CN1548537B - 疫苗制备方法和抗肿瘤疫苗 - Google Patents
疫苗制备方法和抗肿瘤疫苗 Download PDFInfo
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Abstract
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。
Description
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/Projects/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，New York，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.，85：2149-2154，1963)(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，81：3998(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”指氧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、p160erbB3、p180erbB4。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 IDNO 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ViraPowerTM LentiviralExpressionKit(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(ViraPowerTM PackagingMix)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10 l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T4DNA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-T4DNA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-AdFiberI和pSh-AdFiberII。pSh-AdFiberI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AdFiberI/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-AdFiberI；而pSh-AdFiberII用AvrII酶切-T4DNA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’ACTCCCGGGAGTTGTGTCTCCTGTTTCCTG 3’)、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)。
附图说明
图1同源分子疫苗的作用原理示意图
图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3EGFR重组蛋白疫苗的构建流程示意图
图4EGFR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图
图5RGD修饰的腺病毒重组EGFR疫苗构建流程示意图
图6纳米靶向EGFR分子疫苗抗肿瘤作用原理示意图
图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11EGFR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(H22andMMT-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12EGFR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13EGFR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。
具体实施方式
实施例1EGFR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2EGFR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3EGFR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4EGFR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1×109PFU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5EGFR重组Lentivirus病毒疫苗
如前文所述，本发明利用ViraPowerTM LentiviralExpression 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(ViraPowerTM PackagingMix)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10 l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7RGD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T4DNA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-T4DNA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-AdFiberI和pSh-AdFiberII。pSh-AdFiberI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AdFiberI/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-AdFiberI；而pSh-AdFiber II用AvrII酶切-T4DNA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-AdFiber II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。
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。
序列表
<110>深圳市清华源兴生物医药科技有限公司
<120>疫苗制备方法和抗肿瘤疫苗
<130>CGCNS31755
<150>CN02159602.6
<151>2002-12-27
<160>27
<170>PatentIn version 3.1
<210>1
<211>3633
<212>DNA
<213>人(homo sapiens)
<220>
<221>CDS
<222>(1)..(3630)
<223>
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Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
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Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
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Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
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Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
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Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
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Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
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Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
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tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat 384
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
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aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg 432
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
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His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
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tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
atc aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg gac 1248
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
405 410 415
ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc agg acc aag caa 1296
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445
gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att tca 1392
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg 1440
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
485 490 495
aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
500 505 510
gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
515 520 525
gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
530 535 540
gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
610 615 620
acc tac gga tgc act ggg cca ggt ctt gaa ggc tgt cca acg aat ggg 1920
Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly
625 630 635 640
cct aag atc ccg tcc atc gcc act ggg atg gtg ggg gcc ctc ctc ttg 1968
Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu
645 650 655
ctg ctg gtg gtg gcc ctg ggg atc ggc ctc ttc atg cga agg cgc cac 2016
Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His
660 665 670
atc gtt cgg aag cgc acg ctg cgg agg ctg ctg cag gag agg gag ctt 2064
Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu
675 680 685
gtg gag cct ctt aca ccc agt gga gaa gct ccc aac caa gct ctc ttg 2112
Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu
690 695 700
agg atc ttg aag gaa act gaa ttc aaa aag atc aaa gtg ctg ggc tcc 2160
Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser
705 710 715 720
ggt gcg ttc ggc acg gtg tat aag gga ctc tgg atc cca gaa ggt gag 2208
Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu
725 730 735
aaa gtt aaa att ccc gtc gct atc aag gaa tta aga gaa gca aca tct 2256
Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser
740 745 750
ccg aaa gcc aac aag gaa atc ctc gat gaa gcc tac gtg atg gcc agc 2304
Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser
755 760 765
gtg gac aac ccc cac gtg tgc cgc ctg ctg ggc atc tgc ctc acc tcc 2352
Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser
770 775 780
acc gtg cag ctc atc acg cag ctc atg ccc ttc ggc tgc ctc ctg gac 2400
Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp
785 790 795 800
tat gtc cgg gaa cac aaa gac aat att ggc tcc cag tac ctg ctc aac 2448
Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn
805 810 815
tgg tgt gtg cag atc gca aag ggc atg aac tac ttg gag gac cgt cgc 2496
Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg
820 825 830
ttg gtg cac cgc gac ctg gca gcc agg aac gta ctg gtg aaa aca ccg 2544
Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro
835 840 845
cag cat gtc aag atc aca gat ttt ggg ctg gcc aaa ctg ctg ggt gcg 2592
Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala
850 855 860
gaa gag aaa gaa tac cat gca gaa gga ggc aaa gtg cct atc aag tgg 2640
Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp
865 870 875 880
atg gca ttg gaa tca att tta cac aga atc tat acc cac cag agt gat 2688
Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp
885 890 895
gtc tgg agc tac ggg gtg acc gtt tgg gag ttg atg acc ttt gga tcc 2736
Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser
900 905 910
aag cca tat gac gga atc cct gcc agc gag atc tcc tcc atc ctg gag 2784
Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu
915 920 925
aaa gga gaa cgc ctc cct cag cca ccc ata tgt acc atc gat gtc tac 2832
Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr
930 935 940
atg atc atg gtc aag tgc tgg atg ata gac gca gat agt cgc cca aag 2880
Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys
945 950 955 960
ttc cgt gag ttg atc atc gaa ttc tcc aaa atg gcc cga gac ccc cag 2928
Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln
965 970 975
cgc tac ctt gtc att cag ggg gat gaa aga atg cat ttg cca agt cct 2976
Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro
980 985 990
aca gac tcc aac ttc tac cgt gcc ctg atg gat gaa gaa gac atg gac 3024
Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp
995 1000 1005
gac gtg gtg gat gcc gac gag tac ctc atc cca cag cag ggc ttc 3069
Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe
1010 1015 1020
ttc agc agc ccc tcc acg tca cgg act ccc ctc ctg agc tct ctg 3114
Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu
1025 1030 1035
agt gca acc agc aac aat tcc acc gtg gct tgc att gat aga aat 3159
Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn
1040 1045 1050
ggg ctg caa agc tgt ccc atc aag gaa gac agc ttc ttg cag cga 3204
Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg
1055 1060 1065
tac agc tca gac ccc aca ggc gcc ttg act gag gac agc ata gac 3249
Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp
1070 1075 1080
gac acc ttc ctc cca gtg cct gaa tac ata aac cag tcc gtt ccc 3294
Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro
1085 1090 1095
aaa agg ccc gct ggc tct gtg cag aat cct gtc tat cac aat cag 3339
Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln
1100 1105 1110
cct ctg aac ccc gcg ccc agc aga gac cca cac tac cag gac ccc 3384
Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro
1115 1120 1125
cac agc act gca gtg ggc aac ccc gag tat ctc aac act gtc cag 3429
His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln
1130 1135 1140
ccc acc tgt gtc aac agc aca ttc gac agc cct gcc cac tgg gcc 3474
Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala
1145 1150 1155
cag aaa ggc agc cac caa att agc ctg gac aac cct gac tac cag 3519
Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln
1160 1165 1170
cag gac ttc ttt ccc aag gaa gcc aag cca aat ggc atc ttt aag 3564
Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys
1175 1180 1185
ggc tcc aca gct gaa aat gca gaa tac cta agg gtc gcg cca caa 3609
Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln
1190 1195 1200
agc agt gaa ttt att gga gca tga 3633
Ser Ser Glu Phe Ile Gly Ala
1205 1210
<210>2
<211>1210
<212>PRT
<213>人(homo sapiens)
<400>2
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 155 160
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
405 410 415
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
485 490 495
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
500 505 510
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
515 520 525
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
530 535 540
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
610 615 620
Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly
625 630 635 640
Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu
645 650 655
Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His
660 665 670
Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu
675 680 685
Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu
690 695 700
Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser
705 710 715 720
Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu
725 730 735
Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser
740 745 750
Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser
755 760 765
Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser
770 775 780
Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp
785 790 795 800
Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn
805 810 815
Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg
820 825 830
Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro
835 840 845
Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala
850 855 860
Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp
865 870 875 880
Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp
885 890 895
Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser
900 905 910
Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu
915 920 925
Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr
930 935 940
Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys
945 950 955 960
Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln
965 970 975
Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro
980 985 990
Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp
995 1000 1005
Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe
1010 1015 1020
Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu
1025 1030 1035
Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn
1040 1045 1050
Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg
1055 1060 1065
Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp
1070 1075 1080
Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro
1085 1090 1095
Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln
1100 1105 1110
Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro
1115 1120 1125
His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln
1130 1135 1140
Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala
1145 1150 1155
Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln
1160 1165 1170
Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys
1175 1180 1185
Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln
1190 1195 1200
Ser Ser Glu Phe Ile Gly Ala
1205 1210
<210>3
<211>1974
<212>DNA
<213>人(homo sapiens)
<220>
<221>CDS
<222>(1)..(1971)
<223>
<400>3
atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa 96
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt 144
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat 192
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag 240
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg 288
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac 336
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat 384
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125
aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg 432
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag 480
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 155 160
agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
atc aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg gac 1248
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
405 410 415
ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc agg acc aag caa 1296
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445
gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att tca 1392
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg 1440
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
485 490 495
aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
500 505 510
gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
515 520 525
gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
530 535 540
gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
610 615 620
acc tac gga tgc act ggg cca ggt ctt gaa ggc tgt cca acg aat gga 1920
Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly
625 630 635 640
agc tac ata gtg tct cac ttt cca aga tca ttc tac aag atg tca gtg 1968
Ser Tyr Ile Val Ser His Phe Pro Arg Ser Phe Tyr Lys Met Ser Val
645 650 655
cac tga 1974
His
<210>4
<211>657
<212>PRT
<213>人(homo sapiens)
<400>4
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 155 160
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
405 410 415
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
485 490 495
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
500 505 510
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
515 520 525
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
530 535 540
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
610 615 620
Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly
625 630 635 640
Ser Tyr Ile Val Ser His Phe Pro Arg Ser Phe Tyr Lys Met Ser Val
645 650 655
His
<210>5
<211>2118
<212>DNA
<213>人(homo sapiens)
<220>
<221>CDS
<222>(1)..(2115)
<223>
<400>5
atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa 96
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt 144
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat 192
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag 240
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg 288
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac 336
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat 384
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125
aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg 432
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag 480
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 155 160
agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
atc aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg gac 1248
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
405 410 415
ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc agg acc aag caa 1296
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445
gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att tca 1392
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg 1440
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
485 490 495
aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
500 505 510
gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
515 520 525
gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
530 535 540
gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
610 615 620
acc tac ggg cca gga aat gag agt ctc aaa gcc atg tta ttc tgc ctt 1920
Thr Tyr Gly Pro Gly Asn Glu Ser Leu Lys Ala Met Leu Phe Cys Leu
625 630 635 640
ttt aaa cta tca tcc tgt aat caa agt aat gat ggc agc gtg tcc cac 1968
Phe Lys Leu Ser Ser Cys Asn Gln Ser Asn Asp Gly Ser Val Ser His
645 650 655
cag agc ggg agc cca gct gct cag gag tca tgc tta gga tgg atc cct 2016
Gln Ser Gly Ser Pro Ala Ala Gln Glu Ser Cys Leu Gly Trp Ile Pro
660 665 670
tct ctt ctg ccg tca gag ttt cag ctg ggt tgg ggt gga tgc agc cac 2064
Ser Leu Leu Pro Ser Glu Phe Gln Leu Gly Trp Gly Gly Cys Ser His
675 680 685
ctc cat gcc tgg cct tct gca tct gtg atc atc acg gcc tcc tcc tgc 2112
Leu His Ala Trp Pro Ser Ala Ser Val Ile Ile Thr Ala Ser Ser Cys
690 695 700
cac tga 2118
His
705
<210>6
<211>705
<212>PRT
<213>人(homo sapiens)
<400>6
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 155 160
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
405 410 415
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
485 490 495
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
500 505 510
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
515 520 525
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
530 535 540
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
610 615 620
Thr Tyr Gly Pro Gly Asn Glu Ser Leu Lys Ala Met Leu Phe Cys Leu
625 630 635 640
Phe Lys Leu Ser Ser Cys Asn Gln Ser Asn Asp Gly Ser Val Ser His
645 650 655
Gln Ser Gly Ser Pro Ala Ala Gln Glu Ser Cys Leu Gly Trp Ile Pro
660 665 670
Ser Leu Leu Pro Ser Glu Phe Gln Leu Gly Trp Gly Gly Cys Ser His
675 680 685
Leu His Ala Trp Pro Ser Ala Ser Val Ile Ile Thr Ala Ser Ser Cys
690 695 700
His
705
<210>7
<211>1887
<212>DNA
<213>人(homo sapiens)
<220>
<221>CDS
<222>(1)..(1884)
<223>
<400>7
atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa 96
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt 144
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat 192
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag 240
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg 288
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac 336
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat 384
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125
aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg 432
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag 480
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 155 160
agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
atc aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg gac 1248
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
405 410 415
ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc agg acc aag caa 1296
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445
gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att tca 1392
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg 1440
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
485 490 495
aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
500 505 510
gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
515 520 525
gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
530 535 540
gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
610 615 620
acc tac ggg tcc taa 1887
Thr Tyr Gly Ser
625
<210>8
<211>628
<212>PRT
<213>人(homo sapiens)
<400>8
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 155 160
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
405 410 415
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
485 490 495
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
500 505 510
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
515 520 525
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
530 535 540
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
610 615 620
Thr Tyr Gly Ser
625
<210>9
<211>1218
<212>DNA
<213>人(homo sapiens)
<220>
<221>CDS
<222>(1)..(1215)
<223>
<400>9
atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa 96
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt 144
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat 192
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag 240
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg 288
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac 336
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat 384
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125
aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg 432
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag 480
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 155 160
agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
atc aca ggt ttg agc tga 1218
Ile Thr Gly Leu Ser
405
<210>10
<211>405
<212>PRT
<213>人(homo sapiens)
<400>10
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 155 160
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
Ile Thr Gly Leu Ser
405
<210>11
<211>2829
<212>DNA
<213>人(homo sapiens)
<220>
<221>CDS
<222>(1)..(2826)
<223>
<400>11
atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag cgt aat tat gtg 96
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Arg Asn Tyr Val
20 25 30
gtg aca gat cac ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat 144
Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr
35 40 45
gag atg gag gaa gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct 192
Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro
50 55 60
tgc cgc aaa gtg tgt aac gga ata ggt att ggt gaa ttt aaa gac tca 240
Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser
65 70 75 80
ctc tcc ata aat gct acg aat att aaa cac ttc aaa aac tgc acc tcc 288
Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser
85 90 95
atc agt ggc gat ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc 336
Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser
100 105 110
ttc aca cat act cct cct ctg gat cca cag gaa ctg gat att ctg aaa 384
Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys
115 120 125
acc gta aag gaa atc aca ggg ttt ttg ctg att cag gct tgg cct gaa 432
Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu
130 135 140
aac agg acg gac ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc 480
Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly
145 150 155 160
agg acc aag caa cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac 528
Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn
165 170 175
ata aca tcc ttg gga tta cgc tcc ctc aag gag ata agt gat gga gat 576
Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp
180 185 190
gtg ata att tca gga aac aaa aat ttg tgc tat gca aat aca ata aac 624
Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn
195 200 205
tgg aaa aaa ctg ttt ggg acc tcc ggt cag aaa acc aaa att ata agc 672
Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser
210 215 220
aac aga ggt gaa aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc 720
Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala
225 230 235 240
ttg tgc tcc ccc gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc 768
Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val
245 250 255
tct tgc cgg aat gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac 816
Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn
260 265 270
ctt ctg gag ggt gag cca agg gag ttt gtg gag aac tct gag tgc ata 864
Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile
275 280 285
cag tgc cac cca gag tgc ctg cct cag gcc atg aac atc acc tgc aca 912
Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr
290 295 300
gga cgg gga cca gac aac tgt atc cag tgt gcc cac tac att gac ggc 960
Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly
305 310 315 320
ccc cac tgc gtc aag acc tgc ccg gca gga gtc atg gga gaa aac aac 1008
Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn
325 330 335
acc ctg gtc tgg aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc 1056
Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys
340 345 350
cat cca aac tgc acc tac gga tgc act ggg cca ggt ctt gaa ggc tgt 1104
His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys
355 360 365
cca acg aat ggg cct aag atc ccg tcc atc gcc act ggg atg gtg ggg 1152
Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly
370 375 380
gcc ctc ctc ttg ctg ctg gtg gtg gcc ctg ggg atc ggc ctc ttc atg 1200
Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
385 390 395 400
cga agg cgc cac atc gtt cgg aag cgc acg ctg cgg agg ctg ctg cag 1248
Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln
405 410 415
gag agg gag ctt gtg gag cct ctt aca ccc agt gga gaa gct ccc aac 1296
Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn
420 425 430
caa gct ctc ttg agg atc ttg aag gaa act gaa ttc aaa aag atc aaa 1344
Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys
435 440 445
gtg ctg ggc tcc ggt gcg ttc ggc acg gtg tat aag gga ctc tgg atc 1392
Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile
450 455 460
cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gaa tta aga 1440
Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg
465 470 475 480
gaa gca aca tct ccg aaa gcc aac aag gaa atc ctc gat gaa gcc tac 1488
Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr
485 490 495
gtg atg gcc agc gtg gac aac ccc cac gtg tgc cgc ctg ctg ggc atc 1536
Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile
500 505 510
tgc ctc acc tcc acc gtg cag ctc atc acg cag ctc atg ccc ttc ggc 1584
Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly
515 520 525
tgc ctc ctg gac tat gtc cgg gaa cac aaa gac aat att ggc tcc cag 1632
Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln
530 535 540
tac ctg ctc aac tgg tgt gtg cag atc gca aag ggc atg aac tac ttg 1680
Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu
545 550 555 560
gag gac cgt cgc ttg gtg cac cgc gac ctg gca gcc agg aac gta ctg 1728
Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu
565 570 575
gtg aaa aca ccg cag cat gtc aag atc aca gat ttt ggg ctg gcc aaa 1776
Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys
580 585 590
ctg ctg ggt gcg gaa gag aaa gaa tac cat gca gaa gga ggc aaa gtg 1824
Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val
595 600 605
cct atc aag tgg atg gca ttg gaa tca att tta cac aga atc tat acc 1872
Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr
610 615 620
cac cag agt gat gtc tgg agc tac ggg gtg acc gtt tgg gag ttg atg 1920
His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met
625 630 635 640
acc ttt gga tcc aag cca tat gac gga atc cct gcc agc gag atc tcc 1968
Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser
645 650 655
tcc atc ctg gag aaa gga gaa cgc ctc cct cag cca ccc ata tgt acc 2016
Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr
660 665 670
atc gat gtc tac atg atc atg gtc aag tgc tgg atg ata gac gca gat 2064
Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp
675 680 685
agt cgc cca aag ttc cgt gag ttg atc atc gaa ttc tcc aaa atg gcc 2112
Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala
690 695 700
cga gac ccc cag cgc tac ctt gtc att cag ggg gat gaa aga atg cat 2160
Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His
705 710 715 720
ttg cca agt cct aca gac tcc aac ttc tac cgt gcc ctg atg gat gaa 2208
Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu
725 730 735
gaa gac atg gac gac gtg gtg gat gcc gac gag tac ctc atc cca cag 2256
Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln
740 745 750
cag ggc ttc ttc agc agc ccc tcc acg tca cgg act ccc ctc ctg agc 2304
Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser
755 760 765
tct ctg agt gca acc agc aac aat tcc acc gtg gct tgc att gat aga 2352
Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg
770 775 780
aat ggg ctg caa agc tgt ccc atc aag gaa gac agc ttc ttg cag cga 2400
Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg
785 790 795 800
tac agc tca gac ccc aca ggc gcc ttg act gag gac agc ata gac gac 2448
Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp
805 810 815
acc ttc ctc cca gtg cct gaa tac ata aac cag tcc gtt ccc aaa agg 2496
Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg
820 825 830
ccc gct ggc tct gtg cag aat cct gtc tat cac aat cag cct ctg aac 2544
Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn
835 840 845
ccc gcg ccc agc aga gac cca cac tac cag gac ccc cac agc act gca 2592
Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala
850 855 860
gtg ggc aac ccc gag tat ctc aac act gtc cag ccc acc tgt gtc aac 2640
Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn
865 870 875 880
agc aca ttc gac agc cct gcc cac tgg gcc cag aaa ggc agc cac caa 2688
Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln
885 890 895
att agc ctg gac aac cct gac tac cag cag gac ttc ttt ccc aag gaa 2736
Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu
900 905 910
gcc aag cca aat ggc atc ttt aag ggc tcc aca gct gaa aat gca gaa 2784
Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu
915 920 925
tac cta agg gtc gcg cca caa agc agt gaa ttt att gga gca tga 2829
Tyr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala
930 935 940
<210>12
<211>942
<212>PRT
<213>人(homo sapiens)
<400>12
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Arg Asn Tyr Val
20 25 30
Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr
35 40 45
Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro
50 55 60
Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser
65 70 75 80
Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser
85 90 95
Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser
100 105 110
Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys
115 120 125
Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu
130 135 140
Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly
145 150 155 160
Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn
165 170 175
Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile 5er Asp Gly Asp
180 185 190
Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn
195 200 205
Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser
210 215 220
Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala
225 230 235 240
Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val
245 250 255
Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn
260 265 270
Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile
275 280 285
Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr
290 295 300
Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly
305 310 315 320
Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn
325 330 335
Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys
340 345 350
His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys
355 360 365
Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly
370 375 380
Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
385 390 395 400
Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln
405 410 415
Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn
420 425 430
Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys
435 440 445
Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile
450 455 460
Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg
465 470 475 480
Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr
485 490 495
Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile
500 505 510
Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly
515 520 525
Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln
530 535 540
Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu
545 550 555 560
Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu
565 570 575
Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys
580 585 590
Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val
595 600 605
Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr
610 615 620
His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met
625 630 635 640
Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser
645 650 655
Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr
660 665 670
Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp
675 680 685
Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala
690 695 700
Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His
705 710 715 720
Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu
725 730 735
Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln
740 745 750
Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser
755 760 765
Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg
770 775 780
Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg
785 790 795 800
Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp
805 810 815
Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg
820 825 830
Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn
835 840 845
Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala
850 855 860
Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn
865 870 875 880
Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln
885 890 895
Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu
900 905 910
Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu
915 920 925
Tyr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala
930 935 940
<210>13
<211>3633
<212>DNA
<213>小鼠(Mus musculus)
<220>
<221>CDS
<222>(1)..(3630)
<223>
<400>13
atg cga ccc tca ggg acc gcg aga acc aca ctg ctg gtg ctg ctg acc 48
Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr
1 5 10 15
gcg ctc tgc gcc gca ggt ggg gcg ttg gag gaa aag aaa gtc tgc caa 96
Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
ggc aca agt aac agg ctc acc caa ctg ggc act ttt gaa gac cac ttt 144
Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
ctg agc ctg cag agg atg tac aac aac tgt gaa gtg gtc ctt ggg aac 192
Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
ttg gaa att acc tat gtg caa agg aat tac gac ctt tcc ttc tta aag 240
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
acc atc cag gag gtg gcc ggc tat gtc ctc att gcc ctc aac acc gtg 288
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
gag aga atc cct ttg gag aac ctg cag atc atc agg gga aat gct ctt 336
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu
100 105 110
tat gaa aac acc tat gcc tta gcc atc ctg tcc aac tat ggg aca aac 384
Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn
115 120 125
aga act ggg ctt agg gaa ctg ccc atg cgg aac tta cag gaa atc ctg 432
Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
att ggt gct gtg cga ttc agc aac aac ccc atc ctc tgc aat atg gat 480
Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp
145 150 155 160
act atc cag tgg agg gac atc gtc caa aac gtc ttt atg agc aac atg 528
Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met
165 170 175
tca atg gac tta cag agc cat ccg agc agt tgc ccc aaa tgt gat cca 576
Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro
180 185 190
agc tgt ccc aat gga agc tgc tgg gga gga gga gag gag aac tgc cag 624
Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln
195 200 205
aaa ttg acc aaa atc atc tgt gcc cag caa tgt tcc cat cgc tgt cgt 672
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg
210 215 220
ggc agg tcc ccc agt gac tgc tgc cac aac caa tgt gct gcg ggg tgt 720
Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
aca ggg ccc cga gag agt gac tgt ctg gtc tgc caa aag ttc caa gat 768
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp
245 250 255
gag gcc aca tgc aaa gac acc tgc cca cca ctc atg ctg tac aac ccc 816
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
acc acc tat cag atg gat gtc aac cct gaa ggg aag tac agc ttt ggt 864
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
gcc acc tgt gtg aag aag tgc ccc cga aac tac gtg gtg aca gat cat 912
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
ggc tca tgt gtc cga gcc tgt ggg cct gac tac tac gaa gtg gaa gaa 960
Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu
305 310 315 320
gat ggc atc cgc aag tgt aaa aaa tgt gat ggg ccc tgt cgc aaa gtt 1008
Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val
325 330 335
tgt aat ggc ata ggc att ggt gaa ttt aaa gac aca ctc tcc ata aat 1056
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn
340 345 350
gct aca aac atc aaa cac ttc aaa tac tgc act gcc atc agc ggg gac 1104
Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp
355 360 365
ctt cac atc ctg cca gtg gcc ttt aag ggg gat tct ttc acg cgc act 1152
Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr
370 375 380
cct cct cta gac cca cga gaa cta gaa att cta aaa acc gta aag gaa 1200
Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu
385 390 395 400
ata aca ggc ttt ttg ctg att cag gct tgg cct gat aac tgg act gac 1248
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp
405 410 415
ctc cat gct ttc gag aac cta gaa ata ata cgt ggc aga aca aag caa 1296
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
cat ggt cag ttt tct ttg gcg gtc gtt ggc ctg aac atc aca tca ctg 1344
His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu
435 440 445
ggg ctg cgt tcc ctc aag gag atc agt gat ggg gat gtg atc att tct 1392
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
gga aac cga aat ttg tgc tac gca aac aca ata aac tgg aaa aaa ctc 1440
Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
ttc ggg aca ccc aat cag aaa acc aaa atc atg aac aac aga gct gag 1488
Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu
485 490 495
aaa gac tgc aag gcc gtg aac cac gtc tgc aat cct tta tgc tcc tcg 1536
Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser
500 505 510
gaa ggc tgc tgg ggc cct gag ccc agg gac tgt gtc tcc tgc cag aat 1584
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn
515 520 525
gtg agc aga ggc agg gag tgc gtg gag aaa tgc aac atc ctg gag ggg 1632
Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly
530 535 540
gaa cca agg gag ttt gtg gaa aat tct gaa tgc atc cag tgc cat cca 1680
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
gaa tgt ctg ccc cag gcc atg aac atc acc tgt aca ggc agg ggg cca 1728
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
gac aac tgc atc cag tgt gcc cac tac att gat ggc cca cac tgt gtc 1776
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
aag acc tgc cca gct ggc atc atg gga gag aac aac act ctg gtc tgg 1824
Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
aag tat gca gat gcc aat aat gtc tgc cac cta tgc cac gcc aac tgt 1872
Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys
610 615 620
acc tat gga tgt gct ggg cca ggt ctt caa gga tgt gaa gtg tgg cca 1920
Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro
625 630 635 640
tct ggg cca aag ata cca tct att gcc act ggg att gtg ggt ggc ctc 1968
Ser Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Ile Val Gly Gly leu
645 650 655
ctc ttc ata gtg gtg gtg gcc ctt ggg att ggc cta ttc atg cga aga 2016
Leu Phe Ile Val Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg
660 665 670
cgt cac att gtt cga aag cgt aca cta cgc cgc ctg ctt caa gag aga 2064
Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg
675 680 685
gag ctc gtg gaa cct ctc aca ccc agc gga gaa gct cca aac caa gcc 2112
Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala
690 695 700
cac ttg agg ata tta aag gaa aca gaa ttc aaa aag atc aaa gtt ctg 2160
His Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu
705 710 715 720
ggt tcg gga gca ttt ggc aca gtg tat aag ggt ctc tgg atc cca gaa 2208
Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu
725 730 735
ggt gag aaa gta aaa atc ccg gtg gcc atc aag gag tta aga gaa gcc 2256
Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala
740 745 750
aca tct cca aaa gcc aac aaa gaa atc ctt gac gaa gcc tat gtg atg 2304
Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met
755 760 765
gct agt gtg gac aac cct cat gta tgc cgc ctc ctg ggc atc tgt ctg 2352
Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu
770 775 780
acc tcc act gtc cag ctc att aca cag ctc atg ccc tac ggt tgc ctc 2400
Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys Leu
785 790 795 800
ctg gac tac gtc cga gaa cac aag gac aac att ggc tcc cag tac ctc 2448
Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu
805 810 815
ctc aac tgg tgt gtg cag att gca aag ggc atg aac tac ctg gaa gat 2496
Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp
820 825 830
cgg cgt ttg gtg cac cgt gac ttg gca gcc agg aat gta ctg gtg aag 2544
Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys
835 840 845
aca cca cag cat gtc aag atc aca gat ttt ggg ctg gcc aaa ctg ctt 2592
Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu
850 855 860
ggt gct gaa gag aaa gaa tat cat gcc gag ggg ggc aaa gtg cct atc 2640
Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile
865 870 875 880
aag tgg atg gct ttg gaa tca att tta cac cga att tat aca cac caa 2688
Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln
885 890 895
agt gat gtc tgg agc tat ggt gtc act gtg tgg gaa ctg atg acc ttt 2736
Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe
900 905 910
ggg tcc aag cct tat gat gga atc cca gca agt gac atc tca tcc atc 2784
Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Asp Ile Ser Ser Ile
915 920 925
cta gag aaa gga gaa cgc ctt cca cag cca cct atc tgc acc atc gat 2832
Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp
930 935 940
gtc tac atg atc atg gtc aag tgc tgg atg ata gat gct gat agc cgc 2880
Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg
945 950 955 960
cca aag ttc cga gag ttg att ctt gaa ttc tcc aaa atg gcc cga gac 2928
Pro Lys Phe Arg Glu Leu Ile Leu Glu Phe Ser Lys Met Ala Arg Asp
965 970 975
cca cag cgc tac ctt gtt atc cag ggg gat gaa aga atg cat ttg cca 2976
Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro
980 985 990
agc cct aca gac tcc aac ttt tac cga gcc ctg atg gat gaa gag gac 3024
Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp
995 1000 1005
atg gag gat gta gtt gat gct gat gag tat ctt acc cca cag caa 3069
Met Glu Asp Val Val Asp Ala Asp Glu Tyr Leu Thr Pro Gln Gln
1010 1015 1020
ggc ttc ttc aac agc ccg tcc acg tcg agg act ccc ctc ttg agt 3114
Gly Phe Phe Asn Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser
1025 1030 1035
tct ctg agt gca act agc aac aat tcc act gtg gct tgc att aat 3159
Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asn
1040 1045 1050
aga aat ggg agc tgc cgt gtc aaa gaa gac gcc ttc ttg cag cgg 3204
Arg Asn Gly Ser Cys Arg Val Lys Glu Asp Ala Phe Leu Gln Arg
1055 1060 1065
tac agc tcc gac ccc aca ggt gct gta aca gag gac aac ata gat 3249
Tyr Ser Ser Asp Pro Thr Gly Ala Val Thr Glu Asp Asn Ile Asp
1070 1075 1080
gac gca ttc ctc cct gta cct gaa tat gta aac caa tct gtt ccc 3294
Asp Ala Phe Leu Pro Val Pro Glu Tyr Val Asn Gln Ser Val Pro
1085 1090 1095
aag agg cca gca ggc tct gtg cag aac cct gtc tat cac aat cag 3339
Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln
1100 1105 1110
ccc ctg cat cca gct cct gga aga gac ctg cat tat caa aat ccc 3384
Pro Leu His Pro Ala Pro Gly Arg Asp Leu His Tyr Gln Asn Pro
1115 1120 1125
cac agc aat gca gtg ggc aac cct gag tat ctc aac act gcc cag 3429
His Ser Asn Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Ala Gln
1130 1135 1140
cct acc tgt ctc agt agt ggg ttt aac agc cct gca ctc tgg atc 3474
Pro Thr Cys Leu Ser Ser Gly Phe Asn Ser Pro Ala Leu Trp Ile
1145 1150 1155
cag aaa ggc agt cac caa atg agc cta gac aac cct gac tac cag 3519
Gln Lys Gly Ser His Gln Met Ser Leu Asp Asn Pro Asp Tyr Gln
1160 1165 1170
cag gac ttc ttc ccc aag gaa acc aag cca aat ggc ata ttt aag 3564
Gln Asp Phe Phe Pro Lys Glu Thr Lys Pro Asn Gly Ile Phe Lys
1175 1180 1185
ggc ccc aca gct gaa aat gca gag tac cta cgg gtg gca cct cca 3609
Gly Pro Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Pro
1190 1195 1200
agc agc gag ttt att gga gca tga 3633
Ser Ser Glu Phe Ile Gly Ala
1205 1210
<210>14
<211>1210
<212>PRT
<213>小鼠(Mus musculus)
<400>14
Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr
1 5 10 15
Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu
100 105 110
Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn
115 120 125
Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp
145 150 155 160
Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met
165 170 175
Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro
180 185 190
Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln
195 200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg
210 215 220
Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp
245 250 255
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu
305 310 315 320
Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val
325 330 335
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn
340 345 350
Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp
355 360 365
Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr
370 375 380
Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu
385 390 395 400
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp
405 410 415
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu
435 440 445
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu
485 490 495
Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser
500 505 510
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn
515 520 525
Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly
530 535 540
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys
610 615 620
Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro
625 630 635 640
Ser Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Ile Val Gly Gly Leu
645 650 655
Leu Phe Ile Val Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg
660 665 670
Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg
675 680 685
Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala
690 695 700
His Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu
705 710 715 720
Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu
725 730 735
Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala
740 745 750
Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met
755 760 765
Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu
770 775 780
Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys Leu
785 790 795 800
Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu
805 810 815
Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp
820 825 830
Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys
835 840 845
Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu
850 855 860
Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile
865 870 875 880
Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln
885 890 895
Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe
900 905 910
Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Asp Ile Ser Ser Ile
915 920 925
Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp
930 935 940
Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg
945 950 955 960
Pro Lys Phe Arg Glu Leu Ile Leu Glu Phe Ser Lys Met Ala Arg Asp
965 970 975
Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro
980 985 990
Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp
995 1000 1005
Met Glu Asp Val Val Asp Ala Asp Glu Tyr Leu Thr Pro Gln Gln
1010 1015 1020
Gly Phe Phe Asn Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser
1025 1030 1035
Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asn
1040 1045 1050
Arg Asn Gly Ser Cys Arg Val Lys Glu Asp Ala Phe Leu Gln Arg
1055 1060 1065
Tyr Ser Ser Asp Pro Thr Gly Ala Val Thr Glu Asp Asn Ile Asp
1070 1075 1080
Asp Ala Phe Leu Pro Val Pro Glu Tyr Val Asn Gln Ser Val Pro
1085 1090 1095
Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln
1100 1105 1110
Pro Leu His Pro Ala Pro Gly Arg Asp Leu His Tyr Gln Asn Pro
1115 1120 1125
His Ser Asn Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Ala Gln
1130 1135 1140
Pro Thr Cys Leu Ser Ser Gly Phe Asn Ser Pro Ala Leu Trp Ile
1145 1150 1155
Gln Lys Gly Ser His Gln Met Ser Leu Asp Asn Pro Asp Tyr Gln
1160 1165 1170
Gln Asp Phe Phe Pro Lys Glu Thr Lys Pro Asn Gly Ile Phe Lys
1175 1180 1185
Gly Pro Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Pro
1190 1195 1200
Ser Ser Glu Phe Ile Gly Ala
1205 1210
<210>15
<211>1932
<212>DNA
<213>小鼠(Mus musculus)
<220>
<221>CDS
<222>(1)..(1929)
<223>
<400>15
atg cga ccc tca ggg acc gcg aga acc aca ctg ctg gtg ctg ctg acc 48
Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr
1 5 10 15
gcg ctc tgc gcc gca ggt ggg gcg ttg gag gaa aag aaa gtc tgc caa 96
Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
ggc aca agt aac agg ctc acc caa ctg ggc act ttt gaa gac cac ttt 144
Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
ctg agc ctg cag agg atg tac aac aac tgt gaa gtg gtc ctt ggg aac 192
Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
ttg gaa att acc tat gtg caa agg aat tac gac ctt tcc ttc tta aag 240
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
acc atc cag gag gtg gcc ggc tat gtc ctc att gcc ctc aac acc gtg 288
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
gag aga atc cct ttg gag aac ctg cag atc atc agg gga aat gct ctt 336
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu
100 105 110
tat gaa aac acc tat gcc tta gcc atc ctg tcc aac tat ggg aca aac 384
Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn
115 120 125
aga act ggg ctt agg gaa ctg ccc atg cgg aac tta cag gaa atc ctg 432
Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
att ggt gct gtg cga ttc agc aac aac ccc atc ctc tgc aat atg gat 480
Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp
145 150 155 160
act atc cag tgg agg gac atc gtc caa aac gtc ttt atg agc aac atg 528
Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met
165 170 175
tca atg gac tta cag agc cat ccg agc agt tgc ccc aaa tgt gat cca 576
Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro
180 185 190
agc tgt ccc aat gga agc tgc tgg gga gga gga gag gag aac tgc cag 624
Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln
195 200 205
aaa ttg acc aaa atc atc tgt gcc cag caa tgt tcc cat cgc tgt cgt 672
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg
210 215 220
ggc agg tcc ccc agt gac tgc tgc cac aac caa tgt gct gcg ggg tgt 720
Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
aca ggg ccc cga gag agt gac tgt ctg gtc tgc caa aag ttc caa gat 768
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp
245 250 255
gag gcc aca tgc aaa gac acc tgc cca cca ctc atg ctg tac aac ccc 816
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
acc acc tat cag atg gat gtc aac cct gaa ggg aag tac agc ttt ggt 864
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
gcc acc tgt gtg aag aag tgc ccc cga aac tac gtg gtg aca gat cat 912
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
ggc tca tgt gtc cga gcc tgt ggg cct gac tac tac gaa gtg gaa gaa 960
Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu
305 310 315 320
gat ggc atc cgc aag tgt aaa aaa tgt gat ggg ccc tgt cgc aaa gtt 1008
Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val
325 330 335
tgt aat ggc ata ggc att ggt gaa ttt aaa gac aca ctc tcc ata aat 1056
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn
340 345 350
gct aca aac atc aaa cac ttc aaa tac tgc act gcc atc agc ggg gac 1104
Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp
355 360 365
ctt cac atc ctg cca gtg gcc ttt aag ggg gat tct ttc acg cgc act 1152
Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr
370 375 380
cct cct cta gac cca cga gaa cta gaa att cta aaa acc gta aag gaa 1200
Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu
385 390 395 400
ata aca ggc ttt ttg ctg att cag gct tgg cct gat aac tgg act gac 1248
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp
405 410 415
ctc cat gct ttc gag aac cta gaa ata ata cgt ggc aga aca aag caa 1296
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
cat ggt cag ttt tct ttg gcg gtc gtt ggc ctg aac atc aca tca ctg 1344
His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu
435 440 445
ggg ctg cgt tcc ctc aag gag atc agt gat ggg gat gtg atc att tct 1392
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
gga aac cga aat ttg tgc tac gca aac aca ata aac tgg aaa aaa ctc 1440
Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
ttc ggg aca ccc aat cag aaa acc aaa atc atg aac aac aga gct gag 1488
Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu
485 490 495
aaa gac tgc aag gcc gtg aac cac gtc tgc aat cct tta tgc tcc tcg 1536
Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser
500 505 510
gaa ggc tgc tgg ggc cct gag ccc agg gac tgt gtc tcc tgc cag aat 1584
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn
515 520 525
gtg agc aga ggc agg gag tgc gtg gag aaa tgc aac atc ctg gag ggg 1632
Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly
530 535 540
gaa cca agg gag ttt gtg gaa aat tct gaa tgc atc cag tgc cat cca 1680
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
gaa tgt ctg ccc cag gcc atg aac atc acc tgt aca ggc agg ggg cca 1728
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
gac aac tgc atc cag tgt gcc cac tac att gat ggc cca cac tgt gtc 1776
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
aag acc tgc cca gct ggc atc atg gga gag aac aac act ctg gtc tgg 1824
Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
aag tat gca gat gcc aat aat gtc tgc cac cta tgc cac gcc aac tgt 1872
Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys
610 615 620
acc tat gga gta atg gtt cct gaa atg ttg ctc caa agt atc att tta 1920
Thr Tyr Gly Val Met Val Pro Glu Met Leu Leu Gln Ser Ile Ile Leu
625 630 635 640
aaa cct att tga 1932
Lys Pro Ile
<210>16
<211>643
<212>PRT
<213>小鼠(Mus musculus)
<400>16
Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr
1 5 10 15
Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu
100 105 110
Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn
115 120 125
Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp
145 150 155 160
Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met
165 170 175
Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro
180 185 190
Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln
195 200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg
210 215 220
Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp
245 250 255
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu
305 310 315 320
Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val
325 330 335
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn
340 345 350
Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp
355 360 365
Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr
370 375 380
Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu
385 390 395 400
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp
405 410 415
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu
435 440 445
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu
485 490 495
Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser
500 505 510
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn
515 520 525
Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly
530 535 540
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys
610 615 620
Thr Tyr Gly Val Met Val Pro Glu Met Leu Leu Gln Ser Ile Ile Leu
625 630 635 640
Lys Pro Ile
<210>17
<211>1968
<212>DNA
<2113>小鼠(Mus musculus)
<220>
<221>CDS
<222>(1)..(1965)
<223>
<400>17
atg cga ccc tca ggg acc gcg aga acc aca ctg ctg gtg ctg ctg acc 48
Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr
1 5 10 15
gcg ctc tgc gcc gca ggt ggg gcg ttg gag gaa aag aaa gtc tgc caa 96
Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
ggc aca agt aac agg ctc acc caa ctg ggc act ttt gaa gac cac ttt 144
Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
ctg agc ctg cag agg atg tac aac aac tgt gaa gtg gtc ctt ggg aac 192
Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
ttg gaa att acc tat gtg caa agg aat tac gac ctt tcc ttc tta aag 240
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
acc atc cag gag gtg gcc ggc tat gtc ctc att gcc ctc aac acc gtg 288
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
gag aga atc cct ttg gag aac ctg cag atc atc agg gga aat gct ctt 336
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu
100 105 110
tat gaa aac acc tat gcc tta gcc atc ctg tcc aac tat ggg aca aac 384
Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn
115 120 125
aga act ggg ctt agg gaa ctg ccc atg cgg aac tta cag gaa atc ctg 432
Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
att ggt gct gtg cga ttc agc aac aac ccc atc ctc tgc aat atg gat 480
Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp
145 150 155 160
act atc cag tgg agg gac atc gtc caa aac gtc ttt atg agc aac atg 528
Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met
165 170 175
tca atg gac tta cag agc cat ccg agc agt tgc ccc aaa tgt gat cca 576
Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro
180 185 190
agc tgt ccc aat gga agc tgc tgg gga gga gga gag gag aac tgc cag 624
Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln
195 200 205
aaa ttg acc aaa atc atc tgt gcc cag caa tgt tcc cat cgc tgt cgt 672
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg
210 215 220
ggc agg tcc ccc agt gac tgc tgc cac aac caa tgt gct gcg ggg tgt 720
Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
aca ggg ccc cga gag agt gac tgt ctg gtc tgc caa aag ttc caa gat 768
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp
245 250 255
gag gcc aca tgc aaa gac acc tgc cca cca ctc atg ctg tac aac ccc 816
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
acc acc tat cag atg gat gtc aac cct gaa ggg aag tac agc ttt ggt 864
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
gcc acc tgt gtg aag aag tgc ccc cga aac tac gtg gtg aca gat cat 912
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
ggc tca tgt gtc cga gcc tgt ggg cct gac tac tac gaa gtg gaa gaa 960
Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu
305 310 315 320
gat ggc atc cgc aag tgt aaa aaa tgt gat ggg ccc tgt cgc aaa gtt 1008
Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val
325 330 335
tgt aat ggc ata ggc att ggt gaa ttt aaa gac aca ctc tcc ata aat 1056
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn
340 345 350
gct aca aac atc aaa cac ttc aaa tac tgc act gcc atc agc ggg gac 1104
Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp
355 360 365
ctt cac atc ctg cca gtg gcc ttt aag ggg gat tct ttc acg cgc act 1152
Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr
370 375 380
cct cct cta gac cca cga gaa cta gaa att cta aaa acc gta aag gaa 1200
Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu
385 390 395 400
ata aca ggc ttt ttg ctg att cag gct tgg cct gat aac tgg act gac 1248
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp
405 410 415
ctc cat gct ttc gag aac cta gaa ata ata cgt ggc aga aca aag caa 1296
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
cat ggt cag ttt tct ttg gcg gtc gtt ggc ctg aac atc aca tca ctg 1344
His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu
435 440 445
ggg ctg cgt tcc ctc aag gag atc agt gat ggg gat gtg atc att tct 1392
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
gga aac cga aat ttg tgc tac gca aac aca ata aac tgg aaa aaa ctc 1440
Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
ttc ggg aca ccc aat cag aaa acc aaa atc atg aac aac aga gct gag 1488
Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu
485 490 495
aaa gac tgc aag gcc gtg aac cac gtc tgc aat cct tta tgc tcc tcg 1536
Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser
500 505 510
gaa ggc tgc tgg ggc cct gag ccc agg gac tgt gtc tcc tgc cag aat 1584
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn
515 520 525
gtg agc aga ggc agg gag tgc gtg gag aaa tgc aac atc ctg gag ggg 1632
Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly
530 535 540
gaa cca agg gag ttt gtg gaa aat tct gaa tgc atc cag tgc cat cca 1680
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
gaa tgt ctg ccc cag gcc atg aac atc acc tgt aca ggc agg ggg cca 1728
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
gac aac tgc atc cag tgt gcc cac tac att gat ggc cca cac tgt gtc 1776
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
aag acc tgc cca gct ggc atc atg gga gag aac aac act ctg gtc tgg 1824
Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
aag tat gca gat gcc aat aat gtc tgc cac cta tgc cac gcc aac tgt 1872
Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys
610 615 620
acc tat gga tgt gct ggg cca ggt ctt caa gga tgt gaa gtg tgg cca 1920
Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro
625 630 635 640
tct ggg tac gtt caa tgg cag tgg atc tta aag acc ttt tgg atc taa 1968
Ser Gly Tyr Val Gln Trp Gln Trp Ile Leu Lys Thr Phe Trp Ile
645 650 655
<210>18
<211>655
<212>PRT
<213>小鼠(Mus musculus)
<400>18
Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr
1 5 10 15
Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu
100 105 110
Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn
115 120 125
Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp
145 150 155 160
Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met
165 170 175
Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro
180 185 190
Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln
195 200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg
210 215 220
Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp
245 250 255
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu
305 310 315 320
Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val
325 330 335
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn
340 345 350
Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp
355 360 365
Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr
370 375 380
Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu
385 390 395 400
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp
405 410 415
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu
435 440 445
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu
485 490 495
Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser
500 505 510
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn
515 520 525
Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly
530 535 540
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys
610 615 620
Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro
625 630 635 640
Ser Gly Tyr Val Gln Trp Gln Trp Ile Leu Lys Thr Phe Trp Ile
645 650 655
<210>19
<211>1962
<212>DNA
<213>鸡
<220>
<221>CDS
<222>(1)..(1962)
<223>
<400>19
atg ggt gta cgc agc ccc ctg tcc gcc tct ggg cct cgc ggg gcc gct 48
Met Gly Val Arg Ser Pro Leu Ser Ala Ser Gly Pro Arg Gly Ala Ala
1 5 10 15
gtc ctg gtg cta ctg ctg ctg ggc gtc gcg ctg tgt tcc gcc gtg gag 96
Val Leu Val Leu Leu Leu Leu Gly Val Ala Leu Cys Ser Ala Val Glu
20 25 30
gag aag aaa gtt tgt caa ggg aca aat aac aag ttg acc cag ctg ggg 144
Glu Lys Lys Val Cys Gln Gly Thr Asn Asn Lys Leu Thr Gln Leu Gly
35 40 45
cac gtg gaa gac cat ttc acc agc ctg cag aga atg tac aac aac tgc 192
His Val Glu Asp His Phe Thr Ser Leu Gln Arg Met Tyr Asn Asn Cys
50 55 60
gaa gtg gta ctg agt aac ctg gag att acc tac gtg gag cat aat cgc 240
Glu Val Val Leu Ser Asn Leu Glu Ile Thr Tyr Val Glu His Asn Arg
65 70 75 80
gat ctc acc ttc ctt aag acc ata cag gag gtt gca ggc tat gtg ctc 288
Asp Leu Thr Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr Val Leu
85 90 95
att gcg ctt aac atg gtg gac gtc att ccc tta gaa aac ctc cag att 336
Ile Ala Leu Asn Met Val Asp Val Ile Pro Leu Glu Asn Leu Gln Ile
100 105 110
atc cga ggg aat gtg ctt tat gac aac tct ttt gcc ctg gca gtt tta 384
Ile Arg Gly Asn Val Leu Tyr Asp Asn Ser Phe Ala Leu Ala Val Leu
115 120 125
tcc aat tac cac atg aat aaa acc cag gga ctt cga gag ctg cca atg 432
Ser Asn Tyr His Met Asn Lys Thr Gln Gly Leu Arg Glu Leu Pro Met
130 135 140
aaa cgg cta tca gaa att ctc aat gga ggt gtt aaa atc agc aac aac 480
Lys Arg Leu Ser Glu Ile Leu Asn Gly Gly Val Lys Ile Ser Asn Asn
145 150 155 160
ccc aaa ctg tgc aac atg gac act gtt ctc tgg aat gac atc att gat 528
Pro Lys Leu Cys Asn Met Asp Thr Val Leu Trp Asn Asp Ile Ile Asp
165 170 175
aca agc agg aag cct ctc aca gta ctt gac ttt gca agc aat ctt tct 576
Thr Ser Arg Lys Pro Leu Thr Val Leu Asp Phe Ala Ser Asn Leu Ser
180 185 190
tct tgt cca aaa tgc cat ccg aac tgc aca gaa gac cac tgc tgg ggt 624
Ser Cys Pro Lys Cys His Pro Asn Cys Thr Glu Asp His Cys Trp Gly
195 200 205
gct ggt gaa cag aac tgc cag act tta aca aaa gtc atc tgt gcc cag 672
Ala Gly Glu Gln Asn Cys Gln Thr Leu Thr Lys Val Ile Cys Ala Gln
210 215 220
caa tgc tct ggc cgg tgc aga gga aag gtg ccc agt gac tgc tgc cac 720
Gln Cys Ser Gly Arg Cys Arg Gly Lys Val Pro Ser Asp Cys Cys His
225 230 235 240
aat cag tgt gct gca ggg tgc aca gga cct cgg gag agt gac tgc ctg 768
Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys Leu
245 250 255
gca tgc cgc aag ttt cgg gat gat gct acc tgc aag gac aca tgt ccc 816
Ala Cys Arg Lys Phe Arg Asp Asp Ala Thr Cys Lys Asp Thr Cys Pro
260 265 270
cca ctg gtc ctc tat aac ccc acc acc tat caa atg gat gtc aac cct 864
Pro Leu Val Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn Pro
275 280 285
gag gga aaa tac agc ttt gga gcc act tgt gtg agg gaa tgt cca cac 912
Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Arg Glu Cys Pro His
290 295 300
aac tat gtg gtg aca gat cat ggc tcc tgc gtt cgc tcg tgt aat act 960
Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ser Cys Asn Thr
305 310 315 320
gat act tac gaa gtg gaa gaa aat ggt gtt cgg aag tgt aaa aaa tgt 1008
Asp Thr Tyr Glu Val Glu Glu Asn Gly Val Arg Lys Cys Lys Lys Cys
325 330 335
gat ggg cta tgt agc aaa gtg tgc aat ggc att gga ata ggt gaa ctt 1056
Asp Gly Leu Cys Ser Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Leu
340 345 350
aaa ggg atc cta tcc ata aat gcc aca aac atc gac tcc ttc aaa aac 1104
Lys Gly Ile Leu Ser Ile Asn Ala Thr Asn Ile Asp Ser Phe Lys Asn
355 360 365
tgt acg aag atc aat ggg gat gtc agc att ctt cct gtt gca ttt cta 1152
Cys Thr Lys Ile Asn Gly Asp Val Ser Ile Leu Pro Val Ala Phe Leu
370 375 380
ggg gat gcc ttc aca aag aca cta ccc ctt gac cct aag aag ctg gat 1200
Gly Asp Ala Phe Thr Lys Thr Leu Pro Leu Asp Pro Lys Lys Leu Asp
385 390 395 400
gtc ttt aga aca gtc aaa gaa ata tca gga ttt ttg ttg att cag gcc 1248
Val Phe Arg Thr Val Lys Glu Ile Ser Gly Phe Leu Leu Ile Gln Ala
405 410 415
tgg cct gat aat gct act gat ctc tat gct ttt gaa aat ctg gag att 1296
Trp Pro Asp Asn Ala Thr Asp Leu Tyr Ala Phe Glu Asn Leu Glu Ile
420 425 430
atc cga ggc cga acc aag cag cac ggc cag tat tcc ctt gct gtt gtt 1344
Ile Arg Gly Arg Thr Lys Gln His Gly Gln Tyr Ser Leu Ala Val Val
435 440 445
aac ttg aaa ata cag tcg ttg ggg ctg cgc tcc ctc aag gaa ata agt 1392
Asn Leu Lys Ile Gln Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser
450 455 460
gat gga gac att gcc att atg aag aac aag aac ctc tgc tat gct gac 1440
Asp Gly Asp Ile Ala Ile Met Lys Asn Lys Asn Leu Cys Tyr Ala Asp
465 470 475 480
acc atg aac tgg cgc agc ttg ttt gct act cag agt cag aaa aca aaa 1488
Thr Met Asn Trp Arg Ser Leu Phe Ala Thr Gln Ser Gln Lys Thr Lys
485 490 495
att ata cag aac aga aat aaa aat gat tgt act gct gac agg cat gtg 1536
Ile Ile Gln Asn Arg Asn Lys Asn Asp Cys Thr Ala Asp Arg His Val
500 505 510
tgt gac ccg ctg tgc tcg gac gtg ggc tgc tgg ggc cca ggg ccc ttc 1584
Cys Asp Pro Leu Cys Ser Asp Val Gly Cys Trp Gly Pro Gly Pro Phe
515 520 525
cac tgc ttt tcc tgc agg ttt ttc agt cgc cag aag gag tgt gta aaa 1632
His Cys Phe Ser Cys Arg Phe Phe Ser Arg Gln Lys Glu Cys Val Lys
530 535 540
cag tgc aac atc ctg caa ggg gag cca cgt gag ttt gaa aga gac tcc 1680
Gln Cys Asn Ile Leu Gln Gly Glu Pro Arg Glu Phe Glu Arg Asp Ser
545 550 555 560
aaa tgc ctc ccc tgc cac tca gag tgt ctg gta cag aac tcc act gca 1728
Lys Cys Leu Pro Cys His Ser Glu Cys Leu Val Gln Asn Ser Thr Ala
565 570 575
tac aac aca acc tgc tct gga ccg ggc cca gac cac tgc atg aag tgt 1776
Tyr Asn Thr Thr Cys Ser Gly Pro Gly Pro Asp His Cys Met Lys Cys
580 585 590
gcc cat ttt ata gat ggt ccc cac tgt gtg aag gcc tgc ccc gct ggg 1824
Ala His Phe Ile Asp Gly Pro His Cys Val Lys Ala Cys Pro Ala Gly
595 600 605
gtc ctg ggt gag aat gat acc ctg gtc tgg aag tat gca gat gcc aat 1872
Val Leu Gly Glu Asn Asp Thr Leu Val Trp Lys Tyr Ala Asp Ala Asn
610 615 620
gct gtt tgc cag ctc tgc cat cca aac tgt aca cga ggg tgc aaa ggg 11920
Ala Val Cys Gln Leu Cys His Pro Asn Cys Thr Arg Gly Cys Lys Gly
625 630 635 640
cca ggt ctt gaa gga tgt cca aat ggc tcc aaa act cca tct 1962
Pro Gly Leu Glu Gly Cys Pro Asn Gly Ser Lys Thr Pro Ser
645 650
<210>20
<211>654
<212>PRT
<213>鸡
<400>20
Met Gly Val Arg Ser Pro Leu Ser Ala Ser Gly Pro Arg Gly Ala Ala
1 5 10 15
Val Leu Val Leu Leu Leu Leu Gly Val Ala Leu Cys Ser Ala Val Glu
20 25 30
Glu Lys Lys Val Cys Gln Gly Thr Asn Asn Lys Leu Thr Gln Leu Gly
35 40 45
His Val Glu Asp His Phe Thr Ser Leu Gln Arg Met Tyr Asn Asn Cys
50 55 60
Glu Val Val Leu Ser Asn Leu Glu Ile Thr Tyr Val Glu His Asn Arg
65 70 75 80
Asp Leu Thr Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr Val Leu
85 90 95
Ile Ala Leu Asn Met Val Asp Val Ile Pro Leu Glu Asn Leu Gln Ile
100 105 110
Ile Arg Gly Asn Val Leu Tyr Asp Asn Ser Phe Ala Leu Ala Val Leu
115 120 125
Ser Asn Tyr His Met Asn Lys Thr Gln Gly Leu Arg Glu Leu Pro Met
130 135 140
Lys Arg Leu Ser Glu Ile Leu Asn Gly Gly Val Lys Ile Ser Asn Asn
145 150 155 160
Pro Lys Leu Cys Asn Met Asp Thr Val Leu Trp Asn Asp Ile Ile Asp
165 170 175
Thr Ser Arg Lys Pro Leu Thr Val Leu Asp Phe Ala Ser Asn Leu Ser
180 185 190
Ser Cys Pro Lys Cys His Pro Asn Cys Thr Glu Asp His Cys Trp Gly
195 200 205
Ala Gly Glu Gln Asn Cys Gln Thr Leu Thr Lys Val Ile Cys Ala Gln
210 215 220
Gln Cys Ser Gly Arg Cys Arg Gly Lys Val Pro Ser Asp Cys Cys His
225 230 235 240
Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys Leu
245 250 255
Ala Cys Arg Lys Phe Arg Asp Asp Ala Thr Cys Lys Asp Thr Cys Pro
260 265 270
Pro Leu Val Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn Pro
275 280 285
Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Arg Glu Cys Pro His
290 295 300
Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ser Cys Asn Thr
305 310 315 320
Asp Thr Tyr Glu Val Glu Glu Asn Gly Val Arg Lys Cys Lys Lys Cys
325 330 335
Asp Gly Leu Cys Ser Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Leu
340 345 350
Lys Gly Ile Leu Ser Ile Asn Ala Thr Asn Ile Asp Ser Phe Lys Asn
355 360 365
Cys Thr Lys Ile Asn Gly Asp Val Ser Ile Leu Pro Val Ala Phe Leu
370 375 380
Gly Asp Ala Phe Thr Lys Thr Leu Pro Leu Asp Pro Lys Lys Leu Asp
385 390 395 400
Val Phe Arg Thr Val Lys Glu Ile Ser Gly Phe Leu Leu Ile Gln Ala
405 410 415
Trp Pro Asp Asn Ala Thr Asp Leu Tyr Ala Phe Glu Asn Leu Glu Ile
420 425 430
Ile Arg Gly Arg Thr Lys Gln His Gly Gln Tyr Ser Leu Ala Val Val
435 440 445
Asn Leu Lys Ile Gln Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser
450 455 460
Asp Gly Asp Ile Ala Ile Met Lys Asn Lys Asn Leu Cys Tyr Ala Asp
465 470 475 480
Thr Met Asn Trp Arg Ser Leu Phe Ala Thr Gln Ser Gln Lys Thr Lys
485 490 495
Ile Ile Gln Asn Arg Asn Lys Asn Asp Cys Thr Ala Asp Arg His Val
500 505 510
Cys Asp Pro Leu Cys Ser Asp Val Gly Cys Trp Gly Pro Gly Pro Phe
515 520 525
His Cys Phe Ser Cys Arg Phe Phe Ser Arg Gln Lys Glu Cys Val Lys
530 535 540
Gln Cys Asn Ile Leu Gln Gly Glu Pro Arg Glu Phe Glu Arg Asp Ser
545 550 555 560
Lys Cys Leu Pro Cys His Ser Glu Cys Leu Val Gln Asn Ser Thr Ala
565 570 575
Tyr Asn Thr Thr Cys Ser Gly Pro Gly Pro Asp His Cys Met Lys Cys
580 585 590
Ala His Phe Ile Asp Gly Pro His Cys Val Lys Ala Cys Pro Ala Gly
595 600 605
Val Leu Gly Glu Asn Asp Thr Leu Val Trp Lys Tyr Ala Asp Ala Asn
610 615 620
Ala Val Cys Gln Leu Cys His Pro Asn Cys Thr Arg Gly Cys Lys Gly
625 630 635 640
Pro Gly Leu Glu Gly Cys Pro Asn Gly Ser Lys Thr Pro Ser
645 650
<210>21
<211>4134
<212>DNA
<213>果蝇(Drosophila melanogaster)
<220>
<221>CDS
<222>(1)..(3630)
<223>
<400>21
atg atg att atc agc atg tgg atg agc ata tcg cga gga ttg tgg gac 48
Met Met Ile Ile Ser Met Trp Met Ser Ile Ser Arg Gly Leu Trp Asp
1 5 10 15
agc agc tcc atc ttg tcg gtg ctg ctg atc ctc gcc tgc atg gca tcc 96
Ser Ser Ser Ile Leu Ser Val Leu Leu Ile Leu Ala Cys Met Ala Ser
20 25 30
atc acc aca agc tca tcg gtc agc aat gcc ggc tat gtg gat aat ggc 144
Ile Thr Thr Ser Ser Ser Val Ser Asn Ala Gly Tyr Val Asp Asn Gly
35 40 45
aat atg aaa gtc tgc atc ggc act aaa tct cgg ctc tcc gtg ccc tcc 192
Asn Met Lys Val Cys Ile Gly Thr Lys Ser Arg Leu Ser Val Pro Ser
50 55 60
aac aag gaa cat cat tac cga aac ctc aga gat cgg tac acg aac tgt 240
Asn Lys Glu His His Tyr Arg Asn Leu Arg Asp Arg Tyr Thr Asn Cys
65 70 75 80
acg tat gtg gat ggc aac ttg aaa ctg acc tgg cta ccc aac gag aat 288
Thr Tyr Val Asp Gly Asn Leu Lys Leu Thr Trp Leu Pro Asn Glu Asn
85 90 95
ttg gac ctc agc ttc cta gac aac ata cgg gag gtc acc ggc tat att 336
Leu Asp Leu Ser Phe Leu Asp Asn Ile Arg Glu Val Thr Gly Tyr Ile
100 105 110
ctg atc agt cat gtg gac gtt aag aaa gtg gtg ttt ccc aaa cta caa 384
Leu Ile Ser His Val Asp Val Lys Lys Val Val Phe Pro Lys Leu Gln
115 120 125
atc att cgc gga cgc acg ctg ttc agc tta tcc gtg gag gag gag aag 432
Ile Ile Arg Gly Arg Thr Leu Phe Ser Leu Ser Val Glu Glu Glu Lys
130 135 140
tat gcc ttg ttc gtc act tat tcc aaa atg tac acg ctg gag att ccc 480
Tyr Ala Leu Phe Val Thr Tyr Ser Lys Met Tyr Thr Leu Glu Ile Pro
145 150 155 160
gat cta cgc gat gtc tta aat ggc caa gtg ggc ttc cac aac aac tac 528
Asp Leu Arg Asp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn Tyr
165 170 175
aat ctc tgc cac atg cga acg atc cag tgg tcg gag att gta tcc aac 576
Asn Leu Cys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser Asn
180 185 190
ggc acg gat gca tac tac aac tac gac ttt act gct ccg gag cgc gag 624
Gly Thr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg Glu
195 200 205
tgt ccc aag tgc cac gag agc tgc acg cac gga tgt tgg ggc gag ggt 672
Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu Gly
210 215 220
ccc aag aat tgc cag aag ttc agc aag ctc acc tgc tcg cca cag tgt 720
Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser Pro Gln Cys
225 230 235 240
gcc gga ggt cgt tgc tat gga cca aag ccg cgg gag tgt tgt cac ctc 768
Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu Cys Cys His Leu
245 250 255
ttc tgc gcc gga gga tgc act ggt ccc acg caa aag gat tgc atc gcc 816
Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln Lys Asp Cys Ile Ala
260 265 270
tgc aag aac ttc ttc gac gag gca gta tca aag gag gaa tgc ccg ccc 864
Cys Lys Asn Phe Phe Asp Glu Ala Val Ser Lys Glu Glu Cys Pro Pro
275 280 285
atg cgc aag tac aat ccc acc acc tat gtt ctt gaa acg aat cct gag 912
Met Arg Lys Tyr Asn Pro Thr Thr Tyr Val Leu Glu Thr Asn Pro Glu
290 295 300
gga aag tat gcc tat ggt gcc acc tgc gtc aag gag tgt ccc ggt cat 960
Gly Lys Tyr Ala Tyr Gly Ala Thr Cys Val Lys Glu Cys Pro Gly His
305 310 315 320
ctg ttg cgg gat aat ggc gcc tgc gtg cgc agc tgt ccc cag gac aag 1008
Leu Leu Arg Asp Asn Gly Ala Cys Val Arg Ser Cys Pro Gln Asp Lys
325 330 335
atg gac aag ggg ggc gag tgt gtg ccc tgc aat gga ccg tgc ccc aaa 1056
Met Asp Lys Gly Gly Glu Cys Val Pro Cys Asn Gly Pro Cys Pro Lys
340 345 350
acc tgc ccg ggc gtt act gtc ctg cat gcc ggc aac att gac tcg ttc 1104
Thr Cys Pro Gly Val Thr Val Leu His Ala Gly Asn Ile Asp Ser Phe
355 360 365
cgg aat tgt acg gtg atc gat ggc aac att cgc att ttg gat cag acc 1152
Arg Asn Cys Thr Val Ile Asp Gly Asn Ile Arg Ile Leu Asp Gln Thr
370 375 380
ttc tcg ggc ttc cag gat gtc tat gcc aac tac acg atg gga cca cga 1200
Phe Ser Gly Phe Gln Asp Val Tyr Ala Asn Tyr Thr Met Gly Pro Arg
385 390 395 400
tac ata ccg ctg gat ccc gag cga cgg gag gtg ttc tcc acg gtg aag 1248
Tyr Ile Pro Leu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val Lys
405 410 415
gag atc acc ggg tat ctg aat atc gag gga acc cac ccg cag ttc cgg 1296
Glu Ile Thr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe Arg
420 425 430
aat ctg tcg tac ttt cgc aat ctg gaa aca att cat ggc cgc cag ctg 1344
Asn Leu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln Leu
435 440 445
atg gag agc atg ttt gcc gct ttg gcg atc gtt aag tca tcc ctg tac 1392
Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu Tyr
450 455 460
agc ctg gag atg cgc aat ctg aag cag att agt tcc ggc agt gtg gtc 1440
Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val Val
465 470 475 480
atc cag cat aat aga gac ctc tgc tac gta agc aat atc cgt tgg ccg 1488
Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp Pro
485 490 495
gcc att cag aag gag ccc gaa cag aag gtg tgg gtc aac gag aat ctc 1536
Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp Val Asn Glu Asn Leu
500 505 510
agg gcg gat cta tgc gag aaa aat gga acc att tgc tcg gat cag tgc 1584
Arg Ala Asp Leu Cys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln Cys
515 520 525
aac gag gac ggc tgc tgg gga gct ggc acg gat cag tgc ctt acc tgc 1632
Asn Glu Asp Gly Cys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr Cys
530 535 540
aag aac ttc aat ttc aat ggc acc tgc atc gcc gac tgt ggt tat ata 1680
Lys Asn Phe Asn Phe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr Ile
545 550 555 560
tcc aat gcc tac aag ttt gac aat aga acg tgc aag ata tgc cat cca 1728
Ser Asn Ala Tyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His Pro
565 570 575
gag tgc cgg act tgc aat gga gct gga gca gat cac tgc cag gag tgc 1776
Glu Cys Arg Thr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu Cys
580 585 590
gtc cat gtg agg gac ggt cag cac tgt gtg tcc gag tgc ccg aag aac 1824
Val His Val Arg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys Asn
595 600 605
aag tac aac gat cgt ggt gtc tgc cga gag tgc cac gcc acc tgc gat 1872
Lys Tyr Asn Asp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys Asp
610 615 620
gga tgc act ggg ccc aag gac acc atc ggc att gga gcg tgt acg acg 1920
Gly Cys Thr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr Thr
625 630 635 640
tgc aat ttg gcc att atc aac aat gac gcc aca gta aaa cgc tgc ctg 1968
Cys Asn Leu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys Leu
645 650 655
ctg aag gac gac aag tgc ccc gat ggg tat ttc tgg gag tat gtg cat 2016
Leu Lys Asp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val His
660 665 670
ccg caa gag cag gga tcg ctg aag cca ttg gcc ggc aga gca gtt tgc 2064
Pro Gln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val Cys
675 680 685
cga aag tgc cat ccc ctt tgc gag ctg tgc acg aac tac gga tac cat 2112
Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr His
690 695 700
gaa cag gtg tgc tcc aag tgc acc cac tac aag cga cgg gag cag tgc 2160
Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln Cys
705 710 715 720
gag acc gag tgt ccg gcc gat cac tac acg gat gag gag cag cgc gag 2208
Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg Glu
725 730 735
tgc ttc cag cgc cac ccg gaa tgc aat ggt tgc acg ggt ccg ggt gcc 2256
Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly Ala
740 745 750
gac gat tgc aag tct tgc cgc aac ttt aag ttg ttc gac gcg aat gag 2304
Asp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn Glu
755 760 765
acg ggt ccc tat gtg aac tcc acg atg ttc aat tgc acc tcg aag tgt 2352
Thr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys Cys
770 775 780
ccc ttg gag atg cga cat gtg aac tat cag tac acg gcc att gga ccc 2400
Pro Leu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly Pro
785 790 795 800
tac tgc gca gct agt ccg ccg agg agc agc aag ata act gcc aat ctg 2448
Tyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn Leu
805 810 815
gat gtg aac atg atc ttc att atc act ggt gct gtt ctg gtg ccg acg 2496
Asp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro Thr
820 825 830
atc tgc atc ctc tgc gtg gtc aca tac att tgt cgg caa aag caa aag 2544
Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln Lys
835 840 845
gcc aag aag gaa aca gtc aag atg acc atg gct ctg tct ggc tgc gag 2592
Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys Glu
850 855 860
gat tcc gag ccg ctg cgt ccc tcg aac att gga gcc aac cta tgc aag 2640
Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys Lys
865 870 875 880
ttg cgc att gtc aag gac gcc gag ttg cgc aag ggc gga gtc ctt gga 2688
Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu Gly
885 890 895
atg gga gcc ttt gga cga gtg tac aag ggc gtt tgg gtg ccg gag ggt 2736
Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu Gly
900 905 910
gag aac gtc aag att cca gtg gcc att aag gag ctg ctc aag tcc aca 2784
Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser Thr
915 920 925
ggc gcc gag tca agc gaa gag ttc ctc cgc gaa gcc tac atc atg gcc 2832
Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met Ala
930 935 940
tct gag gag cac gtt aat ctg ctg aag ctc ctg gcc gtg tgc atg tcc 2880
Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met Ser
945 950 955 960
tca caa atg atg cta atc acg caa ctg atg ccg ctt ggc tgc ctg ttg 2928
Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys Leu Leu
965 970 975
gac tat gtg cga aat aac cgg gac aag atc ggc tct aag gct ctg ctc 2976
Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys Ala Leu Leu
980 985 990
aac tgg agc acg caa atc gcc aag ggc atg tcg tat ctg gag gag aag 3024
Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr Leu Glu Glu Lys
995 1000 1005
cga ctg gtc cac aga gac ttg gct gcc cgc aat gtc ctg gtg cag 3069
Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Gln
1010 1015 1020
act ccc tcg ctg gtg aag atc acc gac ttt ggg ctg gcc aag ttg 3114
Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu
1025 1030 1035
ctg agc agc gat tcc aat gag tac aag gct gct ggc ggc aag atg 3159
Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala Gly Gly Lys Met
1040 1045 1050
ccc atc aag tgg ttg gca ctg gag tgc atc cgc aat cgt gta ttc 3204
Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg Asn Arg Val Phe
1055 1060 1065
acc agc aag tcc gat gtc tgg gcc ttt ggt gtg acc att tgg gaa 3249
Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val Thr Ile Trp Glu
1070 1075 1080
ctg ctg acc ttt ggc cag cgt cca cac gag aac atc cca gct aag 3294
Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn Ile Pro Ala Lys
1085 1090 1095
gat att ccc gat ctt att gaa gtc ggt ctg aag ctg gag cag ccg 3339
Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys Leu Glu Gln Pro
1100 1105 1110
gag att tgt tcg ctg gac att tac tgt aca ctg ctc tcg tgc tgg 3384
Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu Leu Ser Cys Trp
1115 1120 1125
cac ttg gat gcc gcc atg cgt cca acc ttc aag cag ctg act acg 3429
His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys Gln Leu Thr Thr
1130 1135 1140
gtc ttt gct gag ttc gcc aga gat ccg ggt cgc tat ctg gcc att 3474
Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg Tyr Leu Ala Ile
1145 1150 1155
ccc ggg gat aag ttc acc cgg ctg ccg gct tac acg agt cag gat 3519
Pro Gly Asp Lys Phe Thr Arg Leu Pro Ala Tyr Thr Ser Gln Asp
1160 1165 1170
gag aag gat ctc atc cga aaa ttg gct ccc acc acc gat ggg tcc 3564
Glu Lys Asp Leu Ile Arg Lys Leu Ala Pro Thr Thr Asp Gly Ser
1175 1180 1185
gaa gcc att gcg aaa ccc gat gac tac ctg caa ccc aag gca gca 3609
Glu Ala Ile Ala Lys Pro Asp Asp Tyr Leu Gln Pro Lys Ala Ala
1190 1195 1200
cct ggt cct agt cac aga acc gactgcacgg atgagatgcc caagctgaac 3660
Pro Gly Pro Ser His Arg Thr
1205 1210
cgctactgca aggatcctag taacaagaat tcgagtaccg gagacgatga gagggattcg 3720
agtgcccggg aagtgggcgt gggtaatctg cgcctcgatc taccagtcga tgaggatgat 3780
tatctgatgc ccacttgcca accgggtccc aacaacaaca acaacatgaa taatcccaat 3840
caaaacaata tggcagctgt gggcgtggct gccggctaca tggatctcat cggagtgccc 3900
gttagtgtgg acaatccgga gtatctgcta aacgcgcaga cactgggagt tggggagtcg 3960
ccgataccca cccagaccat cgggataccg gtgatgggag gcccgggcac catggaggtc 4020
aaggtgccaa tgccaggcag tgagccaacc agctccgatc acgagtacta caatgatacc 4080
caacgggagt tgcagccact gcatcgaaac cgcaacacgg agacgagggt gtag 4134
<210>22
<211>1210
<212>PRT
<213>果蝇(Drosophila melanogaster)
<400>22
Met Met Ile Ile Ser Met Trp Met Ser Ile Ser Arg Gly Leu Trp Asp
1 5 10 15
Ser Ser Ser Ile Leu Ser Val Leu Leu Ile Leu Ala Cys Met Ala Ser
20 25 30
Ile Thr Thr Ser Ser Ser Val Ser Asn Ala Gly Tyr Val Asp Asn Gly
35 40 45
Asn Met Lys Val Cys Ile Gly Thr Lys Ser Arg Leu Ser Val Pro Ser
50 55 60
Asn Lys Glu His His Tyr Arg Asn Leu Arg Asp Arg Tyr Thr Asn Cys
65 70 75 80
Thr Tyr Val Asp Gly Asn Leu Lys Leu Thr Trp Leu Pro Asn Glu Asn
85 90 95
Leu Asp Leu Ser Phe Leu Asp Asn Ile Arg Glu Val Thr Gly Tyr Ile
100 105 110
Leu Ile Ser His Val Asp Val Lys Lys Val Val Phe Pro Lys Leu Gln
115 120 125
Ile Ile Arg Gly Arg Thr Leu Phe Ser Leu Ser Val Glu Glu Glu Lys
130 135 140
Tyr Ala Leu Phe Val Thr Tyr Ser Lys Met Tyr Thr Leu Glu Ile Pro
145 150 155 160
Asp Leu Arg Asp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn Tyr
165 170 175
Asn Leu Cys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser Asn
180 185 190
Gly Thr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg Glu
195 200 205
Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu Gly
210 215 220
Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser Pro Gln Cys
225 230 235 240
Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu Cys Cys His Leu
245 250 255
Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln Lys Asp Cys Ile Ala
260 265 270
Cys Lys Asn Phe Phe Asp Glu Ala Val Ser Lys Glu Glu Cys Pro Pro
275 280 285
Met Arg Lys Tyr Asn Pro Thr Thr Tyr Val Leu Glu Thr Asn Pro Glu
290 295 300
Gly Lys Tyr Ala Tyr Gly Ala Thr Cys Val Lys Glu Cys Pro Gly His
305 310 315 320
Leu Leu Arg Asp Asn Gly Ala Cys Val Arg Ser Cys Pro Gln Asp Lys
325 330 335
Met Asp Lys Gly Gly Glu Cys Val Pro Cys Asn Gly Pro Cys Pro Lys
340 345 350
Thr Cys Pro Gly Val Thr Val Leu His Ala Gly Asn Ile Asp Ser Phe
355 360 365
Arg Asn Cys Thr Val Ile Asp Gly Asn Ile Arg Ile Leu Asp Gln Thr
370 375 380
Phe Ser Gly Phe Gln Asp Val Tyr Ala Asn Tyr Thr Met Gly Pro Arg
385 390 395 400
Tyr Ile Pro Leu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val Lys
405 410 415
Glu Ile Thr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe Arg
420 425 430
Asn Leu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln Leu
435 440 445
Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu Tyr
450 455 460
Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val Val
465 470 475 480
Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp Pro
485 490 495
Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp Val Asn Glu Asn Leu
500 505 510
Arg Ala Asp Leu Cys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln Cys
515 520 525
Asn Glu Asp Gly Cys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr Cys
530 535 540
Lys Asn Phe Asn Phe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr Ile
545 550 555 560
Ser Asn Ala Tyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His Pro
565 570 575
Glu Cys Arg Thr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu Cys
580 585 590
Val His Val Arg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys Asn
595 600 605
Lys Tyr Asn Asp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys Asp
610 615 620
Gly Cys Thr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr Thr
625 630 635 640
Cys Asn Leu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys Leu
645 650 655
Leu Lys Asp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val His
660 665 670
Pro Gln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val Cys
675 680 685
Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr His
690 695 700
Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln Cys
705 710 715 720
Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg Glu
725 730 735
Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly Ala
740 745 750
Asp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn Glu
755 760 765
Thr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys Cys
770 775 780
Pro Leu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly Pro
785 790 795 800
Tyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn Leu
805 810 815
Asp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro Thr
820 825 830
Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln Lys
835 840 845
Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys Glu
850 855 860
Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys Lys
865 870 875 880
Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu Gly
885 890 895
Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu Gly
900 905 910
Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser Thr
915 920 925
Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met Ala
930 935 940
Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met Ser
945 950 955 960
Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys Leu Leu
965 970 975
Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys Ala Leu Leu
980 985 990
Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr Leu Glu Glu Lys
995 1000 1005
Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Gln
1010 1015 1020
Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu
1025 1030 1035
Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala Gly Gly Lys Met
1040 1045 1050
Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg Asn Arg Val Phe
1055 1060 1065
Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val Thr Ile Trp Glu
1070 1075 1080
Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn Ile Pro Ala Lys
1085 1090 1095
Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys Leu Glu Gln Pro
1100 1105 1110
Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu Leu Ser Cys Trp
1115 1120 1125
His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys Gln Leu Thr Thr
1130 1135 1140
Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg Tyr Leu Ala Ile
1145 1150 1155
Pro Gly Asp Lys Phe Thr Arg Leu Pro Ala Tyr Thr Ser Gln Asp
1160 1165 1170
Glu Lys Asp Leu Ile Arg Lys Leu Ala Pro Thr Thr Asp Gly Ser
1175 1180 1185
Glu Ala Ile Ala Lys Pro Asp Asp Tyr Leu Gln Pro Lys Ala Ala
1190 1195 1200
Pro Gly Pro Ser His Arg Thr
1205 1210
<210>23
<211>4281
<212>DNA
<213>果蝇(Drosophila melanogaster)
<400>23
atgctgctgc gacggcgcaa cggcccctgc cccttccccc tgctgcttct gctcctggcc 60
cactgcattt gcatttggcc cgcgtcggcg gcccgcgatc gctacgcccg ccagaacaat 120
cgccagcgcc atcaggatat agatcgcgat cgggatcgag atcgattcct ataccgcagc 180
agttcggccc aaaatcgaca gaggggcggg gccaacttcg ccctgggact gggagccaac 240
ggagtcacca ttcccaccag tctggaggat aagaacaaga acgagttcgt caaggggaaa 300
atctgcatcg gcactaaatc tcggctctcc gtgccctcca acaaggaaca tcattaccga 360
aacctcagag atcggtacac gaactgtacg tatgtggatg gcaacttgaa actgacctgg 420
ctacccaacg agaatttgga cctcagcttc ctagacaaca tacgggaggt caccggctat 480
attctgatca gtcatgtgga cgttaagaaa gtggtgtttc ccaaactaca aatcattcgc 540
ggacgcacgc tgttcagctt atccgtggag gaggagaagt atgccttgtt cgtcacttat 600
tccaaaatgt acacgctgga gattcccgat ctacgcgatg tcttaaatgg ccaagtgggc 660
ttccacaaca actacaatct ctgccacatg cgaacgatcc agtggtcgga gattgtatcc 720
aacggcacgg atgcatacta caactacgac tttactgctc cggagcgcga gtgtcccaag 780
tgccacgaga gctgcacgca cggatgttgg ggcgagggtc ccaagaattg ccagaagttc 840
agcaagctca cctgctcgcc acagtgtgcc ggaggtcgtt gctatggacc aaagccgcgg 900
gagtgttgtc acctcttctg cgccggagga tgcactggtc ccacgcaaaa ggattgcatc 960
gcctgcaaga acttcttcga cgaggcagta tcaaaggagg aatgcccgcc catgcgcaag 1020
tacaatccca ccacctatgt tcttgaaacg aatcctgagg gaaagtatgc ctatggtgcc 1080
acctgcgtca aggagtgtcc cggtcatctg ttgcgggata atggcgcctg cgtgcgcagc 1140
tgtccccagg acaagatgga caaggggggc gagtgtgtgc cctgcaatgg accgtgcccc 1200
aaaacctgcc cgggcgttac tgtcctgcat gccggcaaca ttgactcgtt ccggaattgt 1260
acggtgatcg atggcaacat tcgcattttg gatcagacct tctcgggctt ccaggatgtc 1320
tatgccaact acacgatggg accacgatac ataccgctgg atcccgagcg acgggaggtg 1380
ttctccacgg tgaaggagat caccgggtat ctgaatatcg agggaaccca cccgcagttc 1440
cggaatctgt cgtactttcg caatctggaa acaattcatg gccgccagct gatggagagc 1500
atgtttgccg ctttggcgat cgttaagtca tccctgtaca gcctggagat gcgcaatctg 1560
aagcagatta gttccggcag tgtggtcatc cagcataata gagacctctg ctacgtaagc 1620
aatatccgtt ggccggccat tcagaaggag cccgaacaga aggtgtgggt caacgagaat 1680
ctcagggcgg atctatgcga gaaaaatgga accatttgct cggatcagtg caacgaggac 1740
ggctgctggg gagctggcac ggatcagtgc cttacctgca agaacttcaa tttcaatggc 1800
acctgcatcg ccgactgtgg ttatatatcc aatgcctaca agtttgacaa tagaacgtgc 1860
aagatatgcc atccagagtg ccggacttgc aatggagctg gagcagatca ctgccaggag 1920
tgcgtccatg tgagggacgg tcagcactgt gtgtccgagt gcccgaagaa caagtacaac 1980
gatcgtggtg tctgccgaga gtgccacgcc acctgcgatg gatgcactgg gcccaaggac 2040
accatcggca ttggagcgtg tacgacgtgc aatttggcca ttatcaacaa tgacgccaca 2100
gtaaaacgct gcctgctgaa ggacgacaag tgccccgatg ggtatttctg ggagtatgtg 2160
catccgcaag agcagggatc gctgaagcca ttggccggca gagcagtttg ccgaaagtgc 2220
catccccttt gcgagctgtg cacgaactac ggataccatg aacaggtgtg ctccaagtgc 2280
acccactaca agcgacggga gcagtgcgag accgagtgtc cggccgatca ctacacggat 2340
gaggagcagc gcgagtgctt ccagcgccac ccggaatgca atggttgcac gggtccgggt 2400
gccgacgatt gcaagtcttg ccgcaacttt aagttgttcg acgcgaatga gacgggtccc 2460
tatgtgaact ccacgatgtt caattgcacc tcgaagtgtc ccttggagat gcgacatgtg 2520
aactatcagt acacggccat tggaccctac tgcgcagcta gtccgccgag gagcagcaag 2580
ataactgcca atctggatgt gaacatgatc ttcattatca ctggtgctgt tctggtgccg 2640
acgatctgca tcctctgcgt ggtcacatac atttgtcggc aaaagcaaaa ggccaagaag 2700
gaaacagtca agatgaccat ggctctgtct ggctgcgagg attccgagcc gctgcgtccc 2760
tcgaacattg gagccaacct atgcaagttg cgcattgtca aggacgccga gttgcgcaag 2820
ggcggagtcc ttggaatggg agcctttgga cgagtgtaca agggcgtttg ggtgccggag 2880
ggtgagaacg tcaagattcc agtggccatt aaggagctgc tcaagtccac aggcgccgag 2940
tcaagcgaag agttcctccg cgaagcctac atcatggcct ctgaggagca cgttaatctg 3000
ctgaagctcc tggccgtgtg catgtcctca caaatgatgc taatcacgca actgatgccg 3060
cttggctgcc tgttggacta tgtgcgaaat aaccgggaca agatcggctc taaggctctg 3120
ctcaactgga gcacgcaaat cgccaagggc atgtcgtatc tggaggagaa gcgactggtc 3180
cacagagact tggctgcccg caatgtcctg gtgcagactc cctcgctggt gaagatcacc 3240
gactttgggc tggccaagtt gctgagcagc gattccaatg agtacaaggc tgctggcggc 3300
aagatgccca tcaagtggtt ggcactggag tgcatccgca atcgtgtatt caccagcaag 3360
tccgatgtct gggcctttgg tgtgaccatt tgggaactgc tgacctttgg ccagcgtcca 3420
cacgagaaca tcccagctaa ggatattccc gatcttattg aagtcggtct gaagctggag 3480
cagccggaga tttgttcgct ggacatttac tgtacactgc tctcgtgctg gcacttggat 3540
gccgccatgc gtccaacctt caagcagctg actacggtct ttgctgagtt cgccagagat 3600
ccgggtcgct atctggccat tcccggggat aagttcaccc ggctgccggc ttacacgagt 3660
caggatgaga aggatctcat ccgaaaattg gctcccacca ccgatgggtc cgaagccatt 3720
gcgaaacccg atgactacct gcaacccaag gcagcacctg gtcctagtca cagaaccgac 3780
tgcacggatg agatgcccaa gctgaaccgc tactgcaagg atcctagtaa caagaattcg 3840
agtaccggag acgatgagag ggattcgagt gcccgggaag tgggcgtggg taatctgcgc 3900
ctcgatctac cagtcgatga ggatgattat ctgatgccca cttgccaacc gggtcccaac 3960
aacaacaaca acatgaataa tcccaatcaa aacaatatgg cagctgtggg cgtggctgcc 4020
ggctacatgg atctcatcgg agtgcccgtt agtgtggaca atccggagta tctgctaaac 4080
gcgcagacac tgggagttgg ggagtcgccg atacccaccc agaccatcgg gataccggtg 4140
atgggaggcc cgggcaccat ggaggtcaag gtgccaatgc caggcagtga gccaaccagc 4200
tccgatcacg agtactacaa tgatacccaa cgggagttgc agccactgca tcgaaaccgc 4260
aacacggaga cgagggtgta g 4281
<210>24
<211>4281
<212>DNA
<213>大鼠(Rattus norvegicus)
<220>
<221>CDS
<222>(1)..(3627)
<223>
<400>24
atg ctg ctg cga cgg cgc aac ggc ccc tgc ccc ttc ccc ctg ctg ctt 48
Met Leu Leu Arg Arg Arg Asn Gly Pro Cys Pro Phe Pro Leu Leu Leu
1 5 10 15
ctg ctc ctg gcc cac tgc att tgc att tgg ccc gcg tcg gcg gcc cgc 96
Leu Leu Leu Ala His Cys Ile Cys Ile Trp Pro Ala Ser Ala Ala Arg
20 25 30
gat cgc tac gcc cgc cag aac aat cgc cag cgc cat cag gat ata gat 144
Asp Arg Tyr Ala Arg Gln Asn Asn Arg Gln Arg His Gln Asp Ile Asp
35 40 45
cgc gat cgg gat cga gat cga ttc cta tac cgc agc agt tcg gcc caa 192
Arg Asp Arg Asp Arg Asp Arg Phe Leu Tyr Arg Ser Ser Ser Ala Gln
50 55 60
aat cga cag agg ggc ggg gcc aac ttc gcc ctg gga ctg gga gcc aac 240
Asn Arg Gln Arg Gly Gly Ala Asn Phe Ala Leu Gly Leu Gly Ala Asn
65 70 75 80
gga gtc acc att ccc acc agt ctg gag gat aag aac aag aac gag ttc 288
Gly Val Thr Ile Pro Thr Ser Leu Glu Asp Lys Asn Lys Asn Glu Phe
85 90 95
gtc aag ggg aaa atc tgc atc ggc act aaa tct cgg ctc tcc gtg ccc 336
Val Lys Gly Lys Ile Cys Ile Gly Thr Lys Ser Arg Leu Ser Val Pro
100 105 110
tcc aac aag gaa cat cat tac cga aac ctc aga gat cgg tac acg aac 384
Ser Asn Lys Glu His His Tyr Arg Asn Leu Arg Asp Arg Tyr Thr Asn
115 120 125
tgt acg tat gtg gat ggc aac ttg aaa ctg acc tgg cta ccc aac gag 432
Cys Thr Tyr Val Asp Gly Asn Leu Lys Leu Thr Trp Leu Pro Asn Glu
130 135 140
aat ttg gac ctc agc ttc cta gac aac ata cgg gag gtc acc ggc tat 480
Asn Leu Asp Leu Ser Phe Leu Asp Asn Ile Arg Glu Val Thr Gly Tyr
145 150 155 160
att ctg atc agt cat gtg gac gtt aag aaa gtg gtg ttt ccc aaa cta 528
Ile Leu Ile Ser His Val Asp Val Lys Lys Val Val Phe Pro Lys Leu
165 170 175
caa atc att cgc gga cgc acg ctg ttc agc tta tcc gtg gag gag gag 576
Gln Ile Ile Arg Gly Arg Thr Leu Phe Ser Leu Ser Val Glu Glu Glu
180 185 190
aag tat gcc ttg ttc gtc act tat tcc aaa atg tac acg ctg gag att 624
Lys Tyr Ala Leu Phe Val Thr Tyr Ser Lys Met Tyr Thr Leu Glu Ile
195 200 205
ccc gat cta cgc gat gtc tta aat ggc caa gtg ggc ttc cac aac aac 672
Pro Asp Leu Arg Asp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn
210 215 220
tac aat ctc tgc cac atg cga acg atc cag tgg tcg gag att gta tcc 720
Tyr Asn Leu Cys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser
225 230 235 240
aac ggc acg gat gca tac tac aac tac gac ttt act gct ccg gag cgc 768
Asn Gly Thr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg
245 250 255
gag tgt ccc aag tgc cac gag agc tgc acg cac gga tgt tgg ggc gag 816
Glu Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu
260 265 270
ggt ccc aag aat tgc cag aag ttc agc aag ctc acc tgc tcg cca cag 864
Gly Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser Pro Gln
275 280 285
tgt gcc gga ggt cgt tgc tat gga cca aag ccg cgg gag tgt tgt cac 912
Cys Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu Cys Cys His
290 295 300
ctc ttc tgc gcc gga gga tgc act ggt ccc acg caa aag gat tgc atc 960
Leu Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln Lys Asp Cys Ile
305 310 315 320
gcc tgc aag aac ttc ttc gac gag gca gta tca aag gag gaa tgc ccg 1008
Ala Cys Lys Asn Phe Phe Asp Glu Ala Val Ser Lys Glu Glu Cys Pro
325 330 335
ccc atg cgc aag tac aat ccc acc acc tat gtt ctt gaa acg aat cct 1056
Pro Met Arg Lys Tyr Asn Pro Thr Thr Tyr Val Leu Glu Thr Asn Pro
340 345 350
gag gga aag tat gcc tat ggt gcc acc tgc gtc aag gag tgt ccc ggt 1104
Glu Gly Lys Tyr Ala Tyr Gly Ala Thr Cys Val Lys Glu Cys Pro Gly
355 360 365
cat ctg ttg cgg gat aat ggc gcc tgc gtg cgc agc tgt ccc cag gac 1152
His Leu Leu Arg Asp Asn Gly Ala Cys Val Arg Ser Cys Pro Gln Asp
370 375 380
aag atg gac aag ggg ggc gag tgt gtg ccc tgc aat gga ccg tgc ccc 1200
Lys Met Asp Lys Gly Gly Glu Cys Val Pro Cys Asn Gly Pro Cys Pro
385 390 395 400
aaa acc tgc ccg ggc gtt act gtc ctg cat gcc ggc aac att gac tcg 1248
Lys Thr Cys Pro Gly Val Thr Val Leu His Ala Gly Asn Ile Asp Ser
405 410 415
ttc cgg aat tgt acg gtg atc gat ggc aac att cgc att ttg gat cag 1296
Phe Arg Asn Cys Thr Val Ile Asp Gly Asn Ile Arg Ile Leu Asp Gln
420 425 430
acc ttc tcg ggc ttc cag gat gtc tat gcc aac tac acg atg gga cca 1344
Thr Phe Ser Gly Phe Gln Asp Val Tyr Ala Asn Tyr Thr Met Gly Pro
435 440 445
cga tac ata ccg ctg gat ccc gag cga cgg gag gtg ttc tcc acg gtg 1392
Arg Tyr Ile Pro Leu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val
450 455 460
aag gag atc acc ggg tat ctg aat atc gag gga acc cac ccg cag ttc 1440
Lys Glu Ile Thr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe
465 470 475 480
cgg aat ctg tcg tac ttt cgc aat ctg gaa aca att cat ggc cgc cag 1488
Arg Asn Leu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln
485 490 495
ctg atg gag agc atg ttt gcc gct ttg gcg atc gtt aag tca tcc ctg 1536
Leu Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu
500 505 510
tac agc ctg gag atg cgc aat ctg aag cag att agt tcc ggc agt gtg 1584
Tyr Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val
515 520 525
gtc atc cag cat aat aga gac ctc tgc tac gta agc aat atc cgt tgg 1632
Val Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp
530 535 540
ccg gcc att cag aag gag ccc gaa cag aag gtg tgg gtc aac gag aat 1680
Pro Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp Val Asn Glu Asn
545 550 555 560
ctc agg gcg gat cta tgc gag aaa aat gga acc att tgc tcg gat cag 1728
Leu Arg Ala Asp Leu Cys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln
565 570 575
tgc aac gag gac ggc tgc tgg gga gct ggc acg gat cag tgc ctt acc 1776
Cys Asn Glu Asp Gly Cys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr
580 585 590
tgc aag aac ttc aat ttc aat ggc acc tgc atc gcc gac tgt ggt tat 1824
Cys Lys Asn Phe Asn Phe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr
595 600 605
ata tcc aat gcc tac aag ttt gac aat aga acg tgc aag ata tgc cat 1872
Ile Ser Asn Ala Tyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His
610 615 620
cca gag tgc cgg act tgc aat gga gct gga gca gat cac tgc cag gag 1920
Pro Glu Cys Arg Thr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu
625 630 635 640
tgc gtc cat gtg agg gac ggt cag cac tgt gtg tcc gag tgc ccg aag 1968
Cys Val His Val Arg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys
645 650 655
aac aag tac aac gat cgt ggt gtc tgc cga gag tgc cac gcc acc tgc 2016
Asn Lys Tyr Asn Asp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys
660 665 670
gat gga tgc act ggg ccc aag gac acc atc ggc att gga gcg tgt acg 2064
Asp Gly Cys Thr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr
675 680 685
acg tgc aat ttg gcc att atc aac aat gac gcc aca gta aaa cgc tgc 2112
Thr Cys Asn Leu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys
690 695 700
ctg ctg aag gac gac aag tgc ccc gat ggg tat ttc tgg gag tat gtg 2160
Leu Leu Lys Asp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val
705 710 715 720
cat ccg caa gag cag gga tcg ctg aag cca ttg gcc ggc aga gca gtt 2208
His Pro Gln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val
725 730 735
tgc cga aag tgc cat ccc ctt tgc gag ctg tgc acg aac tac gga tac 2256
Cys Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr
740 745 750
cat gaa cag gtg tgc tcc aag tgc acc cac tac aag cga cgg gag cag 2304
His Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln
755 760 765
tgc gag acc gag tgt ccg gcc gat cac tac acg gat gag gag cag cgc 2352
Cys Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg
770 775 780
gag tgc ttc cag cgc cac ccg gaa tgc aat ggt tgc acg ggt ccg ggt 2400
Glu Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly
785 790 795 800
gcc gac gat tgc aag tct tgc cgc aac ttt aag ttg ttc gac gcg aat 2448
Ala Asp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn
805 810 815
gag acg ggt ccc tat gtg aac tcc acg atg ttc aat tgc acc tcg aag 2496
Glu Thr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys
820 825 830
tgt ccc ttg gag atg cga cat gtg aac tat cag tac acg gcc att gga 2544
Cys Pro Leu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly
835 840 845
ccc tac tgc gca gct agt ccg ccg agg agc agc aag ata act gcc aat 2592
Pro Tyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn
850 855 860
ctg gat gtg aac atg atc ttc att atc act ggt gct gtt ctg gtg ccg 2640
Leu Asp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro
865 870 875 880
acg atc tgc atc ctc tgc gtg gtc aca tac att tgt cgg caa aag caa 2688
Thr Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln
885 890 895
aag gcc aag aag gaa aca gtc aag atg acc atg gct ctg tct ggc tgc 2736
Lys Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys
900 905 910
gag gat tcc gag ccg ctg cgt ccc tcg aac att gga gcc aac cta tgc 2784
Glu Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys
915 920 925
aag ttg cgc att gtc aag gac gcc gag ttg cgc aag ggc gga gtc ctt 2832
Lys Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu
930 935 940
gga atg gga gcc ttt gga cga gtg tac aag ggc gtt tgg gtg ccg gag 2880
Gly Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu
945 950 955 960
ggt gag aac gtc aag att cca gtg gcc att aag gag ctg ctc aag tcc 2928
Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser
965 970 975
aca ggc gcc gag tca agc gaa gag ttc ctc cgc gaa gcc tac atc atg 2976
Thr Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met
980 985 990
gcc tct gag gag cac gtt aat ctg ctg aag ctc ctg gcc gtg tgc atg 3024
Ala Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met
995 1000 1005
tcc tca caa atg atg cta atc acg caa ctg atg ccg ctt ggc tgc 3069
Ser Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys
1010 1015 1020
ctg ttg gac tat gtg cga aat aac cgg gac aag atc ggc tct aag 3114
Leu Leu Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys
1025 1030 1035
gct ctg ctc aac tgg agc acg caa atc gcc aag ggc atg tcg tat 3159
Ala Leu Leu Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr
1040 1045 1050
ctg gag gag aag cga ctg gtc cac aga gac ttg gct gcc cgc aat 3204
Leu Glu Glu Lys Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn
1055 1060 1065
gtc ctg gtg cag act ccc tcg ctg gtg aag atc acc gac ttt ggg 3249
Val Leu Val Gln Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly
1070 1075 1080
ctg gcc aag ttg ctg agc agc gat tcc aat gag tac aag gct gct 3294
Leu Ala Lys Leu Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala
1085 1090 1095
ggc ggc aag atg ccc atc aag tgg ttg gca ctg gag tgc atc cgc 3339
Gly Gly Lys Met Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg
1100 1105 1110
aat cgt gta ttc acc agc aag tcc gat gtc tgg gcc ttt ggt gtg 3384
Asn Arg Val Phe Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val
1115 1120 1125
acc att tgg gaa ctg ctg acc ttt ggc cag cgt cca cac gag aac 3429
Thr Ile Trp Glu Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn
1130 1135 1140
atc cca gct aag gat att ccc gat ctt att gaa gtc ggt ctg aag 3474
Ile Pro Ala Lys Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys
1145 1150 1155
ctg gag cag ccg gag att tgt tcg ctg gac att tac tgt aca ctg 3519
Leu Glu Gln Pro Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu
1160 1165 1170
ctc tcg tgc tgg cac ttg gat gcc gcc atg cgt cca acc ttc aag 3564
Leu Ser Cys Trp His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys
1175 1180 1185
cag ctg act acg gtc ttt gct gag ttc gcc aga gat ccg ggt cgc 3609
Gln Leu Thr Thr Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg
1190 1195 1200
tat ctg gcc att ccc ggg gataagttca cccggctgcc ggcttacacg 3657
Tyr Leu Ala Ile Pro Gly
1205
agtcaggatg agaaggatct catccgaaaa ttggctccca ccaccgatgg gtccgaagcc 3717
attgcgaaac ccgatgacta cctgcaaccc aaggcagcac ctggtcctag tcacagaacc 3777
gactgcacgg atgagatgcc caagctgaac cgctactgca aggatcctag taacaagaat 3837
tcgagtaccg gagacgatga gagggattcg agtgcccggg aagtgggcgt gggtaatctg 3897
cgcctcgatc taccagtcga tgaggatgat tatctgatgc ccacttgcca accgggtccc 3957
aacaacaaca acaacatgaa taatcccaat caaaacaata tggcagctgt gggcgtggct 4017
gccggctaca tggatctcat cggagtgccc gttagtgtgg acaatccgga gtatctgcta 4077
aacgcgcaga cactgggagt tggggagtcg ccgataccca cccagaccat cgggataccg 4137
gtgatgggag gcccgggcac catggaggtc aaggtgccaa tgccaggcag tgagccaacc 4197
agctccgatc acgagtacta caatgatacc caacgggagt tgcagccact gcatcgaaac 4257
cgcaacacgg agacgagggt gtag 4281
<210>25
<211>1209
<212>PRT
<213>大鼠(Rattus norvegicus)
<400>25
Met Leu Leu Arg Arg Arg Asn Gly Pro Cys Pro Phe Pro Leu Leu Leu
1 5 10 15
Leu Leu Leu Ala His Cys Ile Cys Ile Trp Pro Ala Ser Ala Ala Arg
20 25 30
Asp Arg Tyr Ala Arg Gln Asn Asn Arg Gln Arg His Gln Asp Ile Asp
35 40 45
Arg Asp Arg Asp Arg Asp Arg Phe Leu Tyr Arg Ser Ser Ser Ala Gln
50 55 60
Asn Arg Gln Arg Gly Gly Ala Asn Phe Ala Leu Gly Leu Gly Ala Asn
65 70 75 80
Gly Val Thr Ile Pro Thr Ser Leu Glu Asp Lys Asn Lys Asn Glu Phe
85 90 95
Val Lys Gly Lys Ile Cys Ile Gly Thr Lys Ser Arg Leu Ser Val Pro
100 105 110
Ser Asn Lys Glu His His Tyr Arg Asn Leu Arg Asp Arg Tyr Thr Asn
115 120 125
Cys Thr Tyr Val Asp Gly Asn Leu Lys Leu Thr Trp Leu Pro Asn Glu
130 135 140
Asn Leu Asp Leu Ser Phe Leu Asp Asn Ile Arg Glu Val Thr Gly Tyr
145 150 155 160
Ile Leu Ile Ser His Val Asp Val Lys Lys Val Val Phe Pro Lys Leu
165 170 175
Gln Ile Ile Arg Gly Arg Thr Leu Phe Ser Leu Ser Val Glu Glu Glu
180 185 190
Lys Tyr Ala Leu Phe Val Thr Tyr Ser Lys Met Tyr Thr Leu Glu Ile
195 200 205
Pro Asp Leu Arg Asp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn
210 215 220
Tyr Asn Leu Cys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser
225 230 235 240
Asn Gly Thr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg
245 250 255
Glu Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu
260 265 270
Gly Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser Pro Gln
275 280 285
Cys Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu Cys Cys His
290 295 300
Leu Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln Lys Asp Cys Ile
305 310 315 320
Ala Cys Lys Asn Phe Phe Asp Glu Ala Val Ser Lys Glu Glu Cys Pro
325 330 335
Pro Met Arg Lys Tyr Asn Pro Thr Thr Tyr Val Leu Glu Thr Asn Pro
340 345 350
Glu Gly Lys Tyr Ala Tyr Gly Ala Thr Cys Val Lys Glu Cys Pro Gly
355 360 365
His Leu Leu Arg Asp Asn Gly Ala Cys Val Arg Ser Cys Pro Gln Asp
370 375 380
Lys Met Asp Lys Gly Gly Glu Cys Val Pro Cys Asn Gly Pro Cys Pro
385 390 395 400
Lys Thr Cys Pro Gly Val Thr Val Leu His Ala Gly Asn Ile Asp Ser
405 410 415
Phe Arg Asn Cys Thr Val Ile Asp Gly Asn Ile Arg Ile Leu Asp Gln
420 425 430
Thr Phe Ser Gly Phe Gln Asp Val Tyr Ala Asn Tyr Thr Met Gly Pro
435 440 445
Arg Tyr Ile Pro Leu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val
450 455 460
Lys Glu Ile Thr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe
465 470 475 480
Arg Asn Leu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln
485 490 495
Leu Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu
500 505 510
Tyr Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val
515 520 525
Val Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp
530 535 540
Pro Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp Val Asn Glu Asn
545 550 555 560
Leu Arg Ala Asp Leu Cys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln
565 570 575
Cys Asn Glu Asp Gly Cys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr
580 585 590
Cys Lys Asn Phe Asn Phe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr
595 600 605
Ile Ser Asn Ala Tyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His
610 615 620
Pro Glu Cys Arg Thr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu
625 630 635 640
Cys Val His Val Arg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys
645 650 655
Asn Lys Tyr Asn Asp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys
660 665 670
Asp Gly Cys Thr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr
675 680 685
Thr Cys Asn Leu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys
690 695 700
Leu Leu Lys Asp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val
705 710 715 720
His Pro Gln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val
725 730 735
Cys Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr
740 745 750
His Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln
755 760 765
Cys Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg
770 775 780
Glu Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly
785 790 795 800
Ala Asp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn
805 810 815
Glu Thr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys
820 825 830
Cys Pro Leu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly
835 840 845
Pro Tyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn
850 855 860
Leu Asp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro
865 870 875 880
Thr Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln
885 890 895
Lys Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys
900 905 910
Glu Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys
915 920 925
Lys Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu
930 935 940
Gly Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu
945 950 955 960
Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser
965 970 975
Thr Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met
980 985 990
Ala Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met
995 1000 1005
Ser Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys
1010 1015 1020
Leu Leu Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys
1025 1030 1035
Ala Leu Leu Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr
1040 1045 1050
Leu Glu Glu Lys Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn
1055 1060 1065
Val Leu Val Gln Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly
1070 1075 1080
Leu Ala Lys Leu Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala
1085 1090 1095
Gly Gly Lys Met Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg
1100 1105 1110
Asn Arg Val Phe Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val
1115 1120 1125
Thr Ile Trp Glu Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn
1130 1135 1140
Ile Pro Ala Lys Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys
1145 1150 1155
Leu Glu Gln Pro Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu
1160 1165 1170
Leu Ser Cys Trp His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys
1175 1180 1185
Gln Leu ThrThr Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg
1190 1195 1200
Tyr Leu Ala Ile Pro Gly
1205
<210>26
<211>3576
<212>DNA
<213>斑马鱼(Danio rerio)
<220>
<221>CDS
<222>(1)..(3573)
<223>
<400>26
atg gca gga cca act gaa atc gga ttg ttt ttt acc tta ttg ctc tcc 48
Met Ala Gly Pro Thr Glu Ile Gly Leu Phe Phe Thr Leu Leu Leu Ser
1 5 10 15
ggg agc ttc tgc gcg acg ccg gaa aag aaa gtg tgt cag gga gca aac 96
Gly Ser Phe Cys Ala Thr Pro Glu Lys Lys Val Cys Gln Gly Ala Asn
20 25 30
aac aaa ctg act ctt ctg gga acg gtg gaa gac cat tat cag gtt ctg 144
Asn Lys Leu Thr Leu Leu Gly Thr Val Glu Asp His Tyr Gln Val Leu
35 40 45
ctc aga atg tac aga aac tgc act gtg gtt ctg gag aac ctg gaa att 192
Leu Arg Met Tyr Arg Asn Cys Thr Val Val Leu Glu Asn Leu Glu Ile
50 55 60
aca cat ata aca gag aaa tat gac ctg tcc ttc ctc aag agc atc cag 240
Thr His Ile Thr Glu Lys Tyr Asp Leu Ser Phe Leu Lys Ser Ile Gln
65 70 75 80
gaa gtt ggt ggc tat gtt ctt atc gcg gtc aat acg gtt tcc aaa atc 288
Glu Val Gly Gly Tyr Val Leu Ile Ala Val Asn Thr Val Ser Lys Ile
85 90 95
cct ctg gag aac ctg cgc atc att cgc gga cac tca ctt tat gaa gac 336
Pro Leu Glu Asn Leu Arg Ile Ile Arg Gly His Ser Leu Tyr Glu Asp
100 105 110
aaa ttt gcc ttg gcc gtc ctg gtg aac ttc aac aac agc atc gaa caa 384
Lys Phe Ala Leu Ala Val Leu Val Asn Phe Asn Asn Ser Ile Glu Gln
115 120 125
ggc gta aaa gag ttg ccg ctg act agt tta act gaa ata ctt aag ggt 432
Gly Val Lys Glu Leu Pro Leu Thr Ser Leu Thr Glu Ile Leu Lys Gly
130 135 140
gga gtc aag ttt tgc agg aac gat tat tta tgt aat gtg ggg acc atc 480
Gly Val Lys Phe Cys Arg Asn Asp Tyr Leu Cys Asn Val Gly Thr Ile
145 150 155 160
gag tgg gcc gac atc ctg aac atg aag agc ctg cct aca atc gtg agc 528
Glu Trp Ala Asp Ile Leu Asn Met Lys Ser Leu Pro Thr Ile Val Ser
165 170 175
cat aat ata agc tat gga aaa aac tgt gga aag tgc gat cca agc tgt 576
His Asn Ile Ser Tyr Gly Lys Asn Cys Gly Lys Cys Asp Pro Ser Cys
180 185 190
ttc aat ggc tcc tgc tgg ggc acc gga ccc gac aag tgc cag aga atg 624
Phe Asn Gly Ser Cys Trp Gly Thr Gly Pro Asp Lys Cys Gln Arg Met
195 200 205
acg aaa gtg atc tgt gcg gag cag tgt tca ggg agg tgt aaa gga ccc 672
Thr Lys Val Ile Cys Ala Glu Gln Cys Ser Gly Arg Cys Lys Gly Pro
210 215 220
aga ccc att gac tgc tgt aat gaa cac tgt gct gct gga tgc act gga 720
Arg Pro Ile Asp Cys Cys Asn Glu His Cys Ala Ala Gly Cys Thr Gly
225 230 235 240
ccc aga cct aca gac tgt ctg gcc tgt aag gac ttc cag gat gaa ggg 768
Pro Arg Pro Thr Asp Cys Leu Ala Cys Lys Asp Phe Gln Asp Glu Gly
245 250 255
aca tgt aag gac gca tgt ccg cgg ctc atg ctc tac gac cca aac aca 816
Thr Cys Lys Asp Ala Cys Pro Arg Leu Met Leu Tyr Asp Pro Asn Thr
260 265 270
cac cag ctc gcg cca aac cca tat ggg aag tac agc ttt ggg gca acg 864
His Gln Leu Ala Pro Asn Pro Tyr Gly Lys Tyr Ser Phe Gly Ala Thr
275 280 285
tgc atc aag aca tgc cca cac aac tat gtg gtg acg gat cac ggg gcc 912
Cys Ile Lys Thr Cys Pro His Asn Tyr Val Val Thr Asp His Gly Ala
290 295 300
tgt gtg aga aca tgc agc cct ggc acc tat gaa gtg gat gag ggt gga 960
Cys Val Arg Thr Cys Ser Pro Gly Thr Tyr Glu Val Asp Glu Gly Gly
305 310 315 320
gtt cgc aaa tgt aag agg tgt gaa ggc ctg tgt cca aaa gtg tgc aat 1008
Val Arg Lys Cys Lys Arg Cys Glu Gly Leu Cys Pro Lys Val Cys Asn
325 330 335
ggg ttg gga atg ggg cct tta gcc aat gtc ctg tca atc aat gcc acc 1056
Gly Leu Gly Met Gly Pro Leu Ala Asn Val Leu Ser Ile Asn Ala Thr
340 345 350
aac atc gac tcc ttt gag aac tgc act aaa atc agc ggc aat gtt gcc 1104
Asn Ile Asp Ser Phe Glu Asn Cys Thr Lys Ile Ser Gly Asn Val Ala
355 360 365
atc ctc agc acc aca ttc aga ggt gac cca cat act aac act tca gga 1152
Ile Leu Ser Thr Thr Phe Arg Gly Asp Pro His Thr Asn Thr Ser Gly
370 375 380
ctg gat cca gca aag ctc agt gta ttg agt act gtc aaa gaa atc act 1200
Leu Asp Pro Ala Lys Leu Ser Val Leu Ser Thr Val Lys Glu Ile Thr
385 390 395 400
ggt tac ctg atg att cag ctg tgg ccg gag agc atg cag tcc ctt agt 1248
Gly Tyr Leu Met Ile Gln Leu Trp Pro Glu Ser Met Gln Ser Leu Ser
405 410 415
gcc ttc gaa aac ctt gag gtc atc cga gga cgg aca aaa aca caa gga 1296
Ala Phe Glu Asn Leu Glu Val Ile Arg Gly Arg Thr Lys Thr Gln Gly
420 425 430
acg tac agc ttt gct gtc acc aag acg gcc atc act cat tta ggc atg 1344
Thr Tyr Ser Phe Ala Val Thr Lys Thr Ala Ile Thr His Leu Gly Met
435 440 445
cgt tct ctg agg gag atc agt gac ggg gac gtg tcc atc gtt aag aat 1392
Arg Ser Leu Arg Glu Ile Ser Asp Gly Asp Val Ser Ile Val Lys Asn
450 455 460
aag aat ctc tgc tac agc agc cct gaa cac tgg aaa cgc ctc ttc aag 1440
Lys Asn Leu Cys Tyr Ser Ser Pro Glu His Trp Lys Arg Leu Phe Lys
465 470 475 480
tcc aaa caa cag tcg gtc aaa atg att gaa aat atg gat gct gcc acc 1488
Ser Lys Gln Gln Ser Val Lys Met Ile Glu Asn Met Asp Ala Ala Thr
485 490 495
tgc gcc aat cag aac agc aca tgt aat gag atg tgc acg gct gac ggc 1536
Cys Ala Asn Gln Asn Ser Thr Cys Asn Glu Met Cys Thr Ala Asp Gly
500 505 510
tgc tgg ggt ccc ggc ccc acc atg tgc ttc ggc tgt gag cat tac agc 1584
Cys Trp Gly Pro Gly Pro Thr Met Cys Phe Gly Cys Glu His Tyr Ser
515 520 525
cgc gga aaa cac tgc gtg gct tcc tgc aac ctg ctg aat ggt gag ccg 1632
Arg Gly Lys His Cys Val Ala Ser Cys Asn Leu Leu Asn Gly Glu Pro
530 535 540
cgt gaa tat gag gtc aat aaa aca tgc atg gaa tgc gat cct gaa tgt 1680
Arg Glu Tyr Glu Val Asn Lys Thr Cys Met Glu Cys Asp Pro Glu Cys
545 550 555 560
ctg ctc atg aat gaa acc cag acc tgc aac ggc cct gga ccc gac aaa 1728
Leu Leu Met Asn Glu Thr Gln Thr Cys Asn Gly Pro Gly Pro Asp Lys
565 570 575
tgt aca gtg tgt gca aac tat aaa gac gga ccg cac tgt gtg cat cgc 1776
Cys Thr Val Cys Ala Asn Tyr Lys Asp Gly Pro His Cys Val His Arg
580 585 590
tgc ccg caa ggt gta cca gga gag aaa gac aca ctc atc tgg aaa tac 1824
Cys Pro Gln Gly Val Pro Gly Glu Lys Asp Thr Leu Ile Trp Lys Tyr
595 600 605
gct gac gtg aca cac gtt tgc cag ccc tgt cat gaa aac tgc acc cag 1872
Ala Asp Val Thr His Val Cys Gln Pro Cys His Glu Asn Cys Thr Gln
610 615 620
gga tgt acg ggg cct gat cta aag gac tgc aaa gat ttc aaa agc tct 1920
Gly Cys Thr Gly Pro Asp Leu Lys Asp Cys Lys Asp Phe Lys Ser Ser
625 630 635 640
ggt ttg ccg atg atc gct gct ggc gtt gtc gga ggt cta ctg gcg ttt 1968
Gly Leu Pro Met Ile Ala Ala Gly Val Val Gly Gly Leu Leu Ala Phe
645 650 655
gtt att ctg gct ctt gga gtg gcc gtt ctc ctg cgc aga cgc cac atc 2016
Val Ile Leu Ala Leu Gly Val Ala Val Leu Leu Arg Arg Arg His Ile
660 665 670
cgg agg aag agg act ctg aga cga ctc ctg caa gag aga gag ctt gtg 2064
Arg Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu Val
675 680 685
gag cct ctg acc ccc agc ggc gaa gcc ccc aat cag gcc tta ctg cgc 2112
Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu Arg
690 695 700
atc ctc aaa gag acg gag ttc aag aag atc aaa gtg ctg ggc tcc ggg 2160
Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser Gly
705 710 715 720
gct ttc ggc act gtg cac aag ggc ctt tgg gtt cca gaa gga gag aat 2208
Ala Phe Gly Thr Val His Lys Gly Leu Trp Val Pro Glu Gly Glu Asn
725 730 735
gtg aag atc cct gtc gcc atc aag gtc tta aga gaa gcc acg tct ccc 2256
Val Lys Ile Pro Val Ala Ile Lys Val Leu Arg Glu Ala Thr Ser Pro
740 745 750
aaa gct aac aag gag ata atg gat gag gcg tac gtc atg gcc agt gtt 2304
Lys Ala Asn Lys Glu Ile Met Asp Glu Ala Tyr Val Met Ala Ser Val
755 760 765
gag cat ccc cat gtg tgt cgt ctg ctg ggc atc tgc ttg acc tcc aca 2352
Glu His Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr
770 775 780
gtg cag ctc atc act cag ctg atg ccc tac ggc tgc ttg ctg gac tat 2400
Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp Tyr
785 790 795 800
gtc aga gaa aac aag gac cgc atc ggc tcc cag cac ctg ctc aac tgg 2448
Val Arg Glu Asn Lys Asp Arg Ile Gly Ser Gln His Leu Leu Asn Trp
805 810 815
tgc gtg cag atc gct aaa ggt atg aat tat cta gaa gag cgc cat ctt 2496
Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Glu Arg His Leu
820 825 830
gtg cac cga gac ctg gca gca cgt aat gtg ttg gta aag acg cct cag 2544
Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro Gln
835 840 845
cat gtc aag att acc gat ttc ggc ctc gcc aag ctg tta aac gcg gac 2592
His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Asn Ala Asp
850 855 860
gag aag gag tat cac gca gat gga gga aag gtt cca att aaa tgg atg 2640
Glu Lys Glu Tyr His Ala Asp Gly Gly Lys Val Pro Ile Lys Trp Met
865 870 875 880
gcg ctg gag tcc atc cag cac agg act tac acc cac cag agt gac gtc 2688
Ala Leu Glu Ser Ile Gln His Arg Thr Tyr Thr His Gln Ser Asp Val
885 890 895
tgg agc tac ggt gtg acc gtc tgg gag ttg atg acg ttt ggg acg aaa 2736
Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Thr Lys
900 905 910
cct tat gat ggg att cct gcc agt gaa atc gcc gga gtt ctg gaa aaa 2784
Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ala Gly Val Leu Glu Lys
915 920 925
gga gaa aga ctt cct caa ccc ccc atc tgc acc att gat gtg tac atg 2832
Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met
930 935 940
atc atg gtc aaa tgt tgg atg att gat gct gag agc aga ccc cgc ttc 2880
Ile Met Val Lys Cys Trp Met Ile Asp Ala Glu Ser Arg Pro Arg Phe
945 950 955 960
agg gag ctc atc gca gag ttc act aaa atg gct cgt gac ccg tcc cgc 2928
Arg Glu Leu Ile Ala Glu Phe Thr Lys Met Ala Arg Asp Pro Ser Arg
965 970 975
tat ctg gtc att cag ggg gac gac cgc atg cat tta ccc agt cct tct 2976
Tyr Leu Val Ile Gln Gly Asp Asp Arg Met His Leu Pro Ser Pro Ser
980 985 990
gac tcc aag ttc tac cgc agc ctg atg agc gga gag ctg gac gag gcc 3024
Asp Ser Lys Phe Tyr Arg Ser Leu Met Ser Gly Glu Leu Asp Glu Ala
995 1000 1005
gtg gac gca gac gag tat tta gtg ccc aat cac agc ttc ttc agc 3069
Val Asp Ala Asp Glu Tyr Leu Val Pro Asn His Ser Phe Phe Ser
1010 1015 1020
agc ccg agc acg tcc cgc aca caa ctg ctg cac tct gtg agc ctg 3114
Ser Pro Ser Thr Ser Arg Thr Gln Leu Leu His Ser Val Ser Leu
1025 1030 1035
aac agc agc ttt gga aac tgt aat agt aga aac ggg aat ggt tat 3159
Asn Ser Ser Phe Gly Asn Cys Asn Ser Arg Asn Gly Asn Gly Tyr
1040 1045 1050
cca gtg agg gag aac agc atg gtc ctg cgc tac atc cca gac ccc 3204
Pro Val Arg Glu Asn Ser Met Val Leu Arg Tyr Ile Pro Asp Pro
1055 1060 1065
aca gag cgc ttt cag gag gga gac ttt cag cct gcg ccg ggt tat 3249
Thr Glu Arg Phe Gln Glu Gly Asp Phe Gln Pro Ala Pro Gly Tyr
1070 1075 1080
aac gaa tat atg aac cag aat gag tcc agc atg atc aac cca gtg 3294
Asn Glu Tyr Met Asn Gln Asn Glu Ser Ser Met Ile Asn Pro Val
1085 1090 1095
tac cag cag ccc cac gga ccc ccg cgg acc ctc ctc cac tcc tcc 3339
Tyr Gln Gln Pro His Gly Pro Pro Arg Thr Leu Leu His Ser Ser
1100 1105 1110
cca gcg ctg gac gag acg gaa gag gag tat ctg aac tgc ttc aag 3384
Pro Ala Leu Asp Glu Thr Glu Glu Glu Tyr Leu Asn Cys Phe Lys
1115 1120 1125
agc ccg gct ccg gct tca gtg gtg gag tat ctg aac acg tcc cac 3429
Ser Pro Ala Pro Ala Ser Val Val Glu Tyr Leu Asn Thr Ser His
1130 1135 1140
aca cag ctg ctc tcc aca aag ccc ttc ttc agc atg gac aac ccc 3474
Thr Gln Leu Leu Ser Thr Lys Pro Phe Phe Ser Met Asp Asn Pro
1145 1150 1155
gac tac cag cag gac ttc tgc ccg ctg gag ctc aaa aca cac acc 3519
Asp Tyr Gln Gln Asp Phe Cys Pro Leu Glu Leu Lys Thr His Thr
1160 1165 1170
aac ggg cac ctg ccg gcc gcg cag aac cag gag tac atg ggc ctg 3564
Asn Gly His Leu Pro Ala Ala Gln Asn Gln Glu Tyr Met Gly Leu
1175 1180 1185
gag gtg cac tag 3576
Glu Val His
1190
<210>27
<211>1191
<212>PRT
<213>斑马鱼(Danio rerio)
<400>27
Met Ala Gly Pro Thr Glu Ile Gly Leu Phe Phe Thr Leu Leu Leu Ser
1 5 10 15
Gly Ser Phe Cys Ala Thr Pro Glu Lys Lys Val Cys Gln Gly Ala Asn
20 25 30
Asn Lys Leu Thr Leu Leu Gly Thr Val Glu Asp His Tyr Gln Val Leu
35 40 45
Leu Arg Met Tyr Arg Asn Cys Thr Val Val Leu Glu Asn Leu Glu Ile
50 55 60
Thr His Ile Thr Glu Lys Tyr Asp Leu Ser Phe Leu Lys Ser Ile Gln
65 70 75 80
Glu Val Gly Gly Tyr Val Leu Ile Ala Val Asn Thr Val Ser Lys Ile
85 90 95
Pro Leu Glu Asn Leu Arg Ile Ile Arg Gly His Ser Leu Tyr Glu Asp
100 105 110
Lys Phe Ala Leu Ala Val Leu Val Asn Phe Asn Asn Ser Ile Glu Gln
115 120 125
Gly Val Lys Glu Leu Pro Leu Thr Ser Leu Thr Glu Ile Leu Lys Gly
130 135 140
Gly Val Lys Phe Cys Arg Asn Asp Tyr Leu Cys Asn Val Gly Thr Ile
145 150 155 160
Glu Trp Ala Asp Ile Leu Asn Met Lys Ser Leu Pro Thr Ile Val Ser
165 170 175
His Asn Ile Ser Tyr Gly Lys Asn Cys Gly Lys Cys Asp Pro Ser Cys
180 185 190
Phe Asn Gly Ser Cys Trp Gly Thr Gly Pro Asp Lys Cys Gln Arg Met
195 200 205
Thr Lys Val Ile Cys Ala Glu Gln Cys Ser Gly Arg Cys Lys Gly Pro
210 215 220
Arg Pro Ile Asp Cys Cys Asn Glu His Cys Ala Ala Gly Cys Thr Gly
225 230 235 240
Pro Arg Pro Thr Asp Cys Leu Ala Cys Lys Asp Phe Gln Asp Glu Gly
245 250 255
Thr Cys Lys Asp Ala Cys Pro Arg Leu Met Leu Tyr Asp Pro Asn Thr
260 265 270
His Gln Leu Ala Pro Asn Pro Tyr Gly Lys Tyr Ser Phe Gly Ala Thr
275 280 285
Cys Ile Lys Thr Cys Pro His Asn Tyr Val Val Thr Asp His Gly Ala
290 295 300
Cys Val Arg Thr Cys Ser Pro Gly Thr Tyr Glu Val Asp Glu Gly Gly
305 310 315 320
Val Arg Lys Cys Lys Arg Cys Glu Gly Leu Cys Pro Lys Val Cys Asn
325 330 335
Gly Leu Gly Met Gly Pro Leu Ala Asn Val Leu Ser Ile Asn Ala Thr
340 345 350
Asn Ile Asp Ser Phe Glu Asn Cys Thr Lys Ile Ser Gly Asn Val Ala
355 360 365
Ile Leu Ser Thr Thr Phe Arg Gly Asp Pro His Thr Asn Thr Ser Gly
370 375 380
Leu Asp Pro Ala Lys Leu Ser Val Leu Ser Thr Val Lys Glu Ile Thr
385 390 395 400
Gly Tyr Leu Met Ile Gln Leu Trp Pro Glu Ser Met Gln Ser Leu Ser
405 410 415
Ala Phe Glu Asn Leu Glu Val Ile Arg Gly Arg Thr Lys Thr Gln Gly
420 425 430
Thr Tyr Ser Phe Ala Val Thr Lys Thr Ala Ile Thr His Leu Gly Met
435 440 445
Arg Ser Leu Arg Glu Ile Ser Asp Gly Asp Val Ser Ile Val Lys Asn
450 455 460
Lys Asn Leu Cys Tyr Ser Ser Pro Glu His Trp Lys Arg Leu Phe Lys
465 470 475 480
Ser Lys Gln Gln Ser Val Lys Met Ile Glu Asn Met Asp Ala Ala Thr
485 490 495
Cys Ala Asn Gln Asn Ser Thr Cys Asn Glu Met Cys Thr Ala Asp Gly
500 505 510
Cys Trp Gly Pro Gly Pro Thr Met Cys Phe Gly Cys Glu His Tyr Ser
515 520 525
Arg Gly Lys His Cys Val Ala Ser Cys Asn Leu Leu Asn Gly Glu Pro
530 535 540
Arg Glu Tyr Glu Val Asn Lys Thr Cys Met Glu Cys Asp Pro Glu Cys
545 550 555 560
Leu Leu Met Asn Glu Thr Gln Thr Cys Asn Gly Pro Gly Pro Asp Lys
565 570 575
Cys Thr Val Cys Ala Asn Tyr Lys Asp Gly Pro His Cys Val His Arg
580 585 590
Cys Pro Gln Gly Val Pro Gly Glu Lys Asp Thr Leu Ile Trp Lys Tyr
595 600 605
Ala Asp Val Thr His Val Cys Gln Pro Cys His Glu Asn Cys Thr Gln
610 615 620
Gly Cys Thr Gly Pro Asp Leu Lys Asp Cys Lys Asp Phe Lys Ser Ser
625 630 635 640
Gly Leu Pro Met Ile Ala Ala Gly Val Val Gly Gly Leu Leu Ala Phe
645 650 655
Val Ile Leu Ala Leu Gly Val Ala Val Leu Leu Arg Arg Arg His Ile
660 665 670
Arg Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu Val
675 680 685
Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu Arg
690 695 700
Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser Gly
705 710 715 720
Ala Phe Gly Thr Val His Lys Gly Leu Trp Val Pro Glu Gly Glu Asn
725 730 735
Val Lys Ile Pro Val Ala Ile Lys Val Leu Arg Glu Ala Thr Ser Pro
740 745 750
Lys Ala Asn Lys Glu Ile Met Asp Glu Ala Tyr Val Met Ala Ser Val
755 760 765
Glu His Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr
770 775 780
Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp Tyr
785 790 795 800
Val Arg Glu Asn Lys Asp Arg Ile Gly Ser Gln His Leu Leu Asn Trp
805 810 815
Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Glu Arg His Leu
820 825 830
Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro Gln
835 840 845
His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Asn Ala Asp
850 855 860
Glu Lys Glu Tyr His Ala Asp Gly Gly Lys Val Pro Ile Lys Trp Met
865 870 875 880
Ala Leu Glu Ser Ile Gln His Arg Thr Tyr Thr His Gln Ser Asp Val
885 890 895
Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Thr Lys
900 905 910
Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ala Gly Val Leu Glu Lys
915 920 925
Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met
930 935 940
Ile Met Val Lys Cys Trp Met Ile Asp Ala Glu Ser Arg Pro Arg Phe
945 950 955 960
Arg Glu Leu Ile Ala Glu Phe Thr Lys Met Ala Arg Asp Pro Ser Arg
965 970 975
Tyr Leu Val Ile Gln Gly Asp Asp Arg Met His Leu Pro Ser Pro Ser
980 985 990
Asp Ser Lys Phe Tyr Arg Ser Leu Met Ser Gly Glu Leu Asp Glu Ala
995 1000 1005
Val Asp Ala Asp Glu Tyr Leu Val Pro Asn His Ser Phe Phe Ser
1010 1015 1020
Ser Pro Ser Thr Ser Arg Thr Gln Leu Leu His Ser Val Ser Leu
1025 1030 1035
Asn Ser Ser Phe Gly Asn Cys Asn Ser Arg Asn Gly Asn Gly Tyr
1040 1045 1050
Pro Val Arg Glu Asn Ser Met Val Leu Arg Tyr Ile Pro Asp Pro
1055 1060 1065
Thr Glu Arg Phe Gln Glu Gly Asp Phe Gln Pro Ala Pro Gly Tyr
1070 1075 1080
Asn Glu Tyr Met Asn Gln Asn Glu Ser Ser Met Ile Asn Pro Val
1085 1090 1095
Tyr Gln Gln Pro His Gly Pro Pro Arg Thr Leu Leu His Ser Ser
1100 1105 1110
Pro Ala Leu Asp Glu Thr Glu Glu Glu Tyr Leu Asn Cys Phe Lys
1115 1120 1125
Ser Pro Ala Pro Ala Ser Val Val Glu Tyr Leu Asn Thr Ser His
1130 1135 1140
Thr Gln Leu Leu Ser Thr Lys Pro Phe Phe Ser Met Asp Asn Pro
1145 1150 1155
Asp Tyr Gln Gln Asp Phe Cys Pro Leu Glu Leu Lys Thr His Thr
1160 1165 1170
Asn Gly His Leu Pro Ala Ala Gln Asn Gln GluTyr Met Gly Leu
1175 1180 1185
Glu Val His
1190
Claims (2)
1.一种制备EGFR重组核酸疫苗的方法，该方法包括：
1)确定编码人体内肿瘤细胞EGFR抗原的多核苷酸序列，如SEQ NO：1中第73个碱基至第1935个碱基所示，其中第73个碱基C替换为A；
2)利用步骤1)所述编码人体内肿瘤细胞EGFR抗原的多核苷酸序列设计引物：5’GACCATGGAGGAAAAGAAAGTTTGC 3’，5’ACGAATTCTTAGGACGGGATCTTAGGCCCA3’；以人肺癌细胞株A431的总RNA为模板进行RT-PCR扩增，电泳收集纯化扩增的EGFR片段，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EGFR片段并纯化，插入到用NcoI和EcoRI双酶切的pORF-MCS载体，筛选，鉴定重组质粒，命名为pORF-hEGFR，所述质粒pORF-hEGFR为EGFR重组核酸疫苗。
2.权利要求1所述方法制备的EGFR重组核酸疫苗，其特征在于：EGFR重组核酸疫苗为质粒pORF-hEGFR。
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2003101130540A CN1548537B (zh) | 2002-12-27 | 2003-12-25 | 疫苗制备方法和抗肿瘤疫苗 |
| US10/749,104 US20040265274A1 (en) | 2002-12-27 | 2003-12-29 | Anti-tumor molecular vaccine and method of making thereof |
| US11/649,155 US20070275049A1 (en) | 2002-12-27 | 2006-12-27 | Anti-tumor molecular vacine and method of making thereof |
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| CN02159602 | 2002-12-27 | ||
| CN02159602.6 | 2002-12-27 | ||
| CN2003101130540A CN1548537B (zh) | 2002-12-27 | 2003-12-25 | 疫苗制备方法和抗肿瘤疫苗 |
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| CN1548537B true CN1548537B (zh) | 2010-05-05 |
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| Country | Link |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2170384B1 (en) | 2007-07-02 | 2016-04-13 | Etubics Corporation | Methods and compositions for producing an adenovirus vector for use with multiple vaccinations |
| WO2009025846A2 (en) | 2007-08-22 | 2009-02-26 | The Regents Of The University Of California | Activatable binding polypeptides and methods of identification and use thereof |
| EP2085479A1 (en) | 2008-01-31 | 2009-08-05 | Institut Pasteur | Reverse genetics of negative-strand rna viruses in yeast |
| WO2010077643A1 (en) * | 2008-12-08 | 2010-07-08 | Tegopharm Corporation | Masking ligands for reversible inhibition of multivalent compounds |
| WO2010081173A2 (en) | 2009-01-12 | 2010-07-15 | Cytomx Therapeutics, Llc | Modified antibody compositions, methods of making and using thereof |
| AU2010215761B2 (en) | 2009-02-23 | 2017-04-06 | Cytomx Therapeutics, Inc | Proproteins and methods of use thereof |
| WO2014031178A1 (en) | 2012-08-24 | 2014-02-27 | Etubics Corporation | Replication defective adenovirus vector in vaccination |
| EP3212784B1 (en) | 2014-10-31 | 2019-05-29 | Abilita Bio, Inc. | Modified membrane spanning proteins and methods for the preparation and use thereof |
| CA2974237C (en) | 2015-01-09 | 2021-07-20 | Etubics Corporation | Methods and compositions for combination immunotherapy |
| KR20170102002A (ko) | 2015-01-09 | 2017-09-06 | 이투빅스 코포레이션 | 에볼라 바이러스 백신접종을 위한 방법 및 조성물 |
| EP3286213B1 (en) | 2015-04-20 | 2021-08-04 | Etubics Corporation | Methods and compositions for combination immunotherapy |
| CN107847611A (zh) * | 2015-05-26 | 2018-03-27 | 阿德瓦希斯公司 | 个性化的基于递送载体的免疫疗法及其用途 |
| AU2017259821B2 (en) * | 2016-05-04 | 2024-07-18 | Abilita Bio, Inc. | Methods and platform for preparing multispanning membrane proteins |
| CN110124020B (zh) | 2019-05-05 | 2023-03-14 | 海南医学院 | 基于胞外诱捕网原理制成的肿瘤细胞疫苗及其制备方法 |
| CN112980840A (zh) * | 2019-12-17 | 2021-06-18 | 南京大学 | 用于癌症治疗的多靶向siRNA |
| CN113072646A (zh) * | 2021-06-04 | 2021-07-06 | 北京承诺未来科技有限公司 | 靶向egfr的肿瘤核酸疫苗 |
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| US5846782A (en) * | 1995-11-28 | 1998-12-08 | Genvec, Inc. | Targeting adenovirus with use of constrained peptide motifs |
| US6080725A (en) * | 1997-05-20 | 2000-06-27 | Galenica Pharmaceuticals, Inc. | Immunostimulating and vaccine compositions employing saponin analog adjuvants and uses thereof |
| US6872518B2 (en) * | 1997-09-22 | 2005-03-29 | University Of Rochester | Methods for selecting polynucleotides encoding T cell epitopes |
| US6852704B1 (en) * | 1999-01-19 | 2005-02-08 | The Children's Hospital Of Philadelphia | Reverse gene therapy |
| DE60128070T2 (de) * | 2000-02-14 | 2008-01-10 | The Regents Of The University Of California, Oakland | Impfstoff spezifisch gegen nierentumore, gerichtet gegen das antigen g-250 des nierentumors |
| US20040002455A1 (en) * | 2002-01-29 | 2004-01-01 | Aventis Pasteur, Ltd. | Targeted immunogens |
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- 2003-12-25 CN CN2003101130540A patent/CN1548537B/zh not_active Expired - Lifetime
- 2003-12-29 US US10/749,104 patent/US20040265274A1/en not_active Abandoned
-
2006
- 2006-12-27 US US11/649,155 patent/US20070275049A1/en not_active Abandoned
Non-Patent Citations (2)
| Title |
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| GenBanK登录号：DD400342.2007, * |
| GenBanK登录号：FB725994.2008, * |
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| Publication number | Publication date |
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| CN1548537A (zh) | 2004-11-24 |
| US20070275049A1 (en) | 2007-11-29 |
| US20040265274A1 (en) | 2004-12-30 |
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