CN113234134A - 一种远端关节挛缩综合症致病基因myh3及其用途 - Google Patents
一种远端关节挛缩综合症致病基因myh3及其用途 Download PDFInfo
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- CN113234134A CN113234134A CN202110483546.7A CN202110483546A CN113234134A CN 113234134 A CN113234134 A CN 113234134A CN 202110483546 A CN202110483546 A CN 202110483546A CN 113234134 A CN113234134 A CN 113234134A
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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
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;
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
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
<400> 1
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<213> 人工合成
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gtggctcgct tgtgggcgga ggtctgggat ctcctggctg ttgctgtctt ctgctctcat 60
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cattgacttc gggatggacc tggctgcctg catcgagctc atcgagaagc ctatgggcat 1680
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ccaagctgaa gaagacaaag tcaattcttt gaacaaaacc aagagcaaac tggaacagca 3180
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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
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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 (10)
1.一种突变MYH3蛋白,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。
2.编码权利要求1所述突变MYH3蛋白的基因。
3.根据权利要求2所述的基因,其特征在于,所述基因的核苷酸序列如SEQ ID NO:4所示。
4.一种表达载体,其特征在于,所述表达载体包含权利要求2所述基因。
5.一种宿主细胞,其特征在于,所述宿主细胞包含权利要求2~3任一所述基因或权利要求4所述表达载体。
6.权利要求2所述基因、权利要求4所述载体或权利要求5所述宿主细胞的用途,其特征在于,所述用途为用于制备远端关节挛缩综合症动物模型。
7.根据权利要求6所述的用途,其特征在于,所述动物包括哺乳动物。
8.一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。
9.根据权利要求8所述的用途,其特征在于,所述引物是如SEQ ID NO:5和SEQ ID NO:6所示的引物对。
10.根据权利要求8所述的用途,其特征在于,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。
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CN110272994A (zh) * | 2019-07-15 | 2019-09-24 | 中国医学科学院北京协和医院 | 诊断cvm的基因突变及其应用 |
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CN110272994A (zh) * | 2019-07-15 | 2019-09-24 | 中国医学科学院北京协和医院 | 诊断cvm的基因突变及其应用 |
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