CN1391608A - 在肿瘤细胞中表达并参与免疫反应的调控之基因的克隆、表达和表征 - Google Patents
在肿瘤细胞中表达并参与免疫反应的调控之基因的克隆、表达和表征 Download PDFInfo
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Abstract
本发明涉及一种在肿瘤中表达并参与免疫反应调控的新基因的鉴定和表征,以及其cDNA和相应多肽的克隆和表征。本发明还涉及含有编码该基因的核酸之载体,由该载体转化的细胞,以及诊断方法,和能够直接或间接与本发明的核酸或多肽相互作用之化学或生物物质的筛选方法。本发明进一步涉及用于治疗免疫疾病,特别是自身免疫疾病和肿瘤的化合物。
Description
本发明涉及一种在肿瘤细胞中表达并参与免疫反应调控的新基因的鉴定和表征,以及其cDNA和相应多肽的克隆和表征。本发明还涉及含有编码该基因的核酸之载体,由该载体转化的细胞,以及诊断方法,和能够直接或间接与本发明的核酸或多肽相互作用之化学或生物化学化合物的筛选方法。本发明进一步涉及用于治疗免疫疾病,特别是自身免疫疾病和癌症的化合物。
多种正常或病理细胞机制涉及差异性地基因表达,有些基因在特定时间和/或以特定强度在给定的组织中表达。
在这些基因中,编码在翻译后的加工过程中或之后转运到亚细胞区室的蛋白质或分泌到细胞外的蛋白质的基因最多。特别可提及的有编码因子蛋白质的基因,诸如细胞因子,或编码膜结合受体的基因,它们构成了疾病治疗靶点的候选物。
大多数这类蛋白质,如膜结合蛋白质具有相应于信号肽的N-端结构域、细胞外结构域和跨膜结构域。
因此,在今天仍需要鉴别和表征这类基因,其表达对某些组织或细胞是特异性的,特别是对于那些参与免疫反应调控的细胞或肿瘤细胞。
这正是本发明的主题。
本发明的一个主题是鉴定和表征优势性地在人的淋巴样组织中特别是未刺激的外周T淋巴细胞和肿瘤细胞中表达的新基因,该基因的翻译产物是膜结合型的。
在第一个方面,本发明的主题是纯化的或分离的核酸,其特征在于其含有选自如下序列的核酸序列:
a)SEQ ID NO:1的序列;
b)缺失了其末端poly A片段的SEQ ID NO:1的序列之至少18个连续核苷酸,优选20,25,30,50,100或200连续核苷酸的片段的序列,优选为SEQ ID NO:3,SEQ ID NO:5,SEQ ID NO:7或SEQ ID NO:9的这样的片段;
c)在优化排比后,具有与a)或b)中定义的序列至少80%,优选90%,95%或99%的百分同一性的核酸序列;
d)与a),b)或c)中定义的序列互补之序列,或RNA序列相应于a),b)或c)中定义的序列之序列。
优选地,本发明涉及一种纯化的或分离的核酸,其特征在于其含有选自如下序列的核酸序列:
a)SEQ ID NO:1的序列;
b)缺失了其末端poly A片段的SEQ ID NO:1的序列之至少18个连续核苷酸的片段的序列;
c)在优化排比后,具有与a)或b)中定义的序列至少80%百分同一性的核酸序列;
d)与a),b)或c)中定义的序列互补之序列,或RNA序列相应于a),b)或c)中定义的序列之序列;
除了WO 99/54461文件中的SEQ ID NO:11的序列,WO/99/06548文件中的SEQ ID NO:51的序列以及除了在EMBL数据库中鉴定号为AI 672868,AA 890726和AI301140的文件中所述的3个序列。
WO 99/54461是一份专利文件,其涉及由子宫内膜肿瘤组织表达的核酸序列。该文件在635个序列中特别描述了SEQ ID NO:11的核酸序列,和其相应的肽序列SEQ ID NO:173,它们分别与本发明的SEQ ID NO:1(nt 1597-3313)和SEQ ID NO:2(aa 519-1013)的序列的片段几乎100%相同。该文件在第153页中显示SEQ ID NO:173的肽不相应于任何已知功能的序列。
WO 99/06548是涉及编码非组织特异性的分泌的蛋白质之EST序列。该文件在545个序列中特别描述了SEQ ID NO:51的核酸序列和肽序列,其aa37-113序列(150个氨基酸)显示与本发明的SEQ IDNO:2之序列的aa1-150片段非常强的同源性(对于150氨基酸约85%相同;对于头135个氨基酸约94%)。该文件还显示相应于此SEQID NO:51的5’EST序列的DNA源于人前列腺肿瘤组织(参见,序列表40页,“来源”)。
“E MBL”文件AI 672868描述了一种mRNA序列,其源于患有Crohn氏疾病的人结肠组织文库,且与互补于本发明之SEQ ID NO:1的nt 441-971 cDNA片段之序列具有相当高的同一性。
“E MBL”文件AA 890726描述了一种mRNA序列,其源于患有散发性腺瘤的人甲状旁腺组织,且与互补于本发明之SEQ ID NO:1的nt 2779-3309 cDNA片段之序列具有相当高的同一性。
最后,“EMBL”文件AI 301140描述了一种mRNA序列,其源于所述的神经内分泌型人肺类瘤文库,且与互补于本发明之SEQ IDNO:1的nt 2736-3299 cDNA片段之序列具有相当高的同一性。
术语“核酸”,“核酸序列”,“多核苷酸”,“寡核苷酸”,“多核苷酸序列”以及“核苷酸序列”等在本发明中的使用没有差别,其意在表示核苷酸的精确链,其可为修饰或未修饰的,从而可定义核酸的片段或区域,其可含有或不含有非天然核苷酸,且其可相应于双链DNA和单链DNA,以及所述DNA转录的产物。
应当理解,本发明不涉及在其天然染色体环境中,即呈天然状态的核苷酸序列。它们是已被分离和/或纯化的序列,即其已被直接或间接地(例如通过拷贝)从其已经被至少部分改变的环境中移出。
优选地,本发明核酸序列的长度少于150kb,更优选少于100kb,50kb,25kb,10kb,或5kb。
为了本发明的目的,术语两个核酸或氨基酸序列之间的“百分同一性”意在表示在优化排比后两个待比较的序列之间核苷酸或氨基酸残基之间的百分比,此百分比是纯粹统计学意义上的,且两个序列间的差异随机分布,且位于其全长上。术语“最佳排比”或“优化排比”意在表示如下确定的百分同一性为最高的排比。两个核苷酸或氨基酸序列之间的序列比较-般在其优化排比后进行两者的比较,所述比较通过区段或“比较窗口”进行,以鉴定和比较序列相似性的局部区域。用于比较的序列之优化排比除手工外可通过Smith和Waterman(1981)[Ad.App.Math.,2:482]的局部同源性算法,或通过Neddleman和Wunsch(1970)[J.Mol.Biol.,48:443]的局部同源性算法,通过Pearson和Lipman(1988)[Proc.Natl.Acad.Sci.,USA,85:2444]的相似性检索,通过利用这些算法的计算机程序(Wisconsin遗传学软件包,Genetics Computer Group,575 Science Dr.,Madison,MI,中的GAP,BESTFIT,BLAST P,BLAST N,FASTA和TFASTA)。
两个核酸或氨基酸序列之间的百分同一性通过比较已优化排比的两序列而确定,其中待比较的核酸或氨基酸序列与用来比对以使此二序列间优化排比的参考序列相比,可含有添加或缺失。可通过计算两序列间相同位置的数目确定百分同一性,其中将两序列间相同的核苷酸或氨基酸残基数除以比较的总位置数,将所得数值乘以100,获得两序列间的百分同一性。
对于“在优化排比后,具有与参比序列至少80%,优选90%,95%或99%的百分同一性的核酸序列”的表述是指与参比序列相比,具有某些修饰,如特别是缺失、截短、延伸、嵌合、融合和/或替换,特别是点替换的核酸序列,以及其在优化排比后,具有与参比序列至少80%,优选90%,95%或99%的同一性的核酸序列。它们优选这样的序列,其与能特异性地与本发明SEQ ID NO:1的序列杂交的序列互补。优选地,特异性或高度严谨杂交条件为这样的条件,其能确保在优化排比后,在两个序列之一与互补于另一序列的序列之间至少80%,优选90%,95%或99%同一性。
在高度严谨条件下杂交意味着选择温度条件和离子强度使得其能在两个互补DNA片段之间维持杂交。以举例的方式,对于上述目的在于确定上述多核苷酸片段之杂交步骤的条件如下详述:
在两步中进行DNA-DNA或RNA-RNA杂交:(1)在磷酸缓冲液中(20mM,pH7.5)42℃预杂交3小时,所述磷酸缓冲液中含有5×SSC(1×SSC相应于0.15M NaCl+0.015M柠檬酸钠溶液),50%甲酰胺,7%十二烷基硫酸钠(SDS),10×Denhardt’s试剂,5%硫酸葡聚糖,和1%鲑精DNA;(2)依据探针的大小决定进行20小时杂交的温度(即:对于大于100核苷酸的探针,42℃),随后是在20℃下用2×SSC+2%SDS进行20分钟的洗涤2次,和在20℃下用0.1×SSC+0.1%SDS进行20分钟的洗涤1次。对于大于100核苷酸的探针最后一次洗涤在60℃下用0.1×SSC+0.1%SDS进行30分钟洗涤。根据Sambrook,等人,(Molecular cloning:a laboratorymanual,Sec.Ed.,cold spring Harbor Lab.,Cold Spring Harbor,NewYork,1989)的教导,上述高度严谨杂交条件对于确定大小的多核苷酸可由本领域技术人员加以调整,以用于大小更大的或更小的寡核苷酸。
在优化排比后,具有与本发明序列序列至少80%,优选90%,95%或99%的百分同一性的诸多核酸序列之中,优选是这样的核酸序列,其为SEQ ID NO:1或其片段的变体,即,均为相应于等位基因变体(即SEQ ID NO:1的核酸序列的各个变体)的核酸序列。这些相当于在哺乳动物(特别是人)中多态形的天然突变的序列,特别是相当于可导致病理状况出现的多态形。本发明优选地涉及这样的变体核酸序列,其中的突变导致多肽之氨基酸序列或其片段的修饰,所述氨基酸由SEQ ID NO:1的正常序列编码。
表述“变体核酸序列”也意指任何由基因组核酸序列的剪接位点突变所得的RNA或cDNA,所述基因组核酸序列的cDNA具有SEQID NO:1的序列。
本发明优选地涉及根据本发明的纯化或分离的核酸,其特征在于其由SEQ ID NO:1组成,由互补于SEQ ID NO:1的序列或相应于SEQ ID NO:1的RNA序列组成,优选地,其片段编码SEQ ID NO:2的肽,或缺失了信号肽aa1-41的SEQ ID NO:2的序列。
本发明优选地涉及根据本发明的纯化或分离的核酸,其特征在于其含有选自如下序列的序列:
a)SEQ ID NO:5,SEQ ID NO:7,SEQ ID NO:15,SEQ ID NO:17和SEQ ID NO:19的序列;
b)至少18个核苷酸的SEQ ID NO:19的序列;
c)在优化排比后,具有与a)或b)中定义的序列至少80%百分同一性的核酸序列;
d)与a),b)或c)中定义的序列互补之序列,或RNA序列相应于a),b)或c)中定义的序列之序列。
本发明优选地涉及纯化或分离的根据本发明的核酸,其特征在于,其序列选自如下序列:
a)SEQ ID NO:1,SEQ ID NO:3,SEQ ID NO:5,SEQ ID NO:7,SEQ ID NO:9,SEQ ID NO:15,SEQ ID NO:17,SEQ ID NO:19和SEQ ID NO:21的序列;
b)在优化排比后,具有与a)中定义的序列至少80%百分同一性的核酸序列;
c)与a),或b)中定义的序列互补之序列,或RNA序列相应于a),a)或b)中定义的序列之序列。
这样的引物或探针也是本发明的一部分,其特征在于其含有根据本发明的核酸的序列。
因此,本发明优选地涉及根据本发明的引物或探针,其可特别是能够区分或显示各个变体核酸序列,或鉴别基因的基因组序列(所述基因的cDNA由SEQ ID NO:1的序列表示),方法是利用特别是扩增的方法,诸如PCR法或相关的方法。
本发明还涉及根据本发明的核酸序列作为探针或引物的用途,用于检测、鉴定、滴定或扩增核酸序列。
根据本发明,可在检测、鉴定、滴定或扩增核酸序列的方法中用作引物或探针的多核苷酸之大小,最少为15个碱基,优选20个碱基,或更好为25-30个碱基。
所有根据本发明的探针及引物可直接或间接地用本领域已知的方法利用放射活性或非放射活性化合物标记,以获得可检测和/或定量的信号。
未标记的根据本发明的多核苷酸可直接用作探针或引物。
通常标记序列以获得能用于多种应用的序列。根据本发明的探针或引物用放射性元素或用非放射性分子标记。
在所用的放射性同位素中,可提及有32p,33p,35S,3H或125I。非放射性基团选自配体,如生物素、抗生物素蛋白、链霉抗生物素蛋白或洋地黄毒苷、半抗原、染料和荧光剂,诸如放射性荧光剂、化学荧光剂、生物荧光剂、荧光素或磷光剂。
根据本发明的多核苷酸可因此在特别是利用PCR技术(聚合酶链反应)的方法中用作引物和/或探针(H.a.Erlich,New York:StocktonPress,1989;M.A.Innis et al.,Academic Press,1990;和A.Roifs et al.,Berlin:Springer_Verlag,1991)。该技术需要选择成对的寡核苷酸引物,以确定需要扩增的片段之边界。参考文献可见例如在美国专利4,683,202中所述的技术。可在如琼脂糖凝胶电泳或聚丙烯酰胺凝胶电泳后鉴定扩增的片段,或在层析技术诸如凝胶过滤或离子交换层析等后鉴定,然后测定序列。扩增的特异性可利用引物(诸如发明的多核苷酸之核苷酸序列)以及基质(含有这些序列的质粒或衍生的扩增产物)来控制。扩增的核苷酸片段可用作杂交反应中的试剂,以证实在生物样品中与所述扩增的核苷酸片段互补之靶核酸序列的存在。
本发明还涉及可利用根据本发明的引物通过扩增获得的核酸。
其他用于扩增靶核酸的技术可有利地作为PCR技术的替代物而利用(PCR-样),方法是使用成对的根据本发明的核苷酸序列之引物。术语“PCR-样”意指所有直接或间接地利用了核酸序列复制的方法,或其中的标记体系已被扩增;这些技术当然为已知的;一般而言,它们包括利用聚合酶的DNA扩增;当样品的来源为RNA时,应事先进行反转录。目前有大量方法用于这类扩增,诸如SDA技术(链置换扩增,G.T.Walker et al.,Nucleic Acids res.,20:1691-1696,1992)、TAS技术(基于转录的扩增体系,D.Y.Kwoh,et al.,1989,Proc.Natl.Acad.Sci.,USA,86:1173-1177)、3SR技术(自维持的序列复制,J.C.Guatelli,et al.,1990,Proc.Natl.Acad.Sci.USA,87:1874-1878,和Cell,85:281-290,1996)、NASBA技术(基于核酸序列的扩增,T.Kievitiset al.,1991,J.Virol.,Methods,35:273-286)、TMA技术(转录介导的扩增)、LCR技术(连接酶链反应,U.Landegren et al.,1988,Science,241:1077-1080)并由F.Barany于1991年利用热温度连接酶加以改进(Proc.Natl.Acad.Sci.USA,88:189-193)、RCR技术(修复链反应,D.Segev,1992,Kessler c.Springer Verlag,Berlin,New York,197-205)、CPR技术(循环探针反应,P.Duck et al.,1990,Biotechniques,9:142-147),以及Q-β-复制酶扩增技术(E.A.Mieleet al.,1983,J.Mol.Biol.,171:281-295),以及特别由B.C.F.Chu et al.,1986,(Nucleic Acid Res.,14:5591-5603)和P.M.Lizardi et al.,1988(Biotechnology,6:1197-1202)以及随后由J.L. Burg et al.,(Mol.andCell.Probes,10:257-271)和由B.B.Stone et al.,(Mol.and Cell Probes,10:359-370)在1996年改进的该方法。
当待检测的靶多核苷酸是mRNA时,可在利用本发明的引物进行扩增反应之前,或在利用本发明的探针进行检测方法之前,有利地使用反转录型酶,以从包含于生物学样品中的mRNA获得cDNA。所得的cDNA然后可用作根据本发明的扩增反应或检测方法中的引物或探针的靶。
探针杂交技术可用多种方法进行(J.A.Matthews et al.,Anal.Biochem.,169:1-25,1988)。最通用的方法中包括将从多种组织的细胞中,或从培养物细胞中提取的核酸固定在支持物(如硝酸纤维素,尼龙或聚苯乙烯)上,和将固定化的靶核酸与探针在充分确定的条件下温育。杂交后,移出过量的探针,利用适当的方法检测形成的杂合分子(测量与探针连接的放射性,荧光或酶活性)。
根据本发明之核酸探针的另一实施方案,后者可用作捕获探针。在这种情况中,称为“捕获探针”的探针被固定于支持物上,用于通过特异性杂交而捕获待检测的获自生物学样品中的靶核酸,然后利用第二种探针(称为“检测探针”,其用方便检测的元件标记)检测靶核酸。
在有用的核酸片段中,可提及特别是反义寡核苷酸,即这样的寡核苷酸,其结构通过与靶序列的杂交能够抑制相应产物的表达。应当提及有义寡核苷酸,其通过与参与调控相应产物表达之蛋白质的相互作用,可诱导对该表达的抑制或激活。
另一方面,发明还包括一种用于筛选基因组DNA或cDNA文库的方法,或用于克隆分离的基因组或cDNA的方法,其特征在于它利用了根据本发明的核酸序列。
在这些方法中,可特别提及的是
-利用根据本发明的核酸序列筛选cDNA文库和克隆分离的cDNA之方法(Sambtook,J.,et al.,1989;Suggs,S.V.,et al.,PNAS78:6613-6617,1981;Woo,S.L.C.,Methods Enzymol.,68:389,1979);
-利用根据本发明的序列筛选基因组文库,例如BAC(Chumakov,I.M.,et al.,1992;Chumakov,I.M.,et al.,Nature,377:175-183,1995)和任选地通过FISH进行遗传分析(Cherif,D.,et al.,Proc.Natl.Acad.Sci.,USA,87:6639-6643,1990),适当分离并染色体定位,然后完全测定基因的序列,其cDNA的序列为SEQ ID NO:1的序列。
特别的,这些根据本发明的方法可在此使用以鉴定并因此获得核酸变体的cDNA。
这些筛选和/或克隆方法可包括特别地用于使本发明的核酸与包含于基因组或cDNA文库中的核酸杂交之步骤。
本发明还包括一种用于鉴定这样的核酸序列的方法,该序列促进和/或调控如下基因的表达,该基因的cDNA具有SEQ ID NO:1的序列,该方法的特征在于其中使用了根据本发明的核酸。
本领域技术人员已知的计算机工具可使其容易地从根据本发明的基因组核酸序列中鉴别必需的和足以控制基因表达的启动子调控盒,特别是TATA盒,CCAAT盒和GC盒,以及刺激性调控序列(“增强子”)或抑制性调控序列(“沉默子”),它们以顺式方式调控本发明基因的表达;这些序列中需要提及的是IRE,MRE和CRE。
本发明还涉及用于鉴定突变的方法,所述突变由编码SEQ ID NO:2序列之多肽的基因特别是负责突变的基因携带,其特征在于它们使用了根据本发明的核酸。
本发明特别涉及一种用于鉴定基因序列中至少18个连续核苷酸的核酸的方法,所述基因编码SEQ ID NO:2序列的多肽,所述核酸包括至少一种突变,优选地,该突变负责该基因的异常表达,其特征在于它使用了根据本发明的核酸序列。
这些用于鉴定此类突变的方法特别地包括如下步骤:(1)从待分析的生物学样品中分离DNA,或从生物学样品中的mRNA产生cDNA;(2)利用根据本发明的引物特异性扩增很可能具有突变的靶DNA;(3)分析扩增产物,特别是相对于参比序列而言,扩增产物的大小和/或序列。
对于根据本发明之基因之启动子和/或调控序列也是本发明的一部分,其具有能修饰相应蛋白质表达的突变。
如下核酸也是本发明的一部分:其特征在于它们是能利用本发明的上述一种方法获得的核酸;或是能在高度严谨条件下(两个序列之一与互补于它的序列之间的百分同一性至少80%)与所述核酸序列杂交的核酸;特别是这样的基因之等位基因变体的核酸,其cDNA具有SEQ ID NO:1的序列,以及其他哺乳动物(如鼠)的同源基因的基因组序列。
根据本发明的核酸序列的作为基因组或cDNA文库筛选中的探针或引物之用途,也当然是本发明主题的一部分。
在另一方面,本发明包括一种纯化或分离的多肽,其由本发明的核酸编码。
在本文的说明书中,术语“多肽”可同样地用于表示蛋白质或肽。
优选地,本发明涉及一种纯化或分离的多肽,其特征在于其含有选自如下的氨基酸序列:
a)SEQ ID NO:2的序列;
b)SEQ ID NO:2的序列之至少6、10、15、30或50个连续氨基酸的片段的序列,优选地是SEQ ID NO:4,SEQ ID NO:6,SEQ IDNO:8或SEQ ID NO:10的这样的序列;
c)在优化排比后,具有与a)或b)中定义的序列至少80%百分同一性的氨基酸序列。
也优选地,本发明的主题是一种SEQ ID NO:2的氨基酸序列,缺失了aa1-41片段的SEQ ID NO:2序列的氨基酸序列,或在优化排比后,具有与sEQ ID NO:2的序列至少80%,优选90%,95%或99%百分同一性的序列。
蛋白质序列分析预期SEQ ID NO:2序列的多肽具有一个信号肽序列,其序列在SEQ ID NO:2序列的位置aa1-41之间;一个胞外结构域,其序列在位置aa 42-911之间;一个跨膜结构域,其序列在位置aa 912-930之间;和一个胞内结构域,其序列在位置aa 931-1013之间。
在根据本发明的多肽中,优选是如下多肽,其序列在SEQ ID NO:2的序列位置aa 42-911之间,相应于膜外结构域;或所述序列aa 42-911的至少6、10、15、30或50个连续氨基酸的任一片段,优选地是SEQ ID NO:4,SEQ ID NO:6,SEQ ID NO:8或SEQ ID NO:10序列的连续氨基酸。
本发明优选地涉及一种纯化或分离的肽,其特征在于它含有选自如下的氨基酸序列:
a)SEQ ID NO:2的序列;
b)SEQ ID NO:22的序列之至少6个连续氨基酸的片段的序列,优选地是SEQ ID NO:20的这样的序列;
c)在优化排比后,具有与a)或b)中定义的序列至少80%百分同一性的氨基酸序列。
本发明优选地涉及一种根据本发明的多肽,其特征在于其序列选自SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20和SEQ ID NO:22,或在优化排比后,具有与这些序列至少80%百分同一性的序列。
应当理解,本发明不涉及天然形式的多肽,即:所述多肽未从其环境中分离。具体地,本发明涉及通过从天然来源中纯化获得的肽,或通过遗传重组获得的肽,或通过化学合成获得的肽,且可能包含非天然氨基酸。利用本发明所述核苷酸序列之一进行的重组多肽的产生特别有利,因为其可获得纯度更高的目标多肽。
表述“在优化排比后,具有与参比序列至少80%,优选90%,95%或99%百分同一性的氨基酸序列的多肽”是指相对于参比序列而言,具有某些修饰的多肽,诸如特别是一种或多种缺失、截短、延伸、嵌合融合和/或一个或多个替换。
对于在优化排比后,具有与根据本发明的SEQ ID NO:2的序列(或其片段)至少80%,优选90%,95%或99%百分同一性的氨基酸序列的多肽而言,优选其中这样的变体多肽,所述变体多肽由上述变体核酸序列编码;特别是这样的多肽,相对于SEQ ID NO:2的序列或其片段而言,其氨基酸序列具有特别是相应于截短、缺失、替换和/或添加至少一个氨基酸残基的至少一种突变;更优选地是这样的变体多肽,其具有与病理条件相关的突变。
本发明还包括克隆和/或表达载体,其含有根据本发明的核酸序列。
这样的根据本发明的载体也是本发明的一部分,其特征在于其含有允许所述序列在宿主细胞中表达和/或分泌的元件,或用于细胞定位的序列。
这样的载体也是本发明的一部分,其特征在于其包含本发明的启动子和/或调控序列。
所述载体优选含有启动子、翻译起始和终止信号、以及用于调控转录的适宜区域。它们可稳定地保持在细胞内,且它们可任选地含有特定的关于翻译的蛋白质分泌之信号。
选择这些多种多样的控制信号以在细胞宿主中有功能。为实现此目的,根据本发明的核酸序列可被插入自主地在所选宿主中复制的载体中,或整合入所选宿主的载体中。
在自主复制的体系中,可根据宿主细胞优选地加以利用质粒或病毒型体系,病毒载体可特别地是腺病毒(Perricaudet,M.,et al.,LaRecherche,23:471-473,1992)、反转录病毒、lentiviruses,poxviruses或疱疹病毒(Epstein,A.,Medecine/sciences,8:902-911,1992)。本领域技术人员知晓可用于这些体系中各个体系的技术。
当期望将序列整合入宿主细胞的染色体中时,可使用例如质粒或病毒型体系,这些病毒例如可为反转录病毒(Temin,H.M.,Retrovirusvectors for gene transfer,Kucherlapati R.,ed,Gene Transfer,NewYork,Plenum Press,149-187,1986)或AAV(Carter,B.J.,Curr.Op.Biotechnology,3:533-539,1993)。
在非病毒型载体中,优选裸多核苷酸,诸如根据由VICAL公司开发的技术之裸DNA或裸RNA,酵母人工染色体(YAC)用于在酵母中表达,小鼠人工染色体(MAC)用于在鼠细胞中表达,且,优选地,人类人工染色体(HAC)用于在人细胞中表达。
这类载体可根据本领域技术人员已知的常用方法制备,其由此得到的克隆可利用标准方法被导入适当的宿主,诸如脂质体转染(Iipofection)、电穿孔或热冲击等。
本发明还包括宿主细胞,特别是真核和原核细胞,其已用根据本发明的载体转化,以及转基因动物(不包括人),其含有根据本发明的所述转化细胞之一。
可用于这些目的的细胞应当提及的当然有细菌细胞(Olins,P.O.,et al.,Curr.Op.Biotechnology,4:520-525,1993),和酵母细胞(Buckholz,R.G.,Curr.Op.Biotechnology,4:538-542,1993),以及动物,特别是哺乳动物细胞培养物(Edwards,C.P.,et al.,Curr.Op.Biotechnology,4:558-563,1993),以及特别是中国仓鼠卵巢细胞(CHO),和昆虫细胞,其中可使用采用杆状病毒的方法例如Luckow,V.A.,Curr.Op.Biotechnology,4:564-572,1993)。COS细胞构成了用于表达本发明的蛋白质之优选的细胞宿主。
在根据本发明的哺乳动物中,表达根据本发明的多肽之诸如小鼠、大鼠或兔的动物是优选的。
在本发明的动物中,优选是这样的动物,如小鼠、大鼠或兔,其特征在于编码SEQ ID NO:2的蛋白质之基因,或由这些动物同源基因编码的序列,是无功能的、或已被敲除、或具有至少一种突变。
这些转基因动物可通过如下获得:例如胚胎干细胞同源重组,这些干细胞向胚胎的转化,在实现可复制细胞系中的嵌合体的选择,以及所述嵌合体的生长。
这些转基因动物(优选小鼠)可过表达编码本发明蛋白质的基因、或其同源基因,或表达这样的基因,其中已经导入了突变。这些转基因动物,特别是小鼠可例如通过转染在强启动子或组织类型选择性的启动子控制下的基因的一个拷贝来获得,所述强启动子是自然界中普遍存在的;或在病毒转染后获得。
这些转基因动物(优选小鼠)可通过利用LOXP/CRE重组酶体系(Rohlmann,A.,et al,Nature Biotech.,14:1562-1565,1996)而失活,使得编码SEQ ID NO:2的序列之多肽的基因缺失,或者利用其他灭活该基因病毒的系统。
根据本发明的细胞和哺乳动物可用于如下述的产生本发明多肽的方法中,也可用作分析模型。
如上述转化的细胞或哺乳动物可因此用作模型,以研究本发明多肽与直接或间接参与本发明多肽之活性的化学或蛋白质化合物之间的相互作用,以此研究涉及的多种机制和相互作用。
其可特别地用于选择这样的产物,所述产物与根据本发明的多肽,尤其是SEQ ID NO:2序列的蛋白质或其根据本发明的变体相互作用,其作为一种辅助因子或作为抑制剂,特别是竞争性抑制剂,或其具有针对本发明多肽的激动剂或拮抗剂活性。优选地,所述转化的细胞或转基因动物可用作模型,特别是用作筛选用于对抗与该基因异常表达相关的疾病状况的产物。
本发明还涉及根据本发明的细胞、哺乳动物或多肽的用途,其用于筛选这样的化学化合物或生物化学化合物,其能够直接或间接与根据本发明多肽相互作用,和/或能够调控这些多肽的表达或活性。
本发明还涉及根据本发明的核酸序列的用途,其用于合成重组多肽。
用于产生呈重组形式的本发明多肽的方法包括例如本发明中的方法,其特征在于,转化的细胞,特别是本发明的细胞或哺乳动物在允许重组多肽表达的条件下生长,所述多肽由根据本发明的核酸序列编码;特征还在于回收所述重组多肽。
特征在于其能利用所述生产方法获得的重组多肽也是本发明的一部分。
如上述获得的重组多肽可呈糖基化和非糖基化形式,且可为或不为天然的四级结构。
重组多肽的序列也可被修饰以改进其稳定性,特别是在水性溶剂中的稳定性。
这类修饰为本领域技术人员周知,诸如,疏水结构域的缺失或疏水性氨基酸用亲水性氨基酸替换。
这些多肽可从上述定义的核酸序列产生,方法是根据本领域已知的用于产生重组多肽的技术。在这些情况中,所用的核酸序列置于允许其在细胞宿主中表达的信号控制之下。
用于产生重组多肽的有效体系需要具有载体和根据本发明的宿主细胞。
这些细胞可通过如下方法而获得:将插入到上述载体中的核苷酸序列导入宿主细胞,然后在允许转染的核苷酸序列复制和/或表达的条件下培养这些细胞。
用于纯化重组多肽的方法为本领域技术人员已知。重组多肽可从细胞裂解物和提取物中纯化,从培养液上清中获得,方法是单独或组合运用如下方法,诸如分级分离、层析方法、利用特异性单克隆抗体或多克隆抗体的免疫亲和技术,等。
根据本发明的多肽可利用化学合成的方法获得,方法是利用众多周知的肽合成方法之一,例如采用固相合成技术或采用部分固相合成技术,通过片段缩合或通过溶液中的常规合成。
固相合成技术是本领域技术人员周知的,参见例如J.M.Stewart,et al.,(Solid phase peptide synthesis,Pierce Chem.,Company,rochford,111,2nd ed.,1984)和M.Bodansky(Priciples of peptidesynthesis,1984)。
由化学合成和可能含有非天然氨基酸的相应的多肽也包括在本发明中。
其特征在于能特异性识别本发明多肽的单克隆抗体或多克隆抗体,或其片段,嵌合抗体或免疫偶联物也是本发明的一部分,同样地还有能够识别文件WO99/54461的SEQ ID NO:173序列或文件WO99/06548的SEQ ID NO:51序列的多肽之所述抗体或其片段。
特异性多克隆抗体可从用本发明多肽minimized动物血清中获得,特别是通过常规方法以遗传重组或以肽合成制备。
特异性识别根据本发明的某些多肽、变体或其免疫原性片段的抗体的优点将特别说明。
其特征在于能特异性识别SEQ ID NO:2序列的多肽之膜外结构域的单克隆抗体或多克隆抗体,或其片段,嵌合抗体或免疫偶联物是特别优选的。
可根据如Kohler和Milstein(Nature,256:495-497,1975)所述的常规方法通过培养杂交瘤获得特异性的单克隆抗体。
根据本发明的抗体为例如嵌合抗体、人抗体或Fab或F(ab’)2片段。其可呈免疫偶联物的形式,或呈标记的抗体形式,以获得可检测和/或定量的信号。
本发明还涉及用于检测和/或纯化根据本发明的多肽的方法,其特征在于它们使用了根据本发明的抗体。
本发明还包括纯化的多肽,其特征在于它们利用本发明的方法获得。
此外,除了其用于纯化本发明的多肽的用途,本发明的抗体,特别是单克隆抗体也可用于检测生物样品中的这些多肽。
因此,它们构成了一种用于免疫化学或免疫组织化学分析本发明多肽,特别是SEQ ID NO:2序列的多肽或其变体在特定组织区段中表达的方法,例如通过免疫荧光或金标记或用酶免疫偶联物。
特别地,它们可用于显示组织或生物学样品中这些多肽的异常表达。
更一般地,本发明的抗体可有利地用于任意情况中,其中正常或突变的本发明多肽的表达必须观察。
用于确定等位基因变化性、突变、缺失、杂合丧失、或任何根据本发明的基因之遗传异常性的方法也是本发明的一部分,其特征在于它们使用了本发明的核酸序列或抗体。
优选地,本发明涉及一种用于诊断与存在至少一种本发明基因的序列上的突变相关之疾病的方法,其中利用了来自患者的生物学样品,其特征在于它包括如下步骤:
a)如适当,从待分析的生物学样品中分离基因组DNA,或从生物学样品的RNA中获得cDNA;
b)利用本发明的引物,特异性扩增可能含有突变的基因之所述DNA序列;
c)分析所得的扩增产物,并将其序列与正常序列比较。
本发明还包括了一种用于诊断与本发明多肽,特别是SEQ ID NO:2序列的多肽或其变体的异常表达相关的疾病的方法,其特征在于将一种或多种根据本发明的抗体与待检测的生物材料在如下条件下接触:该条件使得在所述多肽与所述抗体之间可形成特异性免疫复合物,且其特征在于检测和/或定量可能形成的免疫复合物。
这些诊断方法是例如针对用于检测与编码SEQ ID NO:2序列的多肽之基因的异常表达(如过度表达或表达不足)相关的疾病之方法,方法是利用获自患者的生物学样本,通过确定本发明的序列是否以异常量存在。待分析的核酸序列可为基因组DNA、cDNA或mRNA。
当然,有大量方法用于显示基因中相对于野生型基因的突变。它们基本上可分为两种主要类型。第一种类型的方法是通过比较突变的序列与相应的野生型序列检测突变的存在与否,第二种类型是通过例如由于突变存在造成错配的证据来间接地检测突变存在与否。
这些方法可使用本发明的引物和探针。其为普通地纯化的核酸序列,含有至少15个核苷酸,优选20,25或30-200核苷酸,其特征在于其可特异性地与本发明的核酸序列杂交。
特异性杂交条件优选地如上述定义或见实施例。
在用于确定等位基因变异性、突变、缺失或遗传异常的方法中,优选含有至少一种“PCR”(聚合酶链反应)或“PCR-样”扩增步骤的方法,其用于扩增极有可能带有异常的本发明的靶序列,其中利用本发明寡核苷酸的引物对。在进行检测或用靶定产物滴定前,扩增的产物可用适当的限制性酶处理。
本发明基因的突变可能与其翻译产物的多种修饰有关,这其中包括可用于诊断的修饰。具体地,与这些突变相关的抗原性的修饰可允许开发特异性的抗体。这可通过这类方法区分突变的基因产物。所有这些修饰可用于这样的诊断方法中,其中使用了数种基于单克隆或多克隆抗体的方法,诸如通过RIA或通过ELISA,其中所述抗体能够识别正常的多肽或其突变变体。
另一方面,本发明包括用于筛选能直接或间接地与本发明的多肽(尤其是SEQ ID NO:2序列的多肽)或与本发明的核酸相互作用的化学或生物化学化合物的方法,和/或用于实现调控本发明多肽的表达或活性的方法。
因此,本发明包括用于筛选能体内或体外与本发明的核酸相互作用的化学或生物化学化合物的方法,其特征在于该方法包括将(正常或异常地)表达编码SEQ ID NO:2序列的多肽的基因之细胞与候选化合物接触,以及直接或间接地检测SEQ ID NO:2序列的多肽的表达或活性的修饰。
筛选可用于检测能修饰本发明多肽(尤其是SEQ ID NO:2序列的多肽)的表达的水平和/或特异性之化合物,方法是通过与编码SEQID NO:2序列的多肽之基因的启动子上的转录因子结合位点之竞争性结合,或是通过直接与转录因子结合。
编码SEQ ID NO:2序列的多肽的基因之表达水平及该基因的定位,可通过在溶液中与PCT专利WO 97/05277中所示的大探针杂交来分析。
编码SEQ ID NO:2序列的多肽之基因表达的定量分析可利用DNA矩阵来进行,可能地,包括本发明SEQ ID NO:1的cDNA序列之全部或部分,且其长度足以允许对能与之杂交的mRNA表达的特异性检测。片段优选地含有至少15、25、50、100或500个连续的核酸序列之核苷酸,所述核酸序列如Schena et al.,Science,270:467-470,1995。
编码根据本发明之SEQ ID NO:2序列的多肽的基因之表达的定量分析,可用本发明的DNA在如Pietu et al.,1996中所述的DNA矩阵上分析来进行。根据本发明的DNA或其片段通过PCR扩增,并结合到膜上。从多种组织或细胞中产生的mRNA用放射性核苷酸标记。在受控条件下杂交和洗涤后,用磷成像仪或通过放射自显影检测杂交的mRNA。实验进行双份,并可进行差异表达的mRNA的定量分析。上述提及的技术允许在同一细胞或同一组织中,对编码根据本发明SEQ ID NO:2序列的多肽之基因表达水平的分析,这取决于不同的情况,例如诱导或非诱导型的,以及,允许在可变化的条件下对这种表达的组织特异性的分析。
本发明的另一方面涉及用于鉴定能够与本发明多肽(尤其是SEQID NO:2序列的多肽)结合的分子的方法。这类分子可用于调控这些多肽的生物活性。例如,这类分子可用于刺激或降低这些多肽的活性。
因此,本发明包括用于筛选能调控SEQ ID NO:2序列的多肽的活性的化学或生物化学化合物的方法,其特征在于该方法包括将本发明的多肽或细胞与候选化合物接触,并检测SEQ ID NO:2序列的多肽之活性的修饰。
本发明的一个主题是用于筛选体内或体外能与SEQ ID NO:2序列的多肽结合的化学或生物化学化合物的方法,其特征在于该方法包括将本发明的多肽或细胞与候选化合物接触,并检测候选化合物与所述多肽之间复合物的形成。
有多种用于鉴定本发明的SEQ ID NO:2序列的多肽的配体的方法。
例如,但不限于此,可提及的有用于鉴定能与SEQ ID NO:2序列的多肽相互作用的分子的方法,其利用细菌或酵母双杂交系统,诸如Macchmaker双杂交系统2(参见由Macchmaker双杂交系统2附带的手册,目录号K1604-1,Clontech)。
为研究SEQ ID NO:2序列的多肽与小分子(诸如那些由组合化学产生的)的相互作用,可利用与HPLC偶联的微量透析(microdialysis),如Wang et al.,Chromatographia,44:205-208,1997所述,或利用如Busch et al.,J.Chromatogr.,777:311-328,1997所述的亲和毛细管电泳。
在其他方法中,能够与本发明SEQ ID NO:2序列的多肽相互作用的肽或小分子可以与可检测标记连接,诸如放射活性的、荧光或酶促标记。将这些经标记的分子与所述固定化的本发明多肽在允许特异性相互作用的条件下接触。在去除非特异性连接的分子后,通过适当的方法检测结合的分子。
BIACORE技术也可用于进行能与SEQ ID NO:2序列的多肽相互作用的化合物的筛选。该技术如A.Szabo et al.,Curr.Opin.Struct.Biol.,5:699-705,1995所述,可实时检测分子间的相互作用。该技术基于SPR(表面胞质共振)现象。此方法的优点之一为其可确定SEQ IDNO:2序列的多肽与相互作用分子的结合常数。因此,其可特异性筛选具有高或低结合常数的分子。
与本发明SEQ ID NO:2序列的多肽相互作用的蛋白质或其他分子可利用亲和柱鉴定,所述亲和柱中含有本发明的所述多肽或其片段。SEQ ID NO:2序列的多肽或其片段可利用常规技术与柱连接,包括与适当的柱基质(诸如琼脂糖、Affi胶或其他本领域周知的基质)化学偶联。本发明的另一方面中,亲和柱可含有嵌合蛋白质,其中所述SEQ ID NO:2序列的多肽或其片段可与例如谷胱甘肽-S-转移酶融合。上述待检测的分子然后可上样于柱中。可与SEQ ID NO:2序列的多肽相互作用的分子由柱滞留,然后通过洗脱分离。
本发明还涉及一种用于筛选和/或选择可与基因的启动子和/或调控序列相互作用之化合物的方法,所述基因编码本发明SEQ ID NO:2序列的多肽。
编码与基因的启动子和/或调控序列相互作用之蛋白质的核酸可利用单杂交洗脱筛选和/或选择,诸如在由Macchmaker单杂交系统试剂盒中附带的手册所述的方法(目录号K1603),其中所述基因编码本发明SEQ ID NO:2序列的多肽。
可利用“报道基因”,诸如碱性磷酸酶、β-半乳糖苷酶或GFP(绿色荧光蛋白)筛选能够修饰编码本发明SEQ ID NO:2序列的多肽之基因表达的化合物,所述修饰是通过与其调控和/或启动子序列连接。候选化合物对表达的影响因此可以评估,其中所述表达是由编码本发明SEQ ID NO:2序列的多肽之基因的调控和/或启动子序列产生的。
另一方面,本发明还包括本发明的核酸或多肽的用途,本发明细胞的用途,或本发明哺乳动物的用途,其用于研究本发明SEQ ID NO:2序列的多肽之表达或活性,和/或研究SEQ ID NO:2序列的多肽与能参与其活性之化学或生物化学化合物之间的直接或间接相互作用。
其特征在于是利用上述方法所筛选的化学或生物化学化合物也是本发明的一部分。
在这些化合物中,优选这样的化学或生物化学化合物,其可调控SEQ ID NO:2序列的多肽之表达或活性。
表述“可调控本发明SEQ ID NO:2序列的多肽之表达或活性的化合物’是指这样的化合物,其尤其是可降低、稳定或增加本发明SEQID NO:2序列的多肽的量或特异性;或者促进或抑制该多肽的总体活性或特定结构域之一的活性;或者重建编码本发明SEQ ID NO:2序列的多肽之基因的正常表达,当例如发现遗传异常时。
这些化合物可例如作为配体特异性的与本发明的SEQ ID NO:2序列的多肽或其结构域相互作用,如作为辅因子或抑制剂,尤其是竞争性抑制剂,或具有针对本发明SEQ ID NO:2所述多肽的活性而言的激动剂活性或拮抗剂活性。这些化合物还可通过中和本发明SEQ IDNO:2的所述多肽之天然特异性配体,并因此抑制所述多肽之由配体诱导的活性而起作用;或者通过中和这些天然配体的结合位点而起作用。
本发明还包括本发明的化合物,其特征在于它们是本发明SEQ IDNO:2序列的多肽活性之拮抗剂或激动剂。
本发明还包括本发明的化合物,其特征在于它们能结合于本发明的SEQ ID NO:2序列的多肽。
特别地,在本发明的化合物中,优选如下化合物,其特征在于它们选自:
-根据本发明的抗体;
-根据本发明的多肽,包括分别在文献WO 99/54461和WO99/06458中描述的SEQ ID NO:173和SEQ ID NO:51的多肽;
-根据本发明的多肽,其特征在于其相应于本发明SEQ ID NO:2序列的多肽之片段或可溶性级分;
-根据本发明的核酸,包括文献WO 99/54461的SEQ ID NO:11序列的核酸,文献WO 99/06548的SEQ ID NO:51的序列的核酸,以及EMBL文件中相应鉴别号AI 672868、AA890726或AI30140序列的核酸,其可为有义或反义的,以及
-根据本发明的载体。
在另一方面,本发明包括根据本发明的化合物,作为用于预防和/或治疗疾病的药物产品,所述疾病与本发明SEQ ID NO:2序列的多肽之表达和/或活性异常相关。
特别地,本发明包括根据本发明的化合物,作为用于预防和/或治疗免疫疾病,特别是用于预防和/或治疗自身免疫疾病或癌症的药物产品。
最后,本发明包括根据本发明的化合物,作为用于预防和/或治疗病毒、真菌、细菌或寄生虫型感染的药物产品。
本发明的化合物作为药物产品中的活性成分优选为可溶性的形式,其与药学可接受的载体相组合。
优选地,这些化合物可系统性施用,特别是静脉内、肌肉内、皮内或经口施用。
其最佳制剂形式、剂量和施用方法可根据在建立适于患者的治疗方案中通常考虑的标准来确定,诸如例如患者的年龄、体重,患者的一般状况的严重性,对治疗的耐受性和副作用等。
本发明的其他特征和有利之处用实施例和附图以及下述的附图标记展现在说明书如下的部分。
附图说明
图1A和1B
通过Northern印迹利用市售膜(Multiple Tissue Northern;Clontech,Palo Alto,CA)分析编码SEQ ID NO:2的肽之RNA的表达。表示了用两个不同膜获得的代表性结果。1.胎肝,2.骨髓,3.单核细胞4.胸腺,5.外周淋巴结,6.脾,7.单核细胞,8.肺,9.胎盘,10.小肠,11.肝,12.肾,13.脾,14.胸腺,15.结肠,16.骨骼肌,17.心,18.脑。
编码SEQ ID NO:2序列的多肽之mRNA在单核细胞、脾、肺和小肠中表达。
图2
编码SEQ ID NO:2序列的多肽之RNA的表达通过PCR在多种未受刺激的单核细胞亚群中进行评估:T淋巴细胞(泳道1),B淋巴细胞(泳道2),NK细胞(泳道3)和单核细胞(泳道4),以及在体外产生的树突细胞(泳道5)。RNA的完整性以及合成的cDNA的质量通过扩增编码GAPDH分子的cDNA评估。
编码SEQ ID NO:2序列的多肽之mRNA在单核细胞、脾、肺和小肠中表达。
图3
通过PCR分析在人肿瘤细胞系中编码SEQ ID NO:2序列的多肽之mRNA。所分析的细胞系为:泳道1:A549,泳道2:THP1,泳道3:HTB10,泳道4:T24,泳道5:Daudi,泳道6:A427,泳道7:U373,泳道8:HEP2,泳道9:HT29,泳道10:BT20。
编码SEQ ID NO:2序列的多肽之mRNA在多种人肿瘤细胞系中组成型地表达。
图4
用含有编码SEQ ID NO:2序列的多肽之完整cDNA的载体pCDNA3.1转染COS细胞。通过冻/融法在转染后3天获得细胞的细胞质级分和非细胞质级分。通过SDS-PAGE电泳分离蛋白质提取物;通过Western印迹利用抗-c-myc-抗体评估重组蛋白质的表达。泳道1:非细胞质级分,泳道2:细胞质级分。
SEQ ID NO:2序列的多肽在转染细胞的非细胞质级分中表达。实施例1编码SEQ ID NO:2序列的多肽之cDNA的克隆
借助于在人细胞表面选择性表达的新分子之鉴别,在人EST(表达的序列标签)文库中进行可能含有跨膜基元的序列之搜索。
方法学
编码可能的跨膜基元的人EST序列利用TopPred2程序(用于推测膜结合蛋白质的程序)鉴别,该程序可从网站:http://www.biokemi.su.se/Toppred2获得。在所得的许多序列中,选择EST#AI301140,这是基于如下事实:翻译产物具有跨膜基元(位置14-34,预期得分1,868),和核苷酸序列不与任何已知序列同源。可得核苷酸序列(http://www.ncbi.nih.nlm.gov)具有一个带终止密码子的116个氨基酸的开放阅读框。然后我们在人EST数据库中进行了针对序列#AI301140同源性的搜索。由此,根据与序列#AI301140的同源性,选择了18个新核苷酸序列。这些克隆从Research Genetics(New York,NY)获得。利用适当的抗生素筛选细菌克隆。利用市售的试剂盒纯化质粒DNA(Midiprep DNA提取试剂盒,Qiagen,德国),并用ABI Prism Bigdye Terminator DNA试剂盒(Perkin ElmerApplied Biosystems,Warringtong,英国)依据制造商的指示测序。用ABI Prism377自动测序仪分析序列(Perkin Elmer AppliedBiosystems)。利用测序仪序列分析程序分析这些序列(Genecods,AnnArbor,MI)。在多个序列排比后,确定了具有一个开放阅读框的1100碱基对之DNA片段,其中含有终止密码子和poly-A尾。此片段用相应于排比的序列之5’和3’的特异性引物扩增。利用市售的试剂盒,通过一系列5’末端(5’RACE)的PCR扩增循环获得完整的cDNA(Frohman,M.A.,et al,.Proc.Natl.Acad.Sci.USA,85:8998-9002,1988)。DNA矩阵是从人脑和骨髓中制备的cDNA(Marathon-ReadycDNA:Clontech)。利用从网站
http://www.cbs.dtu.dk/services/SignalP可得的程序确定信号肽的推测。该分子具有预期分子量为113kDa,pI6.5和3个可能的糖基化位点(苏氨酸和丝氨酸)。
结果
通过分析可得的人EST数据库选择了一个cDNA克隆(#AI301140),其编码具有跨膜基元的分子。所得SEQ ID NO:1序列的cDNA为3331碱基对长,未显示与任何数据库中已知的分子同源。其具有一个3042碱基对的开放阅读框,编码推定的1013氨基酸蛋白。起始密码子位于SEQ ID NO:1的位置nt 45-47,终止密码子位于位置nt3084-3086。蛋白质序列的分析预期此SEQ ID NO:2的蛋白质具有序列aa1-41的信号肽,序列aa42-911的细胞外结构域,序列aa912-930的跨膜结构域和序列aa931-1013的细胞内结构域。
SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7和SEQ ID NO:9的序列相应于SEQ ID NO:1的片段,其不与实施例1中所选的EST序列同源。实施例2编码SEQ ID NO:2序列的多肽之mRNA的组织表达
方法学
利用市售膜通过Northern印迹分析SEQ ID NO:2序列的多肽的组织表达,所述方法使得能够在多种组织中评估mRNA的表达(Multiple Tissue Northern或MTN,Clontech)。利用如下特异性引物通过PCR产生相应于nt385-nt1137的cDNA片段,所述引物的序列由SEQ ID NO:1的序列推出。纯化后,将25μg此片段利用市售的试剂盒(Multiprime DNA标记试剂盒,Amersham,Amersham,英国)和放射性核苷酸[α-32P]-dATP进行放射性标记。利用层析纯化标记的探针。将膜在2×SSC溶液(300mM柠檬酸钠,pH7,3M NaCl,0.1%十二烷基硫酸钠)中50℃下带晃动预温育2小时。然后将膜在50℃下带晃动地与放射性标记的探针(其已经在95℃下热变性10分钟)温育过夜。在2×SSC-0.1%SDS溶液中洗涤(55℃下带晃动15分钟)3次后,将膜对放射自显影胶片(Kodak,Rochester,NY)曝光24小时。
结果
示于图1A和1B的Northern印迹膜显示在外周血单核细胞和小肠中有强杂交信号;编码SEQ ID NO:2序列的多肽之mRNA也在胸腺、脾、脑和肺中表达。
因此,编码SEQ ID NO:2序列的多肽之mRNA优势地在人淋巴组织中表达。实施例3编码SEQ ID NO:2序列的多肽之mRNA在多种循环血细胞群中的表达
方法学
分离多种外周血的细胞亚群
从健康志愿者个体中通过leukophoresis(在存在抗凝剂诸如肝素锂的情况下)获取血液。通过在Ficoll-Hypaque(密度1.077,Pharmacia,Uppsala,瑞典)上梯度离心分离单核细胞(MNC)(含有T淋巴细胞,B淋巴细胞和单核细胞)。简言之,在室温下于1500转/分离心30分钟。回收位于Ficoll-血浆界面的MNC,并在RPMI1640培养基存在的条件下洗涤两次(Life technologies)。通过“玫瑰花环(rosetting)”技术纯化T淋巴细胞。简言之,以200×106细胞/ml的浓度重悬MNC,并与1ml含有50%绵羊血红细胞的溶液(BioMerieus,Marcyl’Etoile,法国)混合。在4℃下细胞悬液温育过夜。温和地重悬后,通过在Ficoll-Hypaque离心分离T细胞(室温下于1500转/分离心30分钟)。绵羊血红细胞-T淋巴细胞复合物汇集在试管底部。通过两次连续的低渗透压冲击裂解血红细胞。如此分离的T细胞的纯度通过用FITC抗-CD3抗体标记来评估为>95%。简言之,在FACS缓冲液(10mM磷酸缓冲液,pH7.4,含有1%牛血清白蛋白,和0.01%叠氮钠)中洗涤细胞,然后将其分散于96孔板(圆锥形底部)(Nunc,Roskilde,丹麦)的孔中,浓度为50μl FACS缓冲液体积中2×105个细胞。将FITC标记的抗人CD3抗体(BectonDickinson)或FITC标记的对照同型抗体(Becton Dickinson)加入每一孔。4℃下温育20分钟后,用200μl FACS缓冲液洗涤细胞两次,然后重悬于200μl相同的缓冲液中。通过FACS分析在T淋巴细胞表面的CD3表达。
利用包被有抗人CD14抗体的磁珠通过正筛选技术纯化单核细胞(MACSTM系统;Miltenyi Biotex,Bergish Gladbach,德国)。简言之,将单核细胞(300×106/ml)与60ml珠(Miltenyi)悬浮液4℃下在PBS/2mM EDTA/0.5%牛血清白蛋白(PBS/EDTA/BSA)缓冲液中温育15分钟。洗涤后,将细胞上样于置于磁场中的柱上。用PBS/EDTA/BSA溶液洗涤三次后,将柱移出磁场,通过重力收集单核细胞。通过基于体积/颗粒参数的细胞仪评估,单核细胞的纯度大于95%。
从如上纯化的单核细胞中体外产生未成熟的人树突细胞。以106个细胞/ml的浓度在6-孔培养板(Nunc,Roskilde,丹麦)中在每孔5ml如下培养液(以后称为完全培养液)中培养单核细胞:RPMI1640培养液,其补加了10%胎牛血清(在56℃加热30分钟),2mM L-谷氨酰胺,50U/ml的青霉素和50μm/ml链霉素(Life technologies)。用20ng/ml重组人IL-4和20ng/ml重组人GM-CSF(R&D Systems,Abingdon,英国)活化细胞。在培养6天后(37℃,5%CO2,湿润气氛),通过流式细胞仪分析细胞表型,方法是利用荧光素标记的抗人CD1a抗体或抗人CD83抗体(Becton Dickinson)分析CD1a和CD83分子的表达,所述抗体用荧光素标记的抗鼠免疫球蛋白抗体(Silenus,Hauworth,澳大利亚)检测。未成熟的树突细胞通过CD1a分子的表达(评价荧光强度(MFI)>100)和CD83分子的不表达来表征。
在用荧光素标记的抗CD56抗体(Becton Dickinson)和藻红蛋白标记的抗CD-3抗体标记后,NK细胞(CD56+/CD3-)从MNC中利用流式细胞仪分离。用上述抗体标记细胞。将细胞通过FACSVantage(Becton Dickinson);纯度大于98%。
从手术切除后收集的扁桃体中分离B淋巴细胞。简言之,将扁桃体机械研磨,然后通过具有50μm孔隙的筛子。通过如上述在Ficoll-Hypaque梯度上的离心分离单核细胞。将T淋巴细胞从MNC悬液中移出,方法是利用上述的玫瑰花环技术。利用FITC标记的抗CD20抗体,B-细胞群的纯度由流式细胞仪评估为纯度大于95%。
PCR技术
编码SEQ ID NO:2序列的多肽之RNA的表达通过PCR在多种MNC亚群中分析。简言之,在PBS中洗涤细胞两次,然后在Trizol核酸提取液(Life technologies)中裂解。通过两次连续的酚/氯仿/异戊酸(25∶24∶1)(Life technologies)提取去除蛋白质。将等体积异丙醇加入水相中以沉淀核酸(-20℃过夜)。离心收集核酸(12000转/分,4℃,30分钟)。沉淀用乙醇溶液在70℃洗涤一次。将沉淀于水中重悬,任何通过分光光度计(λ=260nm)定量RNA。变性后(65℃,10分钟),利用oligo-dT引物借助于市售的试剂盒(Promega,Madison,MI)依照制造商指示,反转录2μg总RNA。编码SEQ ID NO:2序列的多肽之cDNA通过PCR利用如下引物扩增:
贴45页11-12行
用于制备cDNA的RNA之完整性通过利用如下引物评价编码GAPDH分子的cDNA之表达来分析:
贴45页18-19行
反应混合物如下:1μl cDNA(相应于25ng总RNA),25pmol引物,200μM dNTP,1.5mM MgCl2,10mM Tris-HCl,pH=8.3,50mM KCl和0.4单位的Taq聚合酶(Perkin Elmer)。PCR条件为:95℃5分钟,30个如下循环:94℃1分钟,55℃1分钟和72℃1分钟。以及最后72℃延伸5分钟。样品加入样品缓冲液中(0.25%溴酚蓝,40%蔗糖)。在含有溴乙锭的1%琼脂糖凝胶上分析样品。在迁移后,通过对凝胶的UV显示目视检测样品。
结果
通过PCR仅在未刺激的外周T淋巴细胞中检测到编码SEQ IDNO:2序列的多肽之mRNA。其在其他检测的单核细胞群(NK细胞,B淋巴细胞,单核细胞)中不存在。在体外产生的人树突细胞中也检测到mRNA。结果见图2
因此,编码SEQ ID NO:2序列的多肽之mRNA选择性地由循环血中的T淋巴细胞表达。实施例4编码SEQ ID NO:2序列的多肽之RNA在多种肿瘤细胞系中的表达
方法学
我们在多种肿瘤细胞系中评估了编码SEQ ID NO:2序列的多肽之mRNA的表达。这些细胞系获自ATCC且根据提供的说明加以培养。如上述进行总RNA提取和cDNA合成。编码SEQ ID NO:2序列的多肽之RNA和GAPDH分子的表达通过上述PCR进行分析。
结果
PCR实验(图3)显示如下人细胞系组成型地表达编码SEQ IDNO:2序列的多肽之mRNA:肺腺瘤(A549系),单核白血病(THP1系),成神经细胞瘤(HTB10系),膀胱癌(T24),Burkitt淋巴瘤(Daudi系),肺癌(A427系),成胶质细胞瘤(U373系),喉癌(HEP2系),结肠癌(HT29系)和乳腺癌(BT20系)。
因此,编码SEQ ID NO:2序列的多肽之RNA在一些肿瘤细胞系中表达。实施例5 SEQ ID NO:2的重组多肽主要在转染的COS细胞之膜级分中表达
方法学
用含有编码SEQ ID NO:2的多肽之插入片段的载体pCDNA3.1(InVitrogen)转染COS细胞。这样的体系可使c-myc表位加入重组分子。转染通过脂质体转染技术进行(Fugene;Boehringer Mannheim,德国)。转染后3天,收集细胞,并用PBS洗涤3次。然后将细胞经过3次迅速的冻/融,4℃下于12000转/分离心10分钟。上清液相应于细胞质级分,而沉淀相应于膜组分和核加细胞器。将沉淀重悬于含有0.5% Nonidet P40和蛋白酶抑制剂混合物(Boehringer Mannheim,德国)的10mM磷酸缓冲液中(pH7.4)4℃下20分钟。然后,将细胞裂解液与4℃下12000转/分离心10分钟,以去除核。相应于膜和细胞质级分的上清液加入等体积的10mM磷酸缓冲液中(含有0.1%溴酚蓝,5%甘油和0.1%巯基乙醇)。然后根据其分子量大小通过聚丙烯酰胺凝胶电泳分离蛋白质(堆积胶5%,分离胶10%)。相应于5×106个细胞的蛋白质的量加入每一泳道。迁移后,通过Western印迹将蛋白质转运到硝酸纤维素膜。在含有10%脱脂牛奶的磷酸溶液中浸泡后,将膜与浓度为10μg/ml抗c-myc抗体(克隆9E10;ATCC,Manassas,VA)在4℃下温育过夜,带有晃动。用含有1 0%脱脂牛奶的磷酸缓冲液洗涤后,将膜与过氧化物酶标记的抗鼠免疫球蛋白抗体(Amersham)在室温下温育2小时,带有晃动。用磷酸缓冲液洗涤膜。通过化学荧光检测利用Amershanm的ECL系统检测结合的抗体。
结果
如图4中Western印迹显示重组蛋白质主要在细胞提取物的非可溶性级分(细胞质膜,加细胞质内细胞器)中表达。这一结果以及可能的跨膜结构域的推定提示重组多肽在转染细胞的表面表达。
因此,SEQ ID NO:2的重组多肽在转染的COS细胞之膜级分(非细胞质部分)中表达。实施例6编码SEQ ID NO:2的基因位于染色体11q33-11q34
方法学
编码SEQ ID NO:2序列的多肽之基因的染色体定位通过搜索Unigene数据库利用EST序列#AI3011410获得。
结果
显示与EST#AI3011410具有98%同源性的基因组DNA序列见于染色体11q的33-34簇的片段中。许多肿瘤易感性基因已经被定位于基因组区域11q23-40(Koreth,J.,et al.,J.Pathol.,187:28-38,1999)。
编码SEQ ID NO:2序列的多肽之基因位于染色体11区域q33-34。
这一结果使得可证实SEQ ID NO:2序列的多肽可参与调控免疫反应,且其表达的修饰可与免疫疾病相关,所述免疫疾病可能导致这样的细胞成为肿瘤性质的,所述细胞显示编码SEQ ID NO:2序列的多肽之基因表达异常。
序列表<110>PIERRE FABRE MEDICAMENT<120>在肿瘤细胞中表达并参与免疫反应的调控之基因的克隆、表达和表征<130>D18391<140><141><1S0>FR 99 13629<151>1999-10-29<160>22<170>PatentIn Ver.2.1<210>1<211>3331<212>ADN<213>人<220><221>CDS<222>(45)..(3083)<400>1gtgattactc actatagggc tcgagcggcc gcccgggcag gtct atg gct gag cct 56
Met Ala Glu Pro
1ggg cac agc cac cat ctc tcc gcc aga gtc agg gga aga act gag agg 104Gly His Ser His His Leu Ser Ala Arg Val Arg Gly Arg Thr Glu Arg5 10 15 20cgc ata ccc cgg ctg tgg cgg ctg ctg ctc tgg gct ggg acc gcc ttc 152Arg Ile Pro Arg Leu Trp Arg Leu Leu Leu Trp Ala Gly Thr Ala Phe
25 30 35cag gtg acc cag gga acg gga ccg gag ctt cat gcc tgc aaa gag tct 200Gln Val Thr Gln Gly Thr Gly Pro Glu Leu His Ala Cys Lys Glu Ser
40 45 50gag tac cac tat gag tac acg gcg tgt gac agc acg ggt tcc agg tgg 248Glu Tyr His Tyr Glu Tyr Thr Ala Cys Asp Ser Thr Gly Ser Arg Trp
55 60 65agg gtc gcc gtg ccg cat acc ccg ggc ctg tgc acc agc ctg cct gac 296Arg Val Ala Val Pro His Thr Pro Gly Leu Cys Thr Ser Leu Pro Asp
70 75 80ccc gtc aag ggc acc gag tgc tcc ttc tcc tgc aac gcc ggg gag ttt 344Pro Val Lys Gly Thr Glu Cys Ser Phe Ser Cys Asn Ala Gly Glu Phe85 90 95 100ctg gat atg aag gac cag tca tgt aag cca tgc gct gag ggc cgc tac 392Leu Asp Met Lys Asp Gln Ser Cys Lys Pro Cys Ala Glu Gly Arg Tyr
105 110 115tcc ctc ggc aca ggc att cgg ttt gat gag tgg gat gag ctg ccc cat 440Ser Leu Gly Thr Gly Ile Arg Phe Asp Glu Trp Asp Glu Leu Pro His
120 125 130ggc ttt gcc agc ctc tca gcc aac atg gag ctg gat gac agt gct gct 488Gly Phe Ala Ser Leu Ser Ala Asn Met Glu Leu Asp Asp Ser Ala Ala
135 140 145gag tcc acc ggg aac tgt act tcg tcc aag tgg gtt ccc cgg ggc gac 536Glu Ser Thr Gly Asn Cys Thr Ser Ser Lys Trp Val Pro Arg Gly Asp
150 155 160tac atc gcc tcc aac acg gac gaa tgc aca gcc aca ctg atg tac gcc 584Tyr Ile Ala Ser Asn Thr Asp Glu Cys Thr Ala Thr Leu Met Tyr Ala165 170 175 180gtc aac ctg aag caa tct ggc acc gtt aac ttc gaa tac tac tat cca 632Val Asn Leu Lys Gln Ser Gly Thr Val Asn Phe Glu Tyr Tyr Tyr Pro
185 190 195gac tcc agc atc atc ttt gag ttt ttc gtt cag aat gac cag tgc cag 680Asp Ser Ser Ile Ile Phe Glu Phe Phe Val Gln Asn Asp Gln Cys Gln
200 205 210ccc aat gca gat gac tcc agg tgg atg aag acc aca gag aaa gga tgg 728Pro Asn Ala Asp Asp Ser Arg Trp Met Lys Thr Thr Glu Lys Gly Trp
215 220 225gaa ttc cac agt gtg gag cta aat cga ggc aat aat gtc ctc tat tgg 776Glu Phe His Ser Val Glu Leu Asn Arg Gly Asn Asn Val Leu Tyr Trp
230 235 240aga acc aca gcc ttc tca gta tgg acc aaa gta ccc aag cct gtg ctg 824Arg Thr Thr Ala Phe Ser Val Trp Thr Lys Val Pro Lys Pro Val Leu245 250 255 260gtg aga aac att gcc ata aca ggg gtg gcc tac act tca gaa tgc ttc 872Val Arg Asn Ile Ala Ile Thr Gly Val Ala Tyr Thr Ser Glu Cys Phe
265 270 275ccc tgc aaa cct ggc acg tat gca gac aag cag ggc tcc tct ttc tgc 920Pro Cys Lys Pro Gly Thr Tyr Ala Asp Lys Gln Gly Ser Ser Phe Cys
280 285 290aaa ctt tgc cca gcc aac tct tat tca aat aaa gga gaa act tct tgc 968Lys Leu Cys Pro Ala Asn Ser Tyr Ser Asn Lys Gly Glu Thr Ser Cys
295 300 305cac cag tgt gac cct gac aaa tac tca gag aaa gga tct tct tcc tgt 1016His Gln Cys Asp Pro Asp Lys Tyr Ser Glu Lys Gly Ser Ser Ser Cys
310 315 320aac gtg cgc cca gct tgc aca gac aaa gat tat ttc tac aca cac acg 1064Asn Val Arg Pro Ala Cys Thr Asp Lys Asp Tyr Phe Tyr Thr His Thr325 330 335 340gcc tgc gat gcc aac gga gag aca caa ctc atg tac aaa tgg gcc aag 1112Ala Cys Asp Ala Asn Gly Glu Thr Gln Leu Met Tyr Lys Trp Ala Lys
345 350 355ccg aaa arc tgt agc gag gac ctt gag ggg gca gtg aag ctg cct gcc 1160Pro Lys Ile Cys Ser Glu Asp Leu Glu Gly Ala Val Lys Leu Pro Ala
360 365 370tct ggt gtg aag acc cac tgc cca ccc tgc aac cca ggc ttc ttc aaa 1208Ser Gly Val Lys Thr His Cys Pro Pro Cys Asn Pro Gly Phe Phe Lys
375 380 385acc aac aac agc acc tgc cag ccc tgc cca tat ggt ccc tac tcc aat 1256Thr Asn Asn Ser Thr Cys Gln Pro Cys Pro Tyr Gly Pro Tyr Ser Asn
390 395 400ggc tca gac tgt acc cgc tgc cct gca ggg act gaa cct gct gtg gga 1304Gly Ser Asp Cys Thr Arg Cys Pro Ala Gly Thr Glu Pro Ala Val Gly405 410 415 420ttt gaa tac aaa tgg tgg aac acg ctg ccc aca aac atg gaa acg acc 1352Phe Glu Tyr Lys Trp Trp Asn Thr Leu Pro Thr Asn Met Glu Thr Thr
425 430 435gtt ctc agt ggg atc aac ttc gag tac aag ggc atg aca ggc tgg gag 1400Val Leu Ser Gly Ile Asn Phe Glu Tyr Lys Gly Met Thr Gly Trp Glu
440 445 450gtg gct ggt gat cac att tac aca gct gct gga gcc tca gac aat gac 1448Val Ala Gly Asp His Ile Tyr Thr Ala Ala Gly Ala Ser Asp Asn Asp
455 460 465ttc atg att ctc act ctg gtt gtg cca gga ttt aga cct ccg cag tcg 1496Phe Met Ile Leu Thr Leu Val Val Pro Gly Phe Arg Pro Pro Gln Ser
470 475 480gtg atg gca gac aca gag aat aaa gag gtg gcc aga atc aca ttt gtc 1544Val Met Ala Asp Thr Glu Asn Lys Glu Val Ala Arg Ile Thr Phe Val485 490 495 500ttt gag acc ctc tgt tct gtg aac tgt gag ctc tac ttc atg gtg ggt 1592Phe Glu Thr Leu Cys Ser Val Asn Cys Glu Leu Tyr Phe Met Val Gly
505 510 515gtg aat tct agg acc aac act cct gtg gag acg tgg aaa ggt tcc aaa 1640Val Asn Ser Arg Thr Asn Thr Pro Val Glu Thr Trp Lys Gly Ser Lys
520 525 530ggc aaa cag tcc tat acc tac atc att gag gag aac act acc acg agc 1688Gly Lys Gln Ser Tyr Thr Tyr Ile Ile Glu Glu Asn Thr Thr Thr Ser
535 540 545ttc acc tgg gcc ttc cag agg acc act ttt cat gag gca agc agg aag 1736Phe Thr Trp Ala Phe Gln Arg Thr Thr Phe His Glu Ala Ser Arg Lys
550 555 560tac acc aat gac gtt gcc aag atc tac tcc atc aat gtc acc aat gtt 1784Tyr Thr Asn Asp Val Ala Lys Ile Tyr Ser Ile Asn Val Thr Asn Val565 570 575 580atg aat ggc gtg gcc tcc tac tgc cgt ccc tgt gcc cta gaa gcc tct 1832Met Asn Gly Val Ala Ser Tyr Cys Arg Pro Cys Ala Leu Glu Ala Ser
585 590 595gat gtg ggc tcc tcc tgc acc tct tgt cct gct ggt tac tat att gac 1880Asp Val Gly Ser Ser Cys Thr Ser Cys Pro Ala Gly Tyr Tyr Ile Asp
600 605 610cga gat tca gga acc tgc cac tcc tgc ccc cct aac aca att ctg aaa 1928Arg Asp Ser Gly Thr Cys His Ser Cys Pro Pro Asn Thr Ile Leu Lys
615 620 625gcc cac cag cct tat ggt gtc cag gcc tgt gtg ccc tgt ggt cca ggg 1976Ala His Gln Pro Tyr Gly Val Gln Ala Cys Val Pro Cys Gly Pro Gly
630 635 640acc aag aac aac aag atc cac tct ctg tgc tac aat gat tgc acc ttc 2024Thr Lys Asn Asn Lys Ile His Ser Leu Cys Tyr Asn Asp Cys Thr Phe645 650 655 660tca cgc aac act cca acc agg act ttc aac tac aac ttc tcc gct ttg 2072Ser Arg Asn Thr Pro Thr Arg Thr Phe Asn Tyr Asn Phe Ser Ala Leu
665 670 675gca aac acc gtc act ctt gct gga ggg cca agc ttc act tcc aaa ggg 2120Ala Asn Thr Val Thr Leu Ala Gly Gly Pro Ser Phe Thr Ser Lys Gly
680 685 690ttg aaa tac ttc cat cac ttt acc ctc agt ctc tgt gga aac cag ggt 2168Leu Lys Tyr Phe His His Phe Thr Leu Ser Leu Cys Gly Asn Gln Gly
695 700 705agg aaa atg tct gtg tgc acc gac aat gtc act gac ctc cgg att cct 2216Arg Lys Met Ser Val Cys Thr Asp Asn Val Thr Asp Leu Arg Ile Pro
710 715 720gag ggt gag tca ggg ttc tcc aaa tct atc aca gcc tac gtc tgc cag 2264Glu Gly Glu Ser Gly Phe Ser Lys Ser Ile Thr Ala Tyr Val Cys Gln725 730 735 740gca gtc atc atc ccc cca gag gtg aca ggc tac aag gcc ggg gtt tcc 2312Ala Val Ile Ile Pro Pro Glu Val Thr Gly Tyr Lys Ala Gly Val Ser
745 750 755tca cag cct gtc agc ctt gct gat cga ctt att ggg gtg aca aca gat 2360Ser Gln Pro Val Ser Leu Ala Asp Arg Leu Ile Gly Val Thr Thr Asp
760 765 770atg act ctg gat gga atc acc tcc cca gct gaa ctt ttc cac ctg gag 2408Met Thr Leu Asp Gly Ile Thr Ser Pro Ala Glu Leu Phe His Leu Glu
775 780 785tcc ttg gga ata ccg gac gtg atc ttc ttt tat agg tcc aat gat gtg 2456Ser Leu Gly Ile Pro Asp Val Ile Phe Phe Tyr Arg Ser Asn Asp Val
790 795 800acc cag tcc tgc agt tct ggg aga tca acc acc atc cgc gtc agg tgc 2504Thr Gln Ser Cys Ser Ser Gly Arg Ser Thr Thr Ile Arg Val Arg Cys805 810 815 820agt cca cag aaa act gtc cct gga agt ttg ctg ctg cca gga acg tgc 2552Ser Pro Gln Lys Thr Val Pro Gly Ser Leu Leu Leu Pro Gly Thr Cys
825 830 835tca gat ggg acc tgt gat ggc tgc aac ttc cac ttc ctg tgg gag agc 2600Ser Asp Gly Thr Cys Asp Gly Cys Asn Phe His Phe Leu Trp Glu Ser
840 845 850gcg gct gct tgc ccg ctc tgc tca gtg gct gac tac cgt gct atc gtc 2648Ala Ala Ala Cys Pro Leu Cys Ser Val Ala Asp Tyr Arg Ala Ile Val
855 860 865agc agc tgt gtg gct ggg atc cag aag act act tac gtg tgg cga gaa 2696Ser Ser Cys Val Ala Gly Ile Gln Lys Thr Thr Tyr Val Trp Arg Glu
870 875 880ccc aag cta tgc tct ggt ggc att tct ctg cct gag cag aga gtc acc 2744Pro Lys Leu Cys Ser Gly Gly Ile Ser Leu Pro Glu Gln Arg Val Thr885 890 895 900atc tgc aaa acc ata gat ttc tgg ctg aaa gtg ggc atc tct gca ggc 2792Ile Cys Lys Thr Ile Asp Phe Trp Leu Lys Val Gly Ile Ser Ala Gly
905 910 915acc tgt act gcc atc ctg ctc acc gtc ttg acc tgc tac ttt tgg aaa 2840Thr Cys Thr Ala Ile Leu Leu Thr Val Leu Thr Cys Tyr Phe Trp Lys
920 925 930aag aat caa aaa cta gag tac aag tac tcc aag ctg gtg atg aat gct 2888Lys Asn Gln Lys Leu Glu Tyr Lys Tyr Ser Lys Leu Val Met Asn Ala
935 940 945act ctc aag gac tgt gac ctg cca gca gct gac agc tgc gcc arc atg 2936Thr Leu Lys Asp Cys Asp Leu Pro Ala Ala Asp Ser Cys Ala Ile Met
950 955 960gaa ggc gag gat gta gag gac gac ctc atc ttt acc agc aag aag tca 2984Glu Gly Glu Asp Val Glu Asp Asp Leu Ile Phe Thr Ser Lys Lys Ser965 970 975 980ctc ttt ggg aag atc aaa tca ttt acc tcc aag agg act cct gat gga 3032Leu Phe Gly Lys Ile Lys Ser Phe Thr Ser Lys Arg Thr Pro Asp Gly
985 990 995ttt gac tca gtg ccg ctg aag aca tcc tca gga ggc ccc gac atg gac 3080Phe Asp Ser Val Pro Leu Lys Thr Ser Ser Gly Gly Pro Asp Met Asp
1000 1005 1010ctg tgagaggcac tgcctgcctc acctgcctcc tcaccttgca tagcaccttt 3133Leugcaagcctgc ggcgatttgg gtgccagcat cctgcaacac ccactgctgg aaatctcttc 3193attgtggcct tatcagatgt ttgaatttca gatctttttt tatagagtac ccaaaccctc 3253ctttctgctt gcctcaaacc tgccaaatat acccacactt tgtttgtaaa aaaaaaaaaa 3313aaaaaaaaaa aaaaaaaa 3331<2l0>2<2ll>1013<212>PRT<213>人<400>2Met Ala Glu Pro Gly His Ser His His Leu Ser Ala Arg Val Arg Gly1 5 10 15Arg Thr Glu Arg Arg Ile Pro Arg Leu Trp Arg Leu Leu Leu Trp Ala
20 25 30Gly Thr Ala Phe Gln Val Thr Gln Gly Thr Gly Pro Glu Leu His Ala
35 40 45Cys Lys Glu Ser Glu Tyr His Tyr Glu Tyr Thr Ala Cys Asp Ser Thr
50 55 60Gly Ser Arg Trp Arg Val Ala Val Pro His Thr Pro Gly Leu Cys Thr65 70 75 80Ser Leu Pro Asp Pro Val Lys Gly Thr Glu Cys Ser Phe Ser Cys Asn
85 90 95Ala Gly Glu Phe Leu Asp Met Lys Asp Gln Ser Cys Lys Pro Cys Ala
100 105 110Glu Gly Arg Tyr Ser Leu Gly Thr Gly Ile Arg Phe Asp Glu Trp Asp
115 120 125Glu Leu Pro His Gly Phe Ala Ser Leu Ser Ala Asn Met Glu Leu Asp
130 135 140Asp Ser Ala Ala Glu Ser Thr Gly Asn Cys Thr Ser Ser Lys Trp Val145 150 155 160Pro Arg Gly Asp Tyr Ile Ala Ser Asn Thr Asp Glu Cys Thr Ala Thr
165 170 175Leu Met Tyr Ala Val Asn Leu Lys Gln Ser Gly Thr Val Asn Phe Glu
180 185 190Tyr Tyr Tyr Pro Asp Ser Ser Ile Ile Phe Glu Phe Phe Val Gln Asn
195 200 205Asp Gln Cys Gln Pro Asn Ala Asp Asp Ser Arg Trp Met Lys Thr Thr
210 215 220Glu Lys Gly Trp Glu Phe His Ser Val Glu Leu Asn Arg Gly Asn Asn225 230 235 240Val Leu Tyr Trp Arg Thr Thr Ala Phe Ser Val Trp Thr Lys Val Pro
245 250 255Lys Pro Val Leu Val Arg Asn Ile Ala Ile Thr Gly Val Ala Tyr Thr
260 265 270Ser Glu Cys Phe Pro Cys Lys Pro Gly Thr Tyr Ala Asp Lys Gln Gly
275 280 285Ser Ser Phe Cys Lys Leu Cys Pro Ala Asn Ser Tyr Ser Asn Lys Gly
290 295 300Glu Thr Ser Cys His Gln Cys Asp Pro Asp Lys Tyr Ser Glu Lys Gly305 310 315 320Ser Ser Ser Cys Asn Val Arg Pro Ala Cys Thr Asp Lys Asp Tyr Phe
325 330 335Tyr Thr His Thr Ala Cys Asp Ala Asn Gly Glu Thr Gln Leu Met Tyr
340 345 350Lys Trp Ala Lys Pro Lys Ile Cys Ser Glu Asp Leu Glu Gly Ala Val
355 360 365Lys Leu Pro Ala Ser Gly Val Lys Thr His Cys Pro Pro Cys Asn Pro
370 375 380Gly Phe Phe Lys Thr Asn Asn Ser Thr Cys Gln Pro Cys Pro Tyr Gly385 390 395 400Pro Tyr Ser Asn Gly Ser Asp Cys Thr Arg Cys Pro Ala Gly Thr Glu
405 410 415Pro Ala Val Gly Phe Glu Tyr Lys Trp Trp Asn Thr Leu Pro Thr Asn
420 425 430Met Glu Thr Thr Val Leu Ser Gly Ile Asn Phe Glu Tyr Lys Gly Met
435 440 445Thr Gly Trp Glu Val Ala Gly Asp His Ile Tyr Thr Ala Ala Gly Ala
450 455 460Ser Asp Asn Asp Phe Met Ile Leu Thr Leu Val Val Pro Gly Phe Arg465 470 475 480Pro Pro Gln Ser Val Met Ala Asp Thr Glu Asn Lys Glu Val Ala Arg
485 490 495Ile Thr Phe Val Phe Glu Thr Leu Cys Ser Val Asn Cys Glu Leu Tyr
500 505 510Phe Met Val Gly Val Asn Ser Arg Thr Asn Thr Pro Val Glu Thr Trp
515 520 525Lys Gly Ser Lys Gly Lys Gln Ser Tyr Thr Tyr Ile Ile Glu Glu Asn
530 535 540Thr Thr Thr Ser Phe Thr Trp Ala Phe Gln Arg Thr Thr Phe His Glu545 550 555 560Ala Ser Arg Lys Tyr Thr Asn Asp Val Ala Lys Ile Tyr Ser Ile Asn
565 570 575Val Thr Asn Val Met Asn Gly Val Ala Ser Tyr Cys Arg Pro Cys Ala
580 585 590Leu Glu Ala Ser Asp Val Gly Ser Ser Cys Thr Ser Cys Pro Ala Gly
595 600 605Tyr Tyr Ile Asp Arg Asp Ser Gly Thr Cys His Ser Cys Pro Pro Asn
610 615 620Thr Ile Leu Lys Ala His Gln Pro Tyr Gly Val Gln Ala Cys Val Pro625 630 635 640Cys Gly Pro Gly Thr Lys Asn Asn Lys Ile His Ser Leu Cys Tyr Asn
645 650 655Asp Cys Thr Phe Ser Arg Asn Thr Pro Thr Arg Thr Phe Asn Tyr Asn
660 665 670Phe Ser Ala Leu Ala Asn Thr Val Thr Leu Ala Gly Gly Pro Ser Phe
675 680 685Thr Ser Lys Gly Leu Lys Tyr Phe His His Phe Thr Leu Ser Leu Cys
690 695 700Gly Asn Gln Gly Arg Lys Met Ser Val Cys Thr Asp Asn Val Thr Asp705 710 715 720Leu Arg Ile Pro Glu Gly Glu Ser Gly Phe Ser Lys Ser Ile Thr Ala
725 730 735Tyr Val Cys Gln Ala Val Ile Ile Pro Pro Glu Val Thr Gly Tyr Lys
740 745 750Ala Gly Val Ser Ser Gln Pro Val Ser Leu Ala Asp Arg Leu Ile Gly
755 760 765Val Thr Thr Asp Met Thr Leu Asp Gly Ile Thr Ser Pro Ala Glu Leu
770 775 780Phe His Leu Glu Ser Leu Gly Ile Pro Asp Val Ile Phe Phe Tyr Arg785 790 795 800Ser Asn Asp Val Thr Gln Ser Cys Ser Ser Gly Arg Ser Thr Thr Ile
805 810 815Arg Val Arg Cys Ser Pro Gln Lys Thr Val Pro Gly Ser Leu Leu Leu
820 825 830Pro Gly Thr Cys Ser Asp Gly Thr Cys Asp Gly Cys Asn Phe His Phe
835 840 845Leu Trp Glu Ser Ala Ala Ala Cys Pro Leu Cys Ser Val Ala Asp Tyr
850 855 860Arg Ala Ile Val Ser Ser Cys Val Ala Gly Ile Gln Lys Thr Thr Tyr865 870 875 880Val Trp Arg Glu Pro Lys Leu Cys Ser Gly Gly Ile Ser Leu Pro Glu
885 890 895Gln Arg Val Thr Ile Cys Lys Thr Ile Asp Phe Trp Leu Lys Val Gly
900 905 910Ile Ser Ala Gly Thr Cys Thr Ala Ile Leu Leu Thr Val Leu Thr Cys
915 920 925Tyr Phe Trp Lys Lys Asn Gln Lys Leu Glu Tyr Lys Tyr Ser Lys Leu
930 935 940Val Met Asn Ala Thr Leu Lys Asp Cys Asp Leu Pro Ala Ala Asp Ser945 950 955 960Cys Ala Ile Met Glu Gly Glu Asp Val Glu Asp Asp Leu Ile Phe Thr
965 970 975Ser Lys Lys Ser Leu Phe Gly Lys Ile Lys Ser Phe Thr Ser Lys Arg
980 985 990Thr Pro Asp Gly Phe Asp Ser Val Pro Leu Lys Thr Ser Ser Gly Gly
995 1000 1005Pro Asp Met Asp Leu1010<210>3<211>181<212>ADN<213>人<220><221>CDS<222>(45)..(179)<400>3gtgattactc actatagggc tcgagcggcc gcccgggcag gtct atg gct gag cct 56
Met Ala Glu Pro
1ggg cac agc cac cat ctc tcc gcc aga gtc agg gga aga act gag agg 104Gly His Ser His His Leu Ser Ala Arg Val Arg Gly Arg Thr Glu Arg5 10 15 20cgc ata ccc cgg ctg tgg cgg ctg ctg ctc tgg gct ggg acc gcc ttc 152Arg Ile Pro Arg Leu Trp Arg Leu Leu Leu Trp Ala Gly Thr Ala Phe
25 30 35cag gtg acc cag gga acg gga ccg gag ct 181Gln Val Thr Gln Gly Thr Gly Pro Glu
40 45<210>4<211>45<212>PRT<213>人<400>4Met Ala Glu Pro Gly His Ser His His Leu Ser Ala Arg Val Arg Gly1 5 10 15Arg Thr Glu Arg Arg Ile Pro Arg Leu Trp Arg Leu Leu Leu Trp Ala
20 25 30Gly Thr Ala Phe Gln Val Thr Gln Gly Thr Gly Pro Glu
35 40 45<210>5<21l>171<212>ADN<213>人<220><221>CDS<222>(2)..(169)<400>5a gga gaa act tct tgc cac cag tgt gac cct gac aaa tac tca gag aaa 49Gly Glu Thr Ser Cys His Gln Cys Asp Pro Asp Lys Tyr Ser Glu Lys
1 5 10 15gga tct tct tcc tgt aac gtg cgc cca gct tgc aca gac aaa gat tat 97Gly Ser Ser Ser Cys Asn Val Arg Pro Ala Cys Thr Asp Lys Asp Tyr
20 25 30ttc tac aca cac acg gcc tgc gat gcc aac gga gag aca caa ctc atg 145Phe Tyr Thr His Thr Ala Cys Asp Ala Asn Gly Glu Thr Gln Leu Met
35 40 45tac aaa tgg gcc aag ccg aaa atc tg 171Tyr Lys Trp Ala Lys Pro Lys Ile
50 55<210>6<211>56<212>PRT<213>人<400>6Gly Glu Thr Ser Cys His Gln Cys Asp Pro Asp Lys Tyr Ser Glu Lys1 5 10 15Gly Ser Ser Ser Cys Asn Val Arg Pro Ala Cys Thr Asp Lys Asp Tyr
20 25 30Phe Tyr Thr His Thr Ala Cys Asp Ala Asn Gly Glu Thr Gln Leu Met
35 40 45Tyr Lys Trp Ala Lys Pro Lys Ile
50 55<210>7<211>158<212>ADN<213>人<220><22l>CDS<222>(3)..(158)<400>7tt ctc act ctg gtt gtg cca gga ttt aga cct ccg cag tcg gtg atg 47Leu Thr Leu Val Val Pro Gly Phe Arg Pro Pro Gln Ser Val Met
1 5 10 15gca gac aca gag aat aaa gag gtg gcc aga atc aca ttt gtc ttt gag 95Ala Asp Thr Glu Asn Lys Glu Val Ala Arg Ile Thr Phe Val Phe Glu
20 25 30acc ctc tgt tct gtg aac tgt gag ctc tac ttc atg gtg ggt gtg aat 143Thr Leu Cys Ser Val Asn Cys Glu Leu Tyr Phe Met Val Gly Val Asn
35 40 45tct agg acc aac act 158Ser Arg Thr Asn Thr
50<210>8<211>52<212>PRT<213>人<400>8Leu Thr Leu Val Val Pro Gly Phe Arg Pro Pro Gln Ser Val Met Ala1 5 10 15Asp Thr Glu Asn Lys Glu Val Ala Arg Ile Thr Phe Val Phe Glu Thr
20 25 30Leu Cys Ser Val Asn Cys Glu Leu Tyr Phe Met Val Gly Val Asn Ser
35 40 45Arg Thr Asn Thr
50<210>9<211>443<212>ADN<213>人<220><221>CDS<222>(1)..(441)<400>9tgc tac aat gat tgc acc ttc tca cgc aac act cca acc agg act ttc 48Cys Tyr Asn Asp Cys Thr Phe Ser Arg Asn Thr Pro Thr Arg Thr Phe1 5 10 15aac tac aac ttc tcc gct ttg gca aac acc gtc act ctt gct gga ggg 96Asn Tyr Asn Phe Ser Ala Leu Ala Asn Thr Val Thr Leu Ala Gly Gly
20 25 30cca agc ttc act tcc aaa ggg ttg aaa tac ttc cat cac ttt acc ctc 144Pro Ser Phe Thr Ser Lys Gly Leu Lys Tyr Phe His His Phe Thr Leu
35 40 45agt ctc tgt gga aac cag ggt agg aaa atg tct gtg tgc acc gac aat 192Ser Leu Cys Gly Asn Gln Gly Arg Lys Met Ser Val Cys Thr Asp Asn
50 55 60gtc act gac ctc cgg att cct gag ggt gag tca ggg ttc tcc aaa tct 240Val Thr Asp Leu Arg Ile Pro Glu Gly Glu Ser Gly Phe Ser Lys Ser65 70 75 80atc aca gcc tac gtc tgc cag gca gtc atc atc ccc cca gag gtg aca 288Ile Thr Ala Tyr Val Cys Gln Ala Val Ile Ile Pro Pro Glu Val Thr
85 90 95ggc tac aag gcc ggg gtt tcc tca cag cct gtc agc ctt gct gat cga 336Gly Tyr Lys Ala Gly Val Ser Ser Gln Pro Val Ser Leu Ala Asp Arg
100 105 110ctt att ggg gtg aca aca gat atg act ctg gat gga atc acc tcc cca 384Leu Ile Gly Val Thr Thr Asp Met Thr Leu Asp Gly Ile Thr Ser Pro
115 120 125gct gaa ctt ttc cac ctg gag tcc ttg gga ata ccg gac gtg atc ttc 432Ala Glu Leu Phe His Leu Glu Ser Leu Gly Ile Pro Asp Val Ile Phe
130 135 140ttt tat agg tc 443Phe Tyr Arg145<210>10<211>147<212>PRT<213>人<400>10Cys Tyr Asn Asp Cys Thr Phe Ser Arg Asn Thr Pro Thr Arg Thr Phe1 5 10 15Asn Tyr Asn Phe Ser Ala Leu Ala Asn Thr Val Thr Leu Ala Gly Gly
20 25 30Pro Ser Phe Thr Ser Lys Gly Leu Lys Tyr Phe His His Phe Thr Leu
35 40 45Ser Leu Cys Gly Asn Gln Gly Arg Lys Met Ser Val Cys Thr Asp Asn
50 55 60Val Thr Asp Leu Arg Ile Pro Glu Gly Glu Ser Gly Phe Ser Lys Ser65 70 75 80Ile Thr Ala Tyr Val Cys Gln Ala Val Ile Ile Pro Pro Glu Val Thr
85 90 95Gly Tyr Lys Ala Gly Val Ser Ser Gln Pro Val Ser Leu Ala Asp Arg
100 105 110Leu Ile Gly Val Thr Thr Asp Met Thr Leu Asp Gly Ile Thr Ser Pro
115 120 125Ala Glu Leu Phe His Leu Glu Ser Leu Gly Ile Pro Asp Val Ile Phe
130 135 140Phe Tyr Arg145<210>11<211>23<212>ADN<213>人工序列<220><223>由来自人的SEQ IDNO:1衍生的引物<400>11tgactcggaa tcacctccca gct 23<210>12<211>28<212>ADN<213>人工序列<220><223>由来自人的SEQ ID NO:1衍生的引物<400>12caagacggtg agcaggatgg cagtacag 28<210>13<211>20<212>ADN<213>人工序列<220><223>由来自人GAPDH序列衍生的引物<400>13tccaccaccc tgttgctgta 20<210>14<211>20<212>ADN<213>人工序列<220><223>由来自人GAPDH序列衍生的引物<400>14accacagtcc atgccatcac 20<210>15<211>2733<212>ADN<213>人<220><221>CDS<222>(1)..(2733)<400>15atg gct gag cct ggg cac agc cac cat ctc tcc gcc aga gtc agg gga 48Met Ala Glu Pro Gly His Ser His His Leu Ser Ala Arg Val Arg Gly1 5 10 15aga act gag agg cgc ata ccc cgg ctg tgg cgg ctg ctg ctc tgg gct 96Arg Thr Glu Arg Arg Ile Pro Arg Leu Trp Arg Leu Leu Leu Trp Ala
20 25 30ggg acc gcc ttc cag gtg acc cag gga acg gga ccg gag ctt cat gcc 144Gly Thr Ala Phe Gln Val Thr Gln Gly Thr Gly Pro Glu Leu His Ala
35 40 45tgc aaa gag tct gag tac cac tat gag tac acg gcg tgt gac agc acg 192Cys Lys Glu Ser Glu Tyr His Tyr Glu Tyr Thr Ala Cys Asp Ser Thr
50 55 60ggt tcc agg tgg agg gtc gcc gtg ccg cat acc ccg ggc ctg tgc acc 240Gly Ser Arg Trp Arg Val Ala Val Pro His Thr Pro Gly Leu Cys Thr65 70 75 80agc ctg cct gac ccc gtc aag ggc acc gag tgc tcc ttc tcc tgc aac 288Ser Leu Pro Asp Pro Val Lys Gly Thr Glu Cys Ser Phe Ser Cys Asn
85 90 95gcc ggg gag ttt ctg gat atg aag gac cag tca tgt aag cca tgc gct 336Ala Gly Glu Phe Leu Asp Met Lys Asp Gln Ser Cys Lys Pro Cys Ala
100 105 110gag ggc cgc tac tcc ctc ggc aca ggc att cgg ttt gat gag tgg gat 384Glu Gly Arg Tyr Ser Leu Gly Thr Gly Ile Arg Phe Asp Glu Trp Asp
115 120 125gag ctg ccc cat ggc ttt gcc agc ctc tca gcc aac atg gag ctg gat 432Glu Leu Pro His Gly Phe Ala Ser Leu Ser Ala Asn Met Glu Leu Asp
130 135 140gac agt gct gct gag tcc acc ggg aac tgt act tcg tcc aag tgg gtt 480Asp Ser Ala Ala Glu Ser Thr Gly Asn Cys Thr Ser Ser Lys Trp Val145 150 155 160ccc cgg ggc gac tac atc gcc tcc aac acg gac gaa tgc aca gcc aca 528Pro Arg Gly Asp Tyr Ile Ala Ser Asn Thr Asp Glu Cys Thr Ala Thr
165 170 175ctg atg tac gcc gtc aac ctg aag caa tct ggc acc gtt aac ttc gaa 576Leu Met Tyr Ala Val Asn Leu Lys Gln Ser Gly Thr Val Asn Phe Glu
180 185 190tac tac tat cca gac tcc agc atc atc ttt gag ttt ttc gtt cag aat 624Tyr Tyr Tyr Pro Asp Ser Ser Ile Ile Phe Glu Phe Phe Val Gln Asn
195 200 205gac cag tgc cag ccc aat gca gat gac tcc agg tgg atg aag acc aca 672Asp Gln Cys Gln Pro Asn Ala Asp Asp Ser Arg Trp Met Lys Thr Thr
210 215 220gag aaa gga tgg gaa ttc cac agt gtg gag cta aat cga ggc aat aat 720Glu Lys Gly Trp Glu Phe His Ser Val Glu Leu Asn Arg Giy Asn Asn225 230 235 240gtc ctc tat tgg aga acc aca gcc ttc tca gta tgg acc aaa gta ccc 768Val Leu Tyr Trp Arg Thr Thr Ala Phe Ser Val Trp Thr Lys Val Pro
245 250 255aag cct gtg ctg gtg aga aac att gcc ata aca ggg gtg gcc tac act 816Lys Pro Val Leu Val Arg Asn Ile Ala Ile Thr Gly Val Ala Tyr Thr
260 265 270tca gaa tgc ttc ccc tgc aaa cct ggc acg tat gca gac aag cag ggc 864Ser Glu Cys Phe Pro Cys Lys Pro Gly Thr Tyr Ala Asp Lys Gln Gly
275 280 285tcc tct ttc tgc aaa ctt tgc cca gcc aac tct tat tca aat aaa gga 912Ser Ser Phe Cys Lys Leu Cys Pro Ala Asn Ser Tyr Ser Asn Lys Gly
290 295 300gaa act tct tgc cac cag tgt gac cct gac aaa tac tca gag aaa gga 960Glu Thr Ser Cys His Gln Cys Asp Pro Asp Lys Tyr Ser Glu Lys Gly305 310 315 320tct tct tcc tgt aac gtg cgc cca gct tgc aca gac aaa gat tat ttc 1008Ser Ser Ser Cys Asn Val Arg Pro Ala Cys Thr Asp Lys Asp Tyr Phe
325 330 335tac aca cac acg gcc tgc gat gcc aac gga gag aca caa ctc atg tac 1056Tyr Thr His Thr Ala Cys Asp Ala Asn Gly Glu Thr Gln Leu Met Tyr
340 345 350aaa tgg gcc aag ccg aaa atc tgt agc gag gac ctt gag ggg gca gtg 1104Lys Trp Ala Lys Pro Lys Ile Cys Ser Glu Asp Leu Glu Gly Ala Val
355 360 365aag ctg cct gcc tct ggt gtg aag acc cac tgc cca ccc tgc aac cca 1152Lys Leu Pro Ala Ser Gly Val Lys Thr His Cys Pro Pro Cys Asn Pro
370 375 380ggc ttc ttc aaa acc aac aac agc acc tgc cag ccc tgc cca tat ggt 1200Gly Phe Phe Lys Thr Asn Asn Ser Thr Cys Gln Pro Cys Pro Tyr Gly385 390 395 400ccc tac tcc aat ggc tca gac tgt acc cgc tgc cct gca ggg act gaa 1248Pro Tyr Ser Asn Gly Ser Asp Cys Thr Arg Cys Pro Ala Gly Thr Glu
405 410 415cct gct gtg gga ttt gaa tac aaa tgg tgg aac acg ctg ccc aca aac 1296Pro Ala Val Gly Phe Glu Tyr Lys Trp Trp Asn Thr Leu Pro Thr Asn
420 425 430atg gaa acg acc gtt ctc agt ggg atc aac ttc gag tac aag ggc atg 1344Met Glu Thr Thr Val Leu Ser Gly Ile Asn Phe Glu Tyr Lys Gly Met
435 440 445aca ggc tgg gag gtg gct ggt gat cac att tac aca gct gct gga gcc 1392Thr Gly Trp Glu Val Ala Gly Asp His Ile Tyr Thr Ala Ala Gly Ala
450 455 460tca gac aat gac ttc atg att ctc act ctg gtt gtg cca gga ttt aga 1440Ser Asp Asn Asp Phe Met Ile Leu Thr Leu Val Val Pro Gly Phe Arg465 470 475 480cct ccg cag tcg gtg atg gca gac aca gag aat aaa gag gtg gcc aga 1488Pro Pro Gln Ser Val Met Ala Asp Thr Glu Asn Lys Glu Val Ala Arg
485 490 495atc aca ttt gtc ttt gag acc ctc tgt tct gtg aac tgt gag ctc tac 1536Ile Thr Phe Val Phe Glu Thr Leu Cys Ser Val Asn Cys Glu Leu Tyr
500 505 510ttc atg gtg ggt gtg aat tct agg acc aac act cct gtg gag acg tgg 1584Phe Met Val Gly Val Asn Ser Arg Thr Asn Thr Pro Val Glu Thr Trp
515 520 525aaa ggt tcc aaa ggc aaa cag tcc tat acc tac atc att gag gag aac 1632Lys Gly Ser Lys Gly Lys Gln Ser Tyr Thr Tyr Ile Ile Glu Glu Asn
530 535 540act acc acg agc ttc acc tgg gcc ttc cag agg acc act ttt cat gag 1680Thr Thr Thr Ser Phe Thr Trp Ala Phe Gln Arg Thr Thr Phe His Glu545 550 555 560gca agc agg aag tac acc aat gac gtt gcc aag atc tac tcc atc aat 1728Ala Ser Arg Lys Tyr Thr Asn Asp Val Ala Lys Ile Tyr Ser Ile Asn
565 570 575gtc acc aat gtt atg aat ggc gtg gcc tcc tac tgc cgt ccc tgt gcc 1776Val Thr Asn Val Met Asn Gly Val Ala Ser Tyr Cys Arg Pro Cys Ala
580 585 590cta gaa gcc tct gat gtg ggc tcc tcc tgc acc tct tgt cct gct ggt 1824Leu Glu Ala Ser Asp Val Gly Ser Ser Cys Thr Ser Cys Pro Ala Gly
595 600 605tac tat att gac cga gat tca gga acc tgc cac tcc tgc ccc cct aac 1872Tyr Tyr Ile Asp Arg Asp Set Gly Thr Cys His Ser Cys Pro Pro Asn
610 615 620aca att ctg aaa gcc cac cag cct tat ggt gtc cag gcc tgt gtg ccc 1920Thr Ile Leu Lys Ala His Gln Pro Tyr Gly Val Gln Ala Cys Val Pro625 630 635 640tgt ggt cca ggg acc aag aac aac aag atc cac tct ctg tgc tac aat 1968Cys Gly Pro Gly Thr Lys Asn Asn Lys Ile His Ser Leu Cys Tyr Asn
645 650 655gat tgc acc ttc tca cgc aac act cca acc agg act ttc aac tac aac 2016Asp Cys Thr Phe Ser Arg Asn Thr Pro Thr Arg Thr Phe Asn Tyr Asn
660 665 670ttc tcc gct ttg gca aac acc gtc act ctt gct gga ggg cca agc ttc 2064Phe Ser Ala Leu Ala Asn Thr Val Thr Leu Ala Gly Gly Pro Ser Phe
675 680 685act tcc aaa ggg ttg aaa tac ttc cat cac ttt acc ctc agt ctc tgt 2112Thr Ser Lys Gly Leu Lys Tyr Phe His His Phe Thr Leu Ser Leu Cys
690 695 700gga aac cag ggt agg aaa atg tct gtg tgc acc gac aat gtc act gac 2160Gly Asn Gln Gly Arg Lys Met Ser Val Cys Thr Asp Asn Val Thr Asp705 710 715 720ctc cgg att cct gag ggt gag tca ggg ttc tcc aaa tct atc aca gcc 2208Leu Arg Ile Pro Glu Gly Glu Ser Gly Phe Ser Lys Ser Ile Thr Ala
725 730 735tac gtc tgc cag gca gtc atc atc ccc cca gag gtg aca ggc tac aag 2256Tyr Val Cys Gln Ala Val Ile Ile Pro Pro Glu Val Thr Gly Tyr Lys
740 745 750gcc ggg gtt tcc tca cag cct gtc agc ctt gct gat cga ctt att ggg 2304Ala Gly Val Ser Ser Gln Pro Val Ser Leu Ala Asp Arg Leu Ile Gly
755 760 765gtg aca aca gat atg act ctg gat gga atc acc tcc cca gct gaa ctt 2352Val Thr Thr Asp Met Thr Leu Asp Gly Ile Thr Ser Pro Ala Glu Leu
770 775 780ttc cac ctg gag tcc ttg gga ata ccg gac gtg atc ttc ttt tat agg 2400Phe His Leu Glu Ser Leu Gly Ile Pro Asp Val Ile Phe Phe Tyr Arg785 790 795 800tcc aat gat gtg acc cag tcc tgc agt tct ggg aga tca acc acc atc 2448Ser Asn Asp Val Thr Gln Ser Cys Ser Ser Gly Arg Ser Thr Thr Ile
805 810 815cgc gtc agg tgc agt cca cag aaa act gtc cct gga agt ttg ctg ctg 2496Arg Val Arg Cys Ser Pro Gln Lys Thr Val Pro Gly Ser Leu Leu Leu
820 825 830cca gga acg tgc tca gat ggg acc tgt gat ggc tgc aac ttc cac ttc 2544Pro Gly Thr Cys Ser Asp Gly Thr Cys Asp Gly Cys Asn Phe His Phe
835 840 845ctg tgg gag agc gcg gct gct tgc ccg ctc tgc tca gtg gct gac tac 2592Leu Trp Glu Ser Ala Ala Ala Cys Pro Leu Cys Ser Val Ala Asp Tyr
850 855 860cgt gct atc gtc agc agc tgt gtg gct ggg atc cag aag act act tac 2640Arg Ala Ile Val Ser Ser Cys Val Ala Gly Ile Gln Lys Thr Thr Tyr865 870 875 880gtg tgg cga gaa ccc aag cta tgc tct ggt ggc att tct ctg cct gag 2688Val Trp Arg Glu Pro Lys Leu Cys Ser Gly Gly Ile Ser Leu Pro Glu
885 890 895cag aga gtc acc atc tgc aaa acc ata gat ttc tgg ctg aaa gtg 2733Gln Arg Val Thr Ile Cys Lys Thr Ile Asp Phe Trp Leu Lys Val
900 905 910<210>16<211>911<212>PRT<213>人<400>16Met Ala Glu Pro Gly His Ser His His Leu Ser Ala Arg Val Arg Gly1 5 10 15Arg Thr Glu Arg Arg Ile Pro Arg Leu Trp Arg Leu Leu Leu Trp Ala
20 25 30Gly Thr Ala Phe Gln Val Thr Gln Gly Thr Gly Pro Glu Leu His Ala
35 40 45Cys Lys Glu Ser Glu Tyr His Tyr Glu Tyr Thr Ala Cys Asp Ser Thr
50 55 60Gly Ser Arg Trp Arg Val Ala Val Pro His Thr Pro Gly Leu Cys Thr65 70 75 80Ser Leu Pro Asp Pro Val Lys Gly Thr Glu Cys Ser Phe Ser Cys Asn
85 90 95Ala Gly Glu Phe Leu Asp Met Lys Asp Gln Ser Cys Lys Pro Cys Ala
100 105 110Glu Gly Arg Tyr Ser Leu Gly Thr Gly Ile Arg Phe Asp Glu Trp Asp
115 120 125Glu Leu Pro His Gly Phe Ala Ser Leu Ser Ala Asn Met Glu Leu Asp
130 135 140Asp Ser Ala Ala Glu Ser Thr Gly Asn Cys Thr Ser Ser Lys Trp Val145 150 155 160Pro Arg Gly Asp Tyr Ile Ala Ser Asn Thr Asp Glu Cys Thr Ala Thr
165 170 175Leu Met Tyr Ala Val Asn Leu Lys Gln Ser Gly Thr Val Asn Phe Glu
180 185 190Tyr Tyr Tyr Pro Asp Ser Ser Ile Ile Phe Glu Phe Phe Val Gln Asn
195 200 205Asp Gln Cys Gln Pro Asn Ala Asp Asp Ser Arg Trp Met Lys Thr Thr
210 215 220Glu Lys Gly Trp Glu Phe His Ser Val Glu Leu Asn Arg Gly Asn Asn225 230 235 240Val Leu Tyr Trp Arg Thr Thr Ala Phe Ser Val Trp Thr Lys Val Pro
245 250 255Lys Pro Val Leu Val Arg Asn Ile Ala Ile Thr Gly Val Ala Tyr Thr
260 265 270Ser Glu Cys Phe Pro Cys Lys Pro Gly Thr Tyr Ala Asp Lys Gln Gly
275 280 285Ser Ser Phe Cys Lys Leu Cys Pro Ala Asn Ser Tyr Ser Asn Lys Gly
290 295 300Glu Thr Ser Cys His Gln Cys Asp Pro Asp Lys Tyr Ser Glu Lys Gly305 310 315 320Ser Ser Ser Cys Asn Val Arg Pro Ala Cys Thr Asp Lys Asp Tyr Phe
325 330 335Tyr Thr His Thr Ala Cys Asp Ala Asn Gly Glu Thr Gln Leu Met Tyr
340 345 350Lys Trp Ala Lys Pro Lys Ile Cys Ser Glu Asp Leu Glu Gly Ala Val
355 360 365Lys Leu Pro Ala Ser Gly Val Lys Thr His Cys Pro Pro Cys Asn Pro
370 375 380Gly Phe Phe Lys Thr Asn Asn Ser Thr Cys Gln Pro Cys Pro Tyr Gly385 390 395 400Pro Tyr Ser Asn Gly Ser Asp Cys Thr Arg Cys Pro Ala Gly Thr Glu
405 410 415Pro Ala Val Gly Phe Glu Tyr Lys Trp Trp Asn Thr Leu Pro Thr Asn
420 425 430Met Glu Thr Thr Val Leu Ser Gly Ile Asn Phe Glu Tyr Lys Gly Met
435 440 445Thr Gly Trp Glu Val Ala Gly Asp His Ile Tyr Thr Ala Ala Gly Ala
450 455 460Ser Asp Asn Asp Phe Met Ile Leu Thr Leu Val Val Pro Gly Phe Arg465 470 475 480Pro Pro Gln Ser Val Met Ala Asp Thr Glu Asn Lys Glu Val Ala Arg
485 490 495Ile Thr Phe Val Phe Glu Thr Leu Cys Ser Val Asn Cys Glu Leu Tyr
500 505 510Phe Met Val Gly Val Asn Ser Arg Thr Asn Thr Pro Val Glu Thr Trp
515 520 525Lys Gly Ser Lys Gly Lys Gln Ser Tyr Thr Tyr Ile Ile Glu Glu Asn
530 535 540Thr Thr Thr Ser Phe Thr Trp Ala Phe Gln Arg Thr Thr Phe His Glu545 550 555 560Ala Ser Arg Lys Tyr Thr Asn Asp Val Ala Lys Ile Tyr Ser Ile Asn
565 570 575Val Thr Asn Val Met Asn Gly Val Ala Ser Tyr Cys Arg Pro Cys Ala
580 585 590Leu Glu Ala Ser Asp Val Gly Ser Ser Cys Thr Ser Cys Pro Ala Gly
595 600 605Tyr Tyr Ile Asp Arg Asp Ser Gly Thr Cys His Ser Cys Pro Pro Asn
610 615 620Thr Ile Leu Lys Ala His Gln Pro Tyr Gly Val Gln Ala Cys Val Pro625 630 635 640Cys Gly Pro Gly Thr Lys Asn Asn Lys Ile His Ser Leu Cys Tyr Asn
645 650 655Asp Cys Thr Phe Ser Arg Asn Thr Pro Thr Arg Thr Phe Asn Tyr Asn
660 665 670Phe Ser Ala Leu Ala Asn Thr Val Thr Leu Ala Gly Gly Pro Ser Phe
675 680 685Thr Ser Lys Gly Leu Lys Tyr Phe His His Phe Thr Leu Ser Leu Cys
690 695 700Gly Asn Gln Gly Arg Lys Met Ser Val Cys Thr Asp Asn Val Thr Asp705 710 715 720Leu Arg Ile Pro Glu Gly Glu Ser Gly Phe Ser Lys Ser Ile Thr Ala
725 730 735Tyr Val Cys Gln Ala Val Ile Ile Pro Pro Glu Val Thr Gly Tyr Lys
740 745 750Ala Gly Val Ser Ser Gln Pro Val Ser Leu Ala Asp Arg Leu Ile Gly
755 760 765Val Thr Thr Asp Met Thr Leu Asp Gly Ile Thr Ser Pro Ala Glu Leu
770 775 780Phe His Leu Glu Ser Leu Gly Ile Pro Asp Val Ile Phe Phe Tyr Arg785 790 795 800Ser Asn Asp Val Thr Gln Ser Cys Ser Ser Gly Arg Ser Thr Thr Ile
805 810 815Arg Val Arg Cys Ser Pro Gln Lys Thr Val Pro Gly Ser Leu Leu Leu
820 825 830Pro Gly Thr Cys Ser Asp Gly Thr Cys Asp Gly Cys Asn Phe His Phe
835 840 845Leu Trp Glu Ser Ala Ala Ala Cys Pro Leu Cys Ser Val Ala Asp Tyr
850 855 860Arg Ala Ile Val Ser Ser Cys Val Ala Gly Ile Gln Lys Thr Thr Tyr865 870 875 880Val Trp Arg Glu Pro Lys Leu Cys Ser Gly Gly Ile Ser Leu Pro Glu
885 890 895Gln Arg Val Thr Ile Cys Lys Thr Ile Asp Phe Trp Leu Lys Val
900 905 910<210>17<211>2610<212>ADN<213>人<220><22l>CDS<222>(1)..(2610)<400>17acg gga ccg gag ctt cat gcc tgc aaa gag tct gag tac cac tat gag 48Thr Gly Pro Glu Leu His Ala Cys Lys Glu Ser Glu Tyr His Tyr Glu1 5 10 15tac acg gcg tgt gac agc acg ggt tcc agg tgg agg gtc gcc gtg ccg 96Tyr Thr Ala Cys Asp Ser Thr Gly Ser Arg Trp Arg Val Ala Val Pro
20 25 30cat acc ccg ggc ctg tgc acc agc ctg cct gac ccc gtc aag ggc acc 144His Thr Pro Gly Leu Cys Thr Ser Leu Pro Asp Pro Val Lys Gly Thr
35 40 45gag tgc tcc ttc tcc tgc aac gcc ggg gag ttt ctg gat atg aag gac 192Glu Cys Ser Phe Ser Cys Asn Ala Gly Glu Phe Leu Asp Met Lys Asp
50 55 60cag tca tgt aag cca tgc gct gag ggc cgc tac tcc ctc ggc aca ggc 240Gln Ser Cys Lys Pro Cys Ala Glu Gly Arg Tyr Ser Leu Gly Thr Gly65 70 75 80att cgg ttt gat gag tgg gat gag ctg ccc cat ggc ttt gcc agc ctc 288Ile Arg Phe Asp Glu Trp Asp Glu Leu Pro His Gly Phe Ala Ser Leu
85 90 95tca gcc aac atg gag ctg gat gac agt gct gct gag tcc acc ggg aac 336Ser Ala Asn Met Glu Leu Asp Asp Ser Ala Ala Glu Ser Thr Gly Asn
100 105 110tgt act tcg tcc aag tgg gtt ccc cgg ggc gac tac atc gcc tcc aac 384Cys Thr Ser Ser Lys Trp Val Pro Arg Gly Asp Tyr Ile Ala Ser Asn
115 120 125acg gac gaa tgc aca gcc aca ctg atg tac gcc gtc aac ctg aag caa 432Thr Asp Glu Cys Thr Ala Thr Leu Met Tyr Ala Val Asn Leu Lys Gln
130 135 140tct ggc acc gtt aac ttc gaa tac tac tat cca gac tcc agc atc atc 480Ser Gly Thr Val Asn Phe Glu Tyr Tyr Tyr Pro Asp Ser Ser Ile Ile145 150 155 160ttt gag ttt ttc gtt cag aat gac cag tgc cag ccc aat gca gat gac 528Phe Glu Phe Phe Val Gln Asn Asp Gln Cys Gln Pro Asn Ala Asp Asp
165 170 175tcc agg tgg atg aag acc aca gag aaa gga tgg gaa ttc cac agt gtg 576Ser Arg Trp Met Lys Thr Thr Glu Lys Gly Trp Glu Phe His Ser Val
180 185 190gag cta aat cga ggc aat aat gtc ctc tat tgg aga acc aca gcc ttc 624Glu Leu Asn Arg Gly Asn Asn Val Leu Tyr Trp Arg Thr Thr Ala Phe
195 200 205tca gta tgg acc aaa gta ccc aag cct gtg ctg gtg aga aac att gcc 672Ser Val Trp Thr Lys Val Pro Lys Pro Val Leu Val Arg Asn Ile Ala
210 215 220ata aca ggg gtg gcc tac act tca gaa tgc ttc ccc tgc aaa cct ggc 720Ile Thr Gly Val Ala Tyr Thr Ser Glu Cys Phe Pro Cys Lys Pro Gly225 230 235 240acg tat gca gac aag cag ggc tcc tct ttc tgc aaa ctt tgc cca gcc 768Thr Tyr Ala Asp Lys Gln Gly Ser Ser Phe Cys Lys Leu Cys Pro Ala
245 250 255aac tct tat tca aat aaa gga gaa act tct tgc cac cag tgt gac cct 816Asn Ser Tyr Ser Asn Lys Gly Glu Thr Ser Cys His Gln Cys Asp Pro
260 265 270gac aaa tac tca gag aaa gga tct tct tcc tgt aac gtg cgc cca gct 864Asp Lys Tyr Ser Glu Lys Gly Ser Ser Ser Cys Asn Val Arg Pro Ala
275 280 285tgc aca gac aaa gat tat ttc tac aca cac acg gcc tgc gat gcc aac 912Cys Thr Asp Lys Asp Tyr Phe Tyr Thr His Thr Ala Cys Asp Ala Asn
290 295 300gga gag aca caa ctc atg tac aaa tgg gcc aag ccg aaa atc tgt agc 960Gly Glu Thr Gln Leu Met Tyr Lys Trp Ala Lys Pro Lys Ile Cys Ser305 310 315 320gag gac ctt gag ggg gca gtg aag ctg cct gcc tct ggt gtg aag acc 1008Glu Asp Leu Glu Gly Ala Val Lys Leu Pro Ala Ser Gly Val Lys Thr
325 330 335cac tgc cca ccc tgc aac cca ggc ttc ttc aaa acc aac aac agc acc 1056His Cys Pro Pro Cys Asn Pro Gly Phe Phe Lys Thr Asn Asn Ser Thr
340 345 350tgc cag ccc tgc cca tat ggt ccc tac tcc aat ggc tca gac tgt acc 1104Cys Gln Pro Cys Pro Tyr Gly Pro Tyr Ser Asn Gly Ser Asp Cys Thr
355 360 365cgc tgc cct gca ggg act gaa cct gct gtg gga ttt gaa tac aaa tgg 1152Arg Cys Pro Ala Gly Thr Glu Pro Ala Val Gly Phe Glu Tyr Lys Trp
370 375 380tgg aac acg ctg ccc aca aac atg gaa acg acc gtt ctc agt ggg atc 1200Trp Asn Thr Leu Pro Thr Asn Met Glu Thr Thr Val Leu Ser Gly Ile385 390 395 400aac ttc gag tac aag ggc atg aca ggc tgg gag gtg gct ggt gat cac 1248Asn Phe Glu Tyr Lys Gly Met Thr Gly Trp Glu Val Ala Gly Asp His
405 410 415att tac aca gct gct gga gcc tca gac aat gac ttc atg att ctc act 1296Ile Tyr Thr Ala Ala Gly Ala Ser Asp Asn Asp Phe Met Ile Leu Thr
420 425 430ctg gtt gtg cca gga ttt aga cct ccg cag tcg gtg atg gca gac aca 1344Leu Val Val Pro Gly Phe Arg Pro Pro Gln Ser Val Met Ala Asp Thr
435 440 445gag aat aaa gag gtg gcc aga atc aca ttt gtc ttt gag acc ctc tgt 1392Glu Asn Lys Glu Val Ala Arg Ile Thr Phe Val Phe Glu Thr Leu Cys
450 455 460tct gtg aac tgt gag ctc tac ttc atg gtg ggt gtg aat tct agg acc 1440Ser Val Asn Cys Glu Leu Tyr Phe Met Val Gly Val Asn Ser Arg Thr465 470 475 480aac act cct gtg gag acg tgg aaa ggt tcc aaa ggc aaa cag tcc tat 1488Asn Thr Pro Val Glu Thr Trp Lys Gly Ser Lys Gly Lys Gln Ser Tyr
485 490 495acc tac atc att gag gag aac act acc acg agc ttc acc tgg gcc ttc 1536Thr Tyr Ile Ile Glu Glu Asn Thr Thr Thr Ser Phe Thr Trp Ala Phe
500 505 510cag agg acc act ttt cat gag gca agc agg aag tac acc aat gac gtt 1584Gln Arg Thr Thr Phe His Glu Ala Ser Arg Lys Tyr Thr Asn Asp Val
515 520 525gcc aag atc tac tcc atc aat gtc acc aat gtt atg aat ggc gtg gcc 1632Ala Lys Ile Tyr Ser Ile Asn Val Thr Asn Val Met Asn Gly Val Ala
530 535 540tcc tac tgc cgt ccc tgt gcc cta gaa gcc tct gat gtg ggc tcc tcc 1680Ser Tyr Cys Arg Pro Cys Ala Leu Glu Ala Ser Asp Val Gly Ser Ser545 550 555 560tgc acc tct tgt cct gct ggt tac tat att gac cga gat tca gga acc 1728Cys Thr Ser Cys Pro Ala Gly Tyr Tyr Ile Asp Arg Asp Ser Gly Thr
565 570 575tgc cac tcc tgc ccc cct aac aca att ctg aaa gcc cac cag cct tat 1776Cys His Ser Cys Pro Pro Asn Thr Ile Leu Lys Ala His Gln Pro Tyr
580 585 590ggt gtc cag gcc tgt gtg ccc tgt ggt cca ggg acc aag aac aac aag 1824Gly Val Gln Ala Cys Val Pro Cys Gly Pro Gly Thr Lys Asn Asn Lys
595 600 605atc cac tct ctg tgc tac aat gat tgc acc ttc tca cgc aac act cca 1872Ile His Ser Leu Cys Tyr Asn Asp Cys Thr Phe Ser Arg Asn Thr Pro
610 615 620acc agg act ttc aac tac aac ttc tcc gct ttg gca aac acc gtc act 1920Thr Arg Thr Phe Asn Tyr Asn Phe Ser Ala Leu Ala Asn Thr Val Thr625 630 635 640ctt gct gga ggg cca agc ttc act tcc aaa ggg ttg aaa tac ttc cat 1968Leu Ala Gly Gly Pro Ser Phe Thr Ser Lys Gly Leu Lys Tyr Phe His
645 650 655cac ttt acc ctc agt ctc tgt gga aac cag ggt agg aaa atg tct gtg 2016His Phe Thr Leu Ser Leu Cys Gly Asn Gln Gly Arg Lys Met Ser Val
660 665 670tgc acc gac aat gtc act gac ctc cgg att cct gag ggt gag tca ggg 2064Cys Thr Asp Asn Val Thr Asp Leu Arg Ile Pro Glu Gly Glu Ser Gly
675 680 685ttc tcc aaa tct atc aca gcc tac gtc tgc cag gca gtc atc atc ccc 2112Phe Ser Lys Ser Ile Thr Ala Tyr Val Cys Gln Ala Val Ile Ile Pro
690 695 700cca gag gtg aca ggc tac aag gcc ggg gtt tcc tca cag cct gtc agc 2160Pro Glu Val Thr Gly Tyr Lys Ala Gly Val Ser Ser Gln Pro Val Ser705 710 715 720ctt gct gat cga ctt att ggg gtg aca aca gat atg act ctg gat gga 2208Leu Ala Asp Arg Leu Ile Gly Val Thr Thr Asp Met Thr Leu Asp Gly
725 730 735atc acc tcc cca gct gaa ctt ttc cac ctg gag tcc ttg gga ata ccg 2256Ile Thr Ser Pro Ala Glu Leu Phe His Leu Glu Ser Leu Gly Ile Pro
740 745 750gac gtg atc ttc ttt tat agg tcc aat gat gtg acc cag tcc tgc agt 2304Asp Val Ile Phe Phe Tyr Arg Ser Asn Asp Val Thr Gln Ser Cys Ser
755 760 765tct ggg aga tca acc acc atc cgc gtc agg tgc agt cca cag aaa act 2352Ser Gly Arg Ser Thr Thr Ile Arg Val Arg Cys Ser Pro Gln Lys Thr
770 775 780gtc cct gga agt ttg ctg ctg cca gga acg tgc tca gat ggg acc tgt 2400Val Pro Gly Ser Leu Leu Leu Pro Gly Thr Cys Ser Asp Gly Thr Cys785 790 795 800gat ggc tgc aac ttc cac ttc ctg tgg gag agc gcg gct gct tgc ccg 2448Asp Gly Cys Asn Phe His Phe Leu Trp Glu Ser Ala Ala Ala Cys Pro
805 810 815ctc tgc tca gtg gct gac tac cgt gct atc gtc agc agc tgt gtg gct 2496Leu Cys Ser Val Ala Asp Tyr Arg Ala Ile Val Ser Ser Cys Val Ala
820 825 830ggg atc cag aag act act tac gtg tgg cga gaa ccc aag cta tgc tct 2544Gly Ile Gln Lys Thr Thr Tyr Val Trp Arg Glu Pro Lys Leu Cys Ser
835 840 845ggt ggc att tct ctg cct gag cag aga gtc acc atc tgc aaa acc ata 2592Gly Gly Ile Ser Leu Pro Glu Gln Arg Val Thr Ile Cys Lys Thr Ile
850 855 860gat ttc tgg ctg aaa gtg 2610Asp Phe Trp Leu Lys Val865 870<210>18<211>870<212>PRT<213>人<400>18Thr Gly Pro Glu Leu His Ala Cys Lys Glu Ser Glu Tyr His Tyr Glu1 5 10 15Tyr Thr Ala Cys Asp Ser Thr Gly Ser Arg Trp Arg Val Ala Val Pro
20 25 30His Thr Pro Gly Leu Cys Thr Ser Leu Pro Asp Pro Val Lys Gly Thr
35 40 45Glu Cys Ser Phe Ser Cys Asn Ala Gly Glu Phe Leu Asp Met Lys Asp
50 55 60Gln Ser Cys Lys Pro Cys Ala Glu Gly Arg Tyr Ser Leu Gly Thr Gly65 70 75 80Ile Arg Phe Asp Glu Trp Asp Glu Leu Pro His Gly Phe Ala Ser Leu
85 90 95Ser Ala Asn Met Glu Leu Asp Asp Ser Ala Ala Glu Ser Thr Gly Asn
100 105 110Cys Thr Ser Ser Lys Trp Val Pro Arg Gly Asp Tyr Ile Ala Ser Asn
115 120 125Thr Asp Glu Cys Thr Ala Thr Leu Met Tyr Ala Val Asn Leu Lys Gln
130 135 140Ser Gly Thr Val Asn Phe Glu Tyr Tyr Tyr Pro Asp Ser Ser Ile Ile145 150 155 160Phe Glu Phe Phe Val Gln Asn Asp Gln Cys Gln Pro Asn Ala Asp Asp
165 170 175Ser Arg Trp Met Lys Thr Thr Glu Lys Gly Trp Glu Phe His Ser Val
180 185 190Glu Leu Asn Arg Gly Asn Asn Val Leu Tyr Trp Arg Thr Thr Ala Phe
195 200 205Ser Val Trp Thr Lys Val Pro Lys Pro Val Leu Val Arg Asn Ile Ala
210 215 220Ile Thr Gly Val Ala Tyr Thr Ser Glu Cys Phe Pro Cys Lys Pro Gly225 230 235 240Thr Tyr Ala Asp Lys Gln Gly Ser Ser Phe Cys Lys Leu Cys Pro Ala
245 250 255Asn Ser Tyr Ser Asn Lys Gly Glu Thr Ser Cys His Gln Cys Asp Pro
260 265 270Asp Lys Tyr Ser Glu Lys Gly Ser Ser Ser Cys Asn Val Arg Pro Ala
275 280 285Cys Thr Asp Lys Asp Tyr Phe Tyr Thr His Thr Ala Cys Asp Ala Asn
290 295 300Gly Glu Thr Gln Leu Met Tyr Lys Trp Ala Lys Pro Lys Ile Cys Ser305 310 315 320Glu Asp Leu Glu Gly Ala Val Lys Leu Pro Ala Ser Gly Val Lys Thr
325 330 335His Cys Pro Pro Cys Asn Pro Gly Phe Phe Lys Thr Asn Asn Ser Thr
340 345 350Cys Gln Pro Cys Pro Tyr Gly Pro Tyr Ser Asn Gly Ser Asp Cys Thr
355 360 365Arg Cys Pro Ala Gly Thr Glu Pro Ala Val Gly Phe Glu Tyr Lys Trp
370 375 380Trp Asn Thr Leu Pro Thr Asn Met Glu Thr Thr Val Leu Ser Gly Ile385 390 395 400Asn Phe Glu Tyr Lys Gly Met Thr Gly Trp Glu Val Ala Gly Asp His
405 410 415Ile Tyr Thr Ala Ala Gly Ala Ser Asp Asn Asp Phe Met Ile Leu Thr
420 425 430Leu Val Val Pro Gly Phe Arg Pro Pro Gln Ser Val Met Ala Asp Thr
435 440 445Glu Asn Lys Glu Val Ala Arg Ile Thr Phe Val Phe Glu Thr Leu Cys
450 455 460Ser Val Asn Cys Glu Leu Tyr Phe Met Val Gly Val Asn Ser Arg Thr465 470 475 480Asn Thr Pro Val Glu Thr Trp Lys Gly Ser Lys Gly Lys Gln Ser Tyr
485 490 495Thr Tyr Ile Ile Glu Glu Asn Thr Thr Thr Ser Phe Thr Trp Ala Phe
500 505 510Gln Arg Thr Thr Phe His Glu Ala Ser Arg Lys Tyr Thr Asn Asp Val
515 520 525Ala Lys Ile Tyr Ser Ile Asn Val Thr Asn Val Met Asn Gly Val Ala
530 535 540Ser Tyr Cys Arg Pro Cys Ala Leu Glu Ala Ser Asp Val Gly Ser Ser545 550 555 560Cys Thr Ser Cys Pro Ala Gly Tyr Tyr Ile Asp Arg Asp Ser Gly Thr
565 570 575Cys His Ser Cys Pro Pro Asn Thr Ile Leu Lys Ala His Gln Pro Tyr
580 585 590Gly Val Gln Ala Cys Val Pro Cys Gly Pro Gly Thr Lys Asn Asn Lys
595 600 605Ile His Ser Leu Cys Tyr Asn Asp Cys Thr Phe Ser Arg Asn Thr Pro
610 615 620Thr Arg Thr Phe Asn Tyr Asn Phe Ser Ala Leu Ala Asn Thr Val Thr625 630 635 640Leu Ala Gly Gly Pro Ser Phe Thr Ser Lys Gly Leu Lys Tyr Phe His
645 650 655His Phe Thr Leu Ser Leu Cys Gly Asn Gln Gly Arg Lys Met Ser Val
660 665 670Cys Thr Asp Asn Val Thr Asp Leu Arg Ile Pro Glu Gly Glu Ser Gly
675 680 685Phe Ser Lys Ser Ile Thr Ala Tyr Val Cys Gln Ala Val Ile Ile Pro
690 695 700Pro Glu Val Thr Gly Tyr Lys Ala Gly Val Ser Ser Gln Pro Val Ser705 710 715 720Leu Ala Asp Arg Leu Ile Gly Val Thr Thr Asp Met Thr Leu Asp Gly
725 730 735Ile Thr Ser Pro Ala Glu Leu Phe His Leu Glu Ser Leu Gly Ile Pro
740 745 750Asp Val Ile Phe Phe Tyr Arg Ser Asn Asp Val Thr Gln Ser Cys Ser
755 760 765Ser Gly Arg Ser Thr Thr Ile Arg Val Arg Cys Ser Pro Gln Lys Thr
770 775 780Val Pro Gly Ser Leu Leu Leu Pro Gly Thr Cys Ser Asp Gly Thr Cys785 790 795 800Asp Gly Cys Asn Phe His Phe Leu Trp Glu Ser Ala Ala Ala Cys Pro
805 810 815Leu Cys Ser Val Ala Asp Tyr Arg Ala Ile Val Ser Ser Cys Val Ala
820 825 830Gly Ile Gln Lys Thr Thr Tyr Val Trp Arg Glu Pro Lys Leu Cys Ser
835 840 845Gly Gly Ile Ser Leu Pro Glu Gln Arg Val Thr Ile Cys Lys Thr Ile
850 855 860Asp Phe Trp Leu Lys Val865 870<2l0>19<211>527<212>ADN<213>人<220><221>CDS<222>(1)..(627)<400>19cag tgt gac cct gac aaa tac tca gag aaa gga tct tct tcc tgt aac 48Gln Cys Asp Pro Asp Lys Tyr Ser Glu Lys Gly Ser Ser Ser Cys Asnl 5 10 15gtg cgc cca gct tgc aca gac aaa gat tat ttc tac aca cac acg gcc 96Val Arg Pro Ala Cys Thr Asp Lys Asp Tyr Phe Tyr Thr His Thr Ala
20 25 30tgc gat gcc aac gga gag aca caa ctc atg tac aaa tgg gcc aag ccg 144Cys Asp Ala Asn Gly Glu Thr Gln Leu Met Tyr Lys Trp Ala Lys Pro
35 40 45aaa atc tgt agc gag gac ctt gag ggg gca gtg aag ctg cct gcc tct 192Lys Ile Cys Ser Glu Asp Leu Glu Gly Ala Val Lys Leu Pro Ala Ser
50 55 60ggt gtg aag acc cac tgc cca ccc tgc aac cca ggc ttc ttc aaa acc 240Gly Val Lys Thr His Cys Pro Pro Cys Asn Pro Gly Phe Phe Lys Thr65 70 75 80aac aac agc acc tgc cag ccc tgc cca tat ggt ccc tac tcc aat ggc 288Asn Asn Ser Thr Cys Gln Pro Cys Pro Tyr Gly Pro Tyr Ser Asn Gly
85 90 95tca gac tgt acc cgc tgc cct gca ggg act gaa cct gct gtg gga ttt 336Ser Asp Cys Thr Arg Cys Pro Ala Gly Thr Glu Pro Ala Val Gly Phe
100 105 110gaa tac aaa tgg tgg aac acg ctg ccc aca aac atg gaa acg acc gtt 384Glu Tyr Lys Trp Trp Asn Thr Leu Pro Thr Asn Met Glu Thr Thr Val
115 120 125ctc agt ggg atc aac ttc gag tac aag ggc atg aca ggc tgg gag gtg 432Leu Ser Gly Ile Asn Phe Glu Tyr Lys Gly Met Thr Gly Trp Glu Val
130 135 140gct ggt gat cac att tac aca gct gct gga gcc rca gac aat gac ttc 480Ala Gly Asp His Ile Tyr Thr Ala Ala Gly Ala Ser Asp Asn Asp Phe145 150 155 160atg att ctc act ctg gtt gtg cca gga ttt aga cct ccg cag tcg gtg 528Met Ile Leu Thr Leu Val Val Pro Gly Phe Arg Pro Pro Gln Ser Val
165 170 175atg gca gac aca gag aat aaa gag gtg gcc aga atc aca ttt gtc ttt 576Met Ala Asp Thr Glu Asn Lys Glu Val Ala Arg Ile Thr Phe Val Phe
180 185 190gag acc ctc tgt tct gtg aac tgt gag ctc tac ttc atg gtg ggt gtg 624Glu Thr Leu Cys Ser Val Asn Cys Glu Leu Tyr Phe Met Val Gly Val
195 200 205aat 627Asn<210>20<211>209<212>PRT<213>人<400>20Gln Cys Asp Pro Asp Lys Tyr Ser Glu Lys Gly Ser Ser Ser Cys Asn1 5 10 15Val Arg Pro Ala Cys Thr Asp Lys Asp Tyr Phe Tyr Thr His Thr Ala
20 25 30Cys Asp Ala Asn Gly Glu Thr Gln Leu Met Tyr Lys Trp Ala Lys Pro
35 40 45Lys Ile Cys Ser Glu Asp Leu Glu Gly Ala Val Lys Leu Pro Ala Ser
50 55 60Gly Val Lys Thr His Cys Pro Pro Cys Asn Pro Gly Phe Phe Lys Thr65 70 75 80Asn Asn Ser Thr Cys Gln Pro Cys Pro Tyr Gly Pro Tyr Ser Asn Gly
85 90 95Ser Asp Cys Thr Arg Cys Pro Ala Gly Thr Glu Pro Ala Val Gly Phe
100 105 110Glu Tyr Lys Trp Trp Asn Thr Leu Pro Thr Asn Met Glu Thr Thr Val
115 120 125Leu Ser Gly Ile Asn Phe Glu Tyr Lys Gly Met Thr Gly Trp Glu Val
130 135 140Ala Gly Asp His Ile Tyr Thr Ala Ala Gly Ala Ser Asp Asn Asp Phe145 150 155 160Met Ile Leu Thr Leu Val Val Pro Gly Phe Arg Pro Pro Gln Ser Val
165 170 175Met Ala Asp Thr Glu Asn Lys Glu Val Ala Arg Ile Thr Phe Val Phe
180 185 190Glu Thr Leu Cys Ser Val Asn Cys Glu Leu Tyr Phe Met Val Gly Val
195 200 205Asn<210>21<2ll>1149<212>ADN<213>人<220><221>CDS<222>(1)..(1149)<400>21agc ctc tca gcc aac atg gag ctg gat gac agt gct gct gag tcc acc 48Ser Leu Ser Ala Asn Met Glu Leu Asp Asp Ser Ala Ala Glu Ser Thr1 5 10 15ggg aac tgt act tcg tcc aag tgg gtt ccc cgg ggc gac tac atc gcc 96Gly Asn Cys Thr Ser Ser Lys Trp Val Pro Arg Gly Asp Tyr Ile Ala
20 25 30tcc aac acg gac gaa tgc aca gcc aca ctg atg tac gcc gtc aac ctg 144Ser Asn Thr Asp Glu Cys Thr Ala Thr Leu Met Tyr Ala Val Asn Leu
35 40 45aag caa tct ggc acc gtt aac ttc gaa tac tac tat cca gac tcc agc 192Lys Gln Ser Gly Thr Val Asn Phe Glu Tyr Tyr Tyr Pro Asp Ser Ser
50 55 60arc atc ttt gag ttt ttc gtt cag aat gac cag tgc cag ccc aat gca 240Ile Ile Phe Glu Phe Phe Val Gln Asn Asp Gln Cys Gln Pro Asn Ala65 70 75 80gat gac tcc agg tgg atg aag acc aca gag aaa gga tgg gaa ttc cac 288Asp Asp Ser Arg Trp Met Lys Thr Thr Glu Lys Gly Trp Glu Phe His
85 90 95agt gtg gag cta aat cga ggc aat aat gtc ctc tat tgg aga acc aca 336Ser Val Glu Leu Asn Arg Gly Asn Asn Val Leu Tyr Trp Arg Thr Thr
100 105 110gcc ttc tca gta tgg acc aaa gta ccc aag cct gtg ctg gtg aga aac 384Ala Phe Ser Val Trp Thr Lys Val Pro Lys Pro Val Leu Val Arg Asn
115 120 125att gcc ata aca ggg gtg gcc tac act tca gaa tgc ttc ccc tgc aaa 432Ile Ala Ile Thr Gly Val Ala Tyr Thr Ser Glu Cys Phe Pro Cys Lys
130 135 140cct ggc acg tat gca gac aag cag ggc tcc tct ttc tgc aaa ctt tgc 480Pro Gly Thr Tyr Ala Asp Lys Gln Gly Ser Ser Phe Cys Lys Leu Cys145 150 155 160cca gcc aac tct tat tca aat aaa gga gaa act tct tgc cac cag tgt 528Pro Ala Asn Ser Tyr Ser Asn Lys Gly Glu Thr Ser Cys His Gln Cys
165 170 175gac cct gac aaa tac tca gag aaa gga tct tct tcc tgt aac gtg cgc 576Asp Pro Asp Lys Tyr Ser Glu Lys Gly Ser Ser Ser Cys Asn Val Arg
180 185 190cca gct tgc aca gac aaa gat tat ttc tac aca cac acg gcc tgc gat 624Pro Ala Cys Thr Asp Lys Asp Tyr Phe Tyr Thr His Thr Ala Cys Asp
195 200 205gcc aac gga gag aca caa ctc atg tac aaa tgg gcc aag ccg aaa atc 672Ala Asn Gly Glu Thr Gln Leu Met Tyr Lys Trp Ala Lys Pro Lys Ile
210 215 220tgt agc gag gac ctt gag ggg gca gtg aag ctg cct gcc tct ggt gtg 720Cys Ser Glu Asp Leu Glu Gly Ala Val Lys Leu Pro Ala Ser Gly Val225 230 235 240aag acc cac tgc cca ccc tgc aac cca ggc ttc ttc aaa acc aac aac 768Lys Thr His Cys Pro Pro Cys Asn Pro Gly Phe Phe Lys Thr Asn Asn
245 250 255agc acc tgc cag ccc tgc cca tat ggt ccc tac tcc aat ggc tca gac 816Ser Thr Cys Gln Pro Cys Pro Tyr Gly Pro Tyr Ser Asn Gly Ser Asp
260 265 270tgt acc cgc tgc cct gca ggg act gaa cct gct gtg gga ttt gaa tac 864Cys Thr Arg Cys Pro Ala Gly Thr Glu Pro Ala Val Gly Phe Glu Tyr
275 280 285aaa tgg tgg aac acg ctg ccc aca aac atg gaa acg acc gtt ctc agt 912Lys Trp Trp Asn Thr Leu Pro Thr Asn Met Glu Thr Thr Val Leu Ser
290 295 300ggg atc aac ttc gag tac aag ggc atg aca ggc tgg gag gtg gct ggt 960Gly Ile Asn Phe Glu Tyr Lys Gly Met Thr Gly Trp Glu Val Ala Gly305 310 315 320gat cac att tac aca gct gct gga gcc tca gac aat gac ttc atg att 1008Asp His Ile Tyr Thr Ala Ala Gly Ala Ser Asp Asn Asp Phe Met Ile
325 330 335ctc act ctg gtt gtg cca gga ttt aga cct ccg cag tcg gtg atg gca 1056Leu Thr Leu Val Val Pro Gly Phe Arg Pro Pro Gln Ser Val Met Ala
340 345 350gac aca gag aat aaa gag gtg gcc aga atc aca ttt gtc ttt gag acc 1104Asp Thr Glu Asn Lys Glu Val Ala Arg Ile Thr Phe Val Phe Glu Thr
355 360 365ctc tgt tct gtg aac tgt gag ctc tac ttc atg gtg ggt gtg aat 1149Leu Cys Ser Val Asn Cys Glu Leu Tyr Phe Met Val Gly Val Asn
370 375 380<210>22<21l>383<212>PRT<213>人<400>22Ser Leu Ser Ala Asn Met Glu Leu Asp Asp Ser Ala Ala Glu Ser Thr1 5 10 15Gly Asn Cys Thr Ser Ser Lys Trp Val Pro Arg Gly Asp Tyr Ile Ala
20 25 30Ser Asn Thr Asp Glu Cys Thr Ala Thr Leu Met Tyr Ala Val Asn Leu
35 40 45Lys Gln Ser Gly Thr Val Asn Phe Glu Tyr Tyr Tyr Pro Asp Ser Ser
50 55 60Ile Ile Phe Glu Phe Phe Val Gln Asn Asp Gln Cys Gln Pro Asn Ala65 70 75 80Asp Asp Ser Arg Trp Met Lys Thr Thr Glu Lys Gly Trp Glu Phe His
85 90 95Ser Val Glu Leu Asn Arg Gly Asn Asn Val Leu Tyr Trp Arg Thr Thr
100 105 110Ala Phe Ser Val Trp Thr Lys Val Pro Lys Pro Val Leu Val Arg Asn
115 120 125Ile Ala Ile Thr Gly Val Ala Tyr Thr Ser Glu Cys Phe Pro Cys Lys
130 135 140Pro Gly Thr Tyr Ala Asp Lys Gln Gly Ser Ser Phe Cys Lys Leu Cys145 150 155 160Pro Ala Asn Ser Tyr Ser Asn Lys Gly Glu Thr Ser Cys His Gln Cys
165 170 175Asp Pro Asp Lys Tyr Ser Glu Lys Gly Ser Ser Ser Cys Asn Val Arg
180 185 190Pro Ala Cys Thr Asp Lys Asp Tyr Phe Tyr Thr His Thr Ala Cys Asp
195 200 205Ala Asn Gly Glu Thr Gln Leu Met Tyr Lys Trp Ala Lys Pro Lys Ile
210 215 220Cys Ser Glu Asp Leu Glu Gly Ala Val Lys Leu Pro Ala Ser Gly Val225 230 235 240Lys Thr His Cys Pro Pro Cys Asn Pro Gly Phe Phe Lys Thr Asn Asn
245 250 255Ser Thr Cys Gln Pro Cys Pro Tyr Gly Pro Tyr Ser Asn Gly Ser Asp
260 265 270Cys Thr Arg Cys Pro Ala Gly Thr Glu Pro Ala Val Gly Phe Glu Tyr
275 280 285Lys Trp Trp Asn Thr Leu Pro Thr Asn Met Glu Thr Thr Val Leu Ser
290 295 300Gly Ile Asn Phe Glu Tyr Lys Gly Met Thr Gly Trp Glu Val Ala Gly305 310 315 320Asp His Ile Tyr Thr Ala Ala Gly Ala Ser Asp Asn Asp Phe Met Ile
325 330 335Leu Thr Leu Val Val Pro Gly Phe Arg Pro Pro Gln Ser Val Met Ala
340 345 350Asp Thr Glu Asn Lys Glu Val Ala Arg Ile Thr Phe Val Phe Glu Thr
355 360 365Leu Cys Ser Val Asn Cys Glu Leu Tyr Phe Met Val Gly Val Asn
370 375 380
Claims (34)
1.一种纯化或分离的核酸,其特征在于其含有选自如下序列的核酸序列:
a)SEQ ID NO:1的序列;
b)缺失了其末端poly A片段的SEQ ID NO:1的序列之至少18个连续核苷酸片段的序列;
c)在优化排比后,具有与a)或b)中定义的序列至少80%百分同一性的核酸序列;
d)与a),b)或c)中定义的序列互补之序列,或RNA序列相应于a),b)或c)中定义的序列之序列;
除了WO 99/54461文件中的SEQ ID NO:11的序列,WO/99/06548文件中的SEQ ID NO:51的序列以及在EMBL数据库中鉴定号为AI672868,AA 890726和AI301140的文件中所述的3个序列。
2.权利要求1中所述的纯化或分离的核酸,其特征在于其含有选自如下序列的序列:
a)SEQ ID NO:5,SEQ ID NO:7,SEQ ID NO:15,SEQ ID NO:17和SEQ ID NO:19的序列;
b)SEQ ID NO:19的至少18个核苷酸的片段之序列;
c)在优化排比后,具有与a)或b)中定义的序列至少80%百分同一性的核酸序列;
d)与a),b)或c)中定义的序列互补之序列,或RNA序列相应于a),b)或c)中定义的序列之序列。
3.权利要求1或2中所述的纯化或分离的核酸,其特征在于其序列选自如下序列:
a)SEQ ID NO:1,SEQ ID NO:3,SEQ ID NO:5,SEQ ID NO:7,SEQ ID NO:9,SEQ ID NO:15,SEQ ID NO:17,SEQ ID NO:19和SEQ ID NO:21的序列;
b)在优化排比后,具有与a)中定义的序列至少80%百分同一性的核酸序列;
c)与a),或b)中定义的序列互补之序列,或RNA序列相应于a),或b)中定义的序列之序列。
4.一种用于筛选基因组cDNA或DNA文库的方法,或用于克隆分离的基因组或cDNA的方法,其特征在于它利用了权利要求1-3中任一项的核酸序列。
5.一种用于鉴定基因序列中至少18个连续核苷酸的核酸的方法,所述基因编码SEQ ID NO:2序列的多肽,所述核酸包括至少一个负责该基因的异常表达之突变,其特征在于它使用了权利要求1-3中任一项的核酸序列。
6.一种用于鉴定编码SEQ ID NO:2序列的多肽之基因的基因组序列,优选地促进和/或调控表达的核酸序列的方法,其特征在于它使用了权利要求1-3中任一项的核酸序列。
7.一种利用如权利要求5或6中任一项的方法鉴定的核酸。
8.一种纯化的或分离的多肽,其由如权利要求1-3或权利要求7中任一项所述的核酸编码。
9.一种纯化或分离的多肽,其特征在于它含有选自如下的氨基酸序列:
a)SEQ ID NO:2的序列;
b)SEQ ID NO:22的序列之至少6个连续氨基酸的片段的序列,优选地是SEQ ID NO:20的这样的序列;
c)在优化排比后,具有与a)或b)中定义的序列至少80%百分同一性的氨基酸序列。
10.权利要求8所述的多肽,其特征在于其序列选自SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20和SEQ ID NO:22,或在优化排比后,具有与这些序列至少80%百分同一性的序列。
11.一种克隆和/或表达载体,其含有如权利要求1-3和7中任一项的核酸序列。
12.权利要求11所述的载体,其特征在于其含有在宿主细胞中表达所需的元件。
13.一种用权利要求11或12所述的载体转化的宿主细胞。
14.一种不包括人的哺乳动物,其特征在于其含有权利要求13所述的细胞。
15.权利要求1-3和7中任一项的核酸序列之用途,其作为探针或引物用于检测和/或扩增核酸序列。
16.权利要求1-3和7中任一项的核酸之用途,其作为有义或反义寡核苷酸。
17.权利要求1-3和7中任一项的核酸序列之用途,其用于产生重组多肽。
18.一种用于产生重组多肽的方法,其特征在于,如权利要求13的转化的细胞在允许该重组多肽表达的条件下培养,以及回收所述重组多肽。
19.一种多肽,其特征在于其利用权利要求18所述的方法获得。
20.一种单克隆抗体或多克隆抗体,或其片段,嵌合抗体或免疫偶联物,其特征在于能特异性识别权利要求8-10和19中任一项所述的多肽,或能够识别文件WO99/54461的SEQ ID NO:173序列或文件WO99/06548的SEQ ID NO:51序列的多肽。
21.一种用于检测和/或纯化多肽的方法,其特征在于其利用了权利要求20所述的抗体。
22.一种用于诊断疾病的方法,所述疾病与编码SEQ ID NO:2序列的多肽之基因的异常表达相关,其特征在于使用了权利要求1-3和7中任一项所述的核酸序列。
23.一种用于诊断疾病的方法,所述疾病与编码SEQ ID NO:2的多肽之基因存在至少一种突变相关,其中利用了来自患者的生物学样品,其特征在于它包括如下步骤:
a)如适当,从待分析的生物学样品中分离基因组DNA,或从生物学样品的RNA中获得cDNA;
b)利用选自如权利要求1-3和7中任一项的核酸的引物,特异性扩增可能含有突变的基因之所述DNA序列;
c)分析所得的扩增产物,并将其序列与相应的正常序列比较。
24.一种用于诊断与SEQ ID NO:2序列的多肽的异常表达相关的疾病的方法,其特征在于将一种或多种权利要求20的抗体与待检测的生物材料在如下条件下接触:该条件使得在所述多肽与所述抗体之间可形成特异性免疫复合物,且其特征在于检测和/或定量可能形成的免疫复合物。
25.一种用于筛选体内或体外能调控SEQ ID NO:2序列的多肽活性的化学或生物化学化合物的方法,其特征在于该方法包括将权利要求8-10和19中任一项的多肽文件WO99/54461的SEQ ID NO:173序列的多肽或文件WO99/06548的SEQ ID NO:51序列的多肽权利要求13所述的细胞或权利要求14所述的哺乳动物与候选化合物接触,并检测SEQ ID NO:2序列的多肽所述活性的修饰。
26.一种筛选能结合SEQ ID NO:2序列的多肽之化学或生物化学化合物的方法,其特征在于该方法包括将权利要求8-10和19中任一项的多肽、文件WO99/54461的SEQ ID NO:173序列的多肽或文件WO99/06548的SEQ ID NO:51序列的多肽、如权利要求13所述的细胞或权利要求14所述的哺乳动物与候选化合物接触,并检测候选化合物与所述多肽之间复合物的形成。
27.一种筛选能体内或体外与权利要求1-3和7中任一项的核酸序列相互作用的化学或生物化学化合物的方法,其特征在于该方法包括将正常或异常地表达编码SEQ ID NO:2序列的多肽的基因之细胞与候选化合物接触,以及直接或间接地检测SEQ ID NO:2序列的多肽的表达或活性的修饰。
28.权利要求8-10和19中任一项所述多肽、文件WO99/54461的SEQ ID NO:173序列的多肽或文件WO99/06548的SEQ ID NO:51序列的多肽、权利要求13所述的细胞或权利要求14所述的哺乳动物的用途,其用于研究SEQ ID NO:2序列的多肽的表达或活性,以及研究SEQ ID NO:2序列所述多肽与可能参与SEQ ID NO:2序列所述多肽活性的化学或生物化学化合物之间的直接或间接相互作用。
29.一种化合物,其特征在于其是利用权利要求25-27所述任一项方法筛选的。
30.一种化合物,其特征在于其选自:
-权利要求20的抗体;
-权利要求8-10和19中任一项的多肽,或分别在文献WO99/54461中描述的SEQ ID NO:173的多肽或WO99/06458中描述的SEQ ID NO:51的多肽;
-权利要求8-10和19中任一项的多肽,或在文献WO 99/54461中描述的SEQ ID NO:173的多肽或WO/06458中描述的SEQ ID NO:51的多肽,其特征在于其相应于SEQ ID NO:2序列的多肽之可溶性片段;
-权利要求11或12所述的载体;
-权利要求1-3和7中任一项的核酸,或文献WO 99/54461的SEQ ID NO:11序列的核酸,文献WO 99/06548的SEQ ID NO:51的序列的核酸,或EMBL文件中相应鉴别号AI 672868、AA890726或AI30140序列的核酸,其可为有义或反义的。
31.权利要求29和30中任一项的化合物,其作为医药产品。
32.权利要求31所述的化合物,其用于预防和/或治疗疾病,所述疾病与本发明SEQ ID NO:2序列的多肽之表达和/或活性异常相关。
33.权利要求31和32中任一项所述的化合物,用于预防和/或治疗免疫疾病,特别是用于预防和/或治疗自身免疫疾病或癌症的药物产品。
34.权利要求31和32中任一项所述的化合物,其用于预防和/或治疗病毒、真菌、细菌或寄生虫型感染。
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Application Number | Priority Date | Filing Date | Title |
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FR9913629A FR2800388A1 (fr) | 1999-10-29 | 1999-10-29 | Clonage, expression et caracterisation d'un gene exprime dans des cellules tumorales et implique dans la regulation de la reponse immune |
FR99/13629 | 1999-10-29 |
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CN1391608A true CN1391608A (zh) | 2003-01-15 |
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CN00816059A Pending CN1391608A (zh) | 1999-10-29 | 2000-10-30 | 在肿瘤细胞中表达并参与免疫反应的调控之基因的克隆、表达和表征 |
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EP (1) | EP1226248A1 (zh) |
JP (1) | JP2003516126A (zh) |
CN (1) | CN1391608A (zh) |
AU (1) | AU1284801A (zh) |
BR (1) | BR0015161A (zh) |
CA (1) | CA2389522A1 (zh) |
FR (1) | FR2800388A1 (zh) |
MX (1) | MXPA02004297A (zh) |
WO (1) | WO2001031003A1 (zh) |
ZA (1) | ZA200203340B (zh) |
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US6951738B2 (en) | 1999-07-16 | 2005-10-04 | Human Genome Sciences, Inc. | Human tumor necrosis factor receptors TR13 and TR14 |
JP2003252801A (ja) * | 2002-02-27 | 2003-09-10 | Japan Science & Technology Corp | 下垂体特異的遺伝子の使用法 |
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ES2267192T3 (es) * | 1997-08-01 | 2007-03-01 | Serono Genetics Institute S.A. | Ests 5' para proteinas secretadas no especificas de tejido. |
JP2001512013A (ja) * | 1997-08-01 | 2001-08-21 | ジェンセット | 前立腺に発現される分泌タンパク質の5’est |
DE19817948A1 (de) * | 1998-04-17 | 1999-10-21 | Metagen Gesellschaft Fuer Genomforschung Mbh | Menschliche Nukleinsäuresequenzen aus Endometrium-Tumor |
TR200102745T2 (tr) * | 1999-03-26 | 2002-05-21 | Smithkline Beecham Biologicals S.A. | Yeni bileşikler |
WO2001002568A2 (en) * | 1999-07-02 | 2001-01-11 | Chiron Corporation | Human genes and gene expression products |
-
1999
- 1999-10-29 FR FR9913629A patent/FR2800388A1/fr active Pending
-
2000
- 2000-10-30 WO PCT/FR2000/003032 patent/WO2001031003A1/fr not_active Application Discontinuation
- 2000-10-30 MX MXPA02004297A patent/MXPA02004297A/es unknown
- 2000-10-30 BR BR0015161-0A patent/BR0015161A/pt not_active Application Discontinuation
- 2000-10-30 CA CA002389522A patent/CA2389522A1/fr not_active Abandoned
- 2000-10-30 JP JP2001533984A patent/JP2003516126A/ja active Pending
- 2000-10-30 EP EP00974612A patent/EP1226248A1/fr not_active Withdrawn
- 2000-10-30 CN CN00816059A patent/CN1391608A/zh active Pending
- 2000-10-30 AU AU12848/01A patent/AU1284801A/en not_active Abandoned
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CA2389522A1 (fr) | 2001-05-03 |
WO2001031003B1 (fr) | 2001-10-11 |
MXPA02004297A (es) | 2002-10-31 |
BR0015161A (pt) | 2002-07-09 |
JP2003516126A (ja) | 2003-05-13 |
ZA200203340B (en) | 2002-12-11 |
AU1284801A (en) | 2001-05-08 |
FR2800388A1 (fr) | 2001-05-04 |
WO2001031003A1 (fr) | 2001-05-03 |
EP1226248A1 (fr) | 2002-07-31 |
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