CN108715838B - 原发性家族性脑钙化症致病基因myorg及其应用 - Google Patents

原发性家族性脑钙化症致病基因myorg及其应用 Download PDF

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CN108715838B
CN108715838B CN201810552232.6A CN201810552232A CN108715838B CN 108715838 B CN108715838 B CN 108715838B CN 201810552232 A CN201810552232 A CN 201810552232A CN 108715838 B CN108715838 B CN 108715838B
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陈万金
王柠
熊志奇
姚香平
程学文
王冲
赵淼
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First Affiliated Hospital of Fujian Medical University
Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
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Abstract

本发明揭示了遗传原发性家族性脑钙化症的一种新的致病基因即MYORG基因，提供了如SEQ ID NO：2‑10所示的致病突变基因的编码序列，并进一步提供了MYORG突变基因的编码蛋白质序列如SEQ ID NO：12‑20所示，可以将这些序列及其突变位点做为PFBC诊断方法检测靶标和防治药物开发的靶点，此外也可以为PFBC发病机制研究以及临床诊治方法和药物开发提供实验基础和理论依据。针对MYORG基因突变的检测，本发明基于PCR扩增和Sanger测序开发了相应的检测试剂盒和检测方法，可以简便有效地对MYORG基因突变进行检测。

Description

原发性家族性脑钙化症致病基因MYORG及其应用
技术领域
本发明涉及人类的变异基因，具体涉及一种原发性家族性脑钙化症新的致病基因MYORG的致病突变形式。本发明还涉及MYORG突变基因编码的蛋白质、突变基因检测试剂盒及其检测方法。
背景技术
原发性家族性脑钙化症(Primary familial brain calcification，PFBC)是一种以基底节或其他脑部位对称性钙化为特征的神经系统遗传病。由于本病在1930年由Fahr学者等做了系统描述，故又称Fahr病。该病多于中年后发病，病程进展较缓慢，无明显性别差异。临床症状呈多种多样，主要表现为运动障碍、精神障碍、认知障碍、癫痫发作、头晕、头痛等。轻者无明显的临床症状，重者则会出现人格及行为改变，终致精神病或痴呆。在不同家系或者同一家系不同的患者之间临床表现可存在明显的异质性。颅脑CT是诊断该病的主要手段，可表现为苍白球、尾状核、齿状核、脑白质、丘脑、皮质等部位的对称性团块状钙化灶，随病程进展钙化灶可连接成大片状。研究发现该病的病理主要为钙盐颗粒(以羟基磷灰石为主要成分)在终末小动脉和静脉、毛细血管中的沉积。
PFBC呈散发或家族性发病，具有高度遗传异质性，常染色体显性遗传研究较多。PFBC危害巨大，然而其治疗主要是对症治疗，无法根治。由于脑部病理性钙化沉积的部位及严重程度导致相应的临床症状。但是目前没有有效的药物能够去除或者减少异常钙沉积。因此只有系统揭示PFBC致病基因，才能一方面指导产前诊断开展优生优育；另一方面实现PFBC患者的早期诊断，进而及早有效控制PFBC患者的临床症状以延缓病程。因此致病基因的发现显得尤为重要，然而现阶段对PFBC的致病基因和发病机制的解析十分有限。
20世纪90年代开始，世界各地的研究人员对PFBC进行了大量遗传学研究，直至2012年，中国武汉科技大学刘静宇教授克隆PFBC第一个致病基因SLC20A2。此后，主要包括PDGFRB、PDGFB和XPR1等在内的致病基因陆续被发现和得到验证。SLC20A2基因编码钠-磷共转运体(PiT2)蛋白，参与细胞外磷的摄取。XPR1基因编码的蛋白参与细胞内磷的排出。PDGFRB基因编码血小板衍生生长因子受体，属于III型受体酪氨酸激酶家族；PDGFB基因则编码血小板衍生生长因子，这两者的结合可激活下游信号通路，参与人脑周细胞的发育，维持血脑屏障的完整性。
本发明首次发现MYORG基因为常染色体隐性遗传PFBC的致病基因，并且MYORG在鼠脑中特异性的表达在S100+的星型胶质细胞中。MYORG，又名KIAA1161或NET37，位于染色体9p13.3，编码一种含714个氨基酸的蛋白，属于糖基水解酶31家族的一员。星型胶质细胞的尾足连同血管周细胞，形成神经血管单元，而这恰好是颅脑病理钙沉积的部位；MYORG基因突变一方面可能导致星型胶质细胞功能障碍，影响与血管周细胞的联系，进而影响神经血单元的功能，导致钙化沉积；另一方面，可能通过影响糖苷酶的活性，导致脑内蛋白质糖基化代谢的异常，导致钙化发生。MYORG与以往发现的4个致病基因不一样的是，作为PFBC新揭示的一个隐性致病基因，提示了一种全新的基因和细胞类型参与PFBC的发病过程。这将对MYORG基因的功能解析及应用提供基础和依据，特别是对PFBC发病机制探究以及诊治和筛查提供新的分子靶标及理论基础。
发明内容
本发明的主要目的在于揭示PFBC新的致病基因MYORG，并提供其相关的突变基因形式；在此基础上，提供MYORG突变基因对应的编码蛋白质、MYORG突变基因检测试剂盒和检测方法，以及包含MYORG突变基因的载体、宿主细胞以及试剂盒，并进一步提供所述突变基因、蛋白、检测试剂盒、检测方法、载体和细胞的应用，尤其是在PFBC中的应用。
本发明解决其技术问题所采用的技术方案如下：
本发明的目的之一在于提供与常染色体隐性遗传PFBC相关的致病基因。
本发明提供了人类MYORG基因的9种突变形式，其编码序列如SED ID NO：2-10所示，正常人的MYORG野生型基因编码序列如SED ID NO：1所示。拥有SEQ ID NO：2-10序列所示任一编码序列的MYORG突变基因的二倍体纯合形式以及SEQ ID NO：2-10序列所示任两个编码序列的MYORG突变基因组成的二倍体杂合形式这两种类型中的任一种的出现均会导致人类原发性家族性脑钙化症的发生。参照SED ID NO：1序列所示的MYORG野生型基因编码序列，SED ID NO：2所示编码序列的MYORG突变基因其突变发生在野生型基因的第225位点，具体为野生型基因的G碱基突变为A碱基即发生c.225G>A突变；SED ID NO：3所示编码序列的MYORG突变基因其突变发生在野生型基因的第1328位点，具体为野生型基因的G碱基突变为A碱基即发生c.1328G>A突变；SED ID NO：4所示编码序列的MYORG突变基因其突变发生在野生型基因的第103位点，具体为野生型基因的A碱基突变为G碱基即发生c.103A>G突变；SEDID NO：5所示编码序列的MYORG突变基因其突变发生在野生型基因的第1321位点，具体为野生型基因的C碱基突变为G碱基即发生c.1321C>G突变；SED ID NO：6所示编码序列的MYORG突变基因其突变发生在野生型基因的第695位点，具体为野生型基因的C碱基突变为T碱基即发生c.695C>T突变；SED ID NO：7所示编码序列的MYORG突变基因其突变发生在野生型基因的第607位点，具体为野生型基因的C碱基突变为T碱基即发生c.607C>T突变；SED ID NO：8所示编码序列的MYORG突变基因其突变发生在野生型基因的第782和783位点，具体为野生型基因的GC碱基突变为TT碱基即发生c.782_783GC>TT突变；SED ID NO：9所示编码序列的MYORG突变基因其突变发生在野生型基因的第348和349位点之间，具体为野生型基因该位置插入CTGGCCTTCCGC这12个碱基即发生c.348_349insCTGGCCTTCCGC突变；SED ID NO：10所示编码序列的MYORG突变基因其突变发生在野生型基因的第1092-1097位点区域，具体为野生型基因此处的6个碱基CTTCGA缺失即发生c.1092_1097delCTTCGA突变。
鉴于MYORG致病基因突变的揭示，可遵循分子生物学方法和操作对包含所述c.225G>A、c.1328G>A、c.103A>G、c.1321C>G、c.695C>T、c.607C>T、c.782_783GC>TT、c.348_349insCTGGCCTTCCGC和c.1092_1097delCTTCGA突变的MYORG基因片段进行扩增或合成，并将突变基因片段导入到载体中获得包含突变基因片段的重组载体，进一步的可以将重组载体导入到细胞中获得包含重组载体的细胞，所述载体和细胞可以在包括但不限于PFBC机制探究、诊治靶标中进行应用。
基因编码序列的改变将引起编码蛋白蛋白序列的变化，与上述MYORG基因的9种突变形式对应的是，本发明还提供了其编码蛋白的蛋白序列。
如SED ID NO：1所示的野生型MYORG基因编码序列，其编码的蛋白蛋白序列如SEDID NO：11所示。以SED ID NO：11所示蛋白序列为参照，对于SED ID NO：2所示MYORG突变基因编码序列，其编码蛋白的蛋白序列如SED ID NO：12序列所示，具体是MYORG野生型基因编码蛋白第75位点的色氨基酸突变为终止密码子，即发生p.W75*突变；对于SED ID NO：3所示MYORG突变基因编码序列，其编码蛋白的蛋白序列如SED ID NO：13序列所示，具体是MYORG野生型基因编码蛋白第443位点的色氨基酸突变为终止密码子，即发生p.W443*突变；对于SED ID NO：4所示MYORG突变基因编码序列，其编码蛋白的蛋白序列如SED ID NO：14序列所示，具体是MYORG野生型基因编码蛋白第35位点的蛋氨基酸突变为缬氨酸，即发生p.M35V突变；对于SED ID NO：5所示MYORG突变基因编码序列，其编码蛋白的蛋白序列如SED ID NO：15序列所示，具体是MYORG野生型基因编码蛋白第441位点的精氨酸突变为甘氨酸，即发生p.R441G突变；对于SED ID NO：6所示MYORG突变基因编码序列，其编码蛋白的蛋白序列如SED ID NO：16序列所示，具体是MYORG野生型基因编码蛋白第232位点的丝氨酸突变为亮氨酸，即发生p.S232L突变；对于SED ID NO：7所示MYORG突变基因编码序列，其编码蛋白的蛋白序列如SED ID NO：17序列所示，具体是MYORG野生型基因编码蛋白第203位点的谷氨酰胺突变为终止密码子，即发生p.Q203*突变；对于SED ID NO：8所示MYORG突变基因编码序列，其编码蛋白的蛋白序列如SED ID NO：18序列所示，具体是MYORG野生型基因编码蛋白第261位点的精氨酸突变为亮氨酸，即发生p.R261L突变；对于SED ID NO：9所示MYORG突变基因编码序列，其编码蛋白的蛋白序列如SED ID NO：19序列所示，具体是MYORG野生型基因编码蛋白第116位点处依次插入了亮氨酸、丙氨酸、苯丙氨酸和精氨酸这4个氨基酸，即发生p.116_117insLAFR突变；对于SED ID NO：10所示MYORG突变基因编码序列，其编码蛋白的蛋白序列如SED ID NO：20序列所示，具体是MYORG野生型基因编码蛋白第365和366位点的苯丙氨酸和天冬氨酸缺失了，即发生p.365_366delFD突变。SEQ ID NO：11序列所示的MYORG野生型基因编码蛋白的缺失，并且SEQ ID NO：12-20序列所示的任一种或一种以上的MYORG突变基因编码蛋白的出现均揭示了原发性家族性脑钙化症的发生。
再者，针对上述MYORG基因的9种突变即SED ID NO：2-10所示序列含有的突变，本发明还提供了相应的突变检测试剂盒。试剂盒在于包含了本发明人设计的用于扩增MYORG基因突变位点所在片段的PCR引物组合，优选地，引物组合序列如SED ID NO：21-26所示，包含SEQ ID NO：21-22、SEQ ID NO：23-24和SEQ ID NO：25-26三对引物。其中，SEQ ID NO：21-22所示引物对用于c.103A>G和c.225G>A突变所在的MYORG基因片段扩增，SEQ ID NO：23-24所示引物对用于c.348_349insCTGGCCTTCCGC、c.607C>T、c.695C>T和c.782_783GC>TT突变所在的MYORG基因片段扩增，SEQ ID NO：25-26所示引物对用于c.1092_1097delCTTCGA、c.1321C>G和c.1328G>A突变所在的MYORG基因片段扩增。此外，试剂盒还包含了PCR的其它必要组分，主要为可用于PCR的DNA聚合酶、PCR缓冲液、dNTP Mix等相关试剂。
最后，本发明还提供基于上述试剂盒的MYORG基因突变检测方法，主要步骤如下：
(1)从生物样本中提取基因组DNA；
(2)采用本发明试剂盒进行MYORG基因突变片段的PCR扩增；
(3)对(2)中的PCR产物进行测序分析，以确定样品中MYORG基因的突变信息。
其中步骤(1)中所述生物样本为体液，包括但不限于血液、血清、羊水和淋巴液。优选地，所述体液为血液。
其中步骤(2)所述PCR扩增其采用的引物组合，优选地，为SED ID NO：21-26。
其中步骤(3)中突变信息参照SEQ ID NO：1所示的MYORG野生型基因编码序列而定。
本发明对于PFBC新的致病基因MYORG的9种致病突变及其对应的编码蛋白序列的揭示，可以将其做为PFBC诊断方法检测靶标和防治药物开发的靶点，此外也可以为PFBC发病机制研究以及临床诊治方法和药物开发提供实验基础和理论依据。针对MYORG基因突变的检测，本发明基于PCR扩增和Sanger测序开发了相应的检测试剂盒和检测方法，可以简便有效地对MYORG基因突变进行检测。
附图说明
图1-6分别是家系A-F的家系图及其相应的MYORG基因复合杂合或纯合突变处的Sanger测序图，其中上半图为家系图，下半图为对应的Sanger测序图。各图中正方形表示男性，圆形表示女性；箭头表示先证者，带斜线表示死亡，带问号表示未行颅脑CT检查个体；实心表示PFBC患病个体，空心表示正常个体；“*”代表送全外显子测序的样本，“－”表示MYORG基因未检出突变，两条线连接表示近亲结婚，I、II和III分别代表第1、2和3代，数字为成员编号；各图中黑色框框内为突变碱基，框内上下碱基相同者为纯合突变。其中图1对应于实施例1中的家系A，为MYORG基因c.225G>A(p.W75*)纯合突变，即具有SED ID NO：2所示突变；图2对应于家系B，为MYORG基因c.1328G>A(p.W443*)和c.103A>G(p.M35V)复合杂合突变，即具有SED ID NO：3，4所示突变；图3对应于家系C，为MYORG基因c.225G>A(p.W75*)和c.1321C>G(p.R441G)复合杂合突变，即具有SED ID NO：2，5所示突变；图4对应于家系D，为MYORG基因c.695C>T(p.S232L)纯合突变，即具有SED ID NO：6所示突变；图5对应于家系E，为MYORG基因c.607C>T(p.Q203*)和c.782_783GC>TT(p.R261L)复合杂合突变，即具有SED ID NO：7，8所示突变；图6对应于家系F，为MYORG基因c.348_349insCTGGCCTTCCGC(p.116_117insLAFR)和c.1092_1097delCTTCGA(p.365_366delFD)复合杂合突变，即具有SED IDNO：9，10所示突变。
图7是实施例1中PFBC患者V7(左半图)和V9(右半图)颅脑CT平扫影像。
具体实施方式
以下结合具体实施例，进一步阐述本发明。
发明人通过对两个常染色体隐性遗传PFBC家系A和B进行全外显子测序以及对其他11个PFBC家系进行Sanger测序，首次揭示出常染色体隐性遗传PFBC的致病基因MYORG的致病突变形式。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件实施。
实施例1运用全外显子测序初步鉴定PFBC家系A和B的致病基因
根据详细的病史和家族史调查情况以及规范的神经系统体格检查和相关辅助检查信息，收集2个PFBC家系样本，进行致病基因检测和分析。
(1)PFBC家系A和B样本信息
发明人首先从2个常染色体隐性遗传的PFBC家系(家系A和B)入手，经检测此2个家系中的PFBC患者并未检出已知致病基因(SLC20A2、PDGFRB、PDGFB及XPR1)的相关突变，提示可能有新的PFBC致病基因。其中家系A中含有2个PFBC患者(编号V7和V9，V9为先证者)，均表现为苍白球及小脑大面积钙化灶(图7)，构音障碍，共济失调，其父母为近亲结婚。家系B中也有2名PFBC患者，先证者(III4)和他的弟弟(III6)的颅脑钙化灶的分布位置及严重程度与家系A高度相似，且其父母(II3，II4)的头颅CT均为阴性。
(2)全外显子组测序分析
选择家系A中的2名PFBC患者(V7，V9)以及无患病的兄长(V5)、家系B中的2名PFBC患者(III4，III6)和未患病的妹妹(III5)进行全外显子测序分析。主要流程为：抽取10mL外周血，采用QIAamp DNA Blood Mini Kit(德国凯杰公司)从中提取基因组DNA，SeqCap EZExome Kit V3(美国罗氏公司)进行全外显子组捕获，在HiSeq 3000二代测序仪(美国Illumina公司)上测序，对比人类参考基因组GRCh38获取基因突变信息。测序结果显示有超过70％的reads位于目标捕获区域，超过99.7％的靶标目的区域被覆盖且平均覆盖深度为82X。
(3)致病基因筛选
根据遗传模式分析，以假定的常染色体隐性遗传方式进行基因筛选，优先考虑纯合突变。从家系A样本的全外显子测序数据中共筛选出5个有共分离现象的基因及其突变，分别是DDX1基因的c.607G<C(p.V203L)，RABGAP1L基因的c.C506C<T(p.T169I)，MYORG基因的c.225G>A(p.W75*)，ZCCHC6基因的3058C>G(p.Q1020E)以及TMEM2基因的c.2971C<T(p.R991W)。结合家系B样本的全外显子组测序结果，按照优先筛选共享于两个病人而不存在于对照的复合杂合突变或纯合突变的基因，发现家系B样本的MYORG基因存在复合杂合突变：其一是截短突变c.1328G>A即p.W443*，其编码序列如SEQ ID NO：3所示；其二是错义突c.103A>G即p.M35V，其编码序列如SEQ ID NO：4所示；MYORG基因野生型序列参见NCBIGene57462的NM_020702序列，编码序列如SEQ ID NO：1所示。
实施例2Sanger法测序验证PFBC家系A和B的MYORG致病基因突变
由于只有MYORG是在这两个家系(家系A和B)中共享的基因，发明人采用本发明提出的试剂盒和方法进行突变所在基因片段的扩增(具体试剂盒和方法参见实施例6)，运用Sanger测序进一步对家系A共12个相关成员(PFBC患者：V7，V9，非PFBC成员：IV6，IV7，V1，V2，V3，V4，V5，V6，VI1，VI2)进行MYORG基因突变验证及检测。发现家系A中的两个患者确实含有MYORG基因的c.225G>A纯合突变，而家系中其余10个正常人均未发现携带该位点纯合突变，说明MYORG基因突变在该家系中与疾病表型共分离。其中，发现的MYORG基因第225位碱基G突变为A即c.225G>A是该基因的一种新突变，使得MYORG基因编码的蛋白序列(SEQ IDNO：12)第75位色氨酸突变为终止密码子(p.W75*)，突变基因编码序列如SEQ ID NO：2所示(图1)。在家系B中，两个复合杂合的MYORG基因突变也存在家系共分离现象。在家系B中发现的两种新的突变—MYORG基因编码序列的第1328位碱基G突变为A即发生了c.1328G>A突变(突变基因编码序列如SEQ ID NO：3所示)，使得基因编码的蛋白序列(SEQ ID NO：13)第443位色氨基酸突变为终止密码子(p.W443*)，另外一个是103位碱基A突变为G即发生了c.103A>G突变(突变基因编码序列如SEQ ID NO：4所示)，使得MYORG基因编码的蛋白序列(SEQ IDNO：14)第35位的蛋氨基酸突变为缬氨酸(p.M35V)(图2)。
实施例3在其他PFBC家系中筛查MYORG致病基因突变
由于只有MYORG是在家系A与B中共享的基因，我们进一步扩大家系样本验证，对8个常染色体隐性遗传的PFBC家系和3个遗传方式不明的家系的先证者进行MYORG全编码区的突变筛查。根据MYORG的野生型基因编码序列SED ID NO：1，设计引物对SED ID NO：21-28扩增全编码区序列，并进行Sanger测序。其中SED ID NO：27-28所示引物对扩增体系和条件同SED ID NO：21-26(具体试剂盒和方法参见实施例6)。其中C、D、E、F四个家系的先证者均含有MYORG基因复合杂合突变或者纯合突变。在C家系中发现MYORG基因两个复合杂合突变，其一是c.225G>A(同家系A)，其二是c.1321C>G即1321位碱基C突变为G，使得MYORG基因编码的蛋白序列(SEQ ID NO：15)第441位精氨酸突变为甘氨酸(p.R441G)，突变基因编码序列如SEQ ID NO：5所示(图3)。在D家系中发现MYORG基因1个纯合突变，在695位碱基C突变为T即c.695C>T，使得MYORG基因编码的蛋白序列(SEQ ID NO：16)第232位丝氨酸突变为亮氨酸(p.S232L)，突变基因编码序列如SEQ ID NO：6所示(图4)。在E家系中发现MYORG基因两个复合杂合突变，一个是在607位碱基C突变为T即c.607C>T，使得MYORG基因编码的蛋白序列(SEQ ID NO：17)第203位谷氨酰胺突变为终止密码子(p.Q203*)，突变基因编码序列如SEQID NO：7所示。另一个是在782和783位碱基GC突变为TT即c.782_783GC>TT，使得MYORG基因编码的蛋白序列(SEQ ID NO：18)第261位精氨酸突变为亮氨酸(p.R261L)，突变基因编码序列如SEQ ID NO：8所示(图5)。在F家系中发现MYORG基因两个复合杂合突变，一个是在348和349位点之间插入12个碱基CTGGCCTTCCGC即c.348_349insCTGGCCTTCCGC，使得MYORG基因编码的蛋白序列(SEQ ID NO：19)第116和117氨基酸位点之间插入4个氨基酸(亮氨酸、丙氨酸、苯丙氨酸及精氨酸，p.116_117insLAFR)，突变基因编码序列如SEQ ID NO：9所示。另一个是在1092-1097位缺失6个碱基CTTCGA即c.1092_1097delCTTCGA，使得MYORG基因编码的蛋白序列(SEQ ID NO：20)第365和366位缺失苯丙氨酸和天冬氨酸(p.365_366delFD)，突变基因具体序列如SEQ ID NO：10所示(图6)。因此，共在6个PFBC家系(家系A-F)样本中检出9种MYORG基因突变(参见表1)。表1中位置和rs ID参考Genome Reference Consortiumhuman genome build 38(GRCh38)获得，gnomAD数据来源于基因组突变频率数据库。
表1发现的MYORG基因突变位点
Figure BDA0001680563840000091
实施例4运用连锁分析进一步证实致病基因MYORG突变在6个家系中与疾病表型成完全共分离现象
通过sanger测序对以上实施例中的6个家系(家系A-F)的所有家系成员均进行MYORG基因以上突变位点的检测，参照文献(Kruglyak，L.，Daly，M.J.，Reeve-Daly，M.P.，and Lander，E.S.,Parametric and nonparametric linkage analysis:a unifiedmultipoint approach.Am J Hum Genet 58,1996,1347-1363.)进行遗传连锁分析，通过假定的常染色体隐性遗传方式，疾病的等位基因频率为0.01，显性纯合子正常等位基因以及杂合的、纯合的疾病等位基因纯合子，分别为0.001、0.999和0.999。MYORG基因突变和脑钙化表型之间呈现完整的共分离现象，概率对数比值为4.91。从而确定MYORG基因是常染色体隐性PFBC的致病基因。
实施例5通过对这9个MYORG突变位点分析，进一步明确致病基因MYORG
在6个PFBC家系中共发9个MYORG基因突变位点，其中3个为无义突变(p.W75*，p.Q203*和p.W443*)，2个为插入或缺失突变(p.116_117insLAFR and p.365_366delFD)，其余4个为高度保守的错义突变(p.M35V，p.S232L，p.R261L和p.R441G)。这些突变均不存在于1000基因组数据库，NHLBI外显子测序数据库，也不存在于基因组突变频率数据库(gnomAD)或者是极低的频率(表1)。发明人同时取1000名非PFBC的健康人对照血样，参照实施例6中方法，进行MYORG基因所有编码区域的Sanger测序，均未发现上述9个突变位点，即未发现任何无义突变，复合杂合或纯合突变，进一步明确这些突变位点的致病性。
实施例6MYORG致病基因检验
基于本发明揭示的PFBC致病基因MYORG的9种致病突变基因形式，本发明进一步提供了所述MYORG基因突变的检测试剂盒和检测方法，并应用于对6个PFBC家系中的个体进行了MYORG基因突变检测，具体如下：
(1)标本采集与处理
对PFBC家系A-F患者及相关成员进行详细的病史和家族史调查，以及规范的神经系统体格检查和相关辅助检查信息，在其签署了知情同意书的情况下，抽取肘静脉血5ml(枸橼酸钠抗凝)，提取基因组DNA。
(2)MYORG突变基因片段扩增和测序
以(1)中基因组DNA为模板，采用本发明试剂盒对MYORG基因片段进行PCR体外扩增。本发明的扩增试剂盒主要组分包括日本Takara公司的rTaq聚合酶(TaKaRa，商品编码为DR100A)以及该rTaq聚合酶配套的10×PCR buffer、dNTP Mix，以及引物组合SED ID NO：21-26(引物由上海生工生物工程有限公司合成)。扩增体系详见表2，其中引物对F和R的选择参见表3，扩增条件为：94℃预变性3min；30个循环：94℃变性30s，60℃退火30s，72℃延伸30s；72℃终延伸5min。
表2扩增体系
编号 组份 添加量/μL
1 10×PCR buffer(Mg<sup>2+</sup>) 2.5
2 dNTP Mix 0.5
3 Primer F(50μM) 0.25
4 Primer R(50μM) 0.25
5 rTaq DNA聚合酶 0.25
6 基因组DNA模板(30ng/μL) 1.0
7 灭菌ddH<sub>2</sub>O 补足至20
(3)MYORG基因突变分析
将(2)中PCR产物送上海生工生物工程股份有限公司进行Sanger测序，分析测序结果，找出突变位点。
表3扩增引物对
Figure BDA0001680563840000111
Figure BDA0001680563840000121
结果(表3)显示A-F这6个家系均含有复合杂合或纯合突变。家系A的所有患者都具有SED ID NO：2所示突变，家系B的所有患者都具有SED ID NO：3和4所示突变，家系C的所有患者都具有SED ID NO：2和5所示突变，家系D的所有患者都具有SED ID NO：6所示突变，家系E的所有患者都具有SED ID NO：7和8所示突变，家系F的所有患者都具有SED ID NO：9和10所示突变。而各家系内正常人个体和1000名正常对照(实施例5)都没有上述突变。证明本发明提供的方法能够精确检测和分析常染色体隐性遗传PFBC。
虽然本发明是通过优选的具体实施例进行说明，但是对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 福建医科大学附属第一医院
中国科学院上海生命科学研究院
<120> 原发性家族性脑钙化症致病基因MYORG及其应用
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<170> SIPOSequenceListing 1.0
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gcggttctgg ggctgctgct tgtgctggcc gcggtggtgg cctggtgcta ctacagcgtc 240
tccctacgca aggcggagcg acttcgcgcg gagctgctgg acctgaaagc tggcggcttc 300
tccatccgca atcagaaggg agagcaggtc ttccgcctgg ccttccgctc cggcgcgctg 360
gaccttgact cctgcagccg cgatggcgcc ctgctgggct gctcgctcac ggccgacggg 420
ctgccgctgc acttcttcat ccagactgtg cggcccaagg acacggtcat gtgctaccgc 480
gtgcgctggg aggaggcagc gccgggccgg gccgtggagc acgccatgtt cttgggcgac 540
gcggcggccc actggtatgg tggcgccgag atgaggacgc aacactggcc catccgcctg 600
gatggccagc aggagcccca gccgttcgtc accagcgatg tctactcctc cgacgccgcg 660
tttgggggca tcctcgagcg ctactggcta tcttcgcgcg cggccgccat caaagtcaat 720
gactcagtgc ccttccacct gggctggaac agcacggagc gctcgctgcg gcttcaggcg 780
cgctaccacg acacgcccta caagccaccc gccggccgcg ccgcagcgcc agagctgagc 840
taccgagtgt gcgtgggctc agacgtcacc tccatccaca agtacatggt gcgtcgctac 900
ttcaacaagc cgtcaagggt gccagcaccc gaggccttcc gagaccccat ttggtccaca 960
tgggcgctgt acgggcgcgc cgtggaccag gacaaggtgc tgcgttttgc ccaacagatc 1020
cgcctgcacc acttcaacag cagccacctg gaaatcgacg acatgtacac acctgcttat 1080
ggcgacttcg acttcgatga ggtcaaattc cccaacgcca gcgacatgtt ccgccgcctg 1140
cgcgacgccg gcttccgcgt cacgctctgg gtgcaccctt ttgtcaacta caactcgtcg 1200
cgcttcggcg agggcgtgga gcgcgagctg ttcgtgcgcg aacccacggg ccggttacct 1260
gcgctggtgc gctggtggaa cggcatcggc gcggtgctag acttcacgca cccaaaggcc 1320
cgcgactggt tccagggaca cctgcggcgg ctgcgctctc gctactccgt ggcttccttc 1380
aagttcgacg cgggcgaggt cagctacctg ccgcgggact tcagcaccta ccggccgctg 1440
ccggacccca gcgtctggag ccggcgctac actgagatgg cgctgccctt cttctcgctg 1500
gcggaggtgc gcgtaggcta ccagtcacag aacatctcct gcttcttccg cctggtggat 1560
cgcgactctg tgtggggcta cgacctgggg ttgcgctcac tcatccccgc ggtgctcacc 1620
gtcagcatgc tgggctaccc attcatccta cccgatatgg tgggcggcaa cgccgtgccc 1680
cagcggacag ccggcggcga tgtgcccgag cgcgagctct acattcgctg gctggaagtg 1740
gccgccttta tgccggccat gcagttctct atcccgccct ggcgctacga cgcggaagtg 1800
gtggccatcg cgcagaagtt cgccgccctg cgggcctcgc ttgtggcacc gctgttgctt 1860
gagctggcgg gcgaggtcac cgacacgggt gaccctatcg tgcgccccct ttggtggatt 1920
gcgcccggcg acgagacagc tcaccgtatc gactcgcagt tccttattgg ggacacgctg 1980
cttgtggccc cggtgctgga gccaggcaag caggagcgcg acgtctattt gcccgccggc 2040
aagtggcgca gctacaaggg tgagcttttc gacaagacgc cggtgctgct caccgattac 2100
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gcggttctgg ggctgctgct tgtgctggcc gcggtggtgg cctgatgcta ctacagcgtc 240
tccctacgca aggcggagcg acttcgcgcg gagctgctgg acctgaaagc tggcggcttc 300
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gaccttgact cctgcagccg cgatggcgcc ctgctgggct gctcgctcac ggccgacggg 420
ctgccgctgc acttcttcat ccagactgtg cggcccaagg acacggtcat gtgctaccgc 480
gtgcgctggg aggaggcagc gccgggccgg gccgtggagc acgccatgtt cttgggcgac 540
gcggcggccc actggtatgg tggcgccgag atgaggacgc aacactggcc catccgcctg 600
gatggccagc aggagcccca gccgttcgtc accagcgatg tctactcctc cgacgccgcg 660
tttgggggca tcctcgagcg ctactggcta tcttcgcgcg cggccgccat caaagtcaat 720
gactcagtgc ccttccacct gggctggaac agcacggagc gctcgctgcg gcttcaggcg 780
cgctaccacg acacgcccta caagccaccc gccggccgcg ccgcagcgcc agagctgagc 840
taccgagtgt gcgtgggctc agacgtcacc tccatccaca agtacatggt gcgtcgctac 900
ttcaacaagc cgtcaagggt gccagcaccc gaggccttcc gagaccccat ttggtccaca 960
tgggcgctgt acgggcgcgc cgtggaccag gacaaggtgc tgcgttttgc ccaacagatc 1020
cgcctgcacc acttcaacag cagccacctg gaaatcgacg acatgtacac acctgcttat 1080
ggcgacttcg acttcgatga ggtcaaattc cccaacgcca gcgacatgtt ccgccgcctg 1140
cgcgacgccg gcttccgcgt cacgctctgg gtgcaccctt ttgtcaacta caactcgtcg 1200
cgcttcggcg agggcgtgga gcgcgagctg ttcgtgcgcg aacccacggg ccggttacct 1260
gcgctggtgc gctggtggaa cggcatcggc gcggtgctag acttcacgca cccaaaggcc 1320
cgcgactggt tccagggaca cctgcggcgg ctgcgctctc gctactccgt ggcttccttc 1380
aagttcgacg cgggcgaggt cagctacctg ccgcgggact tcagcaccta ccggccgctg 1440
ccggacccca gcgtctggag ccggcgctac actgagatgg cgctgccctt cttctcgctg 1500
gcggaggtgc gcgtaggcta ccagtcacag aacatctcct gcttcttccg cctggtggat 1560
cgcgactctg tgtggggcta cgacctgggg ttgcgctcac tcatccccgc ggtgctcacc 1620
gtcagcatgc tgggctaccc attcatccta cccgatatgg tgggcggcaa cgccgtgccc 1680
cagcggacag ccggcggcga tgtgcccgag cgcgagctct acattcgctg gctggaagtg 1740
gccgccttta tgccggccat gcagttctct atcccgccct ggcgctacga cgcggaagtg 1800
gtggccatcg cgcagaagtt cgccgccctg cgggcctcgc ttgtggcacc gctgttgctt 1860
gagctggcgg gcgaggtcac cgacacgggt gaccctatcg tgcgccccct ttggtggatt 1920
gcgcccggcg acgagacagc tcaccgtatc gactcgcagt tccttattgg ggacacgctg 1980
cttgtggccc cggtgctgga gccaggcaag caggagcgcg acgtctattt gcccgccggc 2040
aagtggcgca gctacaaggg tgagcttttc gacaagacgc cggtgctgct caccgattac 2100
ccggtcgacc tggatgagat cgcctacttt acctgggcgt cctga 2145
<210> 3
<211> 2145
<212> DNA
<213> 智人(Homo sapiens)
<400> 3
atgctccaga accctcagga gaagagccag gcctaccccc gccgccgccg gcctggctgc 60
tacgcatacc gtcagaaccc cgaggccatc gcagccgcag ctatgtacac cttcctgccc 120
gacaacttct cacctgccaa gcccaagcct tccaaagacc tgaagccgct gctgggctcc 180
gcggttctgg ggctgctgct tgtgctggcc gcggtggtgg cctggtgcta ctacagcgtc 240
tccctacgca aggcggagcg acttcgcgcg gagctgctgg acctgaaagc tggcggcttc 300
tccatccgca atcagaaggg agagcaggtc ttccgcctgg ccttccgctc cggcgcgctg 360
gaccttgact cctgcagccg cgatggcgcc ctgctgggct gctcgctcac ggccgacggg 420
ctgccgctgc acttcttcat ccagactgtg cggcccaagg acacggtcat gtgctaccgc 480
gtgcgctggg aggaggcagc gccgggccgg gccgtggagc acgccatgtt cttgggcgac 540
gcggcggccc actggtatgg tggcgccgag atgaggacgc aacactggcc catccgcctg 600
gatggccagc aggagcccca gccgttcgtc accagcgatg tctactcctc cgacgccgcg 660
tttgggggca tcctcgagcg ctactggcta tcttcgcgcg cggccgccat caaagtcaat 720
gactcagtgc ccttccacct gggctggaac agcacggagc gctcgctgcg gcttcaggcg 780
cgctaccacg acacgcccta caagccaccc gccggccgcg ccgcagcgcc agagctgagc 840
taccgagtgt gcgtgggctc agacgtcacc tccatccaca agtacatggt gcgtcgctac 900
ttcaacaagc cgtcaagggt gccagcaccc gaggccttcc gagaccccat ttggtccaca 960
tgggcgctgt acgggcgcgc cgtggaccag gacaaggtgc tgcgttttgc ccaacagatc 1020
cgcctgcacc acttcaacag cagccacctg gaaatcgacg acatgtacac acctgcttat 1080
ggcgacttcg acttcgatga ggtcaaattc cccaacgcca gcgacatgtt ccgccgcctg 1140
cgcgacgccg gcttccgcgt cacgctctgg gtgcaccctt ttgtcaacta caactcgtcg 1200
cgcttcggcg agggcgtgga gcgcgagctg ttcgtgcgcg aacccacggg ccggttacct 1260
gcgctggtgc gctggtggaa cggcatcggc gcggtgctag acttcacgca cccaaaggcc 1320
cgcgactagt tccagggaca cctgcggcgg ctgcgctctc gctactccgt ggcttccttc 1380
aagttcgacg cgggcgaggt cagctacctg ccgcgggact tcagcaccta ccggccgctg 1440
ccggacccca gcgtctggag ccggcgctac actgagatgg cgctgccctt cttctcgctg 1500
gcggaggtgc gcgtaggcta ccagtcacag aacatctcct gcttcttccg cctggtggat 1560
cgcgactctg tgtggggcta cgacctgggg ttgcgctcac tcatccccgc ggtgctcacc 1620
gtcagcatgc tgggctaccc attcatccta cccgatatgg tgggcggcaa cgccgtgccc 1680
cagcggacag ccggcggcga tgtgcccgag cgcgagctct acattcgctg gctggaagtg 1740
gccgccttta tgccggccat gcagttctct atcccgccct ggcgctacga cgcggaagtg 1800
gtggccatcg cgcagaagtt cgccgccctg cgggcctcgc ttgtggcacc gctgttgctt 1860
gagctggcgg gcgaggtcac cgacacgggt gaccctatcg tgcgccccct ttggtggatt 1920
gcgcccggcg acgagacagc tcaccgtatc gactcgcagt tccttattgg ggacacgctg 1980
cttgtggccc cggtgctgga gccaggcaag caggagcgcg acgtctattt gcccgccggc 2040
aagtggcgca gctacaaggg tgagcttttc gacaagacgc cggtgctgct caccgattac 2100
ccggtcgacc tggatgagat cgcctacttt acctgggcgt cctga 2145
<210> 4
<211> 2145
<212> DNA
<213> 智人(Homo sapiens)
<400> 4
atgctccaga accctcagga gaagagccag gcctaccccc gccgccgccg gcctggctgc 60
tacgcatacc gtcagaaccc cgaggccatc gcagccgcag ctgtgtacac cttcctgccc 120
gacaacttct cacctgccaa gcccaagcct tccaaagacc tgaagccgct gctgggctcc 180
gcggttctgg ggctgctgct tgtgctggcc gcggtggtgg cctggtgcta ctacagcgtc 240
tccctacgca aggcggagcg acttcgcgcg gagctgctgg acctgaaagc tggcggcttc 300
tccatccgca atcagaaggg agagcaggtc ttccgcctgg ccttccgctc cggcgcgctg 360
gaccttgact cctgcagccg cgatggcgcc ctgctgggct gctcgctcac ggccgacggg 420
ctgccgctgc acttcttcat ccagactgtg cggcccaagg acacggtcat gtgctaccgc 480
gtgcgctggg aggaggcagc gccgggccgg gccgtggagc acgccatgtt cttgggcgac 540
gcggcggccc actggtatgg tggcgccgag atgaggacgc aacactggcc catccgcctg 600
gatggccagc aggagcccca gccgttcgtc accagcgatg tctactcctc cgacgccgcg 660
tttgggggca tcctcgagcg ctactggcta tcttcgcgcg cggccgccat caaagtcaat 720
gactcagtgc ccttccacct gggctggaac agcacggagc gctcgctgcg gcttcaggcg 780
cgctaccacg acacgcccta caagccaccc gccggccgcg ccgcagcgcc agagctgagc 840
taccgagtgt gcgtgggctc agacgtcacc tccatccaca agtacatggt gcgtcgctac 900
ttcaacaagc cgtcaagggt gccagcaccc gaggccttcc gagaccccat ttggtccaca 960
tgggcgctgt acgggcgcgc cgtggaccag gacaaggtgc tgcgttttgc ccaacagatc 1020
cgcctgcacc acttcaacag cagccacctg gaaatcgacg acatgtacac acctgcttat 1080
ggcgacttcg acttcgatga ggtcaaattc cccaacgcca gcgacatgtt ccgccgcctg 1140
cgcgacgccg gcttccgcgt cacgctctgg gtgcaccctt ttgtcaacta caactcgtcg 1200
cgcttcggcg agggcgtgga gcgcgagctg ttcgtgcgcg aacccacggg ccggttacct 1260
gcgctggtgc gctggtggaa cggcatcggc gcggtgctag acttcacgca cccaaaggcc 1320
cgcgactggt tccagggaca cctgcggcgg ctgcgctctc gctactccgt ggcttccttc 1380
aagttcgacg cgggcgaggt cagctacctg ccgcgggact tcagcaccta ccggccgctg 1440
ccggacccca gcgtctggag ccggcgctac actgagatgg cgctgccctt cttctcgctg 1500
gcggaggtgc gcgtaggcta ccagtcacag aacatctcct gcttcttccg cctggtggat 1560
cgcgactctg tgtggggcta cgacctgggg ttgcgctcac tcatccccgc ggtgctcacc 1620
gtcagcatgc tgggctaccc attcatccta cccgatatgg tgggcggcaa cgccgtgccc 1680
cagcggacag ccggcggcga tgtgcccgag cgcgagctct acattcgctg gctggaagtg 1740
gccgccttta tgccggccat gcagttctct atcccgccct ggcgctacga cgcggaagtg 1800
gtggccatcg cgcagaagtt cgccgccctg cgggcctcgc ttgtggcacc gctgttgctt 1860
gagctggcgg gcgaggtcac cgacacgggt gaccctatcg tgcgccccct ttggtggatt 1920
gcgcccggcg acgagacagc tcaccgtatc gactcgcagt tccttattgg ggacacgctg 1980
cttgtggccc cggtgctgga gccaggcaag caggagcgcg acgtctattt gcccgccggc 2040
aagtggcgca gctacaaggg tgagcttttc gacaagacgc cggtgctgct caccgattac 2100
ccggtcgacc tggatgagat cgcctacttt acctgggcgt cctga 2145
<210> 5
<211> 2145
<212> DNA
<213> 智人(Homo sapiens)
<400> 5
atgctccaga accctcagga gaagagccag gcctaccccc gccgccgccg gcctggctgc 60
tacgcatacc gtcagaaccc cgaggccatc gcagccgcag ctatgtacac cttcctgccc 120
gacaacttct cacctgccaa gcccaagcct tccaaagacc tgaagccgct gctgggctcc 180
gcggttctgg ggctgctgct tgtgctggcc gcggtggtgg cctggtgcta ctacagcgtc 240
tccctacgca aggcggagcg acttcgcgcg gagctgctgg acctgaaagc tggcggcttc 300
tccatccgca atcagaaggg agagcaggtc ttccgcctgg ccttccgctc cggcgcgctg 360
gaccttgact cctgcagccg cgatggcgcc ctgctgggct gctcgctcac ggccgacggg 420
ctgccgctgc acttcttcat ccagactgtg cggcccaagg acacggtcat gtgctaccgc 480
gtgcgctggg aggaggcagc gccgggccgg gccgtggagc acgccatgtt cttgggcgac 540
gcggcggccc actggtatgg tggcgccgag atgaggacgc aacactggcc catccgcctg 600
gatggccagc aggagcccca gccgttcgtc accagcgatg tctactcctc cgacgccgcg 660
tttgggggca tcctcgagcg ctactggcta tcttcgcgcg cggccgccat caaagtcaat 720
gactcagtgc ccttccacct gggctggaac agcacggagc gctcgctgcg gcttcaggcg 780
cgctaccacg acacgcccta caagccaccc gccggccgcg ccgcagcgcc agagctgagc 840
taccgagtgt gcgtgggctc agacgtcacc tccatccaca agtacatggt gcgtcgctac 900
ttcaacaagc cgtcaagggt gccagcaccc gaggccttcc gagaccccat ttggtccaca 960
tgggcgctgt acgggcgcgc cgtggaccag gacaaggtgc tgcgttttgc ccaacagatc 1020
cgcctgcacc acttcaacag cagccacctg gaaatcgacg acatgtacac acctgcttat 1080
ggcgacttcg acttcgatga ggtcaaattc cccaacgcca gcgacatgtt ccgccgcctg 1140
cgcgacgccg gcttccgcgt cacgctctgg gtgcaccctt ttgtcaacta caactcgtcg 1200
cgcttcggcg agggcgtgga gcgcgagctg ttcgtgcgcg aacccacggg ccggttacct 1260
gcgctggtgc gctggtggaa cggcatcggc gcggtgctag acttcacgca cccaaaggcc 1320
ggcgactggt tccagggaca cctgcggcgg ctgcgctctc gctactccgt ggcttccttc 1380
aagttcgacg cgggcgaggt cagctacctg ccgcgggact tcagcaccta ccggccgctg 1440
ccggacccca gcgtctggag ccggcgctac actgagatgg cgctgccctt cttctcgctg 1500
gcggaggtgc gcgtaggcta ccagtcacag aacatctcct gcttcttccg cctggtggat 1560
cgcgactctg tgtggggcta cgacctgggg ttgcgctcac tcatccccgc ggtgctcacc 1620
gtcagcatgc tgggctaccc attcatccta cccgatatgg tgggcggcaa cgccgtgccc 1680
cagcggacag ccggcggcga tgtgcccgag cgcgagctct acattcgctg gctggaagtg 1740
gccgccttta tgccggccat gcagttctct atcccgccct ggcgctacga cgcggaagtg 1800
gtggccatcg cgcagaagtt cgccgccctg cgggcctcgc ttgtggcacc gctgttgctt 1860
gagctggcgg gcgaggtcac cgacacgggt gaccctatcg tgcgccccct ttggtggatt 1920
gcgcccggcg acgagacagc tcaccgtatc gactcgcagt tccttattgg ggacacgctg 1980
cttgtggccc cggtgctgga gccaggcaag caggagcgcg acgtctattt gcccgccggc 2040
aagtggcgca gctacaaggg tgagcttttc gacaagacgc cggtgctgct caccgattac 2100
ccggtcgacc tggatgagat cgcctacttt acctgggcgt cctga 2145
<210> 6
<211> 2145
<212> DNA
<213> 智人(Homo sapiens)
<400> 6
atgctccaga accctcagga gaagagccag gcctaccccc gccgccgccg gcctggctgc 60
tacgcatacc gtcagaaccc cgaggccatc gcagccgcag ctatgtacac cttcctgccc 120
gacaacttct cacctgccaa gcccaagcct tccaaagacc tgaagccgct gctgggctcc 180
gcggttctgg ggctgctgct tgtgctggcc gcggtggtgg cctggtgcta ctacagcgtc 240
tccctacgca aggcggagcg acttcgcgcg gagctgctgg acctgaaagc tggcggcttc 300
tccatccgca atcagaaggg agagcaggtc ttccgcctgg ccttccgctc cggcgcgctg 360
gaccttgact cctgcagccg cgatggcgcc ctgctgggct gctcgctcac ggccgacggg 420
ctgccgctgc acttcttcat ccagactgtg cggcccaagg acacggtcat gtgctaccgc 480
gtgcgctggg aggaggcagc gccgggccgg gccgtggagc acgccatgtt cttgggcgac 540
gcggcggccc actggtatgg tggcgccgag atgaggacgc aacactggcc catccgcctg 600
gatggccagc aggagcccca gccgttcgtc accagcgatg tctactcctc cgacgccgcg 660
tttgggggca tcctcgagcg ctactggcta tctttgcgcg cggccgccat caaagtcaat 720
gactcagtgc ccttccacct gggctggaac agcacggagc gctcgctgcg gcttcaggcg 780
cgctaccacg acacgcccta caagccaccc gccggccgcg ccgcagcgcc agagctgagc 840
taccgagtgt gcgtgggctc agacgtcacc tccatccaca agtacatggt gcgtcgctac 900
ttcaacaagc cgtcaagggt gccagcaccc gaggccttcc gagaccccat ttggtccaca 960
tgggcgctgt acgggcgcgc cgtggaccag gacaaggtgc tgcgttttgc ccaacagatc 1020
cgcctgcacc acttcaacag cagccacctg gaaatcgacg acatgtacac acctgcttat 1080
ggcgacttcg acttcgatga ggtcaaattc cccaacgcca gcgacatgtt ccgccgcctg 1140
cgcgacgccg gcttccgcgt cacgctctgg gtgcaccctt ttgtcaacta caactcgtcg 1200
cgcttcggcg agggcgtgga gcgcgagctg ttcgtgcgcg aacccacggg ccggttacct 1260
gcgctggtgc gctggtggaa cggcatcggc gcggtgctag acttcacgca cccaaaggcc 1320
cgcgactggt tccagggaca cctgcggcgg ctgcgctctc gctactccgt ggcttccttc 1380
aagttcgacg cgggcgaggt cagctacctg ccgcgggact tcagcaccta ccggccgctg 1440
ccggacccca gcgtctggag ccggcgctac actgagatgg cgctgccctt cttctcgctg 1500
gcggaggtgc gcgtaggcta ccagtcacag aacatctcct gcttcttccg cctggtggat 1560
cgcgactctg tgtggggcta cgacctgggg ttgcgctcac tcatccccgc ggtgctcacc 1620
gtcagcatgc tgggctaccc attcatccta cccgatatgg tgggcggcaa cgccgtgccc 1680
cagcggacag ccggcggcga tgtgcccgag cgcgagctct acattcgctg gctggaagtg 1740
gccgccttta tgccggccat gcagttctct atcccgccct ggcgctacga cgcggaagtg 1800
gtggccatcg cgcagaagtt cgccgccctg cgggcctcgc ttgtggcacc gctgttgctt 1860
gagctggcgg gcgaggtcac cgacacgggt gaccctatcg tgcgccccct ttggtggatt 1920
gcgcccggcg acgagacagc tcaccgtatc gactcgcagt tccttattgg ggacacgctg 1980
cttgtggccc cggtgctgga gccaggcaag caggagcgcg acgtctattt gcccgccggc 2040
aagtggcgca gctacaaggg tgagcttttc gacaagacgc cggtgctgct caccgattac 2100
ccggtcgacc tggatgagat cgcctacttt acctgggcgt cctga 2145
<210> 7
<211> 2145
<212> DNA
<213> 智人(Homo sapiens)
<400> 7
atgctccaga accctcagga gaagagccag gcctaccccc gccgccgccg gcctggctgc 60
tacgcatacc gtcagaaccc cgaggccatc gcagccgcag ctatgtacac cttcctgccc 120
gacaacttct cacctgccaa gcccaagcct tccaaagacc tgaagccgct gctgggctcc 180
gcggttctgg ggctgctgct tgtgctggcc gcggtggtgg cctggtgcta ctacagcgtc 240
tccctacgca aggcggagcg acttcgcgcg gagctgctgg acctgaaagc tggcggcttc 300
tccatccgca atcagaaggg agagcaggtc ttccgcctgg ccttccgctc cggcgcgctg 360
gaccttgact cctgcagccg cgatggcgcc ctgctgggct gctcgctcac ggccgacggg 420
ctgccgctgc acttcttcat ccagactgtg cggcccaagg acacggtcat gtgctaccgc 480
gtgcgctggg aggaggcagc gccgggccgg gccgtggagc acgccatgtt cttgggcgac 540
gcggcggccc actggtatgg tggcgccgag atgaggacgc aacactggcc catccgcctg 600
gatggctagc aggagcccca gccgttcgtc accagcgatg tctactcctc cgacgccgcg 660
tttgggggca tcctcgagcg ctactggcta tcttcgcgcg cggccgccat caaagtcaat 720
gactcagtgc ccttccacct gggctggaac agcacggagc gctcgctgcg gcttcaggcg 780
cgctaccacg acacgcccta caagccaccc gccggccgcg ccgcagcgcc agagctgagc 840
taccgagtgt gcgtgggctc agacgtcacc tccatccaca agtacatggt gcgtcgctac 900
ttcaacaagc cgtcaagggt gccagcaccc gaggccttcc gagaccccat ttggtccaca 960
tgggcgctgt acgggcgcgc cgtggaccag gacaaggtgc tgcgttttgc ccaacagatc 1020
cgcctgcacc acttcaacag cagccacctg gaaatcgacg acatgtacac acctgcttat 1080
ggcgacttcg acttcgatga ggtcaaattc cccaacgcca gcgacatgtt ccgccgcctg 1140
cgcgacgccg gcttccgcgt cacgctctgg gtgcaccctt ttgtcaacta caactcgtcg 1200
cgcttcggcg agggcgtgga gcgcgagctg ttcgtgcgcg aacccacggg ccggttacct 1260
gcgctggtgc gctggtggaa cggcatcggc gcggtgctag acttcacgca cccaaaggcc 1320
cgcgactggt tccagggaca cctgcggcgg ctgcgctctc gctactccgt ggcttccttc 1380
aagttcgacg cgggcgaggt cagctacctg ccgcgggact tcagcaccta ccggccgctg 1440
ccggacccca gcgtctggag ccggcgctac actgagatgg cgctgccctt cttctcgctg 1500
gcggaggtgc gcgtaggcta ccagtcacag aacatctcct gcttcttccg cctggtggat 1560
cgcgactctg tgtggggcta cgacctgggg ttgcgctcac tcatccccgc ggtgctcacc 1620
gtcagcatgc tgggctaccc attcatccta cccgatatgg tgggcggcaa cgccgtgccc 1680
cagcggacag ccggcggcga tgtgcccgag cgcgagctct acattcgctg gctggaagtg 1740
gccgccttta tgccggccat gcagttctct atcccgccct ggcgctacga cgcggaagtg 1800
gtggccatcg cgcagaagtt cgccgccctg cgggcctcgc ttgtggcacc gctgttgctt 1860
gagctggcgg gcgaggtcac cgacacgggt gaccctatcg tgcgccccct ttggtggatt 1920
gcgcccggcg acgagacagc tcaccgtatc gactcgcagt tccttattgg ggacacgctg 1980
cttgtggccc cggtgctgga gccaggcaag caggagcgcg acgtctattt gcccgccggc 2040
aagtggcgca gctacaaggg tgagcttttc gacaagacgc cggtgctgct caccgattac 2100
ccggtcgacc tggatgagat cgcctacttt acctgggcgt cctga 2145
<210> 8
<211> 2145
<212> DNA
<213> 智人(Homo sapiens)
<400> 8
atgctccaga accctcagga gaagagccag gcctaccccc gccgccgccg gcctggctgc 60
tacgcatacc gtcagaaccc cgaggccatc gcagccgcag ctatgtacac cttcctgccc 120
gacaacttct cacctgccaa gcccaagcct tccaaagacc tgaagccgct gctgggctcc 180
gcggttctgg ggctgctgct tgtgctggcc gcggtggtgg cctggtgcta ctacagcgtc 240
tccctacgca aggcggagcg acttcgcgcg gagctgctgg acctgaaagc tggcggcttc 300
tccatccgca atcagaaggg agagcaggtc ttccgcctgg ccttccgctc cggcgcgctg 360
gaccttgact cctgcagccg cgatggcgcc ctgctgggct gctcgctcac ggccgacggg 420
ctgccgctgc acttcttcat ccagactgtg cggcccaagg acacggtcat gtgctaccgc 480
gtgcgctggg aggaggcagc gccgggccgg gccgtggagc acgccatgtt cttgggcgac 540
gcggcggccc actggtatgg tggcgccgag atgaggacgc aacactggcc catccgcctg 600
gatggccagc aggagcccca gccgttcgtc accagcgatg tctactcctc cgacgccgcg 660
tttgggggca tcctcgagcg ctactggcta tcttcgcgcg cggccgccat caaagtcaat 720
gactcagtgc ccttccacct gggctggaac agcacggagc gctcgctgcg gcttcaggcg 780
ctttaccacg acacgcccta caagccaccc gccggccgcg ccgcagcgcc agagctgagc 840
taccgagtgt gcgtgggctc agacgtcacc tccatccaca agtacatggt gcgtcgctac 900
ttcaacaagc cgtcaagggt gccagcaccc gaggccttcc gagaccccat ttggtccaca 960
tgggcgctgt acgggcgcgc cgtggaccag gacaaggtgc tgcgttttgc ccaacagatc 1020
cgcctgcacc acttcaacag cagccacctg gaaatcgacg acatgtacac acctgcttat 1080
ggcgacttcg acttcgatga ggtcaaattc cccaacgcca gcgacatgtt ccgccgcctg 1140
cgcgacgccg gcttccgcgt cacgctctgg gtgcaccctt ttgtcaacta caactcgtcg 1200
cgcttcggcg agggcgtgga gcgcgagctg ttcgtgcgcg aacccacggg ccggttacct 1260
gcgctggtgc gctggtggaa cggcatcggc gcggtgctag acttcacgca cccaaaggcc 1320
cgcgactggt tccagggaca cctgcggcgg ctgcgctctc gctactccgt ggcttccttc 1380
aagttcgacg cgggcgaggt cagctacctg ccgcgggact tcagcaccta ccggccgctg 1440
ccggacccca gcgtctggag ccggcgctac actgagatgg cgctgccctt cttctcgctg 1500
gcggaggtgc gcgtaggcta ccagtcacag aacatctcct gcttcttccg cctggtggat 1560
cgcgactctg tgtggggcta cgacctgggg ttgcgctcac tcatccccgc ggtgctcacc 1620
gtcagcatgc tgggctaccc attcatccta cccgatatgg tgggcggcaa cgccgtgccc 1680
cagcggacag ccggcggcga tgtgcccgag cgcgagctct acattcgctg gctggaagtg 1740
gccgccttta tgccggccat gcagttctct atcccgccct ggcgctacga cgcggaagtg 1800
gtggccatcg cgcagaagtt cgccgccctg cgggcctcgc ttgtggcacc gctgttgctt 1860
gagctggcgg gcgaggtcac cgacacgggt gaccctatcg tgcgccccct ttggtggatt 1920
gcgcccggcg acgagacagc tcaccgtatc gactcgcagt tccttattgg ggacacgctg 1980
cttgtggccc cggtgctgga gccaggcaag caggagcgcg acgtctattt gcccgccggc 2040
aagtggcgca gctacaaggg tgagcttttc gacaagacgc cggtgctgct caccgattac 2100
ccggtcgacc tggatgagat cgcctacttt acctgggcgt cctga 2145
<210> 9
<211> 2157
<212> DNA
<213> 智人(Homo sapiens)
<400> 9
atgctccaga accctcagga gaagagccag gcctaccccc gccgccgccg gcctggctgc 60
tacgcatacc gtcagaaccc cgaggccatc gcagccgcag ctatgtacac cttcctgccc 120
gacaacttct cacctgccaa gcccaagcct tccaaagacc tgaagccgct gctgggctcc 180
gcggttctgg ggctgctgct tgtgctggcc gcggtggtgg cctggtgcta ctacagcgtc 240
tccctacgca aggcggagcg acttcgcgcg gagctgctgg acctgaaagc tggcggcttc 300
tccatccgca atcagaaggg agagcaggtc ttccgcctgg ccttccgcct ggccttccgc 360
tccggcgcgc tggaccttga ctcctgcagc cgcgatggcg ccctgctggg ctgctcgctc 420
acggccgacg ggctgccgct gcacttcttc atccagactg tgcggcccaa ggacacggtc 480
atgtgctacc gcgtgcgctg ggaggaggca gcgccgggcc gggccgtgga gcacgccatg 540
ttcttgggcg acgcggcggc ccactggtat ggtggcgccg agatgaggac gcaacactgg 600
cccatccgcc tggatggcca gcaggagccc cagccgttcg tcaccagcga tgtctactcc 660
tccgacgccg cgtttggggg catcctcgag cgctactggc tatcttcgcg cgcggccgcc 720
atcaaagtca atgactcagt gcccttccac ctgggctgga acagcacgga gcgctcgctg 780
cggcttcagg cgcgctacca cgacacgccc tacaagccac ccgccggccg cgccgcagcg 840
ccagagctga gctaccgagt gtgcgtgggc tcagacgtca cctccatcca caagtacatg 900
gtgcgtcgct acttcaacaa gccgtcaagg gtgccagcac ccgaggcctt ccgagacccc 960
atttggtcca catgggcgct gtacgggcgc gccgtggacc aggacaaggt gctgcgtttt 1020
gcccaacaga tccgcctgca ccacttcaac agcagccacc tggaaatcga cgacatgtac 1080
acacctgctt atggcgactt cgacttcgat gaggtcaaat tccccaacgc cagcgacatg 1140
ttccgccgcc tgcgcgacgc cggcttccgc gtcacgctct gggtgcaccc ttttgtcaac 1200
tacaactcgt cgcgcttcgg cgagggcgtg gagcgcgagc tgttcgtgcg cgaacccacg 1260
ggccggttac ctgcgctggt gcgctggtgg aacggcatcg gcgcggtgct agacttcacg 1320
cacccaaagg cccgcgactg gttccaggga cacctgcggc ggctgcgctc tcgctactcc 1380
gtggcttcct tcaagttcga cgcgggcgag gtcagctacc tgccgcggga cttcagcacc 1440
taccggccgc tgccggaccc cagcgtctgg agccggcgct acactgagat ggcgctgccc 1500
ttcttctcgc tggcggaggt gcgcgtaggc taccagtcac agaacatctc ctgcttcttc 1560
cgcctggtgg atcgcgactc tgtgtggggc tacgacctgg ggttgcgctc actcatcccc 1620
gcggtgctca ccgtcagcat gctgggctac ccattcatcc tacccgatat ggtgggcggc 1680
aacgccgtgc cccagcggac agccggcggc gatgtgcccg agcgcgagct ctacattcgc 1740
tggctggaag tggccgcctt tatgccggcc atgcagttct ctatcccgcc ctggcgctac 1800
gacgcggaag tggtggccat cgcgcagaag ttcgccgccc tgcgggcctc gcttgtggca 1860
ccgctgttgc ttgagctggc gggcgaggtc accgacacgg gtgaccctat cgtgcgcccc 1920
ctttggtgga ttgcgcccgg cgacgagaca gctcaccgta tcgactcgca gttccttatt 1980
ggggacacgc tgcttgtggc cccggtgctg gagccaggca agcaggagcg cgacgtctat 2040
ttgcccgccg gcaagtggcg cagctacaag ggtgagcttt tcgacaagac gccggtgctg 2100
ctcaccgatt acccggtcga cctggatgag atcgcctact ttacctgggc gtcctga 2157
<210> 10
<211> 2139
<212> DNA
<213> 智人(Homo sapiens)
<400> 10
atgctccaga accctcagga gaagagccag gcctaccccc gccgccgccg gcctggctgc 60
tacgcatacc gtcagaaccc cgaggccatc gcagccgcag ctatgtacac cttcctgccc 120
gacaacttct cacctgccaa gcccaagcct tccaaagacc tgaagccgct gctgggctcc 180
gcggttctgg ggctgctgct tgtgctggcc gcggtggtgg cctggtgcta ctacagcgtc 240
tccctacgca aggcggagcg acttcgcgcg gagctgctgg acctgaaagc tggcggcttc 300
tccatccgca atcagaaggg agagcaggtc ttccgcctgg ccttccgctc cggcgcgctg 360
gaccttgact cctgcagccg cgatggcgcc ctgctgggct gctcgctcac ggccgacggg 420
ctgccgctgc acttcttcat ccagactgtg cggcccaagg acacggtcat gtgctaccgc 480
gtgcgctggg aggaggcagc gccgggccgg gccgtggagc acgccatgtt cttgggcgac 540
gcggcggccc actggtatgg tggcgccgag atgaggacgc aacactggcc catccgcctg 600
gatggccagc aggagcccca gccgttcgtc accagcgatg tctactcctc cgacgccgcg 660
tttgggggca tcctcgagcg ctactggcta tcttcgcgcg cggccgccat caaagtcaat 720
gactcagtgc ccttccacct gggctggaac agcacggagc gctcgctgcg gcttcaggcg 780
cgctaccacg acacgcccta caagccaccc gccggccgcg ccgcagcgcc agagctgagc 840
taccgagtgt gcgtgggctc agacgtcacc tccatccaca agtacatggt gcgtcgctac 900
ttcaacaagc cgtcaagggt gccagcaccc gaggccttcc gagaccccat ttggtccaca 960
tgggcgctgt acgggcgcgc cgtggaccag gacaaggtgc tgcgttttgc ccaacagatc 1020
cgcctgcacc acttcaacag cagccacctg gaaatcgacg acatgtacac acctgcttat 1080
ggcgacttcg atgaggtcaa attccccaac gccagcgaca tgttccgccg cctgcgcgac 1140
gccggcttcc gcgtcacgct ctgggtgcac ccttttgtca actacaactc gtcgcgcttc 1200
ggcgagggcg tggagcgcga gctgttcgtg cgcgaaccca cgggccggtt acctgcgctg 1260
gtgcgctggt ggaacggcat cggcgcggtg ctagacttca cgcacccaaa ggcccgcgac 1320
tggttccagg gacacctgcg gcggctgcgc tctcgctact ccgtggcttc cttcaagttc 1380
gacgcgggcg aggtcagcta cctgccgcgg gacttcagca cctaccggcc gctgccggac 1440
cccagcgtct ggagccggcg ctacactgag atggcgctgc ccttcttctc gctggcggag 1500
gtgcgcgtag gctaccagtc acagaacatc tcctgcttct tccgcctggt ggatcgcgac 1560
tctgtgtggg gctacgacct ggggttgcgc tcactcatcc ccgcggtgct caccgtcagc 1620
atgctgggct acccattcat cctacccgat atggtgggcg gcaacgccgt gccccagcgg 1680
acagccggcg gcgatgtgcc cgagcgcgag ctctacattc gctggctgga agtggccgcc 1740
tttatgccgg ccatgcagtt ctctatcccg ccctggcgct acgacgcgga agtggtggcc 1800
atcgcgcaga agttcgccgc cctgcgggcc tcgcttgtgg caccgctgtt gcttgagctg 1860
gcgggcgagg tcaccgacac gggtgaccct atcgtgcgcc ccctttggtg gattgcgccc 1920
ggcgacgaga cagctcaccg tatcgactcg cagttcctta ttggggacac gctgcttgtg 1980
gccccggtgc tggagccagg caagcaggag cgcgacgtct atttgcccgc cggcaagtgg 2040
cgcagctaca agggtgagct tttcgacaag acgccggtgc tgctcaccga ttacccggtc 2100
gacctggatg agatcgccta ctttacctgg gcgtcctga 2139
<210> 11
<211> 714
<212> PRT
<213> 智人(Homo sapiens)
<400> 11
Met Leu Gln Asn Pro Gln Glu Lys Ser Gln Ala Tyr Pro Arg Arg Arg
1 5 10 15
Arg Pro Gly Cys Tyr Ala Tyr Arg Gln Asn Pro Glu Ala Ile Ala Ala
20 25 30
Ala Ala Met Tyr Thr Phe Leu Pro Asp Asn Phe Ser Pro Ala Lys Pro
35 40 45
Lys Pro Ser Lys Asp Leu Lys Pro Leu Leu Gly Ser Ala Val Leu Gly
50 55 60
Leu Leu Leu Val Leu Ala Ala Val Val Ala Trp Cys Tyr Tyr Ser Val
65 70 75 80
Ser Leu Arg Lys Ala Glu Arg Leu Arg Ala Glu Leu Leu Asp Leu Lys
85 90 95
Ala Gly Gly Phe Ser Ile Arg Asn Gln Lys Gly Glu Gln Val Phe Arg
100 105 110
Leu Ala Phe Arg Ser Gly Ala Leu Asp Leu Asp Ser Cys Ser Arg Asp
115 120 125
Gly Ala Leu Leu Gly Cys Ser Leu Thr Ala Asp Gly Leu Pro Leu His
130 135 140
Phe Phe Ile Gln Thr Val Arg Pro Lys Asp Thr Val Met Cys Tyr Arg
145 150 155 160
Val Arg Trp Glu Glu Ala Ala Pro Gly Arg Ala Val Glu His Ala Met
165 170 175
Phe Leu Gly Asp Ala Ala Ala His Trp Tyr Gly Gly Ala Glu Met Arg
180 185 190
Thr Gln His Trp Pro Ile Arg Leu Asp Gly Gln Gln Glu Pro Gln Pro
195 200 205
Phe Val Thr Ser Asp Val Tyr Ser Ser Asp Ala Ala Phe Gly Gly Ile
210 215 220
Leu Glu Arg Tyr Trp Leu Ser Ser Arg Ala Ala Ala Ile Lys Val Asn
225 230 235 240
Asp Ser Val Pro Phe His Leu Gly Trp Asn Ser Thr Glu Arg Ser Leu
245 250 255
Arg Leu Gln Ala Arg Tyr His Asp Thr Pro Tyr Lys Pro Pro Ala Gly
260 265 270
Arg Ala Ala Ala Pro Glu Leu Ser Tyr Arg Val Cys Val Gly Ser Asp
275 280 285
Val Thr Ser Ile His Lys Tyr Met Val Arg Arg Tyr Phe Asn Lys Pro
290 295 300
Ser Arg Val Pro Ala Pro Glu Ala Phe Arg Asp Pro Ile Trp Ser Thr
305 310 315 320
Trp Ala Leu Tyr Gly Arg Ala Val Asp Gln Asp Lys Val Leu Arg Phe
325 330 335
Ala Gln Gln Ile Arg Leu His His Phe Asn Ser Ser His Leu Glu Ile
340 345 350
Asp Asp Met Tyr Thr Pro Ala Tyr Gly Asp Phe Asp Phe Asp Glu Val
355 360 365
Lys Phe Pro Asn Ala Ser Asp Met Phe Arg Arg Leu Arg Asp Ala Gly
370 375 380
Phe Arg Val Thr Leu Trp Val His Pro Phe Val Asn Tyr Asn Ser Ser
385 390 395 400
Arg Phe Gly Glu Gly Val Glu Arg Glu Leu Phe Val Arg Glu Pro Thr
405 410 415
Gly Arg Leu Pro Ala Leu Val Arg Trp Trp Asn Gly Ile Gly Ala Val
420 425 430
Leu Asp Phe Thr His Pro Lys Ala Arg Asp Trp Phe Gln Gly His Leu
435 440 445
Arg Arg Leu Arg Ser Arg Tyr Ser Val Ala Ser Phe Lys Phe Asp Ala
450 455 460
Gly Glu Val Ser Tyr Leu Pro Arg Asp Phe Ser Thr Tyr Arg Pro Leu
465 470 475 480
Pro Asp Pro Ser Val Trp Ser Arg Arg Tyr Thr Glu Met Ala Leu Pro
485 490 495
Phe Phe Ser Leu Ala Glu Val Arg Val Gly Tyr Gln Ser Gln Asn Ile
500 505 510
Ser Cys Phe Phe Arg Leu Val Asp Arg Asp Ser Val Trp Gly Tyr Asp
515 520 525
Leu Gly Leu Arg Ser Leu Ile Pro Ala Val Leu Thr Val Ser Met Leu
530 535 540
Gly Tyr Pro Phe Ile Leu Pro Asp Met Val Gly Gly Asn Ala Val Pro
545 550 555 560
Gln Arg Thr Ala Gly Gly Asp Val Pro Glu Arg Glu Leu Tyr Ile Arg
565 570 575
Trp Leu Glu Val Ala Ala Phe Met Pro Ala Met Gln Phe Ser Ile Pro
580 585 590
Pro Trp Arg Tyr Asp Ala Glu Val Val Ala Ile Ala Gln Lys Phe Ala
595 600 605
Ala Leu Arg Ala Ser Leu Val Ala Pro Leu Leu Leu Glu Leu Ala Gly
610 615 620
Glu Val Thr Asp Thr Gly Asp Pro Ile Val Arg Pro Leu Trp Trp Ile
625 630 635 640
Ala Pro Gly Asp Glu Thr Ala His Arg Ile Asp Ser Gln Phe Leu Ile
645 650 655
Gly Asp Thr Leu Leu Val Ala Pro Val Leu Glu Pro Gly Lys Gln Glu
660 665 670
Arg Asp Val Tyr Leu Pro Ala Gly Lys Trp Arg Ser Tyr Lys Gly Glu
675 680 685
Leu Phe Asp Lys Thr Pro Val Leu Leu Thr Asp Tyr Pro Val Asp Leu
690 695 700
Asp Glu Ile Ala Tyr Phe Thr Trp Ala Ser
705 710
<210> 12
<211> 74
<212> PRT
<213> 智人(Homo sapiens)
<400> 12
Met Leu Gln Asn Pro Gln Glu Lys Ser Gln Ala Tyr Pro Arg Arg Arg
1 5 10 15
Arg Pro Gly Cys Tyr Ala Tyr Arg Gln Asn Pro Glu Ala Ile Ala Ala
20 25 30
Ala Ala Met Tyr Thr Phe Leu Pro Asp Asn Phe Ser Pro Ala Lys Pro
35 40 45
Lys Pro Ser Lys Asp Leu Lys Pro Leu Leu Gly Ser Ala Val Leu Gly
50 55 60
Leu Leu Leu Val Leu Ala Ala Val Val Ala
65 70
<210> 13
<211> 442
<212> PRT
<213> 智人(Homo sapiens)
<400> 13
Met Leu Gln Asn Pro Gln Glu Lys Ser Gln Ala Tyr Pro Arg Arg Arg
1 5 10 15
Arg Pro Gly Cys Tyr Ala Tyr Arg Gln Asn Pro Glu Ala Ile Ala Ala
20 25 30
Ala Ala Met Tyr Thr Phe Leu Pro Asp Asn Phe Ser Pro Ala Lys Pro
35 40 45
Lys Pro Ser Lys Asp Leu Lys Pro Leu Leu Gly Ser Ala Val Leu Gly
50 55 60
Leu Leu Leu Val Leu Ala Ala Val Val Ala Trp Cys Tyr Tyr Ser Val
65 70 75 80
Ser Leu Arg Lys Ala Glu Arg Leu Arg Ala Glu Leu Leu Asp Leu Lys
85 90 95
Ala Gly Gly Phe Ser Ile Arg Asn Gln Lys Gly Glu Gln Val Phe Arg
100 105 110
Leu Ala Phe Arg Ser Gly Ala Leu Asp Leu Asp Ser Cys Ser Arg Asp
115 120 125
Gly Ala Leu Leu Gly Cys Ser Leu Thr Ala Asp Gly Leu Pro Leu His
130 135 140
Phe Phe Ile Gln Thr Val Arg Pro Lys Asp Thr Val Met Cys Tyr Arg
145 150 155 160
Val Arg Trp Glu Glu Ala Ala Pro Gly Arg Ala Val Glu His Ala Met
165 170 175
Phe Leu Gly Asp Ala Ala Ala His Trp Tyr Gly Gly Ala Glu Met Arg
180 185 190
Thr Gln His Trp Pro Ile Arg Leu Asp Gly Gln Gln Glu Pro Gln Pro
195 200 205
Phe Val Thr Ser Asp Val Tyr Ser Ser Asp Ala Ala Phe Gly Gly Ile
210 215 220
Leu Glu Arg Tyr Trp Leu Ser Ser Arg Ala Ala Ala Ile Lys Val Asn
225 230 235 240
Asp Ser Val Pro Phe His Leu Gly Trp Asn Ser Thr Glu Arg Ser Leu
245 250 255
Arg Leu Gln Ala Arg Tyr His Asp Thr Pro Tyr Lys Pro Pro Ala Gly
260 265 270
Arg Ala Ala Ala Pro Glu Leu Ser Tyr Arg Val Cys Val Gly Ser Asp
275 280 285
Val Thr Ser Ile His Lys Tyr Met Val Arg Arg Tyr Phe Asn Lys Pro
290 295 300
Ser Arg Val Pro Ala Pro Glu Ala Phe Arg Asp Pro Ile Trp Ser Thr
305 310 315 320
Trp Ala Leu Tyr Gly Arg Ala Val Asp Gln Asp Lys Val Leu Arg Phe
325 330 335
Ala Gln Gln Ile Arg Leu His His Phe Asn Ser Ser His Leu Glu Ile
340 345 350
Asp Asp Met Tyr Thr Pro Ala Tyr Gly Asp Phe Asp Phe Asp Glu Val
355 360 365
Lys Phe Pro Asn Ala Ser Asp Met Phe Arg Arg Leu Arg Asp Ala Gly
370 375 380
Phe Arg Val Thr Leu Trp Val His Pro Phe Val Asn Tyr Asn Ser Ser
385 390 395 400
Arg Phe Gly Glu Gly Val Glu Arg Glu Leu Phe Val Arg Glu Pro Thr
405 410 415
Gly Arg Leu Pro Ala Leu Val Arg Trp Trp Asn Gly Ile Gly Ala Val
420 425 430
Leu Asp Phe Thr His Pro Lys Ala Arg Asp
435 440
<210> 14
<211> 714
<212> PRT
<213> 智人(Homo sapiens)
<400> 14
Met Leu Gln Asn Pro Gln Glu Lys Ser Gln Ala Tyr Pro Arg Arg Arg
1 5 10 15
Arg Pro Gly Cys Tyr Ala Tyr Arg Gln Asn Pro Glu Ala Ile Ala Ala
20 25 30
Ala Ala Val Tyr Thr Phe Leu Pro Asp Asn Phe Ser Pro Ala Lys Pro
35 40 45
Lys Pro Ser Lys Asp Leu Lys Pro Leu Leu Gly Ser Ala Val Leu Gly
50 55 60
Leu Leu Leu Val Leu Ala Ala Val Val Ala Trp Cys Tyr Tyr Ser Val
65 70 75 80
Ser Leu Arg Lys Ala Glu Arg Leu Arg Ala Glu Leu Leu Asp Leu Lys
85 90 95
Ala Gly Gly Phe Ser Ile Arg Asn Gln Lys Gly Glu Gln Val Phe Arg
100 105 110
Leu Ala Phe Arg Ser Gly Ala Leu Asp Leu Asp Ser Cys Ser Arg Asp
115 120 125
Gly Ala Leu Leu Gly Cys Ser Leu Thr Ala Asp Gly Leu Pro Leu His
130 135 140
Phe Phe Ile Gln Thr Val Arg Pro Lys Asp Thr Val Met Cys Tyr Arg
145 150 155 160
Val Arg Trp Glu Glu Ala Ala Pro Gly Arg Ala Val Glu His Ala Met
165 170 175
Phe Leu Gly Asp Ala Ala Ala His Trp Tyr Gly Gly Ala Glu Met Arg
180 185 190
Thr Gln His Trp Pro Ile Arg Leu Asp Gly Gln Gln Glu Pro Gln Pro
195 200 205
Phe Val Thr Ser Asp Val Tyr Ser Ser Asp Ala Ala Phe Gly Gly Ile
210 215 220
Leu Glu Arg Tyr Trp Leu Ser Ser Arg Ala Ala Ala Ile Lys Val Asn
225 230 235 240
Asp Ser Val Pro Phe His Leu Gly Trp Asn Ser Thr Glu Arg Ser Leu
245 250 255
Arg Leu Gln Ala Arg Tyr His Asp Thr Pro Tyr Lys Pro Pro Ala Gly
260 265 270
Arg Ala Ala Ala Pro Glu Leu Ser Tyr Arg Val Cys Val Gly Ser Asp
275 280 285
Val Thr Ser Ile His Lys Tyr Met Val Arg Arg Tyr Phe Asn Lys Pro
290 295 300
Ser Arg Val Pro Ala Pro Glu Ala Phe Arg Asp Pro Ile Trp Ser Thr
305 310 315 320
Trp Ala Leu Tyr Gly Arg Ala Val Asp Gln Asp Lys Val Leu Arg Phe
325 330 335
Ala Gln Gln Ile Arg Leu His His Phe Asn Ser Ser His Leu Glu Ile
340 345 350
Asp Asp Met Tyr Thr Pro Ala Tyr Gly Asp Phe Asp Phe Asp Glu Val
355 360 365
Lys Phe Pro Asn Ala Ser Asp Met Phe Arg Arg Leu Arg Asp Ala Gly
370 375 380
Phe Arg Val Thr Leu Trp Val His Pro Phe Val Asn Tyr Asn Ser Ser
385 390 395 400
Arg Phe Gly Glu Gly Val Glu Arg Glu Leu Phe Val Arg Glu Pro Thr
405 410 415
Gly Arg Leu Pro Ala Leu Val Arg Trp Trp Asn Gly Ile Gly Ala Val
420 425 430
Leu Asp Phe Thr His Pro Lys Ala Arg Asp Trp Phe Gln Gly His Leu
435 440 445
Arg Arg Leu Arg Ser Arg Tyr Ser Val Ala Ser Phe Lys Phe Asp Ala
450 455 460
Gly Glu Val Ser Tyr Leu Pro Arg Asp Phe Ser Thr Tyr Arg Pro Leu
465 470 475 480
Pro Asp Pro Ser Val Trp Ser Arg Arg Tyr Thr Glu Met Ala Leu Pro
485 490 495
Phe Phe Ser Leu Ala Glu Val Arg Val Gly Tyr Gln Ser Gln Asn Ile
500 505 510
Ser Cys Phe Phe Arg Leu Val Asp Arg Asp Ser Val Trp Gly Tyr Asp
515 520 525
Leu Gly Leu Arg Ser Leu Ile Pro Ala Val Leu Thr Val Ser Met Leu
530 535 540
Gly Tyr Pro Phe Ile Leu Pro Asp Met Val Gly Gly Asn Ala Val Pro
545 550 555 560
Gln Arg Thr Ala Gly Gly Asp Val Pro Glu Arg Glu Leu Tyr Ile Arg
565 570 575
Trp Leu Glu Val Ala Ala Phe Met Pro Ala Met Gln Phe Ser Ile Pro
580 585 590
Pro Trp Arg Tyr Asp Ala Glu Val Val Ala Ile Ala Gln Lys Phe Ala
595 600 605
Ala Leu Arg Ala Ser Leu Val Ala Pro Leu Leu Leu Glu Leu Ala Gly
610 615 620
Glu Val Thr Asp Thr Gly Asp Pro Ile Val Arg Pro Leu Trp Trp Ile
625 630 635 640
Ala Pro Gly Asp Glu Thr Ala His Arg Ile Asp Ser Gln Phe Leu Ile
645 650 655
Gly Asp Thr Leu Leu Val Ala Pro Val Leu Glu Pro Gly Lys Gln Glu
660 665 670
Arg Asp Val Tyr Leu Pro Ala Gly Lys Trp Arg Ser Tyr Lys Gly Glu
675 680 685
Leu Phe Asp Lys Thr Pro Val Leu Leu Thr Asp Tyr Pro Val Asp Leu
690 695 700
Asp Glu Ile Ala Tyr Phe Thr Trp Ala Ser
705 710
<210> 15
<211> 714
<212> PRT
<213> 智人(Homo sapiens)
<400> 15
Met Leu Gln Asn Pro Gln Glu Lys Ser Gln Ala Tyr Pro Arg Arg Arg
1 5 10 15
Arg Pro Gly Cys Tyr Ala Tyr Arg Gln Asn Pro Glu Ala Ile Ala Ala
20 25 30
Ala Ala Met Tyr Thr Phe Leu Pro Asp Asn Phe Ser Pro Ala Lys Pro
35 40 45
Lys Pro Ser Lys Asp Leu Lys Pro Leu Leu Gly Ser Ala Val Leu Gly
50 55 60
Leu Leu Leu Val Leu Ala Ala Val Val Ala Trp Cys Tyr Tyr Ser Val
65 70 75 80
Ser Leu Arg Lys Ala Glu Arg Leu Arg Ala Glu Leu Leu Asp Leu Lys
85 90 95
Ala Gly Gly Phe Ser Ile Arg Asn Gln Lys Gly Glu Gln Val Phe Arg
100 105 110
Leu Ala Phe Arg Ser Gly Ala Leu Asp Leu Asp Ser Cys Ser Arg Asp
115 120 125
Gly Ala Leu Leu Gly Cys Ser Leu Thr Ala Asp Gly Leu Pro Leu His
130 135 140
Phe Phe Ile Gln Thr Val Arg Pro Lys Asp Thr Val Met Cys Tyr Arg
145 150 155 160
Val Arg Trp Glu Glu Ala Ala Pro Gly Arg Ala Val Glu His Ala Met
165 170 175
Phe Leu Gly Asp Ala Ala Ala His Trp Tyr Gly Gly Ala Glu Met Arg
180 185 190
Thr Gln His Trp Pro Ile Arg Leu Asp Gly Gln Gln Glu Pro Gln Pro
195 200 205
Phe Val Thr Ser Asp Val Tyr Ser Ser Asp Ala Ala Phe Gly Gly Ile
210 215 220
Leu Glu Arg Tyr Trp Leu Ser Ser Arg Ala Ala Ala Ile Lys Val Asn
225 230 235 240
Asp Ser Val Pro Phe His Leu Gly Trp Asn Ser Thr Glu Arg Ser Leu
245 250 255
Arg Leu Gln Ala Arg Tyr His Asp Thr Pro Tyr Lys Pro Pro Ala Gly
260 265 270
Arg Ala Ala Ala Pro Glu Leu Ser Tyr Arg Val Cys Val Gly Ser Asp
275 280 285
Val Thr Ser Ile His Lys Tyr Met Val Arg Arg Tyr Phe Asn Lys Pro
290 295 300
Ser Arg Val Pro Ala Pro Glu Ala Phe Arg Asp Pro Ile Trp Ser Thr
305 310 315 320
Trp Ala Leu Tyr Gly Arg Ala Val Asp Gln Asp Lys Val Leu Arg Phe
325 330 335
Ala Gln Gln Ile Arg Leu His His Phe Asn Ser Ser His Leu Glu Ile
340 345 350
Asp Asp Met Tyr Thr Pro Ala Tyr Gly Asp Phe Asp Phe Asp Glu Val
355 360 365
Lys Phe Pro Asn Ala Ser Asp Met Phe Arg Arg Leu Arg Asp Ala Gly
370 375 380
Phe Arg Val Thr Leu Trp Val His Pro Phe Val Asn Tyr Asn Ser Ser
385 390 395 400
Arg Phe Gly Glu Gly Val Glu Arg Glu Leu Phe Val Arg Glu Pro Thr
405 410 415
Gly Arg Leu Pro Ala Leu Val Arg Trp Trp Asn Gly Ile Gly Ala Val
420 425 430
Leu Asp Phe Thr His Pro Lys Ala Gly Asp Trp Phe Gln Gly His Leu
435 440 445
Arg Arg Leu Arg Ser Arg Tyr Ser Val Ala Ser Phe Lys Phe Asp Ala
450 455 460
Gly Glu Val Ser Tyr Leu Pro Arg Asp Phe Ser Thr Tyr Arg Pro Leu
465 470 475 480
Pro Asp Pro Ser Val Trp Ser Arg Arg Tyr Thr Glu Met Ala Leu Pro
485 490 495
Phe Phe Ser Leu Ala Glu Val Arg Val Gly Tyr Gln Ser Gln Asn Ile
500 505 510
Ser Cys Phe Phe Arg Leu Val Asp Arg Asp Ser Val Trp Gly Tyr Asp
515 520 525
Leu Gly Leu Arg Ser Leu Ile Pro Ala Val Leu Thr Val Ser Met Leu
530 535 540
Gly Tyr Pro Phe Ile Leu Pro Asp Met Val Gly Gly Asn Ala Val Pro
545 550 555 560
Gln Arg Thr Ala Gly Gly Asp Val Pro Glu Arg Glu Leu Tyr Ile Arg
565 570 575
Trp Leu Glu Val Ala Ala Phe Met Pro Ala Met Gln Phe Ser Ile Pro
580 585 590
Pro Trp Arg Tyr Asp Ala Glu Val Val Ala Ile Ala Gln Lys Phe Ala
595 600 605
Ala Leu Arg Ala Ser Leu Val Ala Pro Leu Leu Leu Glu Leu Ala Gly
610 615 620
Glu Val Thr Asp Thr Gly Asp Pro Ile Val Arg Pro Leu Trp Trp Ile
625 630 635 640
Ala Pro Gly Asp Glu Thr Ala His Arg Ile Asp Ser Gln Phe Leu Ile
645 650 655
Gly Asp Thr Leu Leu Val Ala Pro Val Leu Glu Pro Gly Lys Gln Glu
660 665 670
Arg Asp Val Tyr Leu Pro Ala Gly Lys Trp Arg Ser Tyr Lys Gly Glu
675 680 685
Leu Phe Asp Lys Thr Pro Val Leu Leu Thr Asp Tyr Pro Val Asp Leu
690 695 700
Asp Glu Ile Ala Tyr Phe Thr Trp Ala Ser
705 710
<210> 16
<211> 714
<212> PRT
<213> 智人(Homo sapiens)
<400> 16
Met Leu Gln Asn Pro Gln Glu Lys Ser Gln Ala Tyr Pro Arg Arg Arg
1 5 10 15
Arg Pro Gly Cys Tyr Ala Tyr Arg Gln Asn Pro Glu Ala Ile Ala Ala
20 25 30
Ala Ala Met Tyr Thr Phe Leu Pro Asp Asn Phe Ser Pro Ala Lys Pro
35 40 45
Lys Pro Ser Lys Asp Leu Lys Pro Leu Leu Gly Ser Ala Val Leu Gly
50 55 60
Leu Leu Leu Val Leu Ala Ala Val Val Ala Trp Cys Tyr Tyr Ser Val
65 70 75 80
Ser Leu Arg Lys Ala Glu Arg Leu Arg Ala Glu Leu Leu Asp Leu Lys
85 90 95
Ala Gly Gly Phe Ser Ile Arg Asn Gln Lys Gly Glu Gln Val Phe Arg
100 105 110
Leu Ala Phe Arg Ser Gly Ala Leu Asp Leu Asp Ser Cys Ser Arg Asp
115 120 125
Gly Ala Leu Leu Gly Cys Ser Leu Thr Ala Asp Gly Leu Pro Leu His
130 135 140
Phe Phe Ile Gln Thr Val Arg Pro Lys Asp Thr Val Met Cys Tyr Arg
145 150 155 160
Val Arg Trp Glu Glu Ala Ala Pro Gly Arg Ala Val Glu His Ala Met
165 170 175
Phe Leu Gly Asp Ala Ala Ala His Trp Tyr Gly Gly Ala Glu Met Arg
180 185 190
Thr Gln His Trp Pro Ile Arg Leu Asp Gly Gln Gln Glu Pro Gln Pro
195 200 205
Phe Val Thr Ser Asp Val Tyr Ser Ser Asp Ala Ala Phe Gly Gly Ile
210 215 220
Leu Glu Arg Tyr Trp Leu Ser Leu Arg Ala Ala Ala Ile Lys Val Asn
225 230 235 240
Asp Ser Val Pro Phe His Leu Gly Trp Asn Ser Thr Glu Arg Ser Leu
245 250 255
Arg Leu Gln Ala Arg Tyr His Asp Thr Pro Tyr Lys Pro Pro Ala Gly
260 265 270
Arg Ala Ala Ala Pro Glu Leu Ser Tyr Arg Val Cys Val Gly Ser Asp
275 280 285
Val Thr Ser Ile His Lys Tyr Met Val Arg Arg Tyr Phe Asn Lys Pro
290 295 300
Ser Arg Val Pro Ala Pro Glu Ala Phe Arg Asp Pro Ile Trp Ser Thr
305 310 315 320
Trp Ala Leu Tyr Gly Arg Ala Val Asp Gln Asp Lys Val Leu Arg Phe
325 330 335
Ala Gln Gln Ile Arg Leu His His Phe Asn Ser Ser His Leu Glu Ile
340 345 350
Asp Asp Met Tyr Thr Pro Ala Tyr Gly Asp Phe Asp Phe Asp Glu Val
355 360 365
Lys Phe Pro Asn Ala Ser Asp Met Phe Arg Arg Leu Arg Asp Ala Gly
370 375 380
Phe Arg Val Thr Leu Trp Val His Pro Phe Val Asn Tyr Asn Ser Ser
385 390 395 400
Arg Phe Gly Glu Gly Val Glu Arg Glu Leu Phe Val Arg Glu Pro Thr
405 410 415
Gly Arg Leu Pro Ala Leu Val Arg Trp Trp Asn Gly Ile Gly Ala Val
420 425 430
Leu Asp Phe Thr His Pro Lys Ala Arg Asp Trp Phe Gln Gly His Leu
435 440 445
Arg Arg Leu Arg Ser Arg Tyr Ser Val Ala Ser Phe Lys Phe Asp Ala
450 455 460
Gly Glu Val Ser Tyr Leu Pro Arg Asp Phe Ser Thr Tyr Arg Pro Leu
465 470 475 480
Pro Asp Pro Ser Val Trp Ser Arg Arg Tyr Thr Glu Met Ala Leu Pro
485 490 495
Phe Phe Ser Leu Ala Glu Val Arg Val Gly Tyr Gln Ser Gln Asn Ile
500 505 510
Ser Cys Phe Phe Arg Leu Val Asp Arg Asp Ser Val Trp Gly Tyr Asp
515 520 525
Leu Gly Leu Arg Ser Leu Ile Pro Ala Val Leu Thr Val Ser Met Leu
530 535 540
Gly Tyr Pro Phe Ile Leu Pro Asp Met Val Gly Gly Asn Ala Val Pro
545 550 555 560
Gln Arg Thr Ala Gly Gly Asp Val Pro Glu Arg Glu Leu Tyr Ile Arg
565 570 575
Trp Leu Glu Val Ala Ala Phe Met Pro Ala Met Gln Phe Ser Ile Pro
580 585 590
Pro Trp Arg Tyr Asp Ala Glu Val Val Ala Ile Ala Gln Lys Phe Ala
595 600 605
Ala Leu Arg Ala Ser Leu Val Ala Pro Leu Leu Leu Glu Leu Ala Gly
610 615 620
Glu Val Thr Asp Thr Gly Asp Pro Ile Val Arg Pro Leu Trp Trp Ile
625 630 635 640
Ala Pro Gly Asp Glu Thr Ala His Arg Ile Asp Ser Gln Phe Leu Ile
645 650 655
Gly Asp Thr Leu Leu Val Ala Pro Val Leu Glu Pro Gly Lys Gln Glu
660 665 670
Arg Asp Val Tyr Leu Pro Ala Gly Lys Trp Arg Ser Tyr Lys Gly Glu
675 680 685
Leu Phe Asp Lys Thr Pro Val Leu Leu Thr Asp Tyr Pro Val Asp Leu
690 695 700
Asp Glu Ile Ala Tyr Phe Thr Trp Ala Ser
705 710
<210> 17
<211> 202
<212> PRT
<213> 智人(Homo sapiens)
<400> 17
Met Leu Gln Asn Pro Gln Glu Lys Ser Gln Ala Tyr Pro Arg Arg Arg
1 5 10 15
Arg Pro Gly Cys Tyr Ala Tyr Arg Gln Asn Pro Glu Ala Ile Ala Ala
20 25 30
Ala Ala Met Tyr Thr Phe Leu Pro Asp Asn Phe Ser Pro Ala Lys Pro
35 40 45
Lys Pro Ser Lys Asp Leu Lys Pro Leu Leu Gly Ser Ala Val Leu Gly
50 55 60
Leu Leu Leu Val Leu Ala Ala Val Val Ala Trp Cys Tyr Tyr Ser Val
65 70 75 80
Ser Leu Arg Lys Ala Glu Arg Leu Arg Ala Glu Leu Leu Asp Leu Lys
85 90 95
Ala Gly Gly Phe Ser Ile Arg Asn Gln Lys Gly Glu Gln Val Phe Arg
100 105 110
Leu Ala Phe Arg Ser Gly Ala Leu Asp Leu Asp Ser Cys Ser Arg Asp
115 120 125
Gly Ala Leu Leu Gly Cys Ser Leu Thr Ala Asp Gly Leu Pro Leu His
130 135 140
Phe Phe Ile Gln Thr Val Arg Pro Lys Asp Thr Val Met Cys Tyr Arg
145 150 155 160
Val Arg Trp Glu Glu Ala Ala Pro Gly Arg Ala Val Glu His Ala Met
165 170 175
Phe Leu Gly Asp Ala Ala Ala His Trp Tyr Gly Gly Ala Glu Met Arg
180 185 190
Thr Gln His Trp Pro Ile Arg Leu Asp Gly
195 200
<210> 18
<211> 714
<212> PRT
<213> 智人(Homo sapiens)
<400> 18
Met Leu Gln Asn Pro Gln Glu Lys Ser Gln Ala Tyr Pro Arg Arg Arg
1 5 10 15
Arg Pro Gly Cys Tyr Ala Tyr Arg Gln Asn Pro Glu Ala Ile Ala Ala
20 25 30
Ala Ala Met Tyr Thr Phe Leu Pro Asp Asn Phe Ser Pro Ala Lys Pro
35 40 45
Lys Pro Ser Lys Asp Leu Lys Pro Leu Leu Gly Ser Ala Val Leu Gly
50 55 60
Leu Leu Leu Val Leu Ala Ala Val Val Ala Trp Cys Tyr Tyr Ser Val
65 70 75 80
Ser Leu Arg Lys Ala Glu Arg Leu Arg Ala Glu Leu Leu Asp Leu Lys
85 90 95
Ala Gly Gly Phe Ser Ile Arg Asn Gln Lys Gly Glu Gln Val Phe Arg
100 105 110
Leu Ala Phe Arg Ser Gly Ala Leu Asp Leu Asp Ser Cys Ser Arg Asp
115 120 125
Gly Ala Leu Leu Gly Cys Ser Leu Thr Ala Asp Gly Leu Pro Leu His
130 135 140
Phe Phe Ile Gln Thr Val Arg Pro Lys Asp Thr Val Met Cys Tyr Arg
145 150 155 160
Val Arg Trp Glu Glu Ala Ala Pro Gly Arg Ala Val Glu His Ala Met
165 170 175
Phe Leu Gly Asp Ala Ala Ala His Trp Tyr Gly Gly Ala Glu Met Arg
180 185 190
Thr Gln His Trp Pro Ile Arg Leu Asp Gly Gln Gln Glu Pro Gln Pro
195 200 205
Phe Val Thr Ser Asp Val Tyr Ser Ser Asp Ala Ala Phe Gly Gly Ile
210 215 220
Leu Glu Arg Tyr Trp Leu Ser Ser Arg Ala Ala Ala Ile Lys Val Asn
225 230 235 240
Asp Ser Val Pro Phe His Leu Gly Trp Asn Ser Thr Glu Arg Ser Leu
245 250 255
Arg Leu Gln Ala Leu Tyr His Asp Thr Pro Tyr Lys Pro Pro Ala Gly
260 265 270
Arg Ala Ala Ala Pro Glu Leu Ser Tyr Arg Val Cys Val Gly Ser Asp
275 280 285
Val Thr Ser Ile His Lys Tyr Met Val Arg Arg Tyr Phe Asn Lys Pro
290 295 300
Ser Arg Val Pro Ala Pro Glu Ala Phe Arg Asp Pro Ile Trp Ser Thr
305 310 315 320
Trp Ala Leu Tyr Gly Arg Ala Val Asp Gln Asp Lys Val Leu Arg Phe
325 330 335
Ala Gln Gln Ile Arg Leu His His Phe Asn Ser Ser His Leu Glu Ile
340 345 350
Asp Asp Met Tyr Thr Pro Ala Tyr Gly Asp Phe Asp Phe Asp Glu Val
355 360 365
Lys Phe Pro Asn Ala Ser Asp Met Phe Arg Arg Leu Arg Asp Ala Gly
370 375 380
Phe Arg Val Thr Leu Trp Val His Pro Phe Val Asn Tyr Asn Ser Ser
385 390 395 400
Arg Phe Gly Glu Gly Val Glu Arg Glu Leu Phe Val Arg Glu Pro Thr
405 410 415
Gly Arg Leu Pro Ala Leu Val Arg Trp Trp Asn Gly Ile Gly Ala Val
420 425 430
Leu Asp Phe Thr His Pro Lys Ala Arg Asp Trp Phe Gln Gly His Leu
435 440 445
Arg Arg Leu Arg Ser Arg Tyr Ser Val Ala Ser Phe Lys Phe Asp Ala
450 455 460
Gly Glu Val Ser Tyr Leu Pro Arg Asp Phe Ser Thr Tyr Arg Pro Leu
465 470 475 480
Pro Asp Pro Ser Val Trp Ser Arg Arg Tyr Thr Glu Met Ala Leu Pro
485 490 495
Phe Phe Ser Leu Ala Glu Val Arg Val Gly Tyr Gln Ser Gln Asn Ile
500 505 510
Ser Cys Phe Phe Arg Leu Val Asp Arg Asp Ser Val Trp Gly Tyr Asp
515 520 525
Leu Gly Leu Arg Ser Leu Ile Pro Ala Val Leu Thr Val Ser Met Leu
530 535 540
Gly Tyr Pro Phe Ile Leu Pro Asp Met Val Gly Gly Asn Ala Val Pro
545 550 555 560
Gln Arg Thr Ala Gly Gly Asp Val Pro Glu Arg Glu Leu Tyr Ile Arg
565 570 575
Trp Leu Glu Val Ala Ala Phe Met Pro Ala Met Gln Phe Ser Ile Pro
580 585 590
Pro Trp Arg Tyr Asp Ala Glu Val Val Ala Ile Ala Gln Lys Phe Ala
595 600 605
Ala Leu Arg Ala Ser Leu Val Ala Pro Leu Leu Leu Glu Leu Ala Gly
610 615 620
Glu Val Thr Asp Thr Gly Asp Pro Ile Val Arg Pro Leu Trp Trp Ile
625 630 635 640
Ala Pro Gly Asp Glu Thr Ala His Arg Ile Asp Ser Gln Phe Leu Ile
645 650 655
Gly Asp Thr Leu Leu Val Ala Pro Val Leu Glu Pro Gly Lys Gln Glu
660 665 670
Arg Asp Val Tyr Leu Pro Ala Gly Lys Trp Arg Ser Tyr Lys Gly Glu
675 680 685
Leu Phe Asp Lys Thr Pro Val Leu Leu Thr Asp Tyr Pro Val Asp Leu
690 695 700
Asp Glu Ile Ala Tyr Phe Thr Trp Ala Ser
705 710
<210> 19
<211> 718
<212> PRT
<213> 智人(Homo sapiens)
<400> 19
Met Leu Gln Asn Pro Gln Glu Lys Ser Gln Ala Tyr Pro Arg Arg Arg
1 5 10 15
Arg Pro Gly Cys Tyr Ala Tyr Arg Gln Asn Pro Glu Ala Ile Ala Ala
20 25 30
Ala Ala Met Tyr Thr Phe Leu Pro Asp Asn Phe Ser Pro Ala Lys Pro
35 40 45
Lys Pro Ser Lys Asp Leu Lys Pro Leu Leu Gly Ser Ala Val Leu Gly
50 55 60
Leu Leu Leu Val Leu Ala Ala Val Val Ala Trp Cys Tyr Tyr Ser Val
65 70 75 80
Ser Leu Arg Lys Ala Glu Arg Leu Arg Ala Glu Leu Leu Asp Leu Lys
85 90 95
Ala Gly Gly Phe Ser Ile Arg Asn Gln Lys Gly Glu Gln Val Phe Arg
100 105 110
Leu Ala Phe Arg Ser Gly Ala Leu Asp Leu Asp Ser Cys Ser Arg Asp
115 120 125
Gly Ala Leu Leu Gly Cys Ser Leu Thr Ala Asp Gly Leu Pro Leu His
130 135 140
Phe Phe Ile Gln Thr Val Arg Pro Lys Asp Thr Val Met Cys Tyr Arg
145 150 155 160
Val Arg Trp Glu Glu Ala Ala Pro Gly Arg Ala Val Glu His Ala Met
165 170 175
Phe Leu Gly Asp Ala Ala Ala His Trp Tyr Gly Gly Ala Glu Met Arg
180 185 190
Thr Gln His Trp Pro Ile Arg Leu Asp Gly Gln Gln Glu Pro Gln Pro
195 200 205
Phe Val Thr Ser Asp Val Tyr Ser Ser Asp Ala Ala Phe Gly Gly Ile
210 215 220
Leu Glu Arg Tyr Trp Leu Ser Ser Arg Ala Ala Ala Ile Lys Val Asn
225 230 235 240
Asp Ser Val Pro Phe His Leu Gly Trp Asn Ser Thr Glu Arg Ser Leu
245 250 255
Arg Leu Gln Ala Arg Tyr His Asp Thr Pro Tyr Lys Pro Pro Ala Gly
260 265 270
Arg Ala Ala Ala Pro Glu Leu Ser Tyr Arg Val Cys Val Gly Ser Asp
275 280 285
Val Thr Ser Ile His Lys Tyr Met Val Arg Arg Tyr Phe Asn Lys Pro
290 295 300
Ser Arg Val Pro Ala Pro Glu Ala Phe Arg Asp Pro Ile Trp Ser Thr
305 310 315 320
Trp Ala Leu Tyr Gly Arg Ala Val Asp Gln Asp Lys Val Leu Arg Phe
325 330 335
Ala Gln Gln Ile Arg Leu His His Phe Asn Ser Ser Leu Ala Phe Arg
340 345 350
His Leu Glu Ile Asp Asp Met Tyr Thr Pro Ala Tyr Gly Asp Phe Asp
355 360 365
Phe Asp Glu Val Lys Phe Pro Asn Ala Ser Asp Met Phe Arg Arg Leu
370 375 380
Arg Asp Ala Gly Phe Arg Val Thr Leu Trp Val His Pro Phe Val Asn
385 390 395 400
Tyr Asn Ser Ser Arg Phe Gly Glu Gly Val Glu Arg Glu Leu Phe Val
405 410 415
Arg Glu Pro Thr Gly Arg Leu Pro Ala Leu Val Arg Trp Trp Asn Gly
420 425 430
Ile Gly Ala Val Leu Asp Phe Thr His Pro Lys Ala Arg Asp Trp Phe
435 440 445
Gln Gly His Leu Arg Arg Leu Arg Ser Arg Tyr Ser Val Ala Ser Phe
450 455 460
Lys Phe Asp Ala Gly Glu Val Ser Tyr Leu Pro Arg Asp Phe Ser Thr
465 470 475 480
Tyr Arg Pro Leu Pro Asp Pro Ser Val Trp Ser Arg Arg Tyr Thr Glu
485 490 495
Met Ala Leu Pro Phe Phe Ser Leu Ala Glu Val Arg Val Gly Tyr Gln
500 505 510
Ser Gln Asn Ile Ser Cys Phe Phe Arg Leu Val Asp Arg Asp Ser Val
515 520 525
Trp Gly Tyr Asp Leu Gly Leu Arg Ser Leu Ile Pro Ala Val Leu Thr
530 535 540
Val Ser Met Leu Gly Tyr Pro Phe Ile Leu Pro Asp Met Val Gly Gly
545 550 555 560
Asn Ala Val Pro Gln Arg Thr Ala Gly Gly Asp Val Pro Glu Arg Glu
565 570 575
Leu Tyr Ile Arg Trp Leu Glu Val Ala Ala Phe Met Pro Ala Met Gln
580 585 590
Phe Ser Ile Pro Pro Trp Arg Tyr Asp Ala Glu Val Val Ala Ile Ala
595 600 605
Gln Lys Phe Ala Ala Leu Arg Ala Ser Leu Val Ala Pro Leu Leu Leu
610 615 620
Glu Leu Ala Gly Glu Val Thr Asp Thr Gly Asp Pro Ile Val Arg Pro
625 630 635 640
Leu Trp Trp Ile Ala Pro Gly Asp Glu Thr Ala His Arg Ile Asp Ser
645 650 655
Gln Phe Leu Ile Gly Asp Thr Leu Leu Val Ala Pro Val Leu Glu Pro
660 665 670
Gly Lys Gln Glu Arg Asp Val Tyr Leu Pro Ala Gly Lys Trp Arg Ser
675 680 685
Tyr Lys Gly Glu Leu Phe Asp Lys Thr Pro Val Leu Leu Thr Asp Tyr
690 695 700
Pro Val Asp Leu Asp Glu Ile Ala Tyr Phe Thr Trp Ala Ser
705 710 715
<210> 20
<211> 712
<212> PRT
<213> 智人(Homo sapiens)
<400> 20
Met Leu Gln Asn Pro Gln Glu Lys Ser Gln Ala Tyr Pro Arg Arg Arg
1 5 10 15
Arg Pro Gly Cys Tyr Ala Tyr Arg Gln Asn Pro Glu Ala Ile Ala Ala
20 25 30
Ala Ala Met Tyr Thr Phe Leu Pro Asp Asn Phe Ser Pro Ala Lys Pro
35 40 45
Lys Pro Ser Lys Asp Leu Lys Pro Leu Leu Gly Ser Ala Val Leu Gly
50 55 60
Leu Leu Leu Val Leu Ala Ala Val Val Ala Trp Cys Tyr Tyr Ser Val
65 70 75 80
Ser Leu Arg Lys Ala Glu Arg Leu Arg Ala Glu Leu Leu Asp Leu Lys
85 90 95
Ala Gly Gly Phe Ser Ile Arg Asn Gln Lys Gly Glu Gln Val Phe Arg
100 105 110
Leu Ala Phe Arg Ser Gly Ala Leu Asp Leu Asp Ser Cys Ser Arg Asp
115 120 125
Gly Ala Leu Leu Gly Cys Ser Leu Thr Ala Asp Gly Leu Pro Leu His
130 135 140
Phe Phe Ile Gln Thr Val Arg Pro Lys Asp Thr Val Met Cys Tyr Arg
145 150 155 160
Val Arg Trp Glu Glu Ala Ala Pro Gly Arg Ala Val Glu His Ala Met
165 170 175
Phe Leu Gly Asp Ala Ala Ala His Trp Tyr Gly Gly Ala Glu Met Arg
180 185 190
Thr Gln His Trp Pro Ile Arg Leu Asp Gly Gln Gln Glu Pro Gln Pro
195 200 205
Phe Val Thr Ser Asp Val Tyr Ser Ser Asp Ala Ala Phe Gly Gly Ile
210 215 220
Leu Glu Arg Tyr Trp Leu Ser Ser Arg Ala Ala Ala Ile Lys Val Asn
225 230 235 240
Asp Ser Val Pro Phe His Leu Gly Trp Asn Ser Thr Glu Arg Ser Leu
245 250 255
Arg Leu Gln Ala Arg Tyr His Asp Thr Pro Tyr Lys Pro Pro Ala Gly
260 265 270
Arg Ala Ala Ala Pro Glu Leu Ser Tyr Arg Val Cys Val Gly Ser Asp
275 280 285
Val Thr Ser Ile His Lys Tyr Met Val Arg Arg Tyr Phe Asn Lys Pro
290 295 300
Ser Arg Val Pro Ala Pro Glu Ala Phe Arg Asp Pro Ile Trp Ser Thr
305 310 315 320
Trp Ala Leu Tyr Gly Arg Ala Val Asp Gln Asp Lys Val Leu Arg Phe
325 330 335
Ala Gln Gln Ile Arg Leu His His Phe Asn Ser Ser His Leu Glu Ile
340 345 350
Asp Asp Met Tyr Thr Pro Ala Tyr Gly Asp Phe Asp Glu Val Lys Phe
355 360 365
Pro Asn Ala Ser Asp Met Phe Arg Arg Leu Arg Asp Ala Gly Phe Arg
370 375 380
Val Thr Leu Trp Val His Pro Phe Val Asn Tyr Asn Ser Ser Arg Phe
385 390 395 400
Gly Glu Gly Val Glu Arg Glu Leu Phe Val Arg Glu Pro Thr Gly Arg
405 410 415
Leu Pro Ala Leu Val Arg Trp Trp Asn Gly Ile Gly Ala Val Leu Asp
420 425 430
Phe Thr His Pro Lys Ala Arg Asp Trp Phe Gln Gly His Leu Arg Arg
435 440 445
Leu Arg Ser Arg Tyr Ser Val Ala Ser Phe Lys Phe Asp Ala Gly Glu
450 455 460
Val Ser Tyr Leu Pro Arg Asp Phe Ser Thr Tyr Arg Pro Leu Pro Asp
465 470 475 480
Pro Ser Val Trp Ser Arg Arg Tyr Thr Glu Met Ala Leu Pro Phe Phe
485 490 495
Ser Leu Ala Glu Val Arg Val Gly Tyr Gln Ser Gln Asn Ile Ser Cys
500 505 510
Phe Phe Arg Leu Val Asp Arg Asp Ser Val Trp Gly Tyr Asp Leu Gly
515 520 525
Leu Arg Ser Leu Ile Pro Ala Val Leu Thr Val Ser Met Leu Gly Tyr
530 535 540
Pro Phe Ile Leu Pro Asp Met Val Gly Gly Asn Ala Val Pro Gln Arg
545 550 555 560
Thr Ala Gly Gly Asp Val Pro Glu Arg Glu Leu Tyr Ile Arg Trp Leu
565 570 575
Glu Val Ala Ala Phe Met Pro Ala Met Gln Phe Ser Ile Pro Pro Trp
580 585 590
Arg Tyr Asp Ala Glu Val Val Ala Ile Ala Gln Lys Phe Ala Ala Leu
595 600 605
Arg Ala Ser Leu Val Ala Pro Leu Leu Leu Glu Leu Ala Gly Glu Val
610 615 620
Thr Asp Thr Gly Asp Pro Ile Val Arg Pro Leu Trp Trp Ile Ala Pro
625 630 635 640
Gly Asp Glu Thr Ala His Arg Ile Asp Ser Gln Phe Leu Ile Gly Asp
645 650 655
Thr Leu Leu Val Ala Pro Val Leu Glu Pro Gly Lys Gln Glu Arg Asp
660 665 670
Val Tyr Leu Pro Ala Gly Lys Trp Arg Ser Tyr Lys Gly Glu Leu Phe
675 680 685
Asp Lys Thr Pro Val Leu Leu Thr Asp Tyr Pro Val Asp Leu Asp Glu
690 695 700
Ile Ala Tyr Phe Thr Trp Ala Ser
705 710
<210> 21
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ctcaggttcc agtattcatc 20
<210> 22
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
gtagcacatg accgtgtcct t 21
<210> 23
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tccgcaatca gaagggagag 20
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
cgccgatgcc gttccaccag 20
<210> 25
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
cagccacctg gaaatcgacg acat 24
<210> 26
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gcaggagatg ttctgtgact ggta 24
<210> 27
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
cgcgggactt cagcacctac 20
<210> 28
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
aagtcagcag ccacttaatg 20

Claims (6)

1.一种MYORG突变基因，其特征在于：所述MYORG突变基因的编码序列如SEQ ID NO：2所示，而MYORG野生型基因的编码序列如SEQ ID NO：1所示，则SEQ ID NO：2所示MYORG突变基因编码序列所含的基因突变为c.225G>A；SEQ ID NO：2序列所示的MYORG突变基因的二倍体纯合形式以及SEQ ID NO：3-10序列所示的任一个MYORG突变基因和SEQ ID NO：2序列所示的MYORG突变基因组成的二倍体杂合形式这两种类型中的任一种的出现均会导致人类原发性家族性脑钙化症的发生。
2.如权利要求1中所述的MYORG突变基因的编码蛋白，其特征在于：对应于SEQ ID NO：2所示的MYORG突变基因的编码序列，其编码蛋白序列如SEQ ID NO：12所示，而MYORG野生型基因编码蛋白序列如SEQ ID NO：11所示，则相应的氨基酸突变为p.W75*；SEQ ID NO：11序列所示的MYORG野生型基因编码蛋白的缺失，并且SEQ ID NO：12序列所示的MYORG突变基因编码蛋白的出现可以揭示原发性家族性脑钙化症的发生。
3.一种针对权利要求1中所述的MYORG突变基因的MYORG基因突变检测试剂盒，其特征在于：试剂盒包含能够特异性扩增如SEQ ID NO：2所示MYORG突变基因编码序列所含有的c.225G>A突变的PCR引物组合。
4.根据权利要求3中所述的MYORG基因突变检测试剂盒，其特征在于：所述引物组合为SEQ ID NO：21和SEQ ID NO：22所示序列。
5.一种重组载体，其特征在于：所述重组载体含有如SEQ ID NO：2所示的MYORG突变基因片段。
6.一种遗传工程化的宿主细胞，其特征在于：所述宿主细胞为权利要求5所述的载体转化或转导的宿主细胞。
CN201810552232.6A 2018-05-31 2018-05-31 原发性家族性脑钙化症致病基因myorg及其应用 Active CN108715838B (zh)

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CN202110086611.2A CN112779275B (zh) 2018-05-31 2018-05-31 第782-783位点发生突变的人类pfbc致病基因myorg及其应用
CN202110086374.XA CN112662691B (zh) 2018-05-31 2018-05-31 第348-349位点发生突变的人类pfbc致病基因myorg及其应用
CN202011178218.8A CN112342206B (zh) 2018-05-31 2018-05-31 第103位点发生突变的人类pfbc致病基因myorg及其应用
CN202011175905.4A CN112226448B (zh) 2018-05-31 2018-05-31 第695位点发生突变的人类pfbc致病基因myorg及其应用
CN202011178216.9A CN112226449B (zh) 2018-05-31 2018-05-31 第1328位点发生突变的人类pfbc致病基因myorg及其应用
CN202110086373.5A CN112725366B (zh) 2018-05-31 2018-05-31 第1321位点发生突变的人类pfbc致病基因myorg及其应用
CN202110086612.7A CN112680467B (zh) 2018-05-31 2018-05-31 第1092-1097位点发生突变的人类pfbc致病基因myorg及其应用
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CN202110086612.7A Division CN112680467B (zh) 2018-05-31 2018-05-31 第1092-1097位点发生突变的人类pfbc致病基因myorg及其应用
CN202110086374.XA Division CN112662691B (zh) 2018-05-31 2018-05-31 第348-349位点发生突变的人类pfbc致病基因myorg及其应用
CN202011178216.9A Division CN112226449B (zh) 2018-05-31 2018-05-31 第1328位点发生突变的人类pfbc致病基因myorg及其应用
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