln Asn Glu Ser Ser Met Ile Asn Pro Val1085 10901095tac cag cag ccc cac gga ccc ccg cgg acc ctc ctc cac tcc tcc 3339Tyr Gln Gln Pro His Gly Pro Pro Arg Thr Leu Leu His Ser Ser1100 11051110cca gcg ctg gac gag acg gaa gag gag tat ctg aac tgc ttc aag 3384Pro Ala Leu Asp Glu Thr Glu Glu Glu Tyr Leu Asn Cys Phe Lys1115 11201125agc ccg gct ccg gct tca gtg gtg gag tat ctg aac acg tcc cac 3429Ser Pro Ala Pro Ala Ser Val Val Glu Tyr Leu Asn Thr Ser His
1130 1135 1140aca cag ctg ctc tcc aca aag ccc ttc ttc agc atg gac aac ccc 3474Thr Gln Leu Leu Ser Thr Lys Pro Phe Phe Ser Met Asp Asn Pro1145 1150 1155gac tac cag cag gac ttc tgc ccg ctg gag ctc aaa aca cac acc 3519Asp Tyr Gln Gln Asp Phe Cys Pro Leu Glu Leu Lys Thr His Thr1160 1165 1170aac ggg cac ctg ccg gcc gcg cag aac cag gag tac atg ggc ctg 3564Asn Gly His Leu Pro Ala Ala Gln Asn Gln Glu Tyr Met Gly Leu1175 1180 1185gag gtg cac tag 3576Glu Val His1190<210>27<211>1191<212>PRT<213>斑马鱼(Danio reno)<400>27Met Ala Gly Pro Thr Glu Ile Gly Leu Phe Phe Thr Leu Leu Leu Ser1 5 1015Gly Ser Phe Cys Ala Thr Pro Glu Lys Lys Val Cys Gln Gly Ala Asn20 25 30Asn Lys Leu Thr Leu Leu Gly Thr Val Glu Asp His Tyr Gln Val Leu35 4045Leu Arg Met Tyr Arg Asn Cys Thr Val Val Leu Glu Asn Leu Glu Ile505560Thr His Ile Thr Glu Lys Tyr Asp Leu Ser Phe Leu Lys Ser Ile Gln65 70 7580Glu Val Gly Gly Tyr Val Leu Ile Ala Val Asn Thr Val Ser Lys Ile85 90 95Pro Leu Glu Asn Leu Arg Ile Ile Arg Gly His Ser Leu Tyr Glu Asp100 105 110Lys Phe Ala Leu Ala Val Leu Val Asn Phe Asn Asn Ser Ile Glu Gln115 120125Gly Val Lys Glu Leu Pro Leu Thr Ser Leu Thr Glu Ile Leu Lys Gly
130135140Gly Val Lys Phe Cys Arg Asn Asp Tyr Leu Cys Asn Val Gly Thr Ile145 150 155160Glu Trp Ala Asp Ile Leu Asn Met Lys Ser Leu Pro Thr Ile Val Ser165 170175His Asn Ile Ser Tyr Gly Lys Asn Cys Gly Lys Cys Asp Pro Ser Cys180 185190Phe Asn Gly Ser Cys Trp Gly Thr Gly Pro Asp Lys Cys Gln Arg Met195200 205Thr Lys Val Ile Cys Ala Glu Gln Cys Ser Gly Arg Cys Lys Gly Pro210 215220Arg Pro Ile Asp Cys Cys Asn Glu His Cys Ala Ala Gly Cys Thr Gly225 230235 240Pro Arg Pro Thr Asp Cys Leu Ala Cys Lys Asp Phe Gln Asp Glu Gly245250255Thr Cys Lys Asp Ala Cys Pro Arg Leu Met Leu Tyr Asp Pro Asn Thr260265270His Gln Leu Ala Pro Asn Pro Tyr Gly Lys Tyr Ser Phe Gly Ala Thr275 280 285Cys Ile Lys Thr Cys Pro His Asn Tyr Val Val Thr Asp His Gly Ala290 295300Cys Val Arg Thr Cys Ser Pro Gly Thr Tyr Glu Val Asp Glu Gly Gly305310 315320Val Arg Lys Cys Lys Arg Cys Glu Gly Leu Cys Pro Lys Val Cys Asn325330335Gly Leu Gly Met Gly Pro Leu Ala Asn Val Leu Ser Ile Asn Ala Thr340 345 350Asn Ile Asp Ser Phe Glu Asn Cys Thr Lys Ile Ser Gly Asn Val Ala355360365Ile Leu Ser Thr Thr Phe Arg Gly Asp Pro His Thr Asn Thr Ser Gly370375380Leu Asp Pro Ala Lys Leu Ser Val Leu Ser Thr Val Lys Glu Ile Thr
385390 395400Gly Tyr Leu Met Ile Gln Leu Trp Pro Glu Ser Met Gln Ser Leu Ser405 410415Ala Phe Glu Asn Leu Glu Val Ile Arg Gly Arg Thr Lys Thr Gln Gly420425 430Thr Tyr Ser Phe Ala Val Thr Lys Thr Ala Ile Thr His Leu Gly Met435 440 445Arg Ser Leu Arg Glu Ile Ser Asp Gly Asp Val Ser Ile Val Lys Asn450 455 460Lys Asn Leu Cys Tyr Ser Ser Pro Glu His Trp Lys Arg Leu Phe Lys465470475 480Ser Lys Gln Gln Ser Val Lys Met Ile Glu Asn Met Asp Ala Ala Thr485 490495Cys Ala Asn Gln Asn Ser Thr Cys Asn Glu Met Cys Thr Ala Asp Gly500505510Cys Trp Gly Pro Gly Pro Thr Met Cys Phe Gly Cys Glu His Tyr Ser515 520525Arg Gly Lys His Cys Val Ala Ser Cys Asn Leu Leu Asn Gly Glu Pro530535 540Arg Glu Tyr Glu Val Asn Lys Thr Cys Met Glu Cys Asp Pro Glu Cys545 550555560Leu Leu Met Asn Glu Thr Gln Thr Cys Asn Gly Pro Gly Pro Asp Lys565 570 575Cys Thr Val Cys Ala Asn Tyr Lys Asp Gly Pro His Cys Val His Arg580 585590Cys Pro Gln Gly Val Pro Gly Glu Lys Asp Thr Leu Ile Trp Lys Tyr595600 605Ala Asp Val Thr His Val Cys Gln Pro Cys His Glu Asn Cys Thr Gln610 615620Gly Cys Thr Gly Pro Asp Leu Lys Asp Cys Lys Asp Phe Lys Ser Ser625 630635 640Gly Leu Pro Met Ile Ala Ala Gly Val Val Gly Gly Leu Leu Ala Phe
645 650 655Val Ile Leu Ala Leu Gly Val Ala Val Leu Leu Arg Arg Arg His Ile660 665670Arg Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu Val675 680685Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu Arg690695 700Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser Gly705 710 715 720Ala Phe Gly Thr Val His Lys Gly Leu Trp Val Pro Glu Gly Glu Asn725 730735Val Lys Ile Pro Val Ala Ile Lys Val Leu Arg Glu Ala Thr Ser Pro740 745750Lys Ala Asn Lys Glu Ile Met Asp Glu Ala Tyr Val Met Ala Ser Val755 760765Glu His Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr770 775780Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp Tyr785 790795 800Val Arg Glu Asn Lys Asp Arg Ile Gly Ser Gln His Leu Leu Asn Trp805810 815Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Glu Arg His Leu820 825 830Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro Gln835840845His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Asn Ala Asp850 855860Glu Lys Glu Tyr His Ala Asp Gly Gly Lys Val Pro Ile Lys Trp Met865 870 875 880Ala Leu Glu Ser Ile Gln His Arg Thr Tyr Thr His Gln Ser Asp Val885 890895Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Thr Lys
900 905 910Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ala Gly Val Leu Glu Lys915 920 925Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met930935 940Ile Met Val Lys Cys Trp Met Ile Asp Ala Glu Ser Arg Pro Arg Phe945 950 955960Arg Glu Leu Ile Ala Glu Phe Thr Lys Met Ala Arg Asp Pro Ser Arg965 970975Tyr Leu Val Ile Gln Gly Asp Asp Arg Met His Leu Pro Ser Pro Ser980 985990Asp Ser Lys Phe Tyr Arg Ser Leu Met Ser Gly Glu Leu Asp Glu Ala99510001005Val Asp Ala Asp Glu Tyr Leu Val Pro Asn His Ser Phe Phe Ser101010151020Ser Pro Ser Thr Ser Arg Thr Gln Leu Leu His Ser Val Ser Leu102510301035Asn Ser Ser Phe Gly Asn Cys Asn Ser Arg Asn Gly Asn Gly Tyr104010451050Pro Val Arg Glu Asn Ser Met Val Leu Arg Tyr Ile Pro Asp Pro105510601065Thr Glu Arg Phe Gln Glu Gly Asp Phe Gln Pro Ala Pro Gly Tyr107010751080Asn Glu Tyr Met Asn Gln Asn Glu Ser Ser Met Ile Asn Pro Val108510901095Tyr Gln Gln Pro His Gly Pro Pro Arg Thr Leu Leu His Ser Ser110011051110Pro Ala Leu Asp Glu Thr Glu Glu Glu Tyr Leu Asn Cys Phe Lys111511201125Ser Pro Ala Pro Ala Ser Val Val Glu Tyr Leu Asn Thr Ser His113011351140Thr Gln Leu Leu Ser Thr Lys Pro Phe Phe Ser Met Asp Asn Pro
11451150 1155Asp Tyr Gln Gln Asp Phe Cys Pro Leu Glu Leu Lys Thr His Thr11601165 1170Asn Gly His Leu Pro Ala Ala Gln Asn Gln Glu Tyr Met Gly Leu11751180 1185Glu Val His1190
权利要求
1.一种制备疫苗的方法，该方法包括1)分析特定病原体的特异抗原；2)获得编码特异抗原的多核苷酸序列；3)获得与该多核苷酸序列有足够差异的多核苷酸序列；4)利用步骤3)中所得的多核苷酸序列制备疫苗。
2.如权利要求1所述的方法，其中所述病原体选自细菌、病毒、真菌和原生动物。
3.如权利要求1所述的方法，其中步骤1)所述的多核苷酸序列与步骤3)所述的多核苷酸序列的差异，以它们所编码的多肽序列而言，同源性为30％-95％。
4.如权利要求1所述的方法，其中所述疫苗的形式选自重组蛋白疫苗、重组基因疫苗、重组病毒疫苗、基因修饰疫苗及稳定转化共生菌。
5.一种制备肿瘤疫苗的方法，该方法包括1)分析动物体内的肿瘤细胞的特异抗原；2)获得编码特异抗原的多核苷酸序列；3)获得与该多核苷酸序列有足够差异的多核苷酸序列；4)利用步骤3)中所得的多核苷酸序列制备疫苗。
6.如权利要求5所述的方法，其中所述肿瘤细胞发生在动物体内。
7.如权利要求5所述的方法，其中所述肿瘤细胞来源于动物体。
8.如权利要求6或7所述的方法，其中所述动物体包括人体。
9.如权利要求5所述的方法，其中步骤1)所述的多核苷酸序列与步骤3)所述的多核苷酸序列的差异，以它们所编码的多肽序列而言，同源性为30％-95％。
10.如权利要求5所述的方法，其中所述疫苗的形式选自重组蛋白疫苗、重组基因疫苗、重组病毒疫苗、基因修饰疫苗及稳定转化共生菌。
11.一种制备EGFR疫苗的方法，该方法包括1)分析动物体内的肿瘤细胞的EGFR抗原；2)获得编码特异抗原的多核苷酸序列；3)获得与该多核苷酸序列有足够差异的多核苷酸序列；4)利用步骤3)中所得的多核苷酸序列制备疫苗。
12.如权利要求11所述的方法，其中所述肿瘤细胞发生在动物体内。
13.如权利要求11所述的方法，其中所述肿瘤细胞来源于动物体。
14.如权利要求11所述的方法，其中步骤1)所述的多核苷酸序列与步骤3)所述的多核苷酸序列的差异，以它们所编码的多肽序列而言，同源性为30％-95％。
15.如权利要求11所述的方法，其中所述疫苗的形式选自选自重组蛋白疫苗、重组基因疫苗、重组病毒疫苗、基因修饰疫苗及稳定转化共生菌。
16.一种核酸疫苗，其中含有的多核苷酸序列所编码的多肽与病原体中特定抗原的多核苷酸序列所编码的多肽的同源性为30-95％。
17. 一种表皮生长因子受体(EGFR)核酸疫苗，其中含有的多核苷酸序列编码的表皮生长因子受体与生物体内的该表皮生长因子受体中的氨基酸序列的同源性为30-95％。
18.一种表皮生长因子受体(EGFR)核酸疫苗，其中含有的多核苷酸序列编码的表皮生长因子受体与人体内的该表皮生长因子受体中的氨基酸序列的同源性为30-95％。
19.一种制备用于治疗人体肿瘤的EGFR疫苗的方法，该方法包括1)从异种生物获得编码EGFR的多核苷酸序列；和2)利用步骤1)中所得的多核苷酸序列制备疫苗。
20.如权利要求19所述的方法，其中所述异种生物是除人之外的其他生物，如小鼠、大鼠、鸡、鲐、果蝇等。
21.如权利要求19所述的方法，其中所述从异种生物获得编码EGFR的多核苷酸序列选自SEQ.ID.No.13，SEQ.ID.No.15，SEQ.ID.No.17，SEQ.ID.No.19，SEQ.ID.No.21，SEQ.ID.No.23，SEQ.ID.No.24和SEQ.ID.26。
22.如权利要求19所述的方法，其中所述疫苗的形式选自选自重组蛋白疫苗、重组基因疫苗、重组病毒疫苗、基因修饰疫苗及稳定转化共生菌。
23.如权利要求19的方法，其中人体肿瘤选自过量表达EGFR分子的肺癌、乳腺癌、卵巢癌、结肠癌、前列腺癌、胃癌、膀胱癌、头颈部鳞癌和胶质瘤等。
24.一种制备用于治疗人体肿瘤的EGFR疫苗的方法，该方法包括1)修饰人EGFR的多核苷酸序列；和2)从步骤1)中所得的多核苷酸序列中选择修饰后的多核苷酸序列制备疫苗。
25.如权利要求24的方法，其中所述人EGFR的多核苷酸序列选自SEQ.ID.No.1，SEQ.ID.No.3，SEQ.ID.No.5，SEQ.ID.No.7，SEQ.ID.No.9和SEQ.ID.No.11。
26.如权利要求24的方法，其中所述修饰方法选自易错PCR、改组、寡核苷酸介导的诱变、装配PCR、有性PCR诱变、体内诱变、盒式诱变、递归集团诱变、指数集团诱变、位点特异性诱变、基因再装配、GSSM及其任意组合。
27.如权利要求24所述的方法，其中所述疫苗的形式选自选自重组蛋白疫苗、重组基因疫苗、重组病毒疫苗、基因修饰疫苗及稳定转化共生菌。
28.如权利要求24的方法，其中人体肿瘤选自过量表达EGFR分子的肺癌、乳腺癌、卵巢癌、结肠癌、前列腺癌、胃癌、膀胱癌、头颈部鳞癌和胶质瘤等。
29.一种表皮生长因子受体多肽疫苗，其中含有的表皮生长因子受体多肽与人体内的该表皮生长因子受体中的氨基酸序列的同源性为30-95％。
全文摘要
本发明提供了一种制备疫苗的方法，包括1)分析特定病原体的特异抗原；2)获得编码特异抗原的多核苷酸序列；3)获得与该多核苷酸序列有足够差异的多核苷酸序列；4)利用步骤3)中所得的多核苷酸序列制备疫苗。本发明也提供了抗肿瘤疫苗EGFR分子疫苗，可以是常规制剂，如水剂，也可以是利用纳米生物技术制备而成的纳米粒制剂。EGFR分子疫苗的抗肿瘤作用还可在其它免疫促进因子的协同作用下得到增强。
文档编号A61K39/00GK1548537SQ20031011305
公开日2004年11月24日 申请日期2003年12月25日 优先权日2002年12月27日
发明者魏于全, 田聆 申请人:深圳市清华源兴生物医药科技有限公司