CN113234134B

CN113234134B - 一种远端关节挛缩综合症致病基因myh3及其用途

Info

Publication number: CN113234134B
Application number: CN202110483546.7A
Authority: CN
Inventors: 孟怡辰; 张郑; 胡淼; 周许辉
Original assignee: Shanghai Changzheng Hospital
Current assignee: Shanghai Changzheng Hospital
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2022-07-22
Anticipated expiration: 2041-04-30
Also published as: CN113234134A

Abstract

本发明涉及基因检测领域，尤其涉及一种远端关节挛缩综合症致病基因MYH3及其用途。本发明提供的突变MYH3蛋白与野生型MYH3蛋白相比存在一个突变位点p.G232E；野生型MYH3蛋白的氨基酸序列如SEQ ID NO：1所示。本发明发现了一个全新的致病基因MYH3突变位点MYH3(c.695G>A:p.G232E)，该突变位点的检测对于远端关节挛缩综合症患者病因的明确具有重要的意义。

Description

一种远端关节挛缩综合症致病基因MYH3及其用途

技术领域

本发明涉及基因检测领域，尤其涉及一种远端关节挛缩综合症致病基因MYH3及其用途。

背景技术

远端关节挛缩综合症是一组以先天性全身多个关节挛缩为主要特征的疾病。患者通常表现为双手紧握、手指重叠、马蹄内翻足等，同时也可能伴有近端关节的活动困难、面部异常等。研究发现，多数远端关节挛缩综合症患者的发病与编码肌节蛋白的基因突变有关，如TNNI2(肌钙蛋白I)、TPM2(β原肌球蛋白)、MYH3、MYH8等，因此认为远端关节挛缩综合症是一种骨骼肌疾病。

远端关节挛缩综合症是一种具有高度遗传异质性的疾病，同一亚型的疾病可由多种不同的致病基因突变引起，而同一基因不同位点突变对蛋白功能影响不同，患者临床表型会出现差异。因此，虽然已有不少关于远端关节挛缩综合症致病基因及遗传热点区域的相关报道，但发现新的致病基因突变位点对于远端关节挛缩综合症致病机理的研究和寻求该病的治疗方案至关重要。

全基因组外显子测序，也称为外显子组测序(exome sequencing)，是一种经济的测序方法，其主要是对人类基因组的编码区进行测序，从而探测与罕见和常见疾病相关的新基因。由于该方法只测序全部基因组的编码区(占全部基因组的约1％)，因此，该方法相对节省成本，并且伴有较高的覆盖效率和深度。就目前而言，大多数单基因疾病由致病基因的功能变异引起，而大部分的此类功能变异又发生于外显子区域中，因此，全基因组外显子测序被认为是弥补定位克隆技术的重要技术。

发明内容

本发明的目的在于克服现有技术的不足，提供一种远端关节挛缩综合症致病基因MYH3及其用途。本发明发现了一个全新的致病基因MYH3突变位点MYH3(c.695G>A:p.G232E)，该突变位点的检测对于远端关节挛缩综合症患者病因的明确具有重要的意义。

为实现上述目的，本发明采取的技术方案为：提供一种突变MYH3蛋白，所述突变MYH3蛋白与野生型MYH3蛋白相比存在一个突变位点p.G232E；所述野生型MYH3蛋白的氨基酸序列如SEQ ID NO：1所示。

本发明同时提供编码所述突变MYH3蛋白的基因。

作为本发明所述基因的优选实施方式，所述基因的核苷酸序列如SEQ ID NO：4所示。

本发明同时提供一种表达载体，所述表达载体包含所述基因。

本发明同时提供一种宿主细胞，所述宿主细胞包含所述基因或所述表达载体。

本发明同时提供所述基因、所述载体或所述宿主细胞的用途，所述用途为用于制备远端关节挛缩综合症动物模型。

作为本发明所述用途的优选实施方式，所述动物包括哺乳动物，例如小鼠，大鼠，兔或猴。

本发明同时提供一种引物在制备用于诊断远端关节挛缩综合症的诊断剂或试剂盒中的用途，所述引物能够特异性扩增得到包含MYH3基因NM_002470第695位核苷酸的PCR产物。

作为本发明所述用途的优选实施方式，所述引物是如SEQ ID NO:5和SEQ ID NO:6所示的引物对。

作为本发明所述用途的优选实施方式，所述诊断远端关节挛缩综合症包括以下步骤：

(1)采集待测个体的血液、体液或组织，然后提取DNA；

(2)以步骤(1)提取的DNA为模板，加入所述引物进行PCR反应，得到PCR反应产物；

(3)从PCR产物中分离扩增目标片段，对所述目标片段包含的MYH3基因c.695的碱基进行分型鉴定。

本发明的有益效果：

(1)本发明利用全基因组外显子测序技术和Sanger测序技术，鉴定出了远端关节挛缩综合症的致病基因突变位点MYH3(c.695G>A:p.G232E)，构建的杂合突变小鼠动物模型(myh3^+/-)表现出明显的远端挛缩综合症表型。

(2)提供了用于诊断的方法以及包含诊断剂的试剂盒。本发明鉴定的致病基因，为进一步探明疾病病因提供了重要依据，也为疾病治疗提供了靶点。此外，对遗传咨询、产前诊断以及基因治疗具有重要意义。

附图说明

图1：MYH3基因c.695G>A突变在不同物种中的保守性分析。

图2：正常个体MYH3基因测序图和患病个体MYH3基因测序图，其中，箭头所示为突变位置。

图3：扩增产物的电泳图谱，左侧泳道为marker，右侧泳道为扩增的目标序列(500bp)。

图4：MYH3基因定点敲入695G>A点突变小鼠设计策略示意图。

图5：MYH3基因点突变小鼠出现远端挛缩综合症表型。

具体实施方式

为更清楚地表述本发明的技术方案，下面结合具体实施例进一步说明，但不能用于限制本发明，此仅是本发明的部分实施例。

除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法(例如，分子生物学和核酸化学实验方法)。参见例如，Sambrook等人,Molecular Cloning:A Laboratory Manual,第2版,Cold SpringHarbor Laboratory Press,Cold Spring Harbor,N.Y.(1989)；和Ausubel等人,CurrentProtocols in Molecular Biology,Greene Publishing Associates(1992)，其全部通过引用合并入本文。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。

在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、核酸化学和分子生物学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。

如本文中所使用的，术语“MYH3基因”是指编码胚胎型肌球蛋白的基因，具有41个外显子，并且其示例性cDNA序列如SEQ ID NO:2所示，长度为6032bp，翻译出1940个氨基酸。

如本文中所使用的，当用具体的序列来描述基因或核酸时，其不仅包括该具体序列所代表的基因或核酸，而且包括该具体序列的互补序列所代表的基因或核酸。在本申请中，虽然为了方便起见，在多数情况下针对基因或核酸只给出了一条链的序列，然而本领域技术人员可以明确获知其互补链的序列。因此，本申请事实上也公开了所述互补链的序列。

例如，当提及MYH3基因的cDNA序列时，其不仅包括cDNA的实际序列，而且包括所述实际序列的互补序列。又如，当提及SEQ ID NO:2时，其不仅包括SEQ ID NO:2所示的序列，而且包括SEQ ID NO:2的互补序列。

本申请中的核酸序列包括DNA形式和RNA形式。除非上下文特别指明，否则本发明的核酸序列不仅包括DNA形式，而且包括RNA形式。例如，当提及SEQ ID NO:2时，其不仅包括DNA形式(例如，cDNA序列)，而且包括RNA形式(例如，mRNA序列)。

如本文中所使用的，术语“突变”，当用于描述基因或DNA时，是指基因序列或DNA序列中一个或多个(例如，几个)碱基的添加、缺失和/或置换；当用于描述蛋白质时，是指蛋白质氨基酸序列中一个或多个(例如，几个)氨基酸残基的添加、缺失和/或置换。

如本文中所使用的，术语“c.695”是指cDNA序列的第695位碱基(以起始密码子ATG的碱基A为第1位碱基)，其中“c.”表示cDNA，数字“695”表示第695位碱基。本文中所使用的其他类似的术语具有类似的含义。

如本文中所使用的，术语“p.232”是指蛋白质序列的第232位氨基酸残基，其中“p.”表示蛋白质，数字“232”表示第232位氨基酸残基。本文中所使用的其他类似的术语具有类似的含义。

如本文中所使用的，术语“c.695G>A”是指cDNA序列的第695位碱基(以起始密码子ATG的碱基A为第1位碱基)由G突变为A。本文中所使用的其他类似的术语具有类似的含义。

如本文中所使用的，术语“p.G232E”是指蛋白质序列的第232位氨基酸残基由甘氨酸(G，Gly)突变为谷氨酸(E，Glu)。本文中所使用的其他类似的术语具有类似的含义。

如本文中所使用的，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。另外，还用“*”表示终止密码子。

本发明所涉及的序列：

SEQ ID NO：1，野生型MYH3蛋白的氨基酸序列；

SEQ ID NO：2，野生型MYH3蛋白的核苷酸序列；

SEQ ID NO：3，突变MYH3蛋白的氨基酸序列；

SEQ ID NO：4，突变MYH3蛋白的核苷酸序列。

实施例1致病基因突变位点MYH3的鉴定

利用全基因组外显子测序技术和Sanger测序技术，鉴定出了远端关节挛缩综合症的致病基因突变位点MYH3(c.695G>A:p.G232E)。具体鉴定过程如下：

(1)标本收集

本发明的发明人在华东地区收集到一个包括13名患者在内的33人的四代常染色体显性遗传远端关节挛缩综合症家系。远端关节挛缩综合症家系的诊断依据家族史、体格检查以及影像学(X光、CT和核磁共振)检查进行确诊。

(2)全基因组外显子测序

使用

DNA Blood Mini Kit(QIAGEN，Germany)提取家系所有成员静脉血基因组DNA样品。选取家系中5名患者，及一个未患病成员的DNA标本，采用全基因组外显子测序的方法寻找致病基因，主要流程如下：

1)样品DNA质量检测：使用琼脂糖凝胶电泳的方法检测基因组DNA是否完整，要求电泳条带无显著拖尾，清晰；同时应Nanodrop 2000检测DNA浓度及质量，要求浓度≥50ng/μL，总量≥3μg，OD260/280值位于1.8～2.0之间；

2)分选纯化DNA并片段化：使用DNA fragmentase(NEB公司)使DNA片段化并使片段长度位于100-500bp间；

3)末端修复：向上述纯化并片段化的DNA中加入末端补齐体系后使用AgencourtAMpure XP磁珠纯化，从而建立5’端包含磷酸基团的DNA文库；

4)3’末端加“A”和连接测序接头：向体系中加入3’末端加“A”缓冲反应体系以防止文库DNA片段间的自身串连；再加入双链测序接头和连接缓冲剂，使illumina测序接头连接至DNA片段两侧；

5)筛选文库片段并行PCR扩增：使用Agencourt SPRIselect试剂盒中的SPRI磁珠纯去除目标区域双侧的片段最终筛选出片段大小适中的原始DNA文库后，利用PCR富集两端均有illumina测序接头的DNA片段后，利用Qubit测其浓度；

6)全外显子芯片杂交及杂交文库清洗及纯化：利用

My One^TMStreptavidin T1磁珠，通过探针上偶联的生物素基团富集探针富集与探针特异性结合的文库DNA，清洗并纯化；

7)文库的PCR扩增及质量检测：使用高保真的聚合酶在50μL体系中扩增原始DNA文库后，使用Agilent 2100Bioanalyzer及Qubit检测文库浓度及文库DNA片段不同长度的分布情况；DNA文库浓度应＞5ng/μL，长度集中在300-400bp间；

8)高通量测序：使用2×150bp模式在Illumina Hiseq平台上进行高通量双端测序，得到原始Fast Q数据。

(3)Sanger测序

对检测到突变位点的区域进行扩增，通过ABI

3100系统进行Sanger测序分析，鉴定远端关节挛缩综合症的致病基因及突变位点。

结果如图1所示，本发明发现了一个新的远端关节挛缩综合症致病基因突变位点MYH3(c.695G>A:p.G232E)，且经过在不同物种中的保守性分析发现，MYH3的第232位的甘氨酸残基(G)为高度保守的氨基酸残基(图2)。

实施例2验证突变位点MYH3的突变会引起明显的远端挛缩综合症

为了验证突变位点MYH3的突变是否确定会引起明显的远端挛缩综合症，本发明构建得到杂合突变小鼠动物模型(myh3^+/-)，即：利用同源重组原理，采用胚胎干细胞打靶的方式，在MYH3基因定点引入c.695G>A点突变。具体操作过程如下：

(1)通过In-Fusion Cloning的方法构建胚胎干细胞打靶载体(具体操作步骤可参见文献Wu S,Ying G,Wu Q,Capecchi MR.A protocol for constructing gene targetingvectors:generating knockout mice for the cadherin family and beyond.NatProtoc.2008；3(6):1056-76.doi:10.1038/nprot.2008.70.PMID:18546598.或文献ChanW,Costantino N,Li R,Lee SC,Su Q,Melvin D,Court DL,Liu P.Arecombineering basedapproach for high-throughput conditional knockout targeting vectorconstruction.Nucleic Acids Res.2007；35(8):e64.doi:10.1093/nar/gkm163.Epub2007Apr 10.PMID:17426124；PMCID:PMC1885671.)，该载体包含3.0kb5’同源臂、695G>A点突变、PGK-Neo-polyA、3.0kb 3’同源臂和MC1-TK-polyA负筛选标记。

(2)该载体经线性化后，电转转染JM8A3胚胎干细胞。

(3)经G418和Ganc药物筛选后，共获得144个抗性克隆；

(4)经长片段PCR鉴定，共获得2个正确同源重组的阳性克隆，鉴定结果如图3所示。

(5)阳性胚胎干细胞克隆经扩增后，注射入C57BL/6J小鼠的囊胚中，获得嵌合鼠。高比例嵌合小鼠与C57BL/6J小鼠交配获得阳性子代小鼠。

结果如图5所示，MYH3基因点突变小鼠表现出明显的远端挛缩综合症表型，表明本发明发现的突变位点MYH3的突变确定会引起明显的远端挛缩综合症。

实施例3检测MYH3基因c.695G>A突变的试剂盒

本实施例提供一种用于检测MYH3基因c.695G>A突变的试剂盒，包括用于扩增DNA片段的PCR反应试剂，包括PCR引物，该引物扩增的目标片段包含MYH3基因NM_002470：c.695对应的碱基。引物序列为：

MYH3-F：5’TCATCATCTGTTGCCTCTGGTC 3’(SEQ ID NO：5)

MYH3-R：5’GTTGCAGTAAGCCAAGATCGT 3’(SEQ ID NO：6)

实施例4检测MYH3基因c.695G>A突变的方法

本实施例提供一种检测MYH3基因c.695G>A突变的方法，包括以下步骤：

(1)采集待测个体的血液、体液或组织，然后提取DNA；

(2)以步骤(1)提取的DNA为模板，以PCR引物进行PCR反应，得到PCR反应产物，PCR引物序列为：

MYH3-F：5’TCATCATCTGTTGCCTCTGGTC 3’(SEQ ID NO：5)

MYH3-R：5’GTTGCAGTAAGCCAAGATCGT 3’(SEQ ID NO：6)

(3)从PCR产物中分离扩增的目标片段，对该目标片段包含的MYH3基因c.695G>A的碱基进行分型鉴定。

最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

SEQUENCE LISTING

<110> 上海长征医院

<120> 一种远端关节挛缩综合症致病基因MYH3及其用途

<130> 2021.04.30

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 1940

<212> PRT

<213> 人工合成

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Gln Arg Glu Glu Gln Ala Glu Pro Asp Gly Thr Glu Val Ala Asp Lys

370 375 380

Thr Ala Tyr Leu Met Gly Leu Asn Ser Ser Asp Leu Leu Lys Ala Leu

385 390 395 400

Cys Phe Pro Arg Val Lys Val Gly Asn Glu Tyr Val Thr Lys Gly Gln

405 410 415

Thr Val Asp Gln Val His His Ala Val Asn Ala Leu Ser Lys Ser Val

420 425 430

Tyr Glu Lys Leu Phe Leu Trp Met Val Thr Arg Ile Asn Gln Gln Leu

435 440 445

Asp Thr Lys Leu Pro Arg Gln His Phe Ile Gly Val Leu Asp Ile Ala

450 455 460

Gly Phe Glu Ile Phe Glu Tyr Asn Ser Leu Glu Gln Leu Cys Ile Asn

465 470 475 480

Phe Thr Asn Glu Lys Leu Gln Gln Phe Phe Asn His His Met Phe Val

485 490 495

Leu Glu Gln Glu Glu Tyr Lys Lys Glu Gly Ile Glu Trp Thr Phe Ile

500 505 510

Asp Phe Gly Met Asp Leu Ala Ala Cys Ile Glu Leu Ile Glu Lys Pro

515 520 525

Met Gly Ile Phe Ser Ile Leu Glu Glu Glu Cys Met Phe Pro Lys Ala

530 535 540

Thr Asp Thr Ser Phe Lys Asn Lys Leu Tyr Asp Gln His Leu Gly Lys

545 550 555 560

Ser Asn Asn Phe Gln Lys Pro Lys Val Val Lys Gly Arg Ala Glu Ala

565 570 575

His Phe Ser Leu Ile His Tyr Ala Gly Thr Val Asp Tyr Ser Val Ser

580 585 590

Gly Trp Leu Glu Lys Asn Lys Asp Pro Leu Asn Glu Thr Val Val Gly

595 600 605

Leu Tyr Gln Lys Ser Ser Asn Arg Leu Leu Ala His Leu Tyr Ala Thr

610 615 620

Phe Ala Thr Ala Asp Ala Asp Ser Gly Lys Lys Lys Val Ala Lys Lys

625 630 635 640

Lys Gly Ser Ser Phe Gln Thr Val Ser Ala Leu Phe Arg Glu Asn Leu

645 650 655

Asn Lys Leu Met Ser Asn Leu Arg Thr Thr His Pro His Phe Val Arg

660 665 670

Cys Ile Ile Pro Asn Glu Thr Lys Thr Pro Gly Ala Met Glu His Ser

675 680 685

Leu Val Leu His Gln Leu Arg Cys Asn Gly Val Leu Glu Gly Ile Arg

690 695 700

Ile Cys Arg Lys Gly Phe Pro Asn Arg Ile Leu Tyr Gly Asp Phe Lys

705 710 715 720

Gln Arg Tyr Arg Val Leu Asn Ala Ser Ala Ile Pro Glu Gly Gln Phe

725 730 735

Ile Asp Ser Lys Lys Ala Cys Glu Lys Leu Leu Ala Ser Ile Asp Ile

740 745 750

Asp His Thr Gln Tyr Lys Phe Gly His Thr Lys Val Phe Phe Lys Ala

755 760 765

Gly Leu Leu Gly Thr Leu Glu Glu Met Arg Asp Asp Arg Leu Ala Lys

770 775 780

Leu Ile Thr Arg Thr Gln Ala Val Cys Arg Gly Phe Leu Met Arg Val

785 790 795 800

Glu Phe Gln Lys Met Val Gln Arg Arg Glu Ser Ile Phe Cys Ile Gln

805 810 815

Tyr Asn Ile Arg Ser Phe Met Asn Val Lys His Trp Pro Trp Met Lys

820 825 830

Leu Phe Phe Lys Ile Lys Pro Leu Leu Lys Ser Ala Glu Thr Glu Lys

835 840 845

Glu Met Ala Thr Met Lys Glu Glu Phe Gln Lys Thr Lys Asp Glu Leu

850 855 860

Ala Lys Ser Glu Ala Lys Arg Lys Glu Leu Glu Glu Lys Leu Val Thr

865 870 875 880

Leu Val Gln Glu Lys Asn Asp Leu Gln Leu Gln Val Gln Ala Glu Ser

885 890 895

Glu Asn Leu Leu Asp Ala Glu Glu Arg Cys Asp Gln Leu Ile Lys Ala

900 905 910

Lys Phe Gln Leu Glu Ala Lys Ile Lys Glu Val Thr Glu Arg Ala Glu

915 920 925

Asp Glu Glu Glu Ile Asn Ala Glu Leu Thr Ala Lys Lys Arg Lys Leu

930 935 940

Glu Asp Glu Cys Ser Glu Leu Lys Lys Asp Ile Asp Asp Leu Glu Leu

945 950 955 960

Thr Leu Ala Lys Val Glu Lys Glu Lys His Ala Thr Glu Asn Lys Val

965 970 975

Lys Asn Leu Thr Glu Glu Leu Ser Gly Leu Asp Glu Thr Ile Ala Lys

980 985 990

Leu Thr Arg Glu Lys Lys Ala Leu Gln Glu Ala His Gln Gln Ala Leu

995 1000 1005

Asp Asp Leu Gln Ala Glu Glu Asp Lys Val Asn Ser Leu Asn Lys

1010 1015 1020

Thr Lys Ser Lys Leu Glu Gln Gln Val Glu Asp Leu Glu Ser Ser

1025 1030 1035

Leu Glu Gln Glu Lys Lys Leu Arg Val Asp Leu Glu Arg Asn Lys

1040 1045 1050

Arg Lys Leu Glu Gly Asp Leu Lys Leu Ala Gln Glu Ser Ile Leu

1055 1060 1065

Asp Leu Glu Asn Asp Lys Gln Gln Leu Asp Glu Arg Leu Lys Lys

1070 1075 1080

Lys Asp Phe Glu Tyr Cys Gln Leu Gln Ser Lys Val Glu Asp Glu

1085 1090 1095

Gln Thr Leu Gly Leu Gln Phe Gln Lys Lys Ile Lys Glu Leu Gln

1100 1105 1110

Ala Arg Ile Glu Glu Leu Glu Glu Glu Ile Glu Ala Glu Arg Ala

1115 1120 1125

Thr Arg Ala Lys Thr Glu Lys Gln Arg Ser Asp Tyr Ala Arg Glu

1130 1135 1140

Leu Glu Glu Leu Ser Glu Arg Leu Glu Glu Ala Gly Gly Val Thr

1145 1150 1155

Ser Thr Gln Ile Glu Leu Asn Lys Lys Arg Glu Ala Glu Phe Leu

1160 1165 1170

Lys Leu Arg Arg Asp Leu Glu Glu Ala Thr Leu Gln His Glu Ala

1175 1180 1185

Met Val Ala Ala Leu Arg Lys Lys His Ala Asp Ser Val Ala Glu

1190 1195 1200

Leu Gly Glu Gln Ile Asp Asn Leu Gln Arg Val Lys Gln Lys Leu

1205 1210 1215

Glu Lys Glu Lys Ser Glu Phe Lys Leu Glu Ile Asp Asp Leu Ser

1220 1225 1230

Ser Ser Met Glu Ser Val Ser Lys Ser Lys Ala Asn Leu Glu Lys

1235 1240 1245

Ile Cys Arg Thr Leu Glu Asp Gln Leu Ser Glu Ala Arg Gly Lys

1250 1255 1260

Asn Glu Glu Ile Gln Arg Ser Leu Ser Glu Leu Thr Thr Gln Lys

1265 1270 1275

Ser Arg Leu Gln Thr Glu Ala Gly Glu Leu Ser Arg Gln Leu Glu

1280 1285 1290

Glu Lys Glu Ser Ile Val Ser Gln Leu Ser Arg Ser Lys Gln Ala

1295 1300 1305

Phe Thr Gln Gln Thr Glu Glu Leu Lys Arg Gln Leu Glu Glu Glu

1310 1315 1320

Asn Lys Ala Lys Asn Ala Leu Ala His Ala Leu Gln Ser Ser Arg

1325 1330 1335

His Asp Cys Asp Leu Leu Arg Glu Gln Tyr Glu Glu Glu Gln Glu

1340 1345 1350

Gly Lys Ala Glu Leu Gln Arg Ala Leu Ser Lys Ala Asn Ser Glu

1355 1360 1365

Val Ala Gln Trp Arg Thr Lys Tyr Glu Thr Asp Ala Ile Gln Arg

1370 1375 1380

Thr Glu Glu Leu Glu Glu Ala Lys Lys Lys Leu Ala Gln Arg Leu

1385 1390 1395

Gln Asp Ser Glu Glu Gln Val Glu Ala Val Asn Ala Lys Cys Ala

1400 1405 1410

Ser Leu Glu Lys Thr Lys Gln Arg Leu Gln Gly Glu Val Glu Asp

1415 1420 1425

Leu Met Val Asp Val Glu Arg Ala Asn Ser Leu Ala Ala Ala Leu

1430 1435 1440

Asp Lys Lys Gln Arg Asn Phe Asp Lys Val Leu Ala Glu Trp Lys

1445 1450 1455

Thr Lys Cys Glu Glu Ser Gln Ala Glu Leu Glu Ala Ser Leu Lys

1460 1465 1470

Glu Ser Arg Ser Leu Ser Thr Glu Leu Phe Lys Leu Lys Asn Ala

1475 1480 1485

Tyr Glu Glu Ala Leu Asp Gln Leu Glu Thr Val Lys Arg Glu Asn

1490 1495 1500

Lys Asn Leu Glu Gln Glu Ile Ala Asp Leu Thr Glu Gln Ile Ala

1505 1510 1515

Glu Asn Gly Lys Thr Ile His Glu Leu Glu Lys Ser Arg Lys Gln

1520 1525 1530

Ile Glu Leu Glu Lys Ala Asp Ile Gln Leu Ala Leu Glu Glu Ala

1535 1540 1545

Glu Ala Ala Leu Glu His Glu Glu Ala Lys Ile Leu Arg Ile Gln

1550 1555 1560

Leu Glu Leu Thr Gln Val Lys Ser Glu Ile Asp Arg Lys Ile Ala

1565 1570 1575

Glu Lys Asp Glu Glu Ile Glu Gln Leu Lys Arg Asn Tyr Gln Arg

1580 1585 1590

Thr Val Glu Thr Met Gln Ser Ala Leu Asp Ala Glu Val Arg Ser

1595 1600 1605

Arg Asn Glu Ala Ile Arg Leu Lys Lys Lys Met Glu Gly Asp Leu

1610 1615 1620

Asn Glu Ile Glu Ile Gln Leu Ser His Ala Asn Arg Gln Ala Ala

1625 1630 1635

Glu Thr Leu Lys His Leu Arg Ser Val Gln Gly Gln Leu Lys Asp

1640 1645 1650

Thr Gln Leu His Leu Asp Asp Ala Leu Arg Gly Gln Glu Asp Leu

1655 1660 1665

Lys Glu Gln Leu Ala Ile Val Glu Arg Arg Ala Asn Leu Leu Gln

1670 1675 1680

Ala Glu Val Glu Glu Leu Arg Ala Thr Leu Glu Gln Thr Glu Arg

1685 1690 1695

Ala Arg Lys Leu Ala Glu Gln Glu Leu Leu Asp Ser Asn Glu Arg

1700 1705 1710

Val Gln Leu Leu His Thr Gln Asn Thr Ser Leu Ile His Thr Lys

1715 1720 1725

Lys Lys Leu Glu Thr Asp Leu Met Gln Leu Gln Ser Glu Val Glu

1730 1735 1740

Asp Ala Ser Arg Asp Ala Arg Asn Ala Glu Glu Lys Ala Lys Lys

1745 1750 1755

Ala Ile Thr Asp Ala Ala Met Met Ala Glu Glu Leu Lys Lys Glu

1760 1765 1770

Gln Asp Thr Ser Ala His Leu Glu Arg Met Lys Lys Asn Leu Glu

1775 1780 1785

Gln Thr Val Lys Asp Leu Gln His Arg Leu Asp Glu Ala Glu Gln

1790 1795 1800

Leu Ala Leu Lys Gly Gly Lys Lys Gln Ile Gln Lys Leu Glu Thr

1805 1810 1815

Arg Ile Arg Glu Leu Glu Phe Glu Leu Glu Gly Glu Gln Lys Lys

1820 1825 1830

Asn Thr Glu Ser Val Lys Gly Leu Arg Lys Tyr Glu Arg Arg Val

1835 1840 1845

Lys Glu Leu Thr Tyr Gln Ser Glu Glu Asp Arg Lys Asn Val Leu

1850 1855 1860

Arg Leu Gln Asp Leu Val Asp Lys Leu Gln Val Lys Val Lys Ser

1865 1870 1875

Tyr Lys Arg Gln Ala Glu Glu Ala Asp Glu Gln Ala Asn Ala His

1880 1885 1890

Leu Thr Lys Phe Arg Lys Ala Gln His Glu Leu Glu Glu Ala Glu

1895 1900 1905

Glu Arg Ala Asp Ile Ala Glu Ser Gln Val Asn Lys Leu Arg Ala

1910 1915 1920

Lys Thr Arg Asp Phe Thr Ser Ser Arg Met Val Val His Glu Ser

1925 1930 1935

Glu Glu

1940

<210> 2

<211> 6032

<212> DNA

<213> 人工合成

<400> 2

gtggctcgct tgtgggcgga ggtctgggat ctcctggctg ttgctgtctt ctgctctcat 60

cctgcaggtg ggactctcag ctgacaccat gagtagtgac actgaaatgg aagtgttcgg 120

catagctgct cctttcctcc ggaagtcaga aaaggagagg atcgaggctc agaaccagcc 180

ctttgatgcc aagacgtatt gcttcgtggt ggactcaaag gaagaatatg ccaaggggaa 240

aatcaagagt tctcaggatg ggaaggtcac tgtggaaact gaggacaaca ggaccctggt 300

ggtcaaacca gaggatgtgt acgccatgaa cccccccaag ttcgacagga tcgaagacat 360

ggccatgctg acgcacctga atgagccagc cgtgctgtac aacctgaagg accgttacac 420

atcttggatg atctatacct actcaggcct cttctgtgtc actgtcaacc cctacaagtg 480

gctgccggtg tacaaccccg aggtggtgga aggctaccga ggcaaaaagc gccaggaggc 540

cccaccccac atcttctcca tctctgacaa cgcctatcag ttcatgctga ctgatcgtga 600

aaaccagtcc attctgatca ccggagaatc cggggcagga aagactgtga acaccaaacg 660

ggtcatccag tactttgcaa caattgcagc tactggggac ctggccaaga agaaggactc 720

caaaatgaag gggactctgg aagatcaaat catcagtgcc aatcccctgc tggaggcctt 780

tgggaacgcc aagactgtga ggaatgacaa ctcctcccgt tttggcaagt tcatccgaat 840

ccattttgga accactggga agctggcctc tgcagatatt gaaacttatc ttctggaaaa 900

atcaagagtc actttccagc tgaaggctga aagaagctac cacatcttct accagattct 960

ttctaacaag aagcctgagc tcatagagct gctgcttatt acgaccaacc cttacgacta 1020

cccgttcatt agccaggggg agatcctggt ggccagcata gatgatgcag aggagctgct 1080

ggctacagac agcgccattg acatcctggg cttcacccca gaagagaaat ctgggctcta 1140

caagctgacg ggagccgtga tgcactacgg gaacatgaag ttcaagcaga agcagcgaga 1200

ggagcaggcc gagccggatg gcacagaagt ggctgacaaa acagcctatc tgatgggcct 1260

gaactcttcg gacctcctaa aagctttgtg ctttcctaga gtgaaagttg ggaatgagta 1320

cgttaccaaa ggtcaaactg tggatcaggt tcaccatgct gtgaatgctc tttcaaaatc 1380

agtttatgaa aagttgttct tgtggatggt cactcgcatt aaccagcaac tggatacgaa 1440

gcttccaaga caacacttca ttggtgtttt ggacattgca ggctttgaaa tctttgagta 1500

taacagcctg gagcagctgt gcatcaactt caccaatgag aaactgcaac agtttttcaa 1560

ccaccacatg ttcgtgctgg agcaggagga gtacaagaag gaaggcatcg agtggacgtt 1620

cattgacttc gggatggacc tggctgcctg catcgagctc atcgagaagc ctatgggcat 1680

cttctccatc ctggaagagg agtgcatgtt ccccaaggca acagacacct ccttcaagaa 1740

caagctgtat gaccagcatc ttggaaagtc caacaacttc cagaagccca aggtggtcaa 1800

aggcagggcc gaggctcact tctcactgat ccactatgcg ggcaccgtgg actacagtgt 1860

ctcaggttgg ctggagaaga acaaggaccc tctgaacgag actgtggttg ggctgtacca 1920

gaagtcttcc aacaggctcc tggcacacct ctatgccacg tttgccacgg cggatgctga 1980

cagtggaaag aagaaagttg ccaagaagaa gggttcttcc ttccaaactg tctctgccct 2040

tttcagggaa aacctgaaca agctgatgtc aaatttaaga actactcacc ctcattttgt 2100

gcgttgtata attcccaatg aaaccaaaac tccaggggct atggaacaca gccttgttct 2160

gcaccagctg cggtgtaacg gtgtcctgga gggcatccgc atctgcagga aagggttccc 2220

aaacaggatt ctctatggcg attttaaaca aagataccga gtgctgaatg ccagtgcaat 2280

ccctgaggga caattcattg acagcaagaa agcctgtgaa aagcttctgg catccattga 2340

tattgaccac actcagtaca aatttggaca taccaaggtg ttcttcaagg ctggcttgct 2400

gggaaccctg gaagagatgc gggatgaccg cctggccaaa ctaatcaccc ggacacaagc 2460

tgtgtgcaga gggttcctca tgcgtgtgga attccagaag atggtgcaga ggagggagtc 2520

catcttctgc atccagtaca acattcgctc attcatgaac gtcaagcact ggccctggat 2580

gaaactcttc ttcaagatca agcccctcct caagagtgca gagactgaga aagagatggc 2640

caccatgaag gaagaattcc agaaaaccaa agatgaactc gccaagtcgg aggcaaaaag 2700

gaaggagcta gaggaaaaac tggtgactct ggtccaagag aagaatgacc tgcagctcca 2760

agtacaagct gaaagcgaaa atttgttgga tgctgaggaa agatgcgatc agctgatcaa 2820

agccaaattc cagctcgagg ccaagatcaa ggaggtgaca gagagagctg aagatgagga 2880

ggagatcaat gctgagctga cggccaagaa gaggaaactg gaggatgaat gctcagagct 2940

caagaaagac attgatgacc ttgagttgac cctggccaag gttgagaagg agaagcatgc 3000

cacagagaac aaggttaaaa accttactga ggaactctct gggttagatg aaacaattgc 3060

aaagttaacc agagagaaga aggccctcca agaggcgcac cagcaggcct tggatgacct 3120

ccaagctgaa gaagacaaag tcaattcttt gaacaaaacc aagagcaaac tggaacagca 3180

agtggaagac ctggaaagct ccctagaaca agaaaagaag ctccgagtag acctggaaag 3240

gaacaaaagg aaattggaag gagacttgaa gcttgctcaa gagtccatat tagatctgga 3300

gaatgacaag caacagctgg acgaaaggct caagaagaaa gattttgaat attgtcaact 3360

tcaaagcaaa gtggaagatg agcagacact gggcctccag tttcagaaga aaatcaaaga 3420

gttgcaggct cgaattgagg agctggaaga ggagatagag gcggagaggg ccacccgcgc 3480

gaagacagag aaacagcgca gcgactatgc ccgggagctg gaggagctga gcgagcggct 3540

ggaggaggcg ggaggcgtca cctccacgca gatagagctc aacaagaagc gggaggcgga 3600

gttcctgaag ctgcgcaggg acctggagga ggccacactg cagcacgaag ccatggtggc 3660

cgcgctgagg aagaagcatg cggatagtgt ggccgagctt ggggagcaga ttgacaacct 3720

gcagcgggtc aagcagaagc tggagaagga gaagagcgag ttcaagctgg agatcgatga 3780

cctctccagc agcatggaga gtgtgtcgaa atctaaggca aatctggaaa aaatctgccg 3840

aaccctggag gatcagttaa gtgaggccag gggcaagaat gaggaaattc agaggagcct 3900

gagcgagctg accacacaga agtctcgttt gcagaccgag gctggtgagc tgagtcgtca 3960

gctggaagaa aaagaaagca tagtatccca actttccagg agcaagcaag cctttaccca 4020

gcaaacagaa gagctcaaga ggcagctgga ggaagagaac aaggccaaga acgccctggc 4080

gcacgccctg cagtcctccc gccacgactg tgacctgctg cgggaacagt atgaggagga 4140

gcaggaaggc aaagctgagc tgcagagggc gctgtccaag gccaatagtg aggttgccca 4200

gtggagaacc aaatacgaga cggacgccat ccagcgcaca gaagagctgg aggaggccaa 4260

gaaaaaactt gctcagcgcc ttcaagattc cgaggaacag gttgaggcag tgaatgctaa 4320

atgtgcttca ctggagaaga ccaagcagag gctgcaagga gaggtggagg atctgatggt 4380

tgatgttgaa agagccaatt ccttggccgc cgctctggac aagaagcaga ggaactttga 4440

caaggtgttg gcagagtgga agacaaagtg tgaggagagc caagcagagc tggaggcatc 4500

cctgaaggag tcccgctcct tgagcactga gctcttcaaa ctgaaaaatg cctacgagga 4560

agccttagat caacttgaaa ctgtgaaacg ggaaaataag aacttagagc aggagatagc 4620

agatctcaca gaacaaattg ctgaaaatgg caaaaccatc catgaactgg agaaatcaag 4680

aaagcagatt gagctggaaa aggctgatat ccagctggct ctcgaggaag cagaggctgc 4740

tcttgagcat gaagaagcca agatcctccg aatccagctt gaattgacac aagtgaaatc 4800

agaaattgat agaaagatcg ccgagaagga tgaagagatc gagcagctga agaggaacta 4860

ccagagaaca gtggaaacca tgcagagcgc cctggacgcc gaggtgcgga gcaggaatga 4920

agccatccgg ctcaagaaga agatggaggg ggacctgaat gaaatcgaga tccagctgag 4980

ccacgccaac cgccaggcgg cggagaccct caaacacctc aggagtgtcc agggacagct 5040

gaaggatacg cagctccacc tggatgatgc cctccggggc caggaggacc tgaaggagca 5100

gctggcgatt gtggagcgca gagccaacct gctgcaggcc gaggtggagg agctgcgggc 5160

tactctggag cagacggaga gggcccggaa actggcggaa caggagctcc tggactccaa 5220

cgagagggtg cagctgctgc atacccagaa caccagcctc atccacacca agaagaagct 5280

ggagacagac ctcatgcagc tccagagtga ggtagaagat gccagcaggg atgcaaggaa 5340

cgctgaggag aaggccaaga aggccatcac ggacgctgcc atgatggcgg aggagctgaa 5400

gaaggagcag gacaccagcg cccaccttga gcggatgaag aagaacctgg aacagacggt 5460

gaaggacctg cagcatcgtc tagatgaggc cgagcagctg gcgctgaagg gcgggaagaa 5520

gcagatccag aaactggaga ccaggatccg agagctggag tttgaacttg agggagagca 5580

gaagaagaac acagagtctg ttaagggcct gaggaagtat gagcggaggg tcaaggagct 5640

gacgtaccag agtgaagagg acaggaagaa tgtgctgaga ttgcaggatc tggtggataa 5700

actgcaagtg aaagtcaagt cctacaagag gcaggcggag gaggctgatg aacaagccaa 5760

tgctcatctc accaaattcc gaaaggctca gcatgagctg gaggaggccg aggaacgtgc 5820

ggatatcgca gaatctcaag tcaacaagct ccgcgctaag actcgagact tcacctccag 5880

caggatggtg gtccacgaga gtgaagagtg agccagccct tctggagcag gacagaagat 5940

atgcaaaatg tatattttct tgattcctga ccattgatac ttaatgtcca tgtgactctt 6000

tttcacatgc aataaacttt gctttgtttc aa 6032

<210> 3

<211> 1940

<212> PRT

<213> 人工合成

<400> 3

Met Ser Ser Asp Thr Glu Met Glu Val Phe Gly Ile Ala Ala Pro Phe

1 5 10 15

Leu Arg Lys Ser Glu Lys Glu Arg Ile Glu Ala Gln Asn Gln Pro Phe

20 25 30

Asp Ala Lys Thr Tyr Cys Phe Val Val Asp Ser Lys Glu Glu Tyr Ala

35 40 45

Lys Gly Lys Ile Lys Ser Ser Gln Asp Gly Lys Val Thr Val Glu Thr

50 55 60

Glu Asp Asn Arg Thr Leu Val Val Lys Pro Glu Asp Val Tyr Ala Met

65 70 75 80

Asn Pro Pro Lys Phe Asp Arg Ile Glu Asp Met Ala Met Leu Thr His

85 90 95

Leu Asn Glu Pro Ala Val Leu Tyr Asn Leu Lys Asp Arg Tyr Thr Ser

100 105 110

Trp Met Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Thr Val Asn Pro

115 120 125

Tyr Lys Trp Leu Pro Val Tyr Asn Pro Glu Val Val Glu Gly Tyr Arg

130 135 140

Gly Lys Lys Arg Gln Glu Ala Pro Pro His Ile Phe Ser Ile Ser Asp

145 150 155 160

Asn Ala Tyr Gln Phe Met Leu Thr Asp Arg Glu Asn Gln Ser Ile Leu

165 170 175

Ile Thr Gly Glu Ser Gly Ala Gly Lys Thr Val Asn Thr Lys Arg Val

180 185 190

Ile Gln Tyr Phe Ala Thr Ile Ala Ala Thr Gly Asp Leu Ala Lys Lys

195 200 205

Lys Asp Ser Lys Met Lys Gly Thr Leu Glu Asp Gln Ile Ile Ser Ala

210 215 220

Asn Pro Leu Leu Glu Ala Phe Glu Asn Ala Lys Thr Val Arg Asn Asp

225 230 235 240

Asn Ser Ser Arg Phe Gly Lys Phe Ile Arg Ile His Phe Gly Thr Thr

245 250 255

Gly Lys Leu Ala Ser Ala Asp Ile Glu Thr Tyr Leu Leu Glu Lys Ser

260 265 270

Arg Val Thr Phe Gln Leu Lys Ala Glu Arg Ser Tyr His Ile Phe Tyr

275 280 285

Gln Ile Leu Ser Asn Lys Lys Pro Glu Leu Ile Glu Leu Leu Leu Ile

290 295 300

Thr Thr Asn Pro Tyr Asp Tyr Pro Phe Ile Ser Gln Gly Glu Ile Leu

305 310 315 320

Val Ala Ser Ile Asp Asp Ala Glu Glu Leu Leu Ala Thr Asp Ser Ala

325 330 335

Ile Asp Ile Leu Gly Phe Thr Pro Glu Glu Lys Ser Gly Leu Tyr Lys

340 345 350

Leu Thr Gly Ala Val Met His Tyr Gly Asn Met Lys Phe Lys Gln Lys

355 360 365

Gln Arg Glu Glu Gln Ala Glu Pro Asp Gly Thr Glu Val Ala Asp Lys

370 375 380

Thr Ala Tyr Leu Met Gly Leu Asn Ser Ser Asp Leu Leu Lys Ala Leu

385 390 395 400

Cys Phe Pro Arg Val Lys Val Gly Asn Glu Tyr Val Thr Lys Gly Gln

405 410 415

Thr Val Asp Gln Val His His Ala Val Asn Ala Leu Ser Lys Ser Val

420 425 430

Tyr Glu Lys Leu Phe Leu Trp Met Val Thr Arg Ile Asn Gln Gln Leu

435 440 445

Asp Thr Lys Leu Pro Arg Gln His Phe Ile Gly Val Leu Asp Ile Ala

450 455 460

Gly Phe Glu Ile Phe Glu Tyr Asn Ser Leu Glu Gln Leu Cys Ile Asn

465 470 475 480

Phe Thr Asn Glu Lys Leu Gln Gln Phe Phe Asn His His Met Phe Val

485 490 495

Leu Glu Gln Glu Glu Tyr Lys Lys Glu Gly Ile Glu Trp Thr Phe Ile

500 505 510

Asp Phe Gly Met Asp Leu Ala Ala Cys Ile Glu Leu Ile Glu Lys Pro

515 520 525

Met Gly Ile Phe Ser Ile Leu Glu Glu Glu Cys Met Phe Pro Lys Ala

530 535 540

Thr Asp Thr Ser Phe Lys Asn Lys Leu Tyr Asp Gln His Leu Gly Lys

545 550 555 560

Ser Asn Asn Phe Gln Lys Pro Lys Val Val Lys Gly Arg Ala Glu Ala

565 570 575

His Phe Ser Leu Ile His Tyr Ala Gly Thr Val Asp Tyr Ser Val Ser

580 585 590

Gly Trp Leu Glu Lys Asn Lys Asp Pro Leu Asn Glu Thr Val Val Gly

595 600 605

Leu Tyr Gln Lys Ser Ser Asn Arg Leu Leu Ala His Leu Tyr Ala Thr

610 615 620

Phe Ala Thr Ala Asp Ala Asp Ser Gly Lys Lys Lys Val Ala Lys Lys

625 630 635 640

Lys Gly Ser Ser Phe Gln Thr Val Ser Ala Leu Phe Arg Glu Asn Leu

645 650 655

Asn Lys Leu Met Ser Asn Leu Arg Thr Thr His Pro His Phe Val Arg

660 665 670

Cys Ile Ile Pro Asn Glu Thr Lys Thr Pro Gly Ala Met Glu His Ser

675 680 685

Leu Val Leu His Gln Leu Arg Cys Asn Gly Val Leu Glu Gly Ile Arg

690 695 700

Ile Cys Arg Lys Gly Phe Pro Asn Arg Ile Leu Tyr Gly Asp Phe Lys

705 710 715 720

Gln Arg Tyr Arg Val Leu Asn Ala Ser Ala Ile Pro Glu Gly Gln Phe

725 730 735

Ile Asp Ser Lys Lys Ala Cys Glu Lys Leu Leu Ala Ser Ile Asp Ile

740 745 750

Asp His Thr Gln Tyr Lys Phe Gly His Thr Lys Val Phe Phe Lys Ala

755 760 765

Gly Leu Leu Gly Thr Leu Glu Glu Met Arg Asp Asp Arg Leu Ala Lys

770 775 780

Leu Ile Thr Arg Thr Gln Ala Val Cys Arg Gly Phe Leu Met Arg Val

785 790 795 800

Glu Phe Gln Lys Met Val Gln Arg Arg Glu Ser Ile Phe Cys Ile Gln

805 810 815

Tyr Asn Ile Arg Ser Phe Met Asn Val Lys His Trp Pro Trp Met Lys

820 825 830

Leu Phe Phe Lys Ile Lys Pro Leu Leu Lys Ser Ala Glu Thr Glu Lys

835 840 845

Glu Met Ala Thr Met Lys Glu Glu Phe Gln Lys Thr Lys Asp Glu Leu

850 855 860

Ala Lys Ser Glu Ala Lys Arg Lys Glu Leu Glu Glu Lys Leu Val Thr

865 870 875 880

Leu Val Gln Glu Lys Asn Asp Leu Gln Leu Gln Val Gln Ala Glu Ser

885 890 895

Glu Asn Leu Leu Asp Ala Glu Glu Arg Cys Asp Gln Leu Ile Lys Ala

900 905 910

Lys Phe Gln Leu Glu Ala Lys Ile Lys Glu Val Thr Glu Arg Ala Glu

915 920 925

Asp Glu Glu Glu Ile Asn Ala Glu Leu Thr Ala Lys Lys Arg Lys Leu

930 935 940

Glu Asp Glu Cys Ser Glu Leu Lys Lys Asp Ile Asp Asp Leu Glu Leu

945 950 955 960

Thr Leu Ala Lys Val Glu Lys Glu Lys His Ala Thr Glu Asn Lys Val

965 970 975

Lys Asn Leu Thr Glu Glu Leu Ser Gly Leu Asp Glu Thr Ile Ala Lys

980 985 990

Leu Thr Arg Glu Lys Lys Ala Leu Gln Glu Ala His Gln Gln Ala Leu

995 1000 1005

Asp Asp Leu Gln Ala Glu Glu Asp Lys Val Asn Ser Leu Asn Lys

1010 1015 1020

Thr Lys Ser Lys Leu Glu Gln Gln Val Glu Asp Leu Glu Ser Ser

1025 1030 1035

Leu Glu Gln Glu Lys Lys Leu Arg Val Asp Leu Glu Arg Asn Lys

1040 1045 1050

Arg Lys Leu Glu Gly Asp Leu Lys Leu Ala Gln Glu Ser Ile Leu

1055 1060 1065

Asp Leu Glu Asn Asp Lys Gln Gln Leu Asp Glu Arg Leu Lys Lys

1070 1075 1080

Lys Asp Phe Glu Tyr Cys Gln Leu Gln Ser Lys Val Glu Asp Glu

1085 1090 1095

Gln Thr Leu Gly Leu Gln Phe Gln Lys Lys Ile Lys Glu Leu Gln

1100 1105 1110

Ala Arg Ile Glu Glu Leu Glu Glu Glu Ile Glu Ala Glu Arg Ala

1115 1120 1125

Thr Arg Ala Lys Thr Glu Lys Gln Arg Ser Asp Tyr Ala Arg Glu

1130 1135 1140

Leu Glu Glu Leu Ser Glu Arg Leu Glu Glu Ala Gly Gly Val Thr

1145 1150 1155

Ser Thr Gln Ile Glu Leu Asn Lys Lys Arg Glu Ala Glu Phe Leu

1160 1165 1170

Lys Leu Arg Arg Asp Leu Glu Glu Ala Thr Leu Gln His Glu Ala

1175 1180 1185

Met Val Ala Ala Leu Arg Lys Lys His Ala Asp Ser Val Ala Glu

1190 1195 1200

Leu Gly Glu Gln Ile Asp Asn Leu Gln Arg Val Lys Gln Lys Leu

1205 1210 1215

Glu Lys Glu Lys Ser Glu Phe Lys Leu Glu Ile Asp Asp Leu Ser

1220 1225 1230

Ser Ser Met Glu Ser Val Ser Lys Ser Lys Ala Asn Leu Glu Lys

1235 1240 1245

Ile Cys Arg Thr Leu Glu Asp Gln Leu Ser Glu Ala Arg Gly Lys

1250 1255 1260

Asn Glu Glu Ile Gln Arg Ser Leu Ser Glu Leu Thr Thr Gln Lys

1265 1270 1275

Ser Arg Leu Gln Thr Glu Ala Gly Glu Leu Ser Arg Gln Leu Glu

1280 1285 1290

Glu Lys Glu Ser Ile Val Ser Gln Leu Ser Arg Ser Lys Gln Ala

1295 1300 1305

Phe Thr Gln Gln Thr Glu Glu Leu Lys Arg Gln Leu Glu Glu Glu

1310 1315 1320

Asn Lys Ala Lys Asn Ala Leu Ala His Ala Leu Gln Ser Ser Arg

1325 1330 1335

His Asp Cys Asp Leu Leu Arg Glu Gln Tyr Glu Glu Glu Gln Glu

1340 1345 1350

Gly Lys Ala Glu Leu Gln Arg Ala Leu Ser Lys Ala Asn Ser Glu

1355 1360 1365

Val Ala Gln Trp Arg Thr Lys Tyr Glu Thr Asp Ala Ile Gln Arg

1370 1375 1380

Thr Glu Glu Leu Glu Glu Ala Lys Lys Lys Leu Ala Gln Arg Leu

1385 1390 1395

Gln Asp Ser Glu Glu Gln Val Glu Ala Val Asn Ala Lys Cys Ala

1400 1405 1410

Ser Leu Glu Lys Thr Lys Gln Arg Leu Gln Gly Glu Val Glu Asp

1415 1420 1425

Leu Met Val Asp Val Glu Arg Ala Asn Ser Leu Ala Ala Ala Leu

1430 1435 1440

Asp Lys Lys Gln Arg Asn Phe Asp Lys Val Leu Ala Glu Trp Lys

1445 1450 1455

Thr Lys Cys Glu Glu Ser Gln Ala Glu Leu Glu Ala Ser Leu Lys

1460 1465 1470

Glu Ser Arg Ser Leu Ser Thr Glu Leu Phe Lys Leu Lys Asn Ala

1475 1480 1485

Tyr Glu Glu Ala Leu Asp Gln Leu Glu Thr Val Lys Arg Glu Asn

1490 1495 1500

Lys Asn Leu Glu Gln Glu Ile Ala Asp Leu Thr Glu Gln Ile Ala

1505 1510 1515

Glu Asn Gly Lys Thr Ile His Glu Leu Glu Lys Ser Arg Lys Gln

1520 1525 1530

Ile Glu Leu Glu Lys Ala Asp Ile Gln Leu Ala Leu Glu Glu Ala

1535 1540 1545

Glu Ala Ala Leu Glu His Glu Glu Ala Lys Ile Leu Arg Ile Gln

1550 1555 1560

Leu Glu Leu Thr Gln Val Lys Ser Glu Ile Asp Arg Lys Ile Ala

1565 1570 1575

Glu Lys Asp Glu Glu Ile Glu Gln Leu Lys Arg Asn Tyr Gln Arg

1580 1585 1590

Thr Val Glu Thr Met Gln Ser Ala Leu Asp Ala Glu Val Arg Ser

1595 1600 1605

Arg Asn Glu Ala Ile Arg Leu Lys Lys Lys Met Glu Gly Asp Leu

1610 1615 1620

Asn Glu Ile Glu Ile Gln Leu Ser His Ala Asn Arg Gln Ala Ala

1625 1630 1635

Glu Thr Leu Lys His Leu Arg Ser Val Gln Gly Gln Leu Lys Asp

1640 1645 1650

Thr Gln Leu His Leu Asp Asp Ala Leu Arg Gly Gln Glu Asp Leu

1655 1660 1665

Lys Glu Gln Leu Ala Ile Val Glu Arg Arg Ala Asn Leu Leu Gln

1670 1675 1680

Ala Glu Val Glu Glu Leu Arg Ala Thr Leu Glu Gln Thr Glu Arg

1685 1690 1695

Ala Arg Lys Leu Ala Glu Gln Glu Leu Leu Asp Ser Asn Glu Arg

1700 1705 1710

Val Gln Leu Leu His Thr Gln Asn Thr Ser Leu Ile His Thr Lys

1715 1720 1725

Lys Lys Leu Glu Thr Asp Leu Met Gln Leu Gln Ser Glu Val Glu

1730 1735 1740

Asp Ala Ser Arg Asp Ala Arg Asn Ala Glu Glu Lys Ala Lys Lys

1745 1750 1755

Ala Ile Thr Asp Ala Ala Met Met Ala Glu Glu Leu Lys Lys Glu

1760 1765 1770

Gln Asp Thr Ser Ala His Leu Glu Arg Met Lys Lys Asn Leu Glu

1775 1780 1785

Gln Thr Val Lys Asp Leu Gln His Arg Leu Asp Glu Ala Glu Gln

1790 1795 1800

Leu Ala Leu Lys Gly Gly Lys Lys Gln Ile Gln Lys Leu Glu Thr

1805 1810 1815

Arg Ile Arg Glu Leu Glu Phe Glu Leu Glu Gly Glu Gln Lys Lys

1820 1825 1830

Asn Thr Glu Ser Val Lys Gly Leu Arg Lys Tyr Glu Arg Arg Val

1835 1840 1845

Lys Glu Leu Thr Tyr Gln Ser Glu Glu Asp Arg Lys Asn Val Leu

1850 1855 1860

Arg Leu Gln Asp Leu Val Asp Lys Leu Gln Val Lys Val Lys Ser

1865 1870 1875

Tyr Lys Arg Gln Ala Glu Glu Ala Asp Glu Gln Ala Asn Ala His

1880 1885 1890

Leu Thr Lys Phe Arg Lys Ala Gln His Glu Leu Glu Glu Ala Glu

1895 1900 1905

Glu Arg Ala Asp Ile Ala Glu Ser Gln Val Asn Lys Leu Arg Ala

1910 1915 1920

Lys Thr Arg Asp Phe Thr Ser Ser Arg Met Val Val His Glu Ser

1925 1930 1935

Glu Glu

1940

<210> 4

<211> 6032

<212> DNA

<213> 人工合成

<400> 4

gtggctcgct tgtgggcgga ggtctgggat ctcctggctg ttgctgtctt ctgctctcat 60

cctgcaggtg ggactctcag ctgacaccat gagtagtgac actgaaatgg aagtgttcgg 120

catagctgct cctttcctcc ggaagtcaga aaaggagagg atcgaggctc agaaccagcc 180

ctttgatgcc aagacgtatt gcttcgtggt ggactcaaag gaagaatatg ccaaggggaa 240

aatcaagagt tctcaggatg ggaaggtcac tgtggaaact gaggacaaca ggaccctggt 300

ggtcaaacca gaggatgtgt acgccatgaa cccccccaag ttcgacagga tcgaagacat 360

ggccatgctg acgcacctga atgagccagc cgtgctgtac aacctgaagg accgttacac 420

atcttggatg atctatacct actcaggcct cttctgtgtc actgtcaacc cctacaagtg 480

gctgccggtg tacaaccccg aggtggtgga aggctaccga ggcaaaaagc gccaggaggc 540

cccaccccac atcttctcca tctctgacaa cgcctatcag ttcatgctga ctgatcgtga 600

aaaccagtcc attctgatca ccggagaatc cggggcagga aagactgtga acaccaaacg 660

ggtcatccag tactttgcaa caattgcagc tactggggac ctggccaaga agaaggactc 720

caaaatgaag gggactctgg aagatcaaat catcagtgcc aatcccctgc tggaggcctt 780

tgagaacgcc aagactgtga ggaatgacaa ctcctcccgt tttggcaagt tcatccgaat 840

ccattttgga accactggga agctggcctc tgcagatatt gaaacttatc ttctggaaaa 900

atcaagagtc actttccagc tgaaggctga aagaagctac cacatcttct accagattct 960

ttctaacaag aagcctgagc tcatagagct gctgcttatt acgaccaacc cttacgacta 1020

cccgttcatt agccaggggg agatcctggt ggccagcata gatgatgcag aggagctgct 1080

ggctacagac agcgccattg acatcctggg cttcacccca gaagagaaat ctgggctcta 1140

caagctgacg ggagccgtga tgcactacgg gaacatgaag ttcaagcaga agcagcgaga 1200

ggagcaggcc gagccggatg gcacagaagt ggctgacaaa acagcctatc tgatgggcct 1260

gaactcttcg gacctcctaa aagctttgtg ctttcctaga gtgaaagttg ggaatgagta 1320

cgttaccaaa ggtcaaactg tggatcaggt tcaccatgct gtgaatgctc tttcaaaatc 1380

agtttatgaa aagttgttct tgtggatggt cactcgcatt aaccagcaac tggatacgaa 1440

gcttccaaga caacacttca ttggtgtttt ggacattgca ggctttgaaa tctttgagta 1500

taacagcctg gagcagctgt gcatcaactt caccaatgag aaactgcaac agtttttcaa 1560

ccaccacatg ttcgtgctgg agcaggagga gtacaagaag gaaggcatcg agtggacgtt 1620

cattgacttc gggatggacc tggctgcctg catcgagctc atcgagaagc ctatgggcat 1680

cttctccatc ctggaagagg agtgcatgtt ccccaaggca acagacacct ccttcaagaa 1740

caagctgtat gaccagcatc ttggaaagtc caacaacttc cagaagccca aggtggtcaa 1800

aggcagggcc gaggctcact tctcactgat ccactatgcg ggcaccgtgg actacagtgt 1860

ctcaggttgg ctggagaaga acaaggaccc tctgaacgag actgtggttg ggctgtacca 1920

gaagtcttcc aacaggctcc tggcacacct ctatgccacg tttgccacgg cggatgctga 1980

cagtggaaag aagaaagttg ccaagaagaa gggttcttcc ttccaaactg tctctgccct 2040

tttcagggaa aacctgaaca agctgatgtc aaatttaaga actactcacc ctcattttgt 2100

gcgttgtata attcccaatg aaaccaaaac tccaggggct atggaacaca gccttgttct 2160

gcaccagctg cggtgtaacg gtgtcctgga gggcatccgc atctgcagga aagggttccc 2220

aaacaggatt ctctatggcg attttaaaca aagataccga gtgctgaatg ccagtgcaat 2280

ccctgaggga caattcattg acagcaagaa agcctgtgaa aagcttctgg catccattga 2340

tattgaccac actcagtaca aatttggaca taccaaggtg ttcttcaagg ctggcttgct 2400

gggaaccctg gaagagatgc gggatgaccg cctggccaaa ctaatcaccc ggacacaagc 2460

tgtgtgcaga gggttcctca tgcgtgtgga attccagaag atggtgcaga ggagggagtc 2520

catcttctgc atccagtaca acattcgctc attcatgaac gtcaagcact ggccctggat 2580

gaaactcttc ttcaagatca agcccctcct caagagtgca gagactgaga aagagatggc 2640

caccatgaag gaagaattcc agaaaaccaa agatgaactc gccaagtcgg aggcaaaaag 2700

gaaggagcta gaggaaaaac tggtgactct ggtccaagag aagaatgacc tgcagctcca 2760

agtacaagct gaaagcgaaa atttgttgga tgctgaggaa agatgcgatc agctgatcaa 2820

agccaaattc cagctcgagg ccaagatcaa ggaggtgaca gagagagctg aagatgagga 2880

ggagatcaat gctgagctga cggccaagaa gaggaaactg gaggatgaat gctcagagct 2940

caagaaagac attgatgacc ttgagttgac cctggccaag gttgagaagg agaagcatgc 3000

cacagagaac aaggttaaaa accttactga ggaactctct gggttagatg aaacaattgc 3060

aaagttaacc agagagaaga aggccctcca agaggcgcac cagcaggcct tggatgacct 3120

ccaagctgaa gaagacaaag tcaattcttt gaacaaaacc aagagcaaac tggaacagca 3180

agtggaagac ctggaaagct ccctagaaca agaaaagaag ctccgagtag acctggaaag 3240

gaacaaaagg aaattggaag gagacttgaa gcttgctcaa gagtccatat tagatctgga 3300

gaatgacaag caacagctgg acgaaaggct caagaagaaa gattttgaat attgtcaact 3360

tcaaagcaaa gtggaagatg agcagacact gggcctccag tttcagaaga aaatcaaaga 3420

gttgcaggct cgaattgagg agctggaaga ggagatagag gcggagaggg ccacccgcgc 3480

gaagacagag aaacagcgca gcgactatgc ccgggagctg gaggagctga gcgagcggct 3540

ggaggaggcg ggaggcgtca cctccacgca gatagagctc aacaagaagc gggaggcgga 3600

gttcctgaag ctgcgcaggg acctggagga ggccacactg cagcacgaag ccatggtggc 3660

cgcgctgagg aagaagcatg cggatagtgt ggccgagctt ggggagcaga ttgacaacct 3720

gcagcgggtc aagcagaagc tggagaagga gaagagcgag ttcaagctgg agatcgatga 3780

cctctccagc agcatggaga gtgtgtcgaa atctaaggca aatctggaaa aaatctgccg 3840

aaccctggag gatcagttaa gtgaggccag gggcaagaat gaggaaattc agaggagcct 3900

gagcgagctg accacacaga agtctcgttt gcagaccgag gctggtgagc tgagtcgtca 3960

gctggaagaa aaagaaagca tagtatccca actttccagg agcaagcaag cctttaccca 4020

gcaaacagaa gagctcaaga ggcagctgga ggaagagaac aaggccaaga acgccctggc 4080

gcacgccctg cagtcctccc gccacgactg tgacctgctg cgggaacagt atgaggagga 4140

gcaggaaggc aaagctgagc tgcagagggc gctgtccaag gccaatagtg aggttgccca 4200

gtggagaacc aaatacgaga cggacgccat ccagcgcaca gaagagctgg aggaggccaa 4260

gaaaaaactt gctcagcgcc ttcaagattc cgaggaacag gttgaggcag tgaatgctaa 4320

atgtgcttca ctggagaaga ccaagcagag gctgcaagga gaggtggagg atctgatggt 4380

tgatgttgaa agagccaatt ccttggccgc cgctctggac aagaagcaga ggaactttga 4440

caaggtgttg gcagagtgga agacaaagtg tgaggagagc caagcagagc tggaggcatc 4500

cctgaaggag tcccgctcct tgagcactga gctcttcaaa ctgaaaaatg cctacgagga 4560

agccttagat caacttgaaa ctgtgaaacg ggaaaataag aacttagagc aggagatagc 4620

agatctcaca gaacaaattg ctgaaaatgg caaaaccatc catgaactgg agaaatcaag 4680

aaagcagatt gagctggaaa aggctgatat ccagctggct ctcgaggaag cagaggctgc 4740

tcttgagcat gaagaagcca agatcctccg aatccagctt gaattgacac aagtgaaatc 4800

agaaattgat agaaagatcg ccgagaagga tgaagagatc gagcagctga agaggaacta 4860

ccagagaaca gtggaaacca tgcagagcgc cctggacgcc gaggtgcgga gcaggaatga 4920

agccatccgg ctcaagaaga agatggaggg ggacctgaat gaaatcgaga tccagctgag 4980

ccacgccaac cgccaggcgg cggagaccct caaacacctc aggagtgtcc agggacagct 5040

gaaggatacg cagctccacc tggatgatgc cctccggggc caggaggacc tgaaggagca 5100

gctggcgatt gtggagcgca gagccaacct gctgcaggcc gaggtggagg agctgcgggc 5160

tactctggag cagacggaga gggcccggaa actggcggaa caggagctcc tggactccaa 5220

cgagagggtg cagctgctgc atacccagaa caccagcctc atccacacca agaagaagct 5280

ggagacagac ctcatgcagc tccagagtga ggtagaagat gccagcaggg atgcaaggaa 5340

cgctgaggag aaggccaaga aggccatcac ggacgctgcc atgatggcgg aggagctgaa 5400

gaaggagcag gacaccagcg cccaccttga gcggatgaag aagaacctgg aacagacggt 5460

gaaggacctg cagcatcgtc tagatgaggc cgagcagctg gcgctgaagg gcgggaagaa 5520

gcagatccag aaactggaga ccaggatccg agagctggag tttgaacttg agggagagca 5580

gaagaagaac acagagtctg ttaagggcct gaggaagtat gagcggaggg tcaaggagct 5640

gacgtaccag agtgaagagg acaggaagaa tgtgctgaga ttgcaggatc tggtggataa 5700

actgcaagtg aaagtcaagt cctacaagag gcaggcggag gaggctgatg aacaagccaa 5760

tgctcatctc accaaattcc gaaaggctca gcatgagctg gaggaggccg aggaacgtgc 5820

ggatatcgca gaatctcaag tcaacaagct ccgcgctaag actcgagact tcacctccag 5880

caggatggtg gtccacgaga gtgaagagtg agccagccct tctggagcag gacagaagat 5940

atgcaaaatg tatattttct tgattcctga ccattgatac ttaatgtcca tgtgactctt 6000

tttcacatgc aataaacttt gctttgtttc aa 6032

<210> 5

<211> 22

<212> DNA

<213> 人工合成

<400> 5

tcatcatctg ttgcctctgg tc 22

<210> 6

<211> 21

<212> DNA

<213> 人工合成

<400> 6

gttgcagtaa gccaagatcg t 21

Claims

1.编码突变MYH3蛋白的基因在制备远端关节挛缩综合症动物模型中的用途，其特征在于，所述突变MYH3蛋白与野生型MYH3蛋白相比存在一个突变位点p.G232E；所述野生型MYH3蛋白的氨基酸序列如SEQ ID NO：1所示，所述基因的核苷酸序列如SEQ ID NO：4所示；所述动物为小鼠、大鼠、兔或猴。

2.包含编码突变MYH3蛋白的基因的表达载体或宿主细胞在制备远端关节挛缩综合症动物模型中的用途，其特征在于，所述突变MYH3蛋白与野生型MYH3蛋白相比存在一个突变位点p.G232E；所述野生型MYH3蛋白的氨基酸序列如SEQ ID NO：1所示，所述基因的核苷酸序列如SEQ ID NO：4所示；所述动物为小鼠、大鼠、兔或猴。

3.一种引物在制备用于诊断远端关节挛缩综合症的诊断剂或试剂盒中的用途，其特征在于，所述引物能够特异性扩增得到包含MYH3基因NM_002470第695位核苷酸的PCR产物；所述引物是如SEQ ID NO:5和SEQ ID NO:6所示的引物对。