CN1160472C - 人类17号染色体1区3带3亚带区域内人肿瘤相关基因及检测试剂盒 - Google Patents
人类17号染色体1区3带3亚带区域内人肿瘤相关基因及检测试剂盒 Download PDFInfo
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- CN1160472C CN1160472C CNB011426284A CN01142628A CN1160472C CN 1160472 C CN1160472 C CN 1160472C CN B011426284 A CNB011426284 A CN B011426284A CN 01142628 A CN01142628 A CN 01142628A CN 1160472 C CN1160472 C CN 1160472C
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Abstract
本发明提供了一种对个体的肿瘤(如肝癌)易感性进行诊断的方法,它包括步骤:检测该个体的HC56基因、转录本和/或蛋白,并与正常的HC56基因、转录本和/或蛋白相比较,存在差异就表明该个体患肿瘤的可能性高于正常人群。本发明还提供了相应的检测试剂盒。
Description
技术领域
本发明涉及人类17号染色体1区3带3亚带(17p13.3)定点克隆人肿瘤相关基因HC56及应用。更具体地,本发明涉及利用HC56的单核苷酸多态性(SNP)检测肿瘤易感性的方法和试剂盒。
背景技术
恶性肿瘤的死亡率在我国仅次于心、脑血管疾病名列第二。在恶性肿瘤中肝癌的死亡率列在肺癌、胃癌之后居第三位。目前对肝癌(除小肝癌外)缺乏有效的诊治手段。人们普遍认为癌的发生是一种多因子多步骤的复杂事件,是多种癌基因激活和抑癌基因失活的结果。其中抑癌基因可能更为关键。因此寻找肿瘤抑癌基因是目前研究的热点之一。基因经常通过一个等位基因的丢失(杂合子缺失,Loss of Heterozygocity,LOH)而失去一种正常性状,LOH频发部位可能存在抑癌基因。
上海市肿瘤研究所癌基因及相关基因国家重点实验室长期从事人肝癌基因的研究,早在1991年就发现肝癌的发生与p53失活有关,该基因不仅249编码子突变,还有其它编码子出现点突变(Li D.Z;Gu JR et al Carcinogenesis 14(2):169,1993)。p53位于人染色体17p13.1。
另外,对17p13.3区段内的研究国外也有报导。Nishida等(Noashi Nishida etal.,Cancer Research 53:368-372,1993)在17p上选10个多态性探针,研究人肝癌的17p LOH和p53基因突变之间的关系,他认为肝癌的发生可能与17p13.3上未被证明的肿瘤抑制基因有关。Schultz等(David C.Schultz,et al.,Cancer Research56:1997-2002 1996),用等位基因丢失作图和定点克隆方法证明了两个新基因OVCA1和OVCA2,定位于17p13.3,这两个新基因在正常的卵巢上皮细胞有表达,在卵巢瘤或卵巢瘤细胞株中表达减少或无表达。Wales等(Michele M.Waleset al.,Nature Medicine:570-577,1995),通过肿瘤组织DNA高度甲基化分析,在17p13.3区域发现一个后选基因Hic-1,它是一个锌指结构转录因子,在正常组织普遍表达,在肿瘤细胞中表达降低。
然而,上述报告存在以下不足之处:1.没有报导肝癌组织LOH频率的最小范围和位点。2.上述发现的后选基因与肝癌的关系未见报导。
由于癌症是危害人类健康的主要疾病之一。为了有效地治疗和预防肿瘤(如肝癌),目前人们已越来越关注肿瘤的早期诊断和基因治疗。因此,本领域迫切需要开发研究新的癌症相关的和/或具有抑癌功能的人蛋白,以及相应的检测试剂盒。
发明内容
本发明的目的是提供一种新的肿瘤相关蛋白-人HC56蛋白以及其片段、类似物和衍生物。
本发明的另一目的是提供编码这些多肽的多核苷酸。
本发明的另一目的是提供利用HC56检测肿瘤易感性的方法和试剂盒。
在本发明的第一方面,提供了一种分离的正常的人HC56蛋白多肽,它包括具有SEQ ID NO:4所示氨基酸序列的多肽。此外,还提供了正常人HC56相比,因单核苷酸多态性所导致的氨基酸突变。一种具有因单核苷酸多态性所导致的氨基酸突变的HC56序列示于SEQ ID NO:2。
在本发明的第二方面,提供了一种分离的多核苷酸,它编码正常的HC56蛋白多肽(例如SEQ ID NO:3)。还提供了人HC56基因中所存在的单核苷酸多态性。一种具有单核苷酸多态性的HC56序列示于SEQ ID NO:1。
在本发明的第三方面,提供了含有上述多核苷酸的载体,以及被该载体转化或转导的宿主细胞或者被上述多核苷酸直接转化或转导的宿主细胞。
在本发明的第四方面,提供了制备肿瘤相关HC56蛋白活性的多肽的制备方法,该方法包含:(a)在适合表达蛋白的条件下,培养上述被转化或转导的宿主细胞;(b)从培养物中分离出具有肿瘤相关HC56蛋白活性的多肽。
在本发明的第五方面,提供了可用于检测的核酸分子,它含有SEQ ID NO:3中至少20-4750个连续核苷酸,并且还含有选自下组的突变:
SEQ ID NO:3中第2004位处的C→T;
SEQ ID NO:3中第2080位处的A→G;
SEQ ID NO:3中第2655位处的G→C;
SEQ ID NO:3中第3043位处的G→C;
SEQ ID NO:3中第3085位处的A→T;
SEQ ID NO:3中第3358位处的G→A;
SEQ ID NO:3中第4092位处的A→G;
SEQ ID NO:3中第4404位处的T→C;
SEQ ID NO:3中第4564位处的C→G;
SEQ ID NO:3中第4602位处的G→A;
或者,该核酸分子具有SEQ ID NO:1所示的核苷酸序列。这些核酸分子可用作探针、引物、反义核酸片段。
在本发明的第六方面,提供了一种检测肝细胞是否发生癌变或存在癌变易感性的方法,它包括步骤:检测该个体的HC56基因、转录本和/或蛋白,并与正常的HC56基因、转录本和/或蛋白相比较,存在差异就表明该个体患肿瘤的可能性高于正常人群。较佳地,所述的变化是核苷酸的缺失、插入、或置换突变。
在一优选例中,检测的是HC56的基因或转录本,并与正常HC56核苷酸序列比较差异,并且所述的差异选自下组:
SEQ ID NO:3中第2004位处的C→T;
SEQ ID NO:3中第2080位处的A→G;
SEQ ID NO:3中第2655位处的G→C;
SEQ ID NO:3中第3043位处的G→C;
SEQ ID NO:3中第3085位处的A→T;
SEQ ID NO:3中第3358位处的G→A;
SEQ ID NO:3中第4092位处的A→G;
SEQ ID NO:3中第4404位处的T→C;
SEQ ID NO:3中第4564位处的C→G;
SEQ ID NO:3中第4602位处的G→A。
更佳地,所述的差异选自下组:
SEQ ID NO:3中第3043位处的G→C;
SEQ ID NO:3中第3085位处的A→T;
SEQ ID NO:3中第4404位处的T→C;
SEQ ID NO:3中第4602位处的G→A。
在另一优选例中,检测的是HC56蛋白,并与正常HC56蛋白的氨基酸序列比较差异,并且所述的差异选自下组:
SEQ ID NO:4中222位处的Pro→Ser;
SEQ ID NO:4中247位处的Gln→Arg;
SEQ ID NO:4中439位处的Glu→Gln;
SEQ ID NO:4中568位处的Gly→Ala;
SEQ ID NO:4中582位处的Glu→Val;
SEQ ID NO:4中673位处的Arg→Gln;
SEQ ID NO:4中926位处的Asn→Asp;
SEQ ID NO:4中1022位处的Gys→Arg。
更佳地,所述的差异选自下组:
SEQ ID NO:4中568位处的Gly→Ala;
SEQ ID NO:4中582位处的Glu→Val;
SEQ ID NO:4中1022位处的Gys→Arg。
在本发明的第七方面,提供了一种检测肿瘤(如肝癌)易感性的试剂盒,它包括特异性扩增HC56基因或转录本的引物,并且用所述引物扩增出的扩增产物含有选自下组的单核苷酸多态性部位:
SEQ ID NO:3中第2004位处的C→T;
SEQ ID NO:3中第2080位处的A→G;
SEQ ID NO:3中第2655位处的G→C;
SEQ ID NO:3中第3043位处的G→C;
SEQ ID NO:3中第3085位处的A→T;
SEQ ID NO:3中第3358位处的G→A;
SEQ ID NO:3中第4092位处的A→G;
SEQ ID NO:3中第4404位处的T→C;
SEQ ID NO:3中第4564位处的C→G;
SEQ ID NO:3中第4602位处的G→A。
较佳地,所述试剂盒还含有与所述的单核苷酸多态性部位特异性结合的探针和/或特异性识别单核苷酸多态性部位的限制性内切酶,其中所述的单核苷酸多态性部位的核酸变化导致了限制性内切酶的产生或消失。更佳地,所述的限制性内切酶选自:SecI和EarI。
在另一优选例中,所述的突变选自下组:
SEQ ID NO:3中第3043位处的G→C;
SEQ ID NO:3中第3085位处的A→T;
SEQ ID NO:3中第4404位处的T→C;
SEQ ID NO:3中第4602位处的G→A。
本发明的其它方面由于本文的技术的公开,对本领域的技术人员而言是显而易见的。
附图说明
图1和图2显示了HC56克隆在肝癌和癌旁组织中的表达(RT-PCR)。其中,G2、G4、G11、G12、G15、G16、D1、D22、D24为肝癌患者,L:癌旁肝组织;K:癌组织;+:阳性对照:-:阴性对照;β2-M:β-微球蛋白对照探针。
图3显示了HC56在人正常组织中的表达情况,其中1-8泳道分别为:心脏、肝脏、脑、骨骼肌、胎盘、肾、肺、和胰。
图4显示了HC56的Southern杂交结果,其中基因组DNA用MSPI酶切。G7、Q7、D40、G9、D2、D43为肝癌患者,L为癌旁肝组织,K为癌组织。
图5显示了HC56的Southern杂交结果,其中基因组DNA用EcoRI酶切。D12、D13、D14为肝癌患者,L为癌旁肝组织,K为癌组织。
图6显示了HC56的Southern杂交结果,其中基因组DNA用EcoRI酶切。G5、Q9、D10为肝癌患者,L为癌旁肝组织,K为癌组织。
图7显示了PBK-CMV载体转染7721细胞。
图8显示了PBK-CMV-HC56载体转染7721细胞。
图9是重组pBR-CMV-CHx表达载体的构建图。
具体实施方式
在肝癌的研究中,本发明人首先确定了肝癌组织在17p13.3范围内有高频率LOH(60-100%)。最近,通过对肝癌全基因组扫描也证明17p13.3是LOH的最高区域。本发明人对人17号染色体短臂13.3位点的癌相关表达序列(EST)进行了分离和全长克隆。用对应于17p13.3区段内926位点的噬菌体人工染色体(PAC)579号(P579)克隆,筛选人正常肝cDNA文库,得到cDNA克隆,通过RACE方法获得全长核苷酸序列和编码的氨基酸,命名为HC56。通过Northern、RT-PCR和Southern杂交,证明HC56与肿瘤相关。体外实验证明HC56可以抑制肝癌细胞7721的生长。此外,进一步研究揭示HC56的外显子区域中有多点单核苷酸多态性(SNP)。因此,HC56基因可应用于肿瘤的诊断、治疗和预防。
HC56蛋白或多肽有多方面的用途。这些用途包括(但不限于):直接做为药物治疗HC56蛋白功能低下或丧失所致的疾病(如肿瘤),和用于筛选促进HC56蛋白功能的抗体、多肽或其它配体。用表达的重组人HC56蛋白筛选多肽库可用于寻找有治疗价值的能刺激人HC56蛋白功能的多肽分子。
另一方面,本发明还包括对人HC56 DNA或是其片段编码的多肽具有特异性的多克隆抗体和单克隆抗体,尤其是单克隆抗体。这里,“特异性”是指抗体能结合于人HC56基因产物或片段。一种优选的抗体可特异地结合于具有氨基酸序列如SEQ ID NO:2所示的蛋白质,但不结合于氨基酸序列如SEQ ID NO:4所示的蛋白质。
本发明不仅包括完整的单克隆或多克隆抗体,而且还包括具有免疫活性的抗体片段,如Fab′或(Fab)2片段;抗体重链;抗体轻链;遗传工程改造的单链Fv分子;或嵌合抗体。
本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。例如,纯化的人HC56基因产物或者其具有抗原性的片段,可被施用于动物以诱导多克隆抗体的产生。与之相似的,表达人HC56蛋白或其具有抗原性的片段的细胞可用来免疫动物来生产抗体。本发明的抗体也可以是单克隆抗体。此类单克隆抗体可以利用杂交瘤技术来制备。多克隆抗体的生产可用人HC56蛋白或多肽免疫动物,如家兔,小鼠,大鼠等。多种佐剂可用于增强免疫反应,包括但不限于弗氏佐剂等。抗人HC56蛋白的抗体可用于免疫组织化学技术中,检测活检标本中的人HC56蛋白。
本发明还涉及定量和定位检测人HC56蛋白水平的诊断试验方法。这些试验是本领域所熟知的,且包括FISH测定和放射免疫测定。试验中所检测的人HC56蛋白水平,可以用作解释人HC56蛋白在各种疾病中的重要性和用于诊断HC56蛋白起作用的疾病。
一种检测检测样品中是否存在HC56蛋白的方法是利用HC56蛋白的特异性抗体进行检测,它包括:将样品与HC56蛋白特异性抗体接触;观察是否形成抗体复合物,形成了抗体复合物就表示样品中存在HC56蛋白。
HC56蛋白的多聚核苷酸可用于HC56蛋白相关疾病的诊断和治疗。在诊断方面,HC56蛋白的多聚核苷酸可用于检测HC56蛋白的表达与否或在疾病状态下HC56蛋白的异常表达。如HC56 DNA序列可用于对活检标本的杂交以判断HC56蛋白的表达异常。杂交技术包括Southern印迹法,Northern印迹法、原位杂交等。这些技术方法都是公开的成熟技术,相关的试剂盒都可从商业途径得到。本发明的多核苷酸的一部分或全部可作为探针固定在微阵列(microarray)或DNA芯片(又称为“基因芯片”)上,用于分析组织中基因的差异表达分析和基因诊断。用HC56蛋白特异的引物进行RNA-聚合酶链反应(RT-PCR)体外扩增也可检测HC56蛋白的转录产物。
检测HC56基因的突变也可用于诊断HC56蛋白相关的疾病。HC56蛋白突变的形式包括与正常野生型HC56 DNA序列相比的点突变、易位、缺失、重组和其它任何异常等。可用已有的技术如Southern印迹法、DNA序列分析、PCR和原位杂交检测突变。一种优选的在核酸水平检测HC56基因突变的方法是检测SNP的存在与否。另外,突变有可能影响蛋白的表达,因此用Northern印迹法、Western印迹法可间接判断基因有无突变。
本发明还提供了用于在分析物中检测含有HC56基因SNP的试剂盒,该试剂盒包括装在适当容器中的、用于特异性扩增HC56基因或转录本的引物,并且用所述引物扩增出的扩增产物含有本发明所鉴别出的单核苷酸多态性部位,和使用说明。
本发明的特点在于,通过Southern印迹证明肝癌组织中存在HC56的LOH或DNA重排;Northern印迹证明某些肝癌组织中HC56的表达受抑制,说明HC56可能与肝癌发生发展有关,是肝癌抑癌基因的候选者。体外DNA转染证明HC56克隆对肝癌细胞生长有明显抑制作用;SNP检测证明有至少2个位点的SNP在肝癌病人外周血DNA与正常人外周血DNA之间统计学上有明显差异(p<0.01)。因此,据此开发的SNP检测试剂盒可用于检测肝癌易感性。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆:实验室手册(New York:ColdSpring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。
实施例
实施例1 HC56的cDNA克隆
从P579(Genome System公司)菌中抽提DNA,用NotI酶切,回收约100kb的DNA片段作为探针,用32P-dATP标记后,筛选用常规方法构建的人肝cDNA文库。经过预杂交和杂交后,获得阳性克隆。
对cDNA克隆采用双脱氧终止法,在ABI377 DNA自动测序仪上测定其一端近500bp的核苷酸序列。分析后,确定为新基因克隆,进行另一端测序,直到获得全长序列。其中一个克隆被命名为HC56,其核苷酸和氨基酸序列如SEQ ID NO:3和4所示。其中,HC56基因全长为4750核苷酸,开放阅读框位于第1341-4484位,编码全长1047个氨基酸的蛋白。
同源比较结果未发现有与HC56同源的多肽或蛋白。
实施例2 RT-PCR获得全长基因
取正常人肝组织,用Trizol试剂(GIBCO BRL公司)按厂方说明书提取总RNA,用mRNA提纯试剂盒(Pharmacia公司)提取mRNA。用MMLV-RT-Superscript II(GIBCOBRL),反转录酶在42℃进行反转录反应,获得人肝cDNA。根据SEQ ID NO:3序列,设计合成HC56基因的特异引物,按97℃3′1个循环。94℃30″60℃30″72℃1′35个循环,72℃10′1个循环进行PCR扩增,获得含有完整开放阅读框序列的HC56基因的扩增产物。扩增产物经测序验证,与实施例1测得的序列相符。
实施例3 HC56重组表达载体的构建
取实施例1中获得的HC56克隆,抽提DNA后,按图9所示的构建图,经EcoRI酶切后,与用EcoRI酶切的pBK-CMV(Stratagene公司)连接,转化感受态细胞XLI-blue。挑选白色克隆,制备质粒DNA并经酶切验证。结果得到了重组表达载体pBK-CMV-HC56。
实施例4 HC56的半定量RT-PCR
取组织总RNA 1微升,在总反应体积20微升中,用Superscript II RT试剂盒(GIBCO,BRL)合成第一链cDNA。然后用如下引物进行PCR反应:
HC56上游引物:5′-ctgcccacca ccatctgcca-3′(SEQ ID NO:5)
HC56下游引物:5′-agcagcatgg ccaacagggg-3′(SEQ ID NO:6)
并用β2-M作为内参DNA。反应结束后,取3微升PCR产物进行6%PAGE凝胶电泳,结果如图1和2所示。结果表明,HC56在G2、G12、D24中表达条带与β2-M比较,L明显强于K,G15的K强于L,其余的L和K无差异。这提示,HC56在某些肝癌组织中的表达受抑制。
实施例5 HC56的多组织膜Northern杂交
人多组织Northern杂交膜片(MTN)购自Clontech公司,在42℃预杂交3-4小时(预杂交液含有50%甲酰胺,0.05M Tris,1%SDS,5′Denhardt′s 0.1%焦磷酸钠,100mg/ml变性的精子DNA)后,与HC56探针杂交。最后X光片自显影。
结果如图3所示。HC56基因在心、胎盘、和骨骼肌中高表达,肺表达较低,其他组织中等表达表达。
实施例6 HC56的Southern杂交
人肝癌和癌旁组织DNA 10μg用EcoRI内切酶彻底酶切,然后按“分子克隆”一书的方法制成Southern膜片,然后进行预杂交、杂交、洗膜片等程序。
如图4所示,HC56在G7、D2、D43的肝癌组织(K)和癌旁组织(L)中的条带位置不同,说明肝癌组织中DNA有重排。
如图5和6所示,HC56在D13和Q9的癌旁组织(L)有一条带,而癌组织中无条带,说明存在HC56缺失。
表1 HC56基因在肝癌组织中的杂合性缺失(LOH)或DNA重排
cDNA克隆 | 标本(对) | 肝癌中有LOH | 肝癌DNA有重排 | 癌和癌旁无差异 |
HC56 | 15 | 2(D13,Q9) | 4(G7,D2,D43,G6) | 9 |
*注:D12、G7等为样本的编号。
实施例7 HC56基因的cDNA导入肝癌细胞体外试验
在本实施例中,用脂质体试剂盒进行转染肝癌细胞的体外实验。
(1)细胞株:原发肝细胞肝癌细胞系7721。
(2)DNA:来源于表达质粒pBK-CMV-HC56的DNA。
(3)脂质体:LIPOFECT AMINETM Reagent Kit(BRL公司)
(4)培养液:无血清培液,简称SF-DMEM
全培液(10%小牛血清)
含G418的全培液
T25培养瓶。
(5)DNA-脂质体复合物(DNA-liposome complex)的制备:
lipofectin 50μl加50μl SF-DMEM混匀。DNA(20μg在20μl TE中)加80μlSF-DMEM混匀。将稀释的DNA加入稀释的lipofectin溶液中,混匀置室温5-10分钟。加1.3ml SF-DMEM进入DNA-lipofectin复合物中,终体积为1.5ml。
(6)转染细胞:细胞长到80-90%满度为好,实验前换培液一次。加1.5mllipofectin试剂-DNA复合物入细胞表面,轻轻摇动,铺均匀,37℃温育1-3小时。加入1.5ml SF-DMEM混匀,37℃生长过夜。换培液37℃过夜,细胞长到70%满度换含G418培液以后常规换液至克隆出现,数克隆数。
结果如图7和8及表2所示。在图7中,空表达载体(pBK-CMV)转染7721细胞有较多克隆形成,而在图8中,含有HC56基因插入片段的表达载体转染7721细胞无克隆或仅有少数克隆形成。这表明HC56在体外对肝癌细胞生长有抑制作用。
表2 HC56在体外对肝癌细胞生长的抑制作用
cDNA克隆 | 空载体pBK-CMV | 阳性对照p21 | HC56 |
克隆形成数(平均数) | 11 | 0 | 0.3 |
实施例8 SNP检测
构建SEQ ID NO:3的序列巢式PCR引物和测序引物,用ABI3948 oligo仪器合成引物。
按常规方法从肝癌组织、癌旁组织和并入外周血白细胞制备DNA,同时制备正常人外周血白细胞DNA作为对照,50ul反应总体积含50ng基因组DNA,在标准条件下进行PCR反应。用ABI big Dye Terminator Chemistry在ABI 377平板凝胶上测序,或者用ABI3700毛细管测序仪进行测序。然后用SequenceNavigatorTM收集SNP或突变。
对80对肝癌(HCC)和癌旁肝组织进行HC56基因外显子的SNP分析,分析其外显子区有13个位置出现SNP,其中8个SNP变化伴有氨基酸改变。而且一旦3085位置有SNP,必定伴有3043、4404和4602位置的SNP。结果如表3所示。
表3 高频率的cSNP
核苷酸位置 | 氨基酸位置 | 核苷酸变化 | 氨基酸变化 | 连锁 |
2004 | 222 | C→T | Pro→Ser | |
2080 | 247 | A→G | Gln→Arg | |
2655 | 439 | G→C | Glu→Gln | |
3043 | 568 | G→C | Gly→Ala | + |
3085 | 582 | A→T | Glu→Val | |
3358 | 673 | G→A | Arg→Gln | |
4092 | 926 | A→G | Asn→Asp | |
4404 | 1022 | T→C | Gys→Arg | + |
3′UTR | ||||
4564 | C→G | |||
4602 | G→A | + |
检测70例肝癌病人外周血和100例正常人外周血DNA的3043和3085位置的SNP,发现肝癌病人外周血SNP的频率高于正常人,统计学上存在明显差异(p<0.01)。这提示,HC56是肝癌的易感基因。结果如表4所示。
表4 在C-56和肝细胞肝癌(HCC)中的cSNP
SNP 正常PBC(a) 肝细胞癌
(%)
PBC(b) 癌旁肝组织 HCC
(%) (%) (%)
n=100 n=70 n=80 n=80
3043bp G→C
(568aa) (Gly→Ala)
G/G 70.0 41.4(29) 42.5(34) 45.0(36)
G/C 26.0 50.0(35) 50.0(40) 42.5(34)
C/C 4.0 8.6(6) 7.5(6) 12.5(10)
(a Vs b p=0.001)
*3085bp A→T
(582aa) (Glu→Val)
A/A 95.0 80.0(56) 76.3(61) 78.7(63)
A/T 5.0 18.6(13) 22.5(18) 18.8(15)
T/T 0.0 1.4(1) 1.2(1) 2.5(2)
(a Vs b p=0.008)
4404bp T→C
(1022aa) (Cys→Arg)
T/T 57.0 40.0(28) 45.0(36) 43.7(35)
T/C 36.0 51.4(36) 45.0(36) 41.3(33)
C/C 7.0 8.3(6) 10.0(8) 15.0(9)
4602bp G→A
3′UTR G/G 75.0 77.1(54) 75.0(60) 75.0(60)
G/A 22.0 21.4(15) 21.2(17) 17.5(14)
A/A 3.0 1.42(1) 3.8(3) 7.5(6)
*3085位的A→T改变与3043,4404,4602位连锁,在肝和HCC中的多态性。
实施例9检测试剂盒
制备一肝癌易感性检测试剂盒,其中,含有下列可同时扩增出3043位和3085位SNP的引物对:
上游引物:5′-tgtggagggt tttcaggaag-3′(SEQ ID NO:7)(对应于SEQ ID NO:1中2861-2880位)
下游引物:5′-tggagccagt attcctctcg-3′(SEQ ID NO:8)(对应于SEQ ID NO:1中3357-3376位)。
此外,还可含有检测3043位和3085位SNP的限制性内切酶SecI和EarI。对于3043位点SNP,发生G→C变化后,用SecI酶切切不开;而不发生变化的扩增产物可用SecI酶切切开。对于3085位点SNP,发生A→T变化后,用EarI酶切切不开;而不发生变化的扩增产物可用EarI酶切切开。因此,通过对扩增产物进行酶切,该试剂盒可轻易地检测出3043位和3085位的SNP。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110>上海市肿瘤研究所
<120>人类17号染色体1区3带3亚带区域内人肿瘤相关基因及检测试剂盒
<130>013076
<160>8
<170>PatentIn version 3.0
<210>1
<211>4750
<212>DNA
<213>智人(Homo sapiens)
<220>
<221>CDS
<222>(1341)..(4484)
<400>1
tggccgtgtg attgctctgt gcccattacc ctgtggccgt gtgactgctg ctgtgcccat 60
taccctgtgg ccgtgtgatt gctgctgtgc ccattaccct gtggccgtgt gattgctctg 120
tgcccattat cctgtggccg tgtgattgct ctgtgcccat taccctgtgg ccgtgtgatt 180
gctctgtgcc cattaccctg tggccgtgtg attgctctgt gcccattatc ctgtggccgt 240
gtgattgctc tgtgcccatt atcctgtggc cgtgtgattg ctctgtgccc attatcctgt 300
ggccgtgtga ttgctctgtg cccattatcc tgtggccgtg tgattgctgc tgtgcccatt 360
accctgtggc cgtgtgattg ctctgtgccc attatcctgt ggccgtgtga ttgctctgtg 420
cccattatcc tgtggctgtg tgattgctct gtgcccatta tcctgtagcc gtgtgattgc 480
ttctgtgccc attaccctgt ggctgtgtga ttgctgctgt gcccattatc ctgtggctgt 540
gtgattgctg ctgtgcccat taccctgtgg ctgtgtgatg ctctgtgccc attaccctgt 600
ggctgtgtga ttgctctgtc ccattatcct gtggccgtgt gattgctgct gtgcctgtta 660
ccctgtggct gtgtgattgc tctgtgccca ttaccctgtg gctatgctcc cttcatctgt 720
catgagaagc tcagctgtca tgtcctgtgg tacatgctca gtggcccctg tagtttgtac 780
tgtcctccta tttctaaacc cctctcccca catcctctgc tggcccagcc tttgctggag 840
ggtctcgcct cagcagccca gctttttctt ttcacacact tttcctgaag gatcteattc 900
accgtcttgg ttccggtgat cccctctggg cagatacctc tcagaatcac atttcccagc 960
ttacttccct cctgaacttc cacctggcat ttccgttgct ggaggacatc tgtaccttga 1020
tggccaaagc tgaactcatc tttcctcaca cctgctctga ttctcctcca ttccctgtat 1080
gtgatgtcac ctggtggcct cccagttccc aggctggaga gctcggaagc cattctggat 1140
tcctcggcca agtccttctg actccagctg tgcagtggct cttgtagtca tccattctcc 1200
ccgtccgctg atgtccttta aaacccttgt catctcaaac catgacagcc tactaacagc 1260
agtggccatc tggaaacatt ttcactatac tgtcttattt ggcttgtctg tgtgcaggac 1320
ccttgaacat ctgtgaagaa atg act att ctg cat gga ggc ttc ttg ctg gcc 1373
Met Thr Ile Leu His Gly Gly Phe Leu Leu Ala
1 5 10
gag cag ctg ttc cac cct aag gca ctg gca gaa tta aca aag tct gac 1421
Glu Gln Leu Phe His Pro Lys Ala Leu Ala Glu Leu Thr Lys Ser Asp
15 20 25
tgg gaa cgt gtt gga cgg ccc atc gtg gag gcc tta agg gag atc tcc 1469
Trp Glu Arg Val Gly Arg Pro Ile Val Glu Ala Leu Arg Glu Ile Ser
30 35 40
tcg gct gca gca cac tcc cag ccc ttt gcc tgg aag aag aaa gcc ctg 1517
Ser Ala Ala Ala His Ser Gln Pro Phe Ala Trp Lys Lys Lys Ala Leu
45 50 55
atc atc atc tgg gcc aag gtt ctg cag ccg cac ccc gtg acc ccg tcc 1565
Ile Ile Ile Trp Ala Lys Val Leu Gln Pro His Pro Val Thr Pro Ser
60 65 70 75
gac aca gag aca cgg tgg cag gaa gac ctg ttc ttc tcg gtg ggc aac 1613
Asp Thr Glu Thr Arg Trp Gln Glu Asp Leu Phe Phe Ser Val Gly Asn
80 85 90
atg atc ccc acc atc aac cac acc atc ctc ttc gag ctg ctc aaa tcc 1661
Met Ile Pro Thr Ile Asn His Thr Ile Leu Phe Glu Leu Leu Lys Ser
95 100 105
ctg gaa gct tct gga ctc ttt atc cag ctc ctg atg gcc ctg ccc acc 1709
Leu Glu Ala Ser Gly Leu Phe Ile Gln Leu Leu Met Ala Leu Pro Thr
110 115 120
acc atc tgc cat gca gaa cta gag cgc ttt ctg gaa cat gtg acc gtt 1757
Thr Ile Cys His Ala Glu Leu Glu Arg Phe Leu Glu His Val Thr Val
125 130 135
gac act tct gcc gaa gac gtg gcc ttc ttc ctg gac gtc tgg tgg gag 1805
Asp Thr Ser Ala Glu Asp Val Ala Phe Phe Leu Asp Val Trp Trp Glu
140 145 150 155
gtg atg aag cac aag ggt cac ccg cag gac ccc ctg ctc tcc cag ttt 1853
Val Met Lys His Lys Gly His Pro Gln Asp Pro Leu Leu Ser Gln Phe
160 165 170
agt gca atg gcc cat aag tac ctg cct gcc tta gat gag ttc ccc cat 1901
Ser Ala Met Ala His Lys Tyr Leu Pro Ala Leu Asp Glu Phe Pro His
175 180 185
cct cca aag agg ctt agg tca gac cca gac gcg tgc ccc acc atg ccc 1949
Pro Pro Lys Arg Leu Arg Ser Asp Pro Asp Ala Cys Pro Thr Met Pro
190 195 200
ctg ttg gcc atg ctg ctc cgc ggg ctg aca cag atc cag agt cgg atc 1997
Leu Leu Ala Met Leu Leu Arg Gly Leu Thr Gln Ile Gln Ser Arg Ile
205 210 215
ctg ggc ccg ggg agg aag tgc tgt gcg ctg gcc aac ctg gct gac atg 2045
Leu Gly Pro Gly Arg Lys Cys Cys Ala Leu Ala Asn Leu Ala Asp Met
220 225 230 235
ctg act gtg ttt gcg ctg aca gag gac gac ccc cag gag gtg tct gca 2093
Leu Thr Val Phe Ala Leu Thr Glu Asp Asp Pro Gln Glu Val Ser Ala
240 245 250
acc gtg tat ctg gac aaa ctg gcc acg gtg atc tct gtg tgg aac tcg 2141
Thr Val Tyr Leu Asp Lys Leu Ala Thr Val Ile Ser Val Trp Asn Ser
255 260 265
gac acc cag aat ccc tac cac cag cag gcg ctg gca gag aag gtg aag 2189
Asp Thr Gln Asn Pro Tyr His Gln Gln Ala Leu Ala Glu Lys Val Lys
270 275 280
gag gca gaa cgg gat gtc agc ctg acc tcg ctg gcc aaa ctc ccc agt 2237
Glu Ala Glu Arg Asp Val Ser Leu Thr Ser Leu Ala Lys Leu Pro Ser
285 290 295
gag acc att ttc gtg ggc tgc gag ttc ctg cac cac ctg ctg cgg gag 2285
Glu Thr Ile Phe Val Gly Cys Glu Phe Leu His His Leu Leu Arg Glu
300 305 310 315
tgg ggg gag gag ttg cag gcc gtg ctc cgc agc agc cag ggg aca agt 2333
Trp Gly Glu Glu Leu Gln Ala Val Leu Arg Ser Ser Gln Gly Thr Ser
320 325 330
tac gac agc tac cgg ctg tgc gac agt ctg act tcc ttc agc cag aac 2381
Tyr Asp Ser Tyr Arg Leu Cys Asp Ser Leu Thr Ser Phe Ser Gln Asn
335 340 345
gcg acg ctc tac ctg aac cgc acc agc ctg tcc aag gag gac agg cag 2429
Ala Thr Leu Tyr Leu Asn Arg Thr Ser Leu Ser Lys Glu Asp Arg Gln
350 355 360
gtg gtc tct gag ctg gcg gag tgt gtc agg gac ttc ctg agg aaa acg 2477
Val Val Ser Glu Leu Ala Glu Cys Val Arg Asp Phe Leu Arg Lys Thr
365 370 375
agc acg gtg ctg aag aac agg gcc ttg gag gat atc aca gct tcc att 2525
Ser Thr Val Leu Lys Asn Arg Ala Leu Glu Asp Ile Thr Ala Ser Ile
380 385 390 395
gcc atg gcc gtc atc cag cag aag atg gac cgc cat atg gaa gtg tgc 2573
Ala Met Ala Val Ile Gln Gln Lys Met Asp Arg His Met Glu Val Cys
400 405 410
tac att ttt gcc tct gag aag aag tgg gcc ttc tcg gac gag tgg gta 2621
Tyr Ile Phe Ala Ser Glu Lys Lys Trp Ala Phe Ser Asp Glu Trp Val
415 420 425
gcc tgc ctg ggg agt aac agg gcc ctc ttc cga gag cca gac ttg gtg 2669
Ala Cys Leu Gly Ser Asn Arg Ala Leu Phe Arg Glu Pro Asp Leu Val
430 435 440
ttg agg ctg ctg gaa aca gtg ata gac gtc agc aca gct gac aga gcc 2717
Leu Arg Leu Leu Glu Thr Val Ile Asp Val Ser Thr Ala Asp Arg Ala
445 450 455
atc cct gag tct cag atc cgg cag gtg atc cac ctg atc ctg gaa tgt 2765
Ile Pro Glu Ser Gln Ile Arg Gln Val Ile His Leu Ile Leu Glu Cys
460 465 470 475
tac gca gac ctc tcc ctg cca ggt aaa aat aaa gtc ctt gca ggt atc 2813
Tyr Ala Asp Leu Ser Leu Pro Gly Lys Asn Lys Val Leu Ala Gly Ile
480 485 490
ctg cgt tcc tgg ggg cga aag ggc ctc tct gaa aag ttg ctg gct tat 2861
Leu Arg Ser Trp Gly Arg Lys Gly Leu Ser Glu Lys Leu Leu Ala Tyr
495 500 505
gtg gag ggt ttt cag gaa gac ctc aat aca act ttt aac cag ctc act 2909
Val Glu Gly Phe Gln Glu Asp Leu Asn Thr Thr Phe Asn Gln Leu Thr
510 515 520
cag agt gcc tcc gaa cag ggc ttg gca aaa gct gtg gcc tcc gtg gcc 2957
Gln Ser Ala Ser Glu Gln Gly Leu Ala Lys Ala Val Ala Ser Val Ala
525 530 535
cgc ctg gtc ata gtg cac ccg gaa gtc acg gtg aag aaa atg tgc agc 3005
Arg Leu Val Ile Val His Pro Glu Val Thr Val Lys Lys Met Cys Ser
540 545 550 555
ctg gct gtg gtc aat ctc ggc acc cac aag ttc ctg gcc cag att ctc 3053
Leu Ala Val Val Asn Leu Gly Thr His Lys Phe Leu Ala Gln Ile Leu
560 565 570
act gcc ttc cct gcc ctt agg ttt gtg gaa gtg cag ggt ccc aat tca 3101
Thr Ala Phe Pro Ala Leu Arg Phe Val Glu Val Gln Gly Pro Asn Ser
575 580 585
tct gcc act ttc atg gtg tca tgc ctc aaa gaa acc gtc tgg atg aag 3149
Ser Ala Thr Phe Met Val Ser Cys Leu Lys Glu Thr Val Trp Met Lys
590 595 600
ttc tct aca ccc aag gaa gaa aag caa ttt tta gag ctc ctg aac tgc 3197
Phe Ser Thr Pro Lys Glu Glu Lys Gln Phe Leu Glu Leu Leu Asn Cys
605 610 615
ctg atg agt ccc gtg aaa ccc caa ggg att cca gtg gct gct ctt ctt 3245
Leu Met Ser Pro Val Lys Pro Gln Gly Ile Pro Val Ala Ala Leu Leu
620 625 630 635
gag cca gac gag gtg ctg aag gaa ttt gtc ctg cct ttc ttg agg tta 3293
Glu Pro Asp Glu Val Leu Lys Glu Phe Val Leu Pro Phe Leu Arg Leu
640 645 650
gat gtt gaa gag gta gac ctc agt ctg agg atc ttc atc cag act cta 3341
Asp Val Glu Glu Val Asp Leu Ser Leu Arg Ile Phe Ile Gln Thr Leu
655 660 665
gag gca aac gcg tgt cga gag gaa tac tgg ctc cag acc tgc tcc ccg 3389
Glu Ala Asn Ala Cys Arg Glu Glu Tyr Trp Leu Gln Thr Cys Ser Pro
670 675 680
ttt cca ctc ctc ttc agc ttg tgc cag crc ttg gac cgc ttc agc aaa 3437
Phe Pro Leu Leu Phe Ser Leu Cys Gln Leu Leu Asp Arg Phe Ser Lys
685 690 695
tac tgg cag ctt ccc aag gag aag cgg tgc ctc tct ttg gat agg aag 3485
Tyr Trp Gln Leu Pro Lys Glu Lys Arg Cys Leu Ser Leu Asp Arg Lys
700 705 710 715
gat cta gcg atc cat atc ctg gag ctc ctg tgt gag att gta tca gcc 3533
Asp Leu Ala Ile His Ile Leu Glu Leu Leu Cys Glu Ile Val Ser Ala
720 725 730
aat gct gag acc ttc tcc ccg gat gtc tgg atc aag tcc ctg tcc tgg 3581
Asn Ala Glu Thr Phe Ser Pro Asp Val Trp Ile Lys Ser Leu Ser Trp
735 740 745
ctc cac cgc aag tta gaa cag cta gac tgg act gtg ggc ctg agg ctg 3629
Leu His Arg Lys Leu Glu Gln Leu Asp Trp Thr Val Gly Leu Arg Leu
750 755 760
aag agc ttc ttc gag ggg cac ttc aag tgt gaa gtg cca gcc aca ctt 3677
Lys Ser Phe Phe Glu Gly His Phe Lys Cys Glu Val Pro Ala Thr Leu
765 770 775
ttt gag atc tgt aag ctt tca gaa gac gag tgg acc tcc cag gcc cac 3725
Phe Glu Ile Cys Lys Leu Ser Glu Asp Glu Trp Thr Ser Gln Ala His
780 785 790 795
cca ggg tac ggg gct ggc acg ggg ctc ctg gcc tgg atg gag tgc tgc 3773
Pro Gly Tyr Gly Ala Gly Thr Gly Leu Leu Ala Trp Met Glu Cys Cys
800 805 810
tgc gtc tcc agc ggc atc tcg gag agg atg ctg tct ctc ttg gtg gtg 3821
Cys Val Ser Ser Gly Ile Ser Glu Arg Met Leu Ser Leu Leu Val Val
815 820 825
gac gtg ggc aat cct gag gag gtc aga ctg ttc agc aaa ggc ttt ctg 3869
Asp Val Gly Asn Pro Glu Glu Val Arg Leu Phe Ser Lys Gly Phe Leu
830 835 840
gtg gcc ctg gtg caa gtc atg cct tgg tgc agc cct cag gag tgg cag 3917
Val Ala Leu Val Gln Val Met Pro Trp Cys Ser Pro Gln Glu Trp Gln
845 850 855
cgc ctt cac cag ctg acc agg aga ctg ctg gag aag cag ctc ctc cat 3965
Arg Leu His Gln Leu Thr Arg Arg Leu Leu Glu Lys Gln Leu Leu His
860 865 870 875
gtc cct tat agc ctg gaa tat att cag ttt gtt ccc ctg ctc aac ctg 4013
Val Pro Tyr Ser Leu Glu Tyr Ile Gln Phe Val Pro Leu Leu Asn Leu
880 885 890
aag ccc ttt gcc cag gag ttg caa ctc tcc gtc ctc ttc ctg agg act 4061
Lys Pro Phe Ala Gln Glu Leu Gln Leu Ser Val Leu Phe Leu Arg Thr
895 900 905
ttc cag ttt ctc tgc agc cat agc tgt cgt aat tgg ctt cct ctg gaa 4109
Phe Gln Phe Leu Cys Ser His Ser Cys Arg Asn Trp Leu Pro Leu Glu
910 915 920
ggc tgg aac cac gtg gtc aaa ctc ctc tgt ggc agt ctg acc cgc ctc 4157
Gly Trp Asn His Val Val Lys Leu Leu Cys Gly Ser Leu Thr Arg Leu
925 930 935
ctg gac tca gtc agg gcg ata cag gca gct ggc cct tgg gtt caa gga 4205
Leu Asp Ser Val Arg Ala Ile Gln Ala Ala Gly Pro Trp Val Gln Gly
940 945 950 955
cca gag cag gac ctg acc cag gaa gcc ctg ttt gtt tac acc cag gtg 4253
Pro Glu Gln Asp Leu Thr Gln Glu Ala Leu Phe Val Tyr Thr Gln Val
960 965 970
ttc tgc cat gct ctg cac atc atg gcc atg ctc cac ccg gag gtc tgt 4301
Phe Cys His Ala Leu His Ile Met Ala Met Leu His Pro Glu Val Cys
975 980 985
gag cca ctc tac gtt tta gcc ttg gaa acc ctc acc tgc tat gag act 4349
Glu Pro Leu Tyr Val Leu Ala Leu Glu Thr Leu Thr Cys Tyr Glu Thr
990 995 1000
ttg agc aag acc aac cct tct gtc agc tcc ttg ctc cag agg gca 4394
Leu Ser Lys Thr Asn Pro Ser Val Ser Ser Leu Leu Gln Arg Ala
1005 1010 1015
cac gag cag cgc ttc tta aag tcc att gct gag ggc att ggc cct 4439
His Glu Gln Arg Phe Leu Lys Ser Ile Ala Glu Gly Ile Gly Pro
1020 1025 1030
gaa gaa cgg cgc caa acc ctg ttg cag aag atg agc agc ttc tga 4484
Glu Glu Arg Arg Gln Thr Leu Leu Gln Lys Met Ser Ser Phe
1035 1040 1045
cttggcgtgg ggagctgggc cccaacatgg cgggtctgca gaagatcagc agcttcttac 4544
ctgtgcggga gcgaaaaagc tgggcttcaa catggcaggt ctgtaggggt cagacccaag 4604
cagcctggac tttacagtta tgtgaaactg tccacaaaaa gtcatggcaa taatggtgta 4664
aagaaaatag tttcttgggt atttgtaacg tacaaactat cataaaaatt ctcctctttc 4724
ccaaaaaaaa aaaaaaaaaa aaaaaa 4750
<210>2
<211>1047
<212>PRT
<213>智人(Homo sapiens)
<400>2
Met Thr Ile Leu His Gly Gly Phe Leu Leu Ala Glu Gln Leu Phe His
1 5 10 15
Pro Lys Ala Leu Ala Glu Leu Thr Lys Ser Asp Trp Glu Arg Val Gly
20 25 30
Arg Pro Ile Val Glu Ala Leu Arg Glu Ile Ser Ser Ala Ala Ala His
35 40 45
Scr Gln Pro Phc Ala Trp Lys Lys Lys Ala Leu Ile Ile Ile Trp Ala
50 55 60
Lys Val Leu Gln Pro His Pro Val Thr Pro Ser Asp Thr Glu Thr Arg
65 70 75 80
Trp Gln Glu Asp Leu Phe Phe Ser Val Gly Asn Met Ile Pro Thr Ile
85 90 95
Asn His Thr Ile Leu Phe Glu Leu Leu Lys Ser Leu Glu Ala Ser Gly
100 105 110
Leu Phe Ile Gln Leu Leu Met Ala Leu Pro Thr Thr Ile Cys His Ala
115 120 125
Glu Leu Glu Arg Phe Leu Glu His Val Thr Val Asp Thr Ser Ala Glu
130 135 140
Asp Val Ala Phe Phe Leu Asp Val Trp Trp Glu Val Met Lys His Lys
145 150 155 160
Gly His Pro Gln Asp Pro Leu Leu Ser Gln Phe Ser Ala Met Ala His
165 170 175
Lys Tyr Leu Pro Ala Leu Asp Glu Phe Pro His Pro Pro Lys Arg Leu
180 185 190
Arg Ser Asp Pro Asp Ala Cys Pro Thr Met Pro Leu Leu Ala Met Leu
195 200 205
Leu Arg Gly Leu Thr Gln Ile Gln Ser Arg Ile Leu Gly Pro Gly Arg
210 215 220
Lys Cys Cys Ala Leu Ala Asn Leu Ala Asp Met Leu Thr Val Phe Ala
225 230 235 240
Leu Thr Glu Asp Asp Pro Gln Glu Val Ser Ala Thr Val Tyr Leu Asp
245 250 255
Lys Leu Ala Thr Val Ile Ser Val Trp Asn Ser Asp Thr Gln Asn Pro
260 265 270
Tyr His Gln Gln Ala Leu Ala Glu Lys Val Lys Glu Ala Glu Arg Asp
275 280 285
Val Ser Leu Thr Ser Leu Ala Lys Leu Pro Ser Glu Thr Ile Phe Val
290 295 300
Gly Cys Glu Phe Leu His His Leu Leu Arg Glu Trp Gly Glu Glu Leu
305 310 315 320
Gln Ala Val Leu Arg Ser Ser Gln Gly Thr Ser Tyr Asp Ser Tyr Arg
325 330 335
Leu Cys Asp Ser Leu Thr Ser Phe Ser Gln Asn Ala Thr Leu Tyr Leu
340 345 350
Asn Arg Thr Ser Leu Ser Lys Glu Asp Arg Gln Val Val Ser Glu Leu
355 360 365
Ala Glu Cys Val Arg Asp Phe Leu Arg Lys Thr Ser Thr Val Leu Lys
370 375 380
Asn Arg Ala Leu Glu Asp Ile Thr Ala Ser Ile Ala Met Ala Val Ile
385 390 395 400
Gln Gln Lys Met Asp Arg His Met Glu Val Cys Tyr Ile Phe Ala Ser
405 410 415
Glu Lys Lys Trp Ala Phe Ser Asp Glu Trp Val Ala Cys Leu Gly Ser
420 425 430
Asn Arg Ala Leu Phe Arg Glu Pro Asp Leu Val Leu Arg Leu Leu Glu
435 440 445
Thr Val Ile Asp Val Ser Thr Ala Asp Arg Ala Ile Pro Glu Ser Gln
450 455 460
Ile Arg Gln Val Ile His Leu Ile Leu Glu Cys Tyr Ala Asp Leu Ser
465 470 475 480
Leu Pro Gly Lys Asn Lys Val Leu Ala Gly Ile Leu Arg Ser Trp Gly
485 490 495
Arg Lys Gly Leu Ser Glu Lys Leu Leu Ala Tyr Val Glu Gly Phe Gln
500 505 510
Glu Asp Leu Asn Thr Thr Phe Asn Gln Leu Thr Gln Ser Ala Ser Glu
515 520 525
Gln Gly Leu Ala Lys Ala Val Ala Ser Val Ala Arg Leu Val Ile Val
530 535 540
His Pro Glu Val Thr Val Lys Lys Met Cys Ser Leu Ala Val Val Asn
545 550 555 560
Leu Gly Thr His Lys Phe Leu Ala Gln Ile Leu Thr Ala Phe Pro Ala
565 570 575
Leu Arg Phe Val Glu Val Gln Gly Pro Asn Ser Ser Ala Thr Phe Met
580 585 590
Val Ser Cys Leu Lys Glu Thr Val Trp Met Lys Phe Ser Thr Pro Lys
595 600 605
Glu Glu Lys Gln Phe Leu Glu Leu Leu Asn Cys Leu Met Ser Pro Val
610 615 620
Lys Pro Gln Gly Ile Pro Val Ala Ala Leu Leu Glu Pro Asp Glu Val
625 630 635 640
Leu Lys Glu Phe Val Leu Pro Phe Leu Arg Leu Asp Val Glu Glu Val
645 650 655
Asp Leu Ser Leu Arg Ile Phe Ile Gln Thr Leu Glu Ala Asn Ala Cys
660 665 670
Arg Glu Glu Tyr Trp Leu Gln Thr Cys Ser Pro Phe Pro Leu Leu Phe
675 680 685
Ser Leu Cys Gln Leu Leu Asp Arg Phe Ser Lys Tyr Trp Gln Leu Pro
690 695 700
Lys Glu Lys Arg Cys Leu Ser Leu Asp Arg Lys Asp Leu Ala Ile His
705 710 715 720
Ile Leu Glu Leu Leu Cys Glu Ile Val Ser Ala Asn Ala Glu Thr Phe
725 730 735
Ser Pro Asp Val Trp Ile Lys Ser Leu Ser Trp Leu His Arg Lys Leu
740 745 750
Glu Gln Leu Asp Trp Thr Val Gly Leu Arg Leu Lys Ser Phe Phe Glu
755 760 765
Gly His Phe Lys Cys Glu Val Pro Ala Thr Leu Phe Glu Ile Cys Lys
770 775 780
Leu Ser Glu Asp Glu Trp Thr Ser Gln Ala His Pro Gly Tyr Gly Ala
785 790 795 800
Gly Thr Gly Leu Leu Ala Trp Met Glu Cys Cys Cys Val Ser Ser Gly
805 810 815
Ile Ser Glu Arg Met Leu Ser Leu Leu Val Val Asp Val Gly Asn Pro
820 825 830
Glu Glu Val Arg Leu Phe Ser Lys Gly Phe Leu Val Ala Leu Val Gln
835 840 845
Val Met Pro Trp Cys Ser Pro Gln Glu Trp Gln Arg Leu His Gln Leu
850 855 860
Thr Arg Arg Leu Leu Glu Lys Gln Leu Leu His Val Pro Tyr Ser Leu
865 870 875 880
Glu Tyr Ile Gln Phe Val Pro Leu Leu Asn Leu Lys Pro Phe Ala Gln
885 890 895
Glu Leu Gln Leu Ser Val Leu Phe Leu Arg Thr Phe Gln Phe Leu Cys
900 905 910
Ser His Ser Cys Arg Asn Trp Leu Pro Leu Glu Gly Trp Asn His Val
915 920 925
Val Lys Leu Leu Cys Gly Ser Leu Thr Arg Leu Leu Asp Ser Val Arg
930 935 940
Ala Ile Gln Ala Ala Gly Pro Trp Val Gln Gly Pro Glu Gln Asp Leu
945 950 955 960
Thr Gln Glu Ala Leu Phe Val Tyr Thr Gln Val Phe Cys His Ala Leu
965 970 975
His Ile Met Ala Met Leu His Pro Glu Val Cys Glu Pro Leu Tyr Val
980 985 990
Leu Ala Leu Glu Thr Leu Thr Cys Tyr Glu Thr Leu Ser Lys Thr Asn
995 1000 1005
Pro Ser Val Ser Ser Leu Leu Gln Arg Ala His Glu Gln Arg Phe
1010 1015 1020
Leu Lys Ser Ile Ala Glu Gly Ile Gly Pro Glu Glu Arg Arg Gln
1025 1030 1035
Thr Leu Leu Gln Lys Met Ser Ser Phe
1040 1045
<210>3
<211>4750
<212>DNA
<213>智人(Homo sapiens)
<220>
<221>CDS
<222>(1341)..(4484)
<400>3
tggccgtgtg attgctctgt gcccattacc ctgtggccgt gtgactgctg ctgtgcccat 60
taccctgtgg ccgtgtgatt gctgctgtgc ccattaccct gtggccgtgt gattgctctg 120
tgcccattat cctgtggccg tgtgattgct ctgtgcccat taccctgtgg ccgtgtgatt 180
gctctgtgcc cattaccctg tggccgtgtg attgctctgt gcccattatc ctgtggccgt 240
gtgattgctc tgtgcccatt atcctgtggc cgtgtgattg ctctgtgccc attatcctgt 300
ggccgtgtga ttgctctgtg cccattatcc tgtggccgtg tgattgctgc tgtgcccatt 360
accctgtggc cgtgtgattg ctctgtgccc attatcctgt ggccgtgtga ttgctctgtg 420
cccattatcc tgtggctgtg tgattgctct gtgcccatta tcctgtagcc gtgtgattgc 480
ttctgtgccc attaccctgt ggctgtgtga ttgctgctgt gcccattatc ctgtggctgt 540
gtgattgctg ctgtgcccat taccctgtgg ctgtgtgatg ctctgtgccc attaccctgt 600
ggctgtgtga ttgctctgtc ccattatcct gtggccgtgt gattgctgct gtgcctgtta 660
ccctgtggct gtgtgattgc tctgtgccca ttaccctgtg gctatgctcc cttcatctgt 720
catgagaagc tcagctgtca tgtcctgtgg tacatgctca gtggcccctg tagtttgtac 780
tgtcctccta tttctaaacc cctctcccca catcctctgc tggcccagcc tttgctggag 840
ggtctcgcct cagcagccca gctttttctt ttcacacact tttcctgaag gatctcattc 900
accgtcttgg ttccggtgat cccctctggg cagatacctc tcagaatcac atttcccagc 960
ttacttccct cctgaacttc cacctggcat ttccgttgct ggaggacatc tgtaccttga 1020
tggccaaagc tgaactcatc tttcctcaca cctgctctga ttctcctcca ttccctgtat 1080
gtgatgtcac ctggtggcct cccagttccc aggctggaga gctcggaagc cattctggat 1140
tcctcggcca agtccttctg actccagctg tgcagtggct cttgtagtca tccattctcc 1200
ccgtccgctg atgtccttta aaacccttgt catctcaaac catgacagcc tactaacagc 1260
agtggccatc tggaaacatt ttcactatac tgtcttattt ggcttgtctg tgtgcaggac 1320
ccttgaacat ctgtgaagaa atg act att ctg cat gga ggc ttc ttg ctg gcc 1373
Met Thr Ile Leu His Gly Gly Phe Leu Leu Ala
1 5 10
gag cag ctg ttc cac cct aag gca ctg gca gaa tta aca aag tct gac 1421
Glu Gln Leu Phe His Pro Lys Ala Leu Ala Glu Leu Thr Lys Ser Asp
15 20 25
tgg gaa cgt gtt gga cgg ccc atc gtg gag gcc tta agg gag atc tcc 1469
Trp Glu Arg Val Gly Arg Pro Ile Val Glu Ala Leu Arg Glu Ile Ser
30 35 40
tcg get gca gca cac tcc cag ccc ttt gcc tgg aag aag aaa gcc ctg 1517
Ser Ala Ala Ala His Ser Gln Pro Phe Ala Trp Lys Lys Lys Ala Leu
45 50 55
atc atc atc tgg gcc aag gtt ctg cag ccg cac ccc gtg acc ccg tcc 1565
Ile Ile Ile Trp Ala Lys Val Leu Gln Pro His Pro Val Thr Pro Ser
60 65 70 75
gac aca gag aca cgg tgg cag gaa gac ctg ttc ttc tcg gtg ggc aac 1613
Asp Thr Glu Thr Arg Trp Gln Glu Asp Leu Phe Phe Ser Val Gly Asn
80 85 90
atg atc ccc acc atc aac cac acc atc ctc ttc gag ctg ctc aaa tcc 1661
Met Ile Pro Thr Ile Asn His Thr Ile Leu Phe Glu Leu Leu Lys Ser
95 100 105
ctg gaa gct tct gga ctc ttt atc cag ctc ctg atg gcc ctg ccc acc 1709
Leu Glu Ala Ser Gly Leu Phe Ile Gln Leu Leu Met Ala Leu Pro Thr
110 115 120
acc atc tgc cat gca gaa cta gag cgc ttt ctg gaa cat gtg acc gtt 1757
Thr Ile Cys His Ala Glu Leu Glu Arg Phe Leu Glu His Val Thr Val
125 130 135
gac act tct gcc gaa gac gtg gcc ttc ttc ctg gac gtc tgg tgg gag 1805
Asp Thr Ser Ala Glu Asp Val Ala Phe Phe Leu Asp Val Trp Trp Glu
140 145 150 155
gtg atg aag cac aag ggt cac ccg cag gac ccc ctg ctc tcc cag ttt 1853
Val Met Lys His Lys Gly His Pro Gln Asp Pro Leu Leu Ser Gln Phe
160 165 170
agt gca atg gcc cat aag tac ctg cct gcc tta gat gag ttc ccc cat 1901
Ser Ala Met Ala His Lys Tyr Leu Pro Ala Leu Asp Glu Phe Pro His
175 180 185
cct cca aag agg ctt agg tca gac cca gac gcg tgc ccc acc atg ccc 1949
Pro Pro Lys Arg Leu Arg Ser Asp Pro Asp Ala Cys Pro Thr Met Pro
190 195 200
ctg ttg gcc atg ctg ctc cgc ggg ctg aca cag atc cag agt cgg atc 1997
Leu Leu Ala Met Leu Leu Arg Gly Leu Thr Gln Ile Gln Ser Arg Ile
205 210 215
ctg ggc ccg ggg agg aag tgc tgt gcg ctg gcc aac ctg gct gac atg 2045
Leu Gly Pro Gly Arg Lys Cys Cys Ala Leu Ala Asn Leu Ala Asp Met
220 225 230 235
ctg act gtg ttt gcg ctg aca gag gac gac ccc cag gag gtg tct gca 2093
Leu Thr Val Phe Ala Leu Thr Glu Asp Asp Pro Gln Glu Val Ser Ala
240 245 250
acc gtg tat ctg gac aaa ctg gcc acg gtg atc tct gtg tgg aac tcg 2141
Thr Val Tyr Leu Asp Lys Leu Ala Thr Val Ile Ser Val Trp Asn Ser
255 260 265
gac acc cag aat ccc tac cac cag cag gcg ctg gca gag aag gtg aag 2189
Asp Thr Gln Asn Pro Tyr His Gln Gln Ala Leu Ala Glu Lys Val Lys
270 275 280
gag gca gaa cgg gat gtc agc ctg acc tcg ctg gcc aaa ctc ccc agt 2237
Glu Ala Glu Arg Asp Val Ser Leu Thr Ser Leu Ala Lys Leu Pro Ser
285 290 295
gag acc att ttc gtg ggc tgc gag ttc ctg cac cac ctg ctg cgg gag 2285
Glu Thr Ile Phe Val Gly Cys Glu Phe Leu His His Leu Leu Arg Glu
300 305 310 315
tgg ggg gag gag ttg cag gcc gtg ctc cgc agc agc cag ggg aca agt 2333
Trp Gly Glu Glu Leu Gln Ala Val Leu Arg Ser Ser Gln Gly Thr Ser
320 325 330
tac gac agc tac cgg ctg tgc gac agt ctg act tcc ttc agc cag aac 2381
Tyr Asp Ser Tyr Arg Leu Cys Asp Ser Leu Thr Ser Phe Ser Gln Asn
335 340 345
gcg acg ctc tac ctg aac cgc acc agc ctg tcc aag gag gac agg cag 2429
Ala Thr Leu Tyr Leu Asn Arg Thr Ser Leu Ser Lys Glu Asp Arg Gln
350 355 360
gtg gtc tct gag ctg gcg gag tgt gtc agg gac ttc ctg agg aaa acg 2477
Val Val Ser Glu Leu Ala Glu Cys Val Arg Asp Phe Leu Arg Lys Thr
365 370 375
agc acg gtg ctg aag aac agg gcc ttg gag gat atc aca gct tcc att 2525
Ser Thr Val Leu Lys Asn Arg Ala Leu Glu Asp Ile Thr Ala Ser Ile
380 385 390 395
gcc atg gcc gtc atc cag cag aag atg gac cgc cat atg gaa gtg tgc 2573
Ala Met Ala Val Ile Gln Gln Lys Met Asp Arg His Met Glu Val Cys
400 405 410
tac att ttt gcc tct gag aag aag tgg gcc ttc tcg gac gag tgg gta 2621
Tyr Ile Phe Ala Ser Glu Lys Lys Trp Ala Phe Ser Asp Glu Trp Val
415 420 425
gcc tgc ctg ggg agt aac agg gcc ctc ttc cga gag cca gac ttg gtg 2669
Ala Cys Leu Gly Ser Asn Arg Ala Leu Phe Arg Glu Pro Asp Leu Val
430 435 440
ttg agg ctg ctg gaa aca gtg ata gac gtc agc aca gct gac aga gcc 2717
Leu Arg Leu Leu Glu Thr Val Ile Asp Val Ser Thr Ala Asp Arg Ala
445 450 455
atc cct gag tct cag atc cgg cag gtg atc cac ctg atc ctg gaa tgt 2765
Ile Pro Glu Ser Gln Ile Arg Gln Val Ile His Leu Ile Leu Glu Cys
460 465 470 475
tac gca gac ctc tcc ctg cca ggt aaa aat aaa gtc ctt gca ggt atc 2813
Tyr Ala Asp Leu Ser Leu Pro Gly Lys Asn Lys Val Leu Ala Gly Ile
480 485 490
ctg cgt tcc tgg ggg cga aag ggc ctc tct gaa aag ttg ctg gct tat 2861
Leu Arg Ser Trp Gly Arg Lys Gly Leu Ser Glu Lys Leu Leu Ala Tyr
495 500 505
gtg gag ggt ttt cag gaa gac ctc aat aca act ttt aac cag ctc act 2909
Val Glu Gly Phe Gln Glu Asp Leu Asn Thr Thr Phe Asn Gln Leu Thr
510 515 520
cag agt gcc tcc gaa cag ggc ttg gca aaa gct gtg gcc tcc gtg gcc 2957
Gln Ser Ala Ser Glu Gln Gly Leu Ala Lys Ala Val Ala Ser Val Ala
525 530 535
cgc ctg gtc ata gtg cac ccg gaa gtc acg gtg aag aaa atg tgc agc 3005
Arg Leu Val Ile Val His Pro Glu Val Thr Val Lys Lys Met Cys Ser
540 545 550 555
ctg gct gtg gtc aat ctc ggc acc cac aag ttc ctg gcc cag att ctc 3053
Leu Ala Val Val Asn Leu Gly Thr His Lys Phe Leu Ala Gln Ile Leu
560 565 570
act gcc ttc gct gcc ctt agg ttt gtg gaa gag cag ggt ccc aat tca 3101
Thr Ala Phe Ala Ala Leu Arg Phe Val Glu Glu Gln Gly Pro Asn Ser
575 580 585
tct gcc act ttc atg gtg tca tgc ctc aaa gaa acc gtc tgg atg aag 3149
Ser Ala Thr Phe Met Val Ser Cys Leu Lys Glu Thr Val Trp Met Lys
590 595 600
ttc tct aca ccc aag gaa gaa aag caa ttt tta gag ctc ctg aac tgc 3197
Phe Ser Thr Pro Lys Glu Glu Lys Gln Phe Leu Glu Leu Leu Asn Cys
605 610 615
ctg atg agt ccc gtg aaa ccc caa ggg att cca gtg gct gct ctt ctt 3245
Leu Met Ser Pro Val Lys Pro Gln Gly Ile Pro Val Ala Ala Leu Leu
620 625 630 635
gag cca gac gag gtg ctg aag gaa ttt gtc ctg cct ttc ttg agg tta 3293
Glu Pro Asp Glu Val Leu Lys Glu Phe Val Leu Pro Phe Leu Arg Leu
640 645 650
gat gtt gaa gag gta gac ctc agt ctg agg atc ttc atc cag act cta 3341
Asp Val Glu Glu Val Asp Leu Ser Leu Arg Ile Phe Ile Gln Thr Leu
655 660 665
gag gca aac gcg tgt cga gag gaa tac tgg ctc cag acc tgc tcc ccg 3389
Glu Ala Asn Ala Cys Arg Glu Glu Tyr Trp Leu Gln Thr Cys Ser Pro
670 675 680
ttt cca ctc ctc ttc agc ttg tgc cag ctc ttg gac cgc ttc agc aaa 3437
Phe Pro Leu Leu Phe Ser Leu Cys Gln Leu Leu Asp Arg Phe Ser Lys
685 690 695
tac tgg cag ctt ccc aag gag aag cgg tgc ctc tct ttg gat agg aag 3485
Tyr Trp Gln Leu Pro Lys Glu Lys Arg Cys Leu Ser Leu Asp Arg Lys
700 705 710 715
gat cta gcg atc cat atc ctg gag ctc ctg tgt gag att gta tca gcc 3533
Asp Leu Ala Ile His Ile Leu Glu Leu Leu Cys Glu Ile Val Ser Ala
720 725 730
aat gct gag acc ttc tcc ccg gat gtc tgg atc aag tcc ctg tcc tgg 3581
Asn Ala Glu Thr Phe Ser Pro Asp Val Trp Ile Lys Ser Leu Ser Trp
735 740 745
ctc cac cgc aag tta gaa cag cta gac tgg act gtg ggc ctg agg ctg 3629
Leu His Arg Lys Leu Glu Gln Leu Asp Trp Thr Val Gly Leu Arg Leu
750 755 760
aag agc ttc ttc gag ggg cac ttc aag tgt gaa gtg cca gcc aca ctt 3677
Lys Ser Phe Phe Glu Gly His Phe Lys Cys Glu Val Pro Ala Thr Leu
765 770 775
ttt gag atc tgt aag ctt tca gaa gac gag tgg acc tcc cag gcc cac 3725
Phe Glu Ile Cys Lys Leu Ser Glu Asp Glu Trp Thr Ser Gln Ala His
780 785 790 795
cca ggg tac ggg gct ggc acg ggg ctc ctg gcc tgg atg gag tgc tgc 3773
Pro Gly Tyr Gly Ala Gly Thr Gly Leu Leu Ala Trp Met Glu Cys Cys
800 805 810
tgc gtc tcc agc ggc atc tcg gag agg atg ctg tct ctc ttg gtg gtg 3821
Cys Val Ser Ser Gly Ile Ser Glu Arg Met Leu Ser Leu Leu Val Val
815 820 825
gac gtg ggc aat cct gag gag gtc aga ctg ttc agc aaa ggc ttt ctg 3869
Asp Val Gly Asn Pro Glu Glu Val Arg Leu Phe Ser Lys Gly Phe Leu
830 835 840
gtg gcc ctg gtg caa gtc atg cct tgg tgc agc cct cag gag tgg cag 3917
Val Ala Leu Val Gln Val Met Pro Trp Cys Ser Pro Gln Glu Trp Gln
845 850 855
cgc ctt cac cag ctg acc agg aga ctg ctg gag aag cag ctc ctc cat 3965
Arg Leu His Gln Leu Thr Arg Arg Leu Leu Glu Lys Gln Leu Leu His
860 865 870 875
gtc cct tat agc ctg gaa tat att cag ttt gtt ccc ctg ctc aac ctg 4013
Val Pro Tyr Ser Leu Glu Tyr Ile Gln Phe Val Pro Leu Leu Asn Leu
880 885 890
aag ccc ttt gcc cag gag ttg caa ctc tcc gtc ctc ttc ctg agg act 4061
Lys Pro Phe Ala Gln Glu Leu Gln Leu Ser Val Leu Phe Leu Arg Thr
895 900 905
ttc cag ttt ctc tgc agc cat agc tgt cgt aat tgg ctt cct ctg gaa 4109
Phe Gln Phe Leu Cys Ser His Ser Cys Arg Asn Trp Leu Pro Leu Glu
910 915 920
ggc tgg aac cac gtg gtc aaa ctc ctc tgt ggc agt ctg acc cgc ctc 4157
Gly Trp Asn His Val Val Lys Leu Leu Cys Gly Ser Leu Thr Arg Leu
925 930 935
ctg gac tca gtc agg gcg ata cag gca gct ggc cct tgg gtt caa gga 4205
Leu Asp Ser Val Arg Ala Ile Gln Ala Ala Gly Pro Trp Val Gln Gly
940 945 950 955
cca gag cag gac ctg acc cag gaa gcc ctg ttt gtt tac acc cag gtg 4253
Pro Glu Gln Asp Leu Thr Gln Glu Ala Leu Phe Val Tyr Thr Gln Val
960 965 970
ttc tgc cat gct ctg cac atc atg gcc atg ctc cac ccg gag gtc tgt 4301
Phe Cys His Ala Leu His Ile Met Ala Met Leu His Pro Glu Val Cys
975 980 985
gag cca ctc tac gtt tta gcc ttg gaa acc ctc acc tgc tat gag act 4349
Glu Pro Leu Tyr Val Leu Ala Leu Glu Thr Leu Thr Cys Tyr Glu Thr
990 995 1000
ttg agc aag acc aac cct tct gtc agc tcc ttg ctc cag agg gca 4394
Leu Ser Lys Thr Asn Pro Ser Val Ser Ser Leu Leu Gln Arg Ala
1005 1010 1015
cac gag cag tgc ttc tta aag tcc att gct gag ggc att ggc cct 4439
His Glu Gln Cys Phe Leu Lys Ser Ile Ala Glu Gly Ile Gly Pro
1020 1025 1030
gaa gaa cgg cgc caa acc ctg ttg cag aag atg agc agc ttc tga 4484
Glu Glu Arg Arg Gln Thr Leu Leu Gln Lys Met Ser Ser Phe
1035 1040 1045
cttggcgtgg ggagctgggc cccaacatgg cgggtctgca gaagatcagc agcttcttac 4544
ctgtgcggga gcgaaaaagc tgggcttcaa catggcaggt ctgtaggggt cagacccgag 4604
cagcctggac tttacagtta tgtgaaactg tccacaaaaa gtcatggcaa taatggtgta 4664
aagaaaatag tttcttgggt atttgtaacg tacaaactat cataaaaatt ctcctctttc 4724
ccaaaaaaaa aaaaaaaaaa aaaaaa 4750
<210>4
<21l>1047
<212>PRT
<213>智人(Homo sapiens)
<400>4
Met Thr Ile Leu His Gly Gly Phe Leu Leu Ala Glu Gln Leu Phe His
1 5 10 15
Pro Lys Ala Leu Ala Glu Leu Thr Lys Ser Asp Trp Glu Arg Val Gly
20 25 30
Arg Pro Ile Val Glu Ala Leu Arg Glu Ile Ser Ser Ala Ala Ala His
35 40 45
Ser Gln Pro Phe Ala Trp Lys Lys Lys Ala Leu Ile Ile Ile Trp Ala
50 55 60
Lys Val Leu Gln Pro His Pro Val Thr Pro Ser Asp Thr Glu Thr Arg
65 70 75 80
Trp Gln Glu Asp Leu Phe Phe Ser Val Gly Asn Met Ile Pro Thr Ile
85 90 95
Asn His Thr Ile Leu Phe Glu Leu Leu Lys Ser Leu Glu Ala Ser Gly
100 105 110
Leu Phe Ile Gln Leu Leu Met Ala Leu Pro Thr Thr Ile Cys His Ala
115 120 125
Glu Leu Glu Arg Phe Leu Glu His Val Thr Val Asp Thr Ser Ala Glu
130 135 140
Asp Val Ala Phe Phe Leu Asp Val Trp Trp Glu Val Met Lys His Lys
145 150 155 160
Gly His Pro Gln Asp Pro Leu Leu Ser Gln Phe Ser Ala Met Ala His
165 170 175
Lys Tyr Leu Pro Ala Leu Asp Glu Phe Pro His Pro Pro Lys Arg Leu
180 185 190
Arg Ser Asp Pro Asp Ala Cys Pro Thr Met Pro Leu Leu Ala Met Leu
195 200 205
Leu Arg Gly Leu Thr Gln Ile Gln Ser Arg Ile Leu Gly Pro Gly Arg
210 215 220
Lys Cys Cys Ala Leu Ala Asn Leu Ala Asp Met Leu Thr Val Phe Ala
225 230 235 240
Leu Thr Glu Asp Asp Pro Gln Glu Val Ser Ala Thr Val Tyr Leu Asp
245 250 255
Lys Leu Ala Thr Val Ile Ser Val Trp Asn Ser Asp Thr Gln Asn Pro
260 265 270
Tyr His Gln Gln Ala Leu Ala Glu Lys Val Lys Glu Ala Glu Arg Asp
275 280 285
Val Ser Leu Thr Ser Leu Ala Lys Leu Pro Ser Glu Thr Ile Phe Val
290 295 300
Gly Cys Glu Phe Leu His His Leu Leu Arg Glu Trp Gly Glu Glu Leu
305 310 315 320
Gln Ala Val Leu Arg Ser Ser Gln Gly Thr Ser Tyr Asp Ser Tyr Arg
325 330 335
Leu Cys Asp Ser Leu Thr Ser Phe Ser Gln Asn Ala Thr Leu Tyr Leu
340 345 350
Asn Arg Thr Ser Leu Ser Lys Glu Asp Arg Gln Val Val Ser Glu Leu
355 360 365
Ala Glu Cys Val Arg Asp Phe Leu Arg Lys Thr Ser Thr Val Leu Lys
370 375 380
Asn Arg Ala Leu Glu Asp Ile Thr Ala Ser Ile Ala Met Ala Val Ile
385 390 395 400
Gln Gln Lys Met Asp Arg His Met Glu Val Cys Tyr Ile Phe Ala Ser
405 410 415
Glu Lys Lys Trp Ala Phe Ser Asp Glu Trp Val Ala Cys Leu Gly Ser
420 425 430
Asn Arg Ala Leu Phe Arg Glu Pro Asp Leu Val Leu Arg Leu Leu Glu
435 440 445
Thr Val Ile Asp Val Ser Thr Ala Asp Arg Ala Ile Pro Glu Ser Gln
450 455 460
Ile Arg Gln Val Ile His Leu Ile Leu Glu Cys Tyr Ala Asp Leu Ser
465 470 475 480
Leu Pro Gly Lys Asn Lys Val Leu Ala Gly Ile Leu Arg Ser Trp Gly
485 490 495
Arg Lys Gly Leu Ser Glu Lys Leu Leu Ala Tyr Val Glu Gly Phe Gln
500 505 510
Glu Asp Leu Asn Thr Thr Phe Asn Gln Leu Thr Gln Ser Ala Ser Glu
515 520 525
Gln Gly Leu Ala Lys Ala Val Ala Ser Val Ala Arg Leu Val Ile Val
530 535 540
His Pro Glu Val Thr Val Lys Lys Met Cys Ser Leu Ala Val Val Asn
545 550 555 560
Leu Gly Thr His Lys Phe Leu Ala Gln Ile Leu Thr Ala Phe Ala Ala
565 570 575
Leu Arg Phe Val Glu Glu Gln Gly Pro Asn Ser Ser Ala Thr Phe Met
580 585 590
Val Ser Cys Leu Lys Glu Thr Val Trp Met Lys Phe Ser Thr Pro Lys
595 600 605
Glu Glu Lys Gln Phe Leu Glu Leu Leu Asn Cys Leu Met Ser Pro Val
610 615 620
Lys Pro Gln Gly Ile Pro Val Ala Ala Leu Leu Glu Pro Asp Glu Val
625 630 635 640
Leu Lys Glu Phe Val Leu Pro Phe Leu Arg Leu Asp Val Glu Glu Val
645 650 655
Asp Leu Ser Leu Arg Ile Phe Ile Gln Thr Leu Glu Ala Asn Ala Cys
660 665 670
Arg Glu Glu Tyr Trp Leu Gln Thr Cys Ser Pro Phe Pro Leu Leu Phe
675 680 685
Ser Leu Cys Gln Leu Leu Asp Arg Phe Ser Lys Tyr Trp Gln Leu Pro
690 695 700
Lys Glu Lys Arg Cys Leu Ser Leu Asp Arg Lys Asp Leu Ala Ile His
705 710 715 720
Ile Leu Glu Leu Leu Cys Glu Ile Val Ser Ala Asn Ala Glu Thr Phe
725 730 735
Ser Pro Asp Val Trp Ile Lys Ser Leu Ser Trp Leu His Arg Lys Leu
740 745 750
Glu Gln Leu Asp Trp Thr Val Gly Leu Arg Leu Lys Ser Phe Phe Glu
755 760 765
Gly His Phe Lys Cys Glu Val Pro Ala Thr Leu Phe Glu Ile Cys Lys
770 775 780
Leu Ser Glu Asp Glu Trp Thr Ser Gln Ala His Pro Gly Tyr Gly Ala
785 790 795 800
Gly Thr Gly Leu Leu Ala Trp Met Glu Cys Cys Cys Val Ser Ser Gly
805 810 815
Ile Ser Glu Arg Met Leu Ser Leu Leu Val Val Asp Val Gly Asn Pro
820 825 830
Glu Glu Val Arg Leu Phe Ser Lys Gly Phe Leu Val Ala Leu Val Gln
835 840 845
Val Met Pro Trp Cys Ser Pro Gln Glu Trp Gln Arg Leu His Gln Leu
850 855 860
Thr Arg Arg Leu Leu Glu Lys Gln Leu Leu His Val Pro Tyr Ser Leu
865 870 875 880
Glu Tyr Ile Gln Phe Val Pro Leu Leu Asn Leu Lys Pro Phe Ala Gln
885 890 895
Glu Leu Gln Leu Ser Val Leu Phe Leu Arg Thr Phe Gln Phe Leu Cys
900 905 910
Ser His Ser Cys Arg Asn Trp Leu Pro Leu Glu Gly Trp Asn His Val
915 920 925
Val Lys Leu Leu Cys Gly Ser Leu Thr Arg Leu Leu Asp Ser Val Arg
930 935 940
Ala Ile Gln Ala Ala Gly Pro Trp Val Gln Gly Pro Glu Gln Asp Leu
945 950 955 960
Thr Gln Glu Ala Leu Phe Val Tyr Thr Gln Val Phe Cys His Ala Leu
965 970 975
His Ile Met Ala Met Leu His Pro Glu Val Cys Glu Pro Leu Tyr Val
980 985 990
Leu Ala Leu Glu Thr Leu Thr Cys Tyr Glu Thr Leu Ser Lys Thr Asn
995 1000 1005
Pro Ser Val Ser Ser Leu Leu Gln Arg Ala His Glu Gln Cys Phe
1010 1015 1020
Leu Lys Ser Ile Ala Glu Gly Ile Gly Pro Glu Glu Arg Arg Gln
1025 1030 1035
Thr Leu Leu Gln Lys Met Ser Ser Phe
1040 1045
<210>5
<211>20
<212>DNA
<213>引物
<400>5
ctgcccacca ccatctgcca 20
<210>6
<211>20
<212>DNA
<213>引物
<400>6
agcagcatgg ccaacagggg 20
<210>7
<211>20
<212>DNA
<213>引物
<400>7
tgtggagggt tttcaggaag 20
<210>8
<211>20
<212>DNA
<213>引物
<400>8
tggagccagt attcctctcg 20
Claims (9)
1.一种体外检测HC56的基因和转录本是否存在单核苷酸多态性的方法,其特征在于,它包括步骤:
检测所述的HC56基因或转录本,并与正常的HC56基因、转录本的核苷酸序列相比较,存在选自下组的差异就表明HC56的基因和转录本是否存在单核苷酸多态性:
SEQ ID NO:3中第2004位处的C→T;
SEQ ID NO:3中第2080位处的A→G;
SEQ ID NO:3中第2655位处的G→C;
SEQ ID NO:3中第3043位处的G→C;
SEQ ID NO:3中第3085位处的A→T;
SEQ ID NO:3中第3358位处的G→A;
SEQ ID NO:3中第4092位处的A→G;
SEQ ID NO:3中第4404位处的T→C;
SEQ ID NO:3中第4564位处的C→G;
SEQ ID NO:3中第4602位处的G→A。
2.如权利要求1所述的方法,其特征在于,所述的差异选自下组;
SEQ ID NO:3中第3043位处的G→C;
SEQ ID NO:3中第3085位处的A→T;
SEQ ID NO:3中第4404位处的T→C;
SEQ ID NO:3中第4602位处的G→A。
3.一种体外检测HC56蛋白是否存在氨基酸突变的方法,其特征在于,它包括步骤:
检测所述的HC56蛋白,并与正常HC56蛋白的氨基酸序列比较差异,存在选自下组的差异就表明HC56蛋白存在氨基酸突变:
SEQ ID NO:4中222位处的Pro→Ser;
SEQ ID NO:4中247位处的Gln→Arg;
SEQ ID NO:4中439位处的Glu→Gln;
SEQ ID NO:4中568位处的Gly→Ala;
SEQ ID NO:4中582位处的Glu→Val;
SEQ ID NO:4中673位处的Arg→Gln;
SEQ ID NO:4中926位处的Asn→Asp;
SEQ ID NO:4中1022位处的Gys→Arg。
4.如权利要求3所述的方法,其特征在于,所述的差异选自下组:
SEQ ID NO:4中568位处的Gly→Ala;
SEQ ID NO:4中582位处的Glu→Val;
SEQ ID NO:4中1022位处的Gys→Arg。
5.一种检测肿瘤易感性的试剂盒,其特征在于,它包括特异性扩增HC56基因或转录本的引物,并且用所述引物扩增出的扩增产物含有选自下组的单核苷酸多态性部位:
SEQ ID NO:3中第2004位处的C→T;
SEQ ID NO:3中第2080位处的A→G;
SEQ ID NO:3中第2655位处的G→C;
SEQ ID NO:3中第3043位处的G→C;
SEQ ID NO:3中第3085位处的A→T;
SEQ ID NO:3中第3358位处的G→A;
SEQ ID NO:3中第4092位处的A→G;
SEQ ID NO:3中第4404位处的T→C;
SEQ ID NO:3中第4564位处的C→G;
SEQ ID NO:3中第4602位处的G→A。
6.如权利要求5所述的试剂盒,其特征在于,它还含有与所述的单核苷酸多态性部位特异性结合的探针和/或特异性识别单核苷酸多态性部位的限制性内切酶,其中所述的单核苷酸多态性部位的核酸变化导致了限制性内切酶的产生或消失。
7.如权利要求6所述的试剂盒,其特征在于,所述的限制性内切酶选自:SecI和EarI。
8.如权利要求5所述的试剂盒,其特征在于,所述的突变选自下组:
SEQ ID NO:3中第3043位处的G→C;
SEQ ID NO:3中第3085位处的A→T;
SEQ ID NO:3中第4404位处的T→C;
SEQ ID NO:3中第4602位处的G→A。
9.一种分离的核酸,其特征在于,所述的核酸由SEQ ID NO:3中20-4750个连续核苷酸构成,并且还含有选自下组的突变:
SEQ ID NO:3中第2004位处的C→T;
SEQ ID NO:3中第2080位处的A→G;
SEQ ID NO:3中第2655位处的G→C;
SEQ ID NO:3中第3043位处的G→C;
SEQ ID NO:3中第3085位处的A→T;
SEQ ID NO:3中第3358位处的G→A;
SEQ ID NO:3中第4092位处的A→G;
SEQ ID NO:3中第4404位处的T→C;
SEQ ID NO:3中第4564位处的C→G;
SEQ ID NO:3中第4602位处的G→A;
或者,该核酸具有SEQ ID NO:1所示的核苷酸序列。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB011426284A CN1160472C (zh) | 2001-12-12 | 2001-12-12 | 人类17号染色体1区3带3亚带区域内人肿瘤相关基因及检测试剂盒 |
US10/498,584 US7205109B2 (en) | 2001-12-12 | 2002-03-21 | Method for detecting hepatocarcinoma susceptibility by detecting a tumor related gene in the region of human chromosome 17 p. 13. 3 |
PCT/CN2002/000182 WO2003050279A1 (fr) | 2001-12-12 | 2002-03-21 | Gene associe a l'apparition de tumeurs chez l'homme, present dans la region de sous-bande 3 de la bande 3 de la zone 1 du chromosome humain 17 et trousse de detection associee |
AU2002250770A AU2002250770A1 (en) | 2001-12-12 | 2002-03-21 | Human tumor related gene in region of sub-band 3 of band 3 of zone 1 of human chromosome 17 and detection kit |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB011426284A CN1160472C (zh) | 2001-12-12 | 2001-12-12 | 人类17号染色体1区3带3亚带区域内人肿瘤相关基因及检测试剂盒 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1424404A CN1424404A (zh) | 2003-06-18 |
CN1160472C true CN1160472C (zh) | 2004-08-04 |
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CNB011426284A Expired - Fee Related CN1160472C (zh) | 2001-12-12 | 2001-12-12 | 人类17号染色体1区3带3亚带区域内人肿瘤相关基因及检测试剂盒 |
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US (1) | US7205109B2 (zh) |
CN (1) | CN1160472C (zh) |
AU (1) | AU2002250770A1 (zh) |
WO (1) | WO2003050279A1 (zh) |
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US5976790A (en) * | 1992-03-04 | 1999-11-02 | The Regents Of The University Of California | Comparative Genomic Hybridization (CGH) |
WO1994007906A1 (en) * | 1992-10-01 | 1994-04-14 | Board Of Regents, The University Of Texas System | Epidermal surface antigen and uses thereof |
WO1999043196A2 (fr) * | 1998-02-25 | 1999-09-02 | Sumitomo Electric Industries, Ltd. | Procede de diagnostic du cancer fonde sur la detection d'anomalies chromosomiques |
CN1281899A (zh) | 1999-07-23 | 2001-01-31 | 上海市肿瘤研究所 | 人类17号染色体1区3带3亚带区域内人肿瘤相关基因 |
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2001
- 2001-12-12 CN CNB011426284A patent/CN1160472C/zh not_active Expired - Fee Related
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2002
- 2002-03-21 WO PCT/CN2002/000182 patent/WO2003050279A1/zh not_active Application Discontinuation
- 2002-03-21 AU AU2002250770A patent/AU2002250770A1/en not_active Abandoned
- 2002-03-21 US US10/498,584 patent/US7205109B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
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CN1424404A (zh) | 2003-06-18 |
US7205109B2 (en) | 2007-04-17 |
AU2002250770A1 (en) | 2003-06-23 |
US20050042616A1 (en) | 2005-02-24 |
WO2003050279A1 (fr) | 2003-06-19 |
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