CN114107398A - 一种牙本质发育不全的点突变动物模型构建方法及其应用 - Google Patents
一种牙本质发育不全的点突变动物模型构建方法及其应用 Download PDFInfo
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Abstract
本发明涉及基础科研用动物模型领域，特别是涉及一种牙本质发育不全的点突变动物模型的构建方法及其应用。所述牙本质发育不全的点突变动物模型的构建方法，包括将具有DSPP基因c.144+1G>A或c.144+1G>T点突变的孤雄单倍体胚胎干细胞与卵细胞结合获得细胞胚胎；培育所述细胞胚胎即获得DGI疾病动物模型。所述构建方法还包括在c.144+1G>A或c.144+1G>T点突变的DSPP基因上插入有标签。本发明的动物模型的构建方法简洁，为研究病人基因突变对DSPP蛋白表达及功能的影响，以及明确病人杂合突变是如何造成DGI疾病表型提供方法。
Description
技术领域
本发明涉及基础科研用动物模型领域，特别是涉及一种牙本质发育不全的点突变动物模型的构建方法及其应用。
背景技术
牙本质发育不全(dentinogenesis imperfect，DGI)属于牙体硬组织非龋性疾病，具有乳恒牙受累现象，是一种牙本质发育异常的常染色体显性遗传疾病，发病率为1/8000～1/6000。临床上常根据患牙的临床和影像学表现将牙本质发育不全分为3种亚型：Ⅰ型牙本质发育不全(DGI-Ⅰ)，患者常伴有成骨不全症，全身骨骼发育不全。全口牙齿呈半透明的琥珀色，磨耗明显；DGI-Ⅱ又名遗传性乳光牙本质，患者不伴有成骨不全症，无全身骨骼异常。患牙与DGI-Ⅰ牙齿相似，但完全通透；Ⅲ型牙本质发育不全(DGI-Ⅲ)，患牙多表现为空壳状和多发性露髓。
Xiao等在2001年首次发现遗传性牙本质发育异常的致病基因为牙本质涎磷蛋白(dentin sialophosphoprotein，DSPP)基因，DSPP及其蛋白剪切产物—牙本质涎蛋白(dentin sialoprotein，DSP)和牙本质磷蛋白(dentinphosphoprotein，DPP)均属于非胶原蛋白，对牙本质发育和矿化中起着重要作用。此外，DPP可通过形成矿化和结合结构蛋白，进而调节牙本质的生物矿化。目前研究认为DGI-II/III是由于DSPP基因发生突变而产生的，例如人的DSPP基因c.135+1位点杂合突变，不同家系突变位点不同，通常都为杂合突变。目前仅有DSPP纯合敲除的小鼠会出现DGI-III的表型，但杂合敲除小鼠没有疾病表型。而DSPP纯合敲除的小鼠无法研究病人突变对DSPP蛋白表达及功能的影响，也无法明确病人杂合突变是如何造成DGI疾病表型，因此DGI疾病的点突变动物模型和体内分子机制研究为空白。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种DGI疾病动物模型的构建方法，用于解决现有技术中存在的问题。
为实现上述目的及其他相关目的，本发明提供一种DGI疾病动物模型的构建方法，所述方法包括以下步骤：
1)将具有DSPP基因c.144+1G>A或c.144+1G>T点突变的孤雄单倍体胚胎干细胞与卵细胞结合获得细胞胚胎；
2)培育所述细胞胚胎即获得DGI疾病动物模型。
本发明还提供一种DGI疾病动物模型，所述DGI疾病动物模型由前述构建方法制备获得。
本发明还提供所述DGI疾病动物模型在研究DSPP蛋白的时空表达谱式、切割谱式、蛋白定位以及DGI疾病发生发展机制中的用途。
如上所述，本发明的DGI疾病动物模型的构建方法，具有以下有益效果：动物模型的构建方法简洁，为研究病人基因突变对DSPP蛋白表达及功能的影响，以及明确病人杂合突变是如何造成DGI疾病表型提供方法。
附图说明
图1显示为模拟病人突变的Dspp杂合突变小鼠构建与鉴定。
图2显示为Dspp杂合突变小鼠能很好的模拟病人DGI表型。
图3显示为模拟病人突变的Dspp WT-tag、GAmut-tag和GTmut-tag杂合小鼠构建。
图4显示为Dspp杂合突变标签小鼠能很好的模拟病人DGI表型。
图5显示为Dspp蛋白在野生型和突变型小鼠出生后发育前期的蛋白表达和切割谱式。
图6显示为Dspp蛋白在野生型和突变型小鼠出生后发育前期的蛋白表达定位谱式。
图7显示为Dspp杂合突变蛋白引起早期炎症反应进而造成DGI表型。
具体实施方式
除另有说明外，本发明中的术语全称和简称如下：
c.144+1G>A：简称GAmut
c.144+1G>T：简称GTmut
AG-haESCs：孤雄单倍体胚胎干细胞
DKO-AG-haESCs：H19 DMR及IG-DMR双敲除的孤雄单倍体胚胎干细胞(详见申请号为201580024974.6的专利)
ICAHCI：卵胞浆注射(intracytoplasmic AG-haESCs injection)
本发明提供一种DGI疾病动物模型的构建方法，所述方法包括以下步骤：
1)将具有DSPP基因c.144+1G>A或c.144+1G>T点突变的AG-haESCs与卵细胞结合获得细胞胚胎；
2)培育所述细胞胚胎即获得DGI疾病动物模型。
在一种实施方式中，所述AG-haESCs为DKO-AG-haESCs。
所述DSPP基因c.144+1G>A或c.144+1G>T点突变后编码的蛋白质氨基酸序列如SEQID NO.1所示。
所述DSPP基因c.144+1G>A点突变后基因组的核苷酸序列片段如SEQ ID NO.2所示。
所述DSPP基因c.144+1G>T点突变后基因组的核苷酸序列片段如SEQ ID NO.3所示。
由于c.144+1G>A或c.144+1G>T点突变是发生在内含子上，仅能从基因组序列上体现出突变，而由于基因组序列过长，因此本发明仅示出包括点突变位置和同源臂序列的核苷酸片段，即分别如SEQ ID NO.2和SEQ ID NO.3所示。
所述具有c.144+1G>A或c.144+1G>T点突变的DKO-AG-haESCs可以采用基因打靶、同源重组等技术来完成，包括但不限于基于ZFN(锌指核酸酶)、TALEN(转录激活样效应因子核酸酶)和CRISPR/Cas9(成簇规律间隔短回文重复技术)等的基因操作。
在一种实施方式中，所述具有DSPP基因c.144+1G>A或c.144+1G>T点突变的DKO-AG-haESCs通过以下步骤获得：
1)Cas9质粒的构建：针对DSPP基因设计sgRNA，将sgRNA连接到Cas9质粒上，挑出连入sgRNA的阳性Cas9质粒；
2)双链DNA供体的制备：设计包括DSPP基因c.144+1G>A点突变的同源臂和/或c.144+1G>T点突变的同源臂，并将同源臂分别插入到供体载体上；
3)将步骤1)的Cas9质粒和步骤2)的双链DNA供体转染到DKO-AG-haESCs；
4)分选阳性的单倍体细胞进行培养；挑取单克隆，测序确定具有c.144+1G>A或c.144+1G>T点突变的DKO-AG-haESCs。
在一种实施方式中，SEQ ID NO.4所示的序列为突变前的靶位点序列，针对这一序列进行sgRNA设计。
具体的，所述sgRNA的序列可以利用现有的在线软件设计，例如https://chopchop.cbu.uib.no/。在一种实施方式中，所述sgRNA的序列如SEQ ID NO.5所示。
在一种实施方式中，所述Cas9质粒为pX330-mCherry。所述Cas9质粒并不限于用pX330质粒构建表达，只需要适合在哺乳动物细胞中进行外源基因的表达即可。
具体的，所述步骤1)为：将合成好的sgRNA的正向寡核苷酸链和反向寡核苷酸链进行退火获得双链寡核苷酸链(本发明中sgRNA序列均指sgRNA的正向寡核苷酸链序列)然后与酶切好的Cas9质粒进行连接，挑出连入sgRNA的阳性Cas9质粒。
在一种实施方式中，c.144+1G>A点突变的同源臂的左边引物如SEQ ID NO.6和SEQID NO.7所示，右边引物如SEQ ID NO.8和SEQ ID NO.9所示。
在一种实施方式中，c.144+1G>T点突变的同源臂的左边引物如SEQ ID NO.10和SEQ ID NO.11所示，右边引物如SEQ ID NO.12和SEQ ID NO.13所示。
在一种实施方式中，所述供体载体选自pMD19-T。
在一种实施方式中，所述供体载体还可以插入荧光蛋白基因。例如，所述荧光蛋白基因选自mCherry。
在一种实施方式中，所述DKO-AG-haESCs的构建方法同申请号为201580024974.6的专利。所述孤雄单倍体胚胎干细胞来源于哺乳动物，可以为人或非人哺乳动物。较佳的，所述孤雄单倍体胚胎干细胞来源于啮齿动物，如兔、鼠，鼠可以为小鼠、大鼠。在优选的实施例中，所述孤雄单倍体胚胎干细胞来源于小鼠。
在一种实施方式中，所述转染可以通过Lipofectamine 2000转染。
一般情况下，所述卵细胞与所述孤雄单倍体胚胎干细胞来源于同类动物。较佳的，来源于同种动物。所述卵细胞为未经基因改造的卵细胞。
在一种实施方式中，所述卵细胞与所述孤雄单倍体胚胎干细胞结合采用卵胞浆注射法。
较佳的，所述卵胞浆注射前将带点突变的孤雄单倍体胚胎干细胞用含秋水仙素的培养基处理。在一种实施方式中，秋水仙素的浓度为0.03μg/ml～0.06μg/ml。在一种实施方式中，秋水仙素的处理时间为7～9h。孤雄单倍体胚胎干细胞用秋水仙素处理后可使细胞周期同步到M期。
在一种实施方式中，培育所述细胞胚胎是指将细胞胚胎移入到母体中，母鼠怀孕后生产获得点突变杂合鼠即为DGI疾病动物模型。
在一种实施方式中，所述母体为假孕小鼠。较佳的，所述母体为假孕ICR小鼠。更佳的，所述母体为交配0.5天后的假孕ICR小鼠。
在一种实施方式中，母鼠在怀孕19.5天后生产，获得GAmut和GTmut点突变杂合小鼠。
在一种实施方式中，母鼠生产为自然生产。
在一种实施方式中，产出的GAmut和GTmut点突变杂合鼠经清除掉其身上的液体后，放在有氧气的温箱中，存活下来的小鼠后续则由代孕母鼠进行养育。
当然，所述DGI疾病动物模型可以是小鼠模型、大鼠模型或者其他动物模型。
所述DGI疾病动物模型的构建方法还包括以下特征：所述DSPP基因具有c.144+1G>A或c.144+1G>T点突变外，在DSPP基因上还插入有标签。
在一种实施方式中，在DSPP基因组5’端和3’端插入有标签。
具体的，在DSPP蛋白质编码区N端和C端对应的基因组序列位置插入有标签，以使DSPP蛋白质编码区N端和C端分别带有标签。
一般的，5’端和3’端的标签不同。标签的种类没有具体限制，例如，可以分别为Flag标签和HA标签。5’端插入Flag标签前后的核苷酸序列分别如SEQ ID NO.14和SEQ IDNO.15所示；3’端引入HA标签前后的核苷酸序列分别如SEQ ID NO.16和SEQ ID NO.17所示。插入Flag标签和HA标签后的c.144+1G>A或c.144+1G>T点突变DSPP蛋白序列如SEQ IDNO.18所示。
标签插入过程中用到的sgRNA、同源臂、标签序列如下所示：
在一种实施方式中，5’端sgRNA序列如SEQ ID NO.19所示。
在一种实施方式中，5’端左边同源臂PCR引物序列如SEQ ID NO.20和SEQ IDNO.21所示。
在一种实施方式中，5’端右边同源臂PCR引物序列如SEQ ID NO.22和SEQ IDNO.23所示。
在一种实施方式中，Flag标签整合序列如SEQ ID NO.24和SEQ ID NO.25所示。
在一种实施方式中，3’端sgRNA序列如SEQ ID NO.26所示。
在一种实施方式中，3’端左边同源臂PCR引物序列如SEQ ID NO.27和SEQ IDNO.28所示。
在一种实施方式中，3’端右边同源臂PCR引物序列如SEQ ID NO.29和SEQ IDNO.30所示。
在一种实施方式中，HA标签序列较短，直接合成在引物里即可，不需要特别处理。
标签的插入可以在DSPP蛋白被切割成DSP和DPP的时候，可以分别用标签对应的抗体分别检测DSP蛋白和DPP蛋白，以利于进一步研究DSPP蛋白时空表达谱式、切割谱式、蛋白定位或者DGI疾病的机制。
本发明还提供一种DGI疾病动物模型，所述DGI疾病动物模型由前述构建方法制备获得。
本发明还提供所述DGI疾病动物模型在研究DSPP蛋白的时空表达谱式、切割谱式、蛋白定位以及DGI疾病发生发展机制中的用途。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
实施例1模拟病人突变的Dspp杂合突变小鼠构建与鉴定
利用CRISPR/Cas9技术，针对靶位点序列(如SEQ ID NO.4所示)，在引入突变区域附近设计sgRNA(sgRNA核苷酸序列如SEQ ID NO.5所示)，并设计了分别带有c.144+1G>A(核苷酸序列如SEQ ID NO.2所示)和c.144+1G>T点突变(核苷酸序列如SEQ ID NO.3所示)的同源臂，GA左同源臂PCR引物序列如SEQ ID NO.6和SEQ ID NO.7所示，右同源臂PCR引物如SEQID NO.8和SEQ ID NO.9所示。将GA左、右同源臂和pMD19-T供体载体利用同源重组连接起来。GT左同源臂PCR引物如SEQ ID NO.10和SEQ ID NO.11所示，右同源臂PCR引物如SEQ IDNO.12和SEQ ID NO.13所示。GT左、右同源臂和pMD19-T供体载体利用同源重组连接起来。
将CRISPR/Cas9质粒和pMD19-T供体载体用Lipofectamine 2000转染到DKO-AG-haESCs(DKO-AG-haESCs的制备方法详见申请号为201580024974.6的专利)中，次日通过FACS流式将mCherry阳性的单倍体细胞分选出来，以较低的密度铺下。7-9天后，挑取单克隆进行后续的建系和鉴定，通过PCR测序的方法，PCR鉴定使用的引物序列如SEQ ID NO.31和SEQ ID NO.32所示。PCR产物经过测序确定阳性c.144+1G>A(简称GAmut)和c.144+1G>T(简称GTmut)点突变的孤雄单倍体胚胎干细胞系。为获得点突变小鼠，将带点突变的DKO-AG-haESCs用含有0.05μg/ml秋水仙素的培养基处理8h后，进行卵胞浆注射(intracytoplasmicAG-haESCs injection，ICAHCI)。由ICAHCI获得的两细胞胚胎移入到0.5dpc(交配后0.5天)假孕ICR小鼠的子宫中。母鼠在怀孕19.5天后通过自然生产，获得GAmut和GTmut点突变杂合小鼠(图1A)。按照上述方法构建了5株GAmut和4株GTmut点突变的孤雄单倍体胚胎干细胞系。经过ICAHCI注射，获得21只GAmut和4只GTmut点突变杂合小鼠(图1B)。分别取WT、GAmut和GTmut点突变杂合小鼠下颌骨，加入Trizol裂解抽提RNA，将RNA反转录(TOYOBO，CAT#FSQ-301)成cDNA，用KOD-neo(TOYOBO)进行PCR扩增。PCR扩增引物序列如SEQ ID NO.33和SEQ IDNO.34所示，PCR产物经过测序，证明了GAmut和GTmut的杂合突变确实存在594bp条带的野生型Dspp mRNA转录产物，以及507bp条带的exon3缺失的转录产物(图1C)。野生型小鼠DSPP蛋白序列如SEQ ID NO.35所示，GAmut和GTmut的杂合突变的DSPP突变蛋白序列如SEQ IDNO.1所示。
实施例2Dspp杂合突变小鼠能很好的模拟病人DGI表型
分别取8周龄的WT、GAmut和GTmut点突变杂合小鼠下颌骨，分离出小鼠切牙、磨牙和下颌髁突整体完整结构，经过μ-CT与X射线分析，发现GAmut和GTmut点突变杂合小鼠的下颌磨牙的牙髓室相比WT小鼠更大些，并且牙本质相比WT小鼠更薄些(图2A-F)。GAmut和GTmut点突变杂合小鼠的下颌切牙的长度与WT小鼠相比没有明显变化；但牙髓室相比WT小鼠更大些，并且牙本质相比WT小鼠更薄些(图2G-L)。GAmut和GTmut点突变杂合小鼠的表型与Dspp纯合敲除小鼠表型一致，能很好的模拟病人DGI表型。从建立的小鼠模型和病人表型来看，Dspp这个基因确实是对牙本质的形成和矿化起着重要的作用。
实施例3Dspp杂合突变标签小鼠构建与鉴定
为了指示Dspp蛋白质表达、切割成DSP和DPP的过程，在DSPP蛋白的N端(对应Dspp基因组5’端区域)引入Flag标签，该标签的插入位置位于信号肽(SP)后面；Dspp基因组5’端插入Flag标签前后的核苷酸序列分别如SEQ ID NO.14和SEQ ID NO.15所示。同时在DSPP蛋白的C端(对应Dspp基因组3’端区域)引入HA标签；小鼠Dspp基因组C端插入HA标签前后的核苷酸序列分别如SEQ ID NO.16和SEQ ID NO.17所示；利用CRISPR/Cas9技术，标签插入过程中用到的sgRNA、同源臂PCR引物、标签序列如下所示：
N端sgRNA：TGCACTTACCGGAATGGCCC AGG(SEQ ID NO.19)
N端左边同源臂PCR引物
F:GAATTCGAGCTCGGTACCCGGGGCTCCTCCCTCAGTCCGAGA(SEQ ID NO.20)
R:CCGTCATGGTCTTTGTAGTCGGCCCAGGCAGTTGCCCA(SEQ ID NO.21)
N端右边同源臂PCR引物
F:ATGACAAGGCTACCGGTGCAATTCCGGTAAGTGCATCTTCCC(SEQ ID NO.22)
R：CAAGCTTGCATGCCTGCAGGCTCGGAGCCATTCCCATCTC(SEQ ID NO.23)
Flag标签整合PCR引物
F:TTTGGGCAACTGCCTGGGCCGACTACAAAGACCATGACGGTG(SEQ ID NO.24)
R：GAAGATGCACTTACCGGAATTGCACCGGTAGCCTTGTCATCGTCATCCTTGTAG(SEQ IDNO.25)
将N端三个片段和pMD19-T供体载体利用同源重组连接起来。
C端sgRNA：CTCTCTGATCTAATCATCAC TGG(SEQ ID NO.26)
C端左边同源臂PCR引物
F:GAATTCGAGCTCGGTACCCGGGACAAGGACGAATCTGACAGCA(SEQ ID NO.27)
R：TCCGGCACATCATACGGATAAGCGGGCCCTGCATCATCACTGGTTGAGTGG
TTAC(SEQ ID NO.28)
C端右边同源臂PCR引物
F:TATCCGTATGATGTGCCGGATTATGCGTAGATCAGAGAGAACCCATGATAT(SEQ ID NO.29)
R:CAAGCTTGCATGCCTGCAGGACGCGTTTACAGATAGCAGC(SEQ ID NO.30)
将C端左右同源臂和pMD19-T供体载体利用同源重组连接起来。
通过以上方法构建N-SP-Flag-Dspp-C-HA的标签孤雄胚胎干细胞系，简称WT-tag细胞。野生型小鼠DSPP蛋白序列如SEQ ID NO.35所示，插入Flag标签和HA标签后的小鼠DSPP蛋白序列如SEQ ID NO.36所示。这样在DSPP蛋白被切割成DSP和DPP的时候，可以分别用Flag抗体检测DSP和用HA抗体检测DPP(图3A)。另外在以上WT-tag细胞基础上，同样引入GAmut和GTmut，从而构建GAmut-tag细胞和GTmut-tag细胞。突变型小鼠DSPP蛋白序列如SEQID NO.1所示，插入Flag标签和HA标签后的突变型小鼠DSPP蛋白序列分别如SEQ ID NO.36所示。本实施例分别构建了2株WT-tag细胞、4株GAmut-tag细胞和4株GTmut-tag细胞。经过ICAHCI注射，分别获得13只WT-tag杂合小鼠和26只GAmut-tag、24只GTmut-tag的点突变杂合小鼠(图3B)。
实施例4Dspp杂合突变标签小鼠能很好的模拟病人DGI表型
分别取2个月龄的WT-tag、GAmut-tag和GTmut-tag杂合小鼠下颌骨，分离出小鼠切牙、磨牙和下颌髁突整体完整结构，经过本领域熟知的μ-CT与X射线分析，发现GAmut-tag和GTmut-tag杂合小鼠的下颌第一、第二磨牙的髓室顶和髓室底之间的距离相比WT-tag小鼠更小，而髓室角相比WT-tag更高。WT-tag小鼠髓室底较厚且弧度圆滑，GAmut-tag髓室底较薄，GTmut-tag髓室底往两边髓室角方向有些增生。整体表现为GAmut-tag和GTmut-tag杂合小鼠相比WT-tag小鼠牙髓腔更大，并且牙本质更薄(图4A)，表型与图2一致。
另外，分别取2个月龄(图4B)和8个月(图4C)的WT-tag、GAmut-tag和GTmut-tag杂合小鼠下颌骨分离出小鼠磨牙进行石蜡切片和HE染色(均为本领域熟知的实验)观察第一磨牙的结构。如图4B，2个月龄小鼠GAmut-tag和GTmut-tag杂合小鼠的髓角相比WT-tag小鼠更高；而WT-tag小鼠髓室底较厚且弧度圆滑，GAmut-tag髓室底较薄，GTmut-tag髓室底往两边髓角方向有些增生，表明WT-tag小鼠继发性牙本质生成功能正常，GTmut-tag小鼠继发性牙本质生成功能受到明显影响，GAmut-tag小鼠生成功能影响最严重。GAmut-tag和GTmut-tag小鼠相比WT-tag小鼠切牙切片易破损，表明牙本质微结构形成不良，不够致密。整体表现为GAmut-tag和GTmut-tag杂合小鼠相比WT-tag小鼠牙髓腔更大，牙本质更薄，与图4A的μ-CT与X射线结果一致。如图4C，8个月龄小鼠GAmut-tag和GTmut-tag杂合小鼠相比WT-tag小鼠牙尖磨耗更严重，WT-tag小鼠还存在完整的牙釉质结构，而GAmut-tag和GTmut-tag小鼠牙尖已经磨损至牙本质层，牙尖变得平坦。同样WT-tag小鼠继发性牙本质生成功能正常，GTmut-tag小鼠继发性牙本质生成功能受到明显影响，GAmut-tag小鼠生成功能影响最严重。GAmut-tag和GTmut-tag小鼠可见较厚前期牙本质层，表明牙本质矿化较差，并且切片易破损，牙本质结构紊乱，表明牙本质生成受到了影响。以上结果表明，Dspp杂合突变标签小鼠能很好的模拟病人DGI表型，标签的插入不会影响DSPP的功能，并且能利用标签进行DSPP的蛋白指示。
实施例5利用Dspp杂合突变标签小鼠明确野生型和突变型DSPP蛋白的表达谱式
分别取新生(P0)、出生3天(P3)、出生7天(P7)和1个月(1M)的WT-tag、GAmut-tag和GTmut-tag点突变杂合小鼠下颌骨组织，抽提出组织蛋白和蛋白定量，经过本领域熟知的Western blot，用Flag抗体可以检测到全长未切割的DSPP和切割后的DSP，用HA抗体可以检测到全长未切割的DSPP和切割后的DPP。如图5，Flag和HA抗体都能检测到表达谱式相似的蛋白分子量约在240KDa的条带，表明下颌骨组织中仍然存在全长未切割的DSPP，并且全长未切割的DSPP表达量较高。野生型DSPP WT-tag的全长DSPP随着P0、P3、P7、1M逐渐增加，GAmut-tag和GTmut-tag表达趋势同WT-tag相似。之前的文献都认为全长蛋白DSPP一边翻译的过程中就直接被酶切成2个DPP和DSP小蛋白，组织中并不存在全长蛋白。通过构建的Dspp杂合突变标签小鼠证明了小鼠组织中确实存在全长未切割的DSPP。DSP是高度糖基化的蛋白多糖，因此用Flag抗体检测到120KDa左右的弥散条带就是切割后的Flag-DSP。Flag-DSP表达趋势也随P0、P3、P7、1M逐渐增加，并且GAmut-tag和GTmut-tag的DSP蛋白大小确实比WT-tag的小些，表明3号外显子确实缺失。而全长DSPP蛋白由于分子量较大，3号外显子片段较小，突变造成3号外显子缺失导致的分子量变小相比较而言区别并不明显。HA抗体检测到140-120KDa左右的弥散条带就是切割后的DPP-HA，其表达趋势也随P0、P3、P7、1M逐渐增加，并且GAmut-tag和GTmut-tag对DPP蛋白大小没有影响。由于DPP是一富含天门冬氨酸和磷酸丝氨酸的蛋白，高度磷酸化，易与Ca2+结合，在胞外基质脱矿后才有溶解性，在我们常规蛋白提取步骤不容易被检测到，所以我们检测到的DPP蛋白表达量相比DSP少了很多。
本申请首次构建Dspp杂合突变标签小鼠，并且利用该小鼠模型发现了小鼠体内组织中确实存在全长未切割的DSPP，并且表达量较高；野生型DSPP蛋白和突变型DSPP蛋白在小鼠出生后发育前期随着时间不断表达增加并逐步被切割成DSP和DPP的切割谱式，突变型DSPP蛋白表达和切割谱式与野生型DSPP蛋白相似，但信号相比有少量增加。之前对DGI致病机制的主要假说是认为突变型DSPP蛋白导致蛋白合成后积聚在内质网上，影响了正常DSPP蛋白的分泌和稳定性，造成DSPP蛋白被降解而导致的功能缺失。但我们的实验表明野生型DSPP蛋白和突变型DSPP蛋白时空表达谱式相似，DSPP/DSP/DPP蛋白在野生型和突变性小鼠模型中都能被正常表达与切割，突变型DSPP蛋白没有被异常降解，表明DGI表型不是由于ER应激造成DSPP蛋白降解引起的，推翻了之前的假说，也进一步暗示GAmut-tag和GTmut-tag点突变造成的DSP突变蛋白对牙本质发育的影响可能性更大些。本实施例证明了Dspp杂合突变标签小鼠在明确野生型和突变型DSPP蛋白的切割谱式或表达谱式中的用途。
实施例6利用Dspp杂合突变标签小鼠具体确定DSPP/DSP/DPP蛋白的定位
接下来用构建的Dspp杂合突变标签小鼠具体确定DSPP/DSP/DPP蛋白的定位，分别取新生(P0)和出生3天(P3)的WT-tag、GAmut-tag和GTmut-tag杂合小鼠磨牙，进行石蜡包埋和IHC检测(均为本领域熟知的实验)。用Flag抗体可以检测到全长未切割的DSPP和切割后的DSP阳性信号，用HA抗体可以检测到全长未切割的DSPP和切割后的DPP阳性信号。如图6A，在P0的WT-tag、GAmut-tag和GTmut-tag杂合小鼠的第一磨牙中，在发育中的牙尖区域可以检测到Flag和HA的阳性信号，表明DSPP蛋白从牙尖开始表达，其他部分还未开始表达。其中GAmut-tag杂合小鼠相较WT-tag和GTmut-tag的DSPP/DSP/DPP表达进程提前了一些。牙胚的成釉器、牙乳头和牙胚外的信号为样品制备过程中不可避免的血细胞污染的假阳性信号。如图6B，到P3天，WT-tag、GAmut-tag和GTmut-tag杂合小鼠第一磨牙的牙乳头和成釉器之间的大部分区域都能检测到Flag和HA的阳性信号，Flag和HA的阳性信号基本重叠，信号主要集中在成釉细胞、牙釉质基质、前期牙本质/牙本质基质、成牙本质细胞中。与DAPI指示细胞核位置的合并图中，可以看到DSPP被成牙本质细胞和成釉细胞合成分别被分泌到前期牙本质/牙本质基质和牙釉质基质中。GAmut-tag和GTmut-tag杂合小鼠第一磨牙的发育进程稍微早于WT-tag杂合小鼠，结构上除了GAmut-tag和GTmut-tag杂合小鼠磨牙切片较易破碎，其他与WT-tag杂合小鼠没有显著不同。本实施例可以证明Dspp杂合突变标签小鼠能用于具体确定DSPP/DSP/DPP蛋白的定位。
实施例7Dspp杂合突变标签小鼠引起DGI表型的机制研究
我们已证明Dspp杂合突变标签小鼠能很好的模拟病人DGI表型，并且不会造成DSPP的蛋白降解，突变型DSPP蛋白和野生型DSPP蛋白时空表达谱式、切割谱式和蛋白定位均相似，暗示DGI表型是由于DSPP/DSP突变蛋白引起的，因此我们进一步分析研究DSPP/DSP突变蛋白是否有特异性结合的蛋白。我们分别取了新生P0天的WT-tag、GAmut-tag和GTmut-tag杂合小鼠下颌骨，抽提组织蛋白利用Flag抗体和HA抗体进行蛋白免疫共沉淀(co-IP)实验(为本领域熟知的实验)，然后将co-IP得到的复合物进行质谱(MS)检测。以没有携带标签的WT小鼠的IP-MS结果作为阴性对照，我们将WT-tag、GAmut-tag和GTmut-tag下颌骨组织IP-MS获得的蛋白质谱数据与阴性对照相比较，保留结果是阴性对照组中的两倍及以上的Prey阳性蛋白用于后续数据分析。如图7A，我们将WT-tag、GAmut-tag和GTmut-tag下颌骨组织蛋白提取液分别利用Flag抗体和HA抗体co-IP获得的prey蛋白进行比较，画出韦恩图。其中，Set1交集中的prey蛋白为WT-tag小鼠独有的相互作用蛋白，Set6交集中的prey蛋白为GAmut-tag和GTmut-tag小鼠区别于WT-tag小鼠而共有的相互作用蛋白，即为DGI小鼠独有的相互作用蛋白。如图7B，我们进一步将Flag抗体和HA抗体获得的Set1和Set6蛋白群分别取交集，分别获得22个WT-tag小鼠独有的相互作用蛋白，和19个DGI小鼠独有的相互作用蛋白，蛋白对应的基因名字如图7C展示。由于DSPP蛋白是细胞外基质蛋白，所以我们主要关注细胞外基质有定位的蛋白，列在图7C左边第一列字体加粗的基因。其中COL6A2、MATN1、BGN被报道相互形成复合物参与软骨细胞的细胞外基质组成，而TGM2在破骨细胞分化和骨平衡中起着重要作用，表明野生型DSPP/DSP/DPP与细胞外基质相互作用，形成特殊结构促进磷灰石晶体形成来调控牙本质的矿化过程。我们购买了TGM2，BGN，AHSG，SERPINA1A和PGK1的自身抗体，分别检测了它们在在野生型和突变型小鼠出生后发育前期的蛋白表达情况。如图7D，在GAmut-tag和GTmut-tag小鼠下颌骨组织中，TGM2和AHSG两个蛋白的表达谱式与WT-tag小鼠相似。在P0天GAmut-tag和GTmut-tag小鼠的BGN蛋白表达高于WT-tag小鼠，而SERPINA1A和PGK1蛋白表达低于WT-tag小鼠，与IP-MS结果相似。发育到一个月时，BGN和SERPINA1A的表达量与WT-tag小鼠相似。PGK1在WT-tag小鼠中，随着发育的进程蛋白量逐渐增加；而在GAmut-tag和GTmut-tag小鼠中，P0和P3天虽然蛋白表达量高于WT-tag小鼠，但发育到一个月时，蛋白表达量却低于WT-tag小鼠，暗示PGK1可能与DGI表型有关。接着，我们进一步利用IP实验来验证IP-MS的数据。如图7E，用Flag抗体可以拉下DPP-HA蛋白，用HA抗体可以拉下Flag-DSP蛋白，表明DSPP/DSP/DPP直接可以形成复合物。用Flag抗体和HA抗体拉下的DSPP/DSP和DSPP/DPP分别都能检测到与PGK1、SERPINA1A存在相互作用，其中GAmut-tag和GTmut-tag小鼠相比WT-tag小鼠拉到更多蛋白，并且用Flag抗体拉下的蛋白信号强于HA抗体拉下的蛋白信号，表明PGK1、SERPINA1A更倾向于与DSPP/DSP相互作用，与DSPP突变蛋白造成DGI表型的模式一致。PGK1是糖酵解过程中的一个关键酶，催化1,3-二磷酸甘油酸生成3-磷酸甘油酸，并同时生成ATP，因而在细胞能量代谢中发挥着重要功能。除了糖分解代谢功能，PGK1在细胞自噬起始、DNA复制、DNA修复、炎症前期反应、关节增生、促进骨生成等作用都有报道，PGK1在胚胎E10.5到E18.0天的生牙上皮细胞中特异性高表达，暗示它与牙矿化形成有关。并且SERPINA1A在炎症起始阶段表达显著增加，暗示我们DGI小鼠P0天即存在炎症反应，DGI表型可能由于DSPP/DSP突变蛋白引起炎症反应而导致牙本质发育不良、影响矿化造成的表型。
本发明首次构建了Dspp基因的GAmut和GTmut点突变杂合小鼠，并利用该小鼠模型模拟DGI疾病表型。利用在DSPP蛋白的N端和C端原位引入标签蛋白，进一步构建的Dspp杂合突变标签小鼠，揭示了DSPP蛋白在小鼠磨牙发育过程中的时空表达谱式、切割谱式和蛋白定位。探索DGI疾病机制，推翻了之前的主流假说，证明了DGI的致病表型并不是由于突变型DSPP蛋白影响野生型蛋白的稳定性造成DSPP被降解而导致的。利用DGI标签小鼠还发现了新生小鼠Dspp杂合蛋白就出现早期炎症反应，可能是其进展为DGI疾病的原因之一。
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
序列表
<110> 中国科学院分子细胞科学卓越创新中心
<120> 一种牙本质发育不全的点突变动物模型构建方法及其应用
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 916
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Lys Met Lys Ile Ile Ile Tyr Ile Cys Ile Trp Ala Thr Ala Trp
1 5 10 15
Ala Ile Pro Asn Glu Leu Ser Ile Asn Ser Thr Thr Ser Asn Ser Asn
20 25 30
Asp Ser Pro Asp Gly Ser Glu Ile Gly Glu Gln Val Leu Ser Glu Asp
35 40 45
Gly Tyr Lys Arg Asp Gly Asn Gly Ser Glu Ser Ile His Val Gly Gly
50 55 60
Lys Asp Phe Pro Thr Gln Pro Ile Leu Val Asn Glu Gln Gly Asn Thr
65 70 75 80
Ala Glu Glu His Asn Asp Ile Glu Thr Tyr Gly His Asp Gly Val His
85 90 95
Ala Arg Gly Glu Asn Ser Thr Ala Asn Gly Ile Arg Ser Gln Val Gly
100 105 110
Ile Val Glu Asn Ala Glu Glu Ala Glu Ser Ser Val His Gly Gln Ala
115 120 125
Gly Gln Asn Thr Lys Ser Gly Gly Ala Ser Asp Val Ser Gln Asn Gly
130 135 140
Asp Ala Thr Leu Val Gln Glu Asn Glu Pro Pro Glu Ala Ser Ile Lys
145 150 155 160
Asn Ser Thr Asn His Glu Ala Gly Ile His Gly Ser Gly Val Ala Thr
165 170 175
His Glu Thr Thr Pro Gln Arg Glu Gly Leu Gly Ser Glu Asn Gln Gly
180 185 190
Thr Glu Val Thr Pro Ser Ile Gly Glu Asp Ala Gly Leu Asp Asp Thr
195 200 205
Asp Gly Ser Pro Ser Gly Asn Gly Val Glu Glu Asp Glu Asp Thr Gly
210 215 220
Ser Gly Asp Gly Glu Gly Ala Glu Ala Gly Asp Gly Arg Glu Ser His
225 230 235 240
Asp Gly Thr Lys Gly Gln Gly Gly Gln Ser His Gly Gly Asn Thr Asp
245 250 255
His Arg Gly Gln Ser Ser Val Ser Thr Glu Asp Asp Asp Ser Lys Glu
260 265 270
Gln Glu Gly Phe Pro Asn Gly His Asn Gly Asp Asn Ser Ser Glu Glu
275 280 285
Asn Gly Val Glu Glu Gly Asp Ser Thr Gln Ala Thr Gln Asp Asn Gln
290 295 300
Lys Leu Ser Pro Lys Asp Thr Arg Asp Ala Glu Gly Gly Ile Ile Ser
305 310 315 320
Gln Ser Glu Ala Cys Pro Ser Gly Lys Ser Gln Asp Gln Gly Ile Glu
325 330 335
Thr Glu Gly Pro Asn Lys Gly Asn Lys Ser Ile Ile Thr Lys Glu Ser
340 345 350
Gly Lys Leu Ser Gly Ser Lys Asp Ser Asn Gly His Gln Gly Val Glu
355 360 365
Leu Asp Lys Arg Asn Ser Pro Lys Gln Gly Glu Ser Asp Lys Pro Gln
370 375 380
Gly Thr Ala Glu Lys Ser Ala Ala His Ser Asn Leu Gly His Ser Arg
385 390 395 400
Ile Gly Ser Ser Ser Asn Ser Asp Gly His Asp Ser Tyr Glu Phe Asp
405 410 415
Asp Glu Ser Met Gln Gly Asp Asp Pro Lys Ser Ser Asp Glu Ser Asn
420 425 430
Gly Ser Asp Glu Ser Asp Thr Asn Ser Glu Ser Ala Asn Glu Ser Gly
435 440 445
Ser Arg Gly Asp Ala Ser Tyr Thr Ser Asp Glu Ser Ser Asp Asp Asp
450 455 460
Asn Asp Ser Asp Ser His Ala Gly Glu Asp Asp Ser Ser Asp Asp Ser
465 470 475 480
Ser Asp Thr Asp Asp Ser Asp Ser Asn Gly Asp Gly Asp Ser Asp Ser
485 490 495
Asn Gly Asp Gly Asp Ser Glu Ser Glu Asp Lys Asp Glu Ser Asp Ser
500 505 510
Ser Asp His Asp Asn Ser Ser Asp Ser Glu Ser Lys Ser Asp Ser Ser
515 520 525
Asp Ser Ser Asp Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
530 535 540
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
545 550 555 560
Ser Ser Asp Ser Asn Ser Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser
565 570 575
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
580 585 590
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
595 600 605
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser
610 615 620
Ser Asp Ser Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser Asp Ser Ser
625 630 635 640
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Ser
645 650 655
Ser Asp Ser Ser Ser Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser
660 665 670
Asp Ser Ser Ser Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp
675 680 685
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser
690 695 700
Ser Asp Ser Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser Asp Ser Ser
705 710 715 720
Ser Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Glu
725 730 735
Ser Ser Glu Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser
740 745 750
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
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Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp
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Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser
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Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
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Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
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Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
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Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser
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Asp Ser Ser Asp Ser Asp Ser Lys Asp Ser Ser Ser Asp Ser Ser Asp
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Gly Asp Ser Lys Ser Gly Asn Gly Asn Ser Asp Ser Asn Ser Asp Ser
885 890 895
Asn Ser Asp Ser Asp Ser Asp Ser Glu Gly Ser Asp Ser Asn His Ser
900 905 910
Thr Ser Asp Asp
915
<210> 2
<211> 2072
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gaacctgggg tgttttctca cattagtact ttctccttgt cctagaaaag ggccaaatgt 60
aagaccaaaa tattggggta ctgtggctgt catctttcat cttatgaccc gttttgtggt 120
gttctttgtt ctaaacagac attgattact actcataatg aaaatgaaga taattatata 180
tatatgcatt tgggcaactg cctgggccat tccggtaagt gcatcttccc aatcaagcct 240
tcttactttg ctgtatcttt caacccaatg ttgaaatgta catatttcct tatggtttta 300
cagagaagtt gagtctaaac acttaataga aatgttaaga tttgcattgc agctattatg 360
tgatatcata tggggtctcg atgaaggcaa acacatgcac caatgcatgc tccctccatt 420
cctgttgaaa catcctaatg aaagaatgac cctttttttt taaagtttat ccaaattaat 480
tcagtgctcc aaagtcatga agcttgtctg cttcattcca cacgaattcc actgtaatgt 540
caacacactg tattctgttt gggaaaaaac tgaagaaaga acaggagcta aaagtcagat 600
ctttcaatgt ttcatgtgtg catttgtgtg ttcactgtgg gaaatctgga gcatcagaac 660
aagtacaaag gcagaaacat taagaaagtc gatctgtttg tcatttcatc agctggcttc 720
cacatctaac attgtcacag ggcgtcacat aaccagattc tgggttgttc ctgtacttga 780
gaagttttgt aagcactccg agctcactct tgcagggtga gaattatcag ctaccggggc 840
tgcttctcca gtggtccact ctcatgttgc tttaggggtt tggggctgat cgacaacaac 900
attataaaaa tcctcacttt ctctgcctga aaccccacat aagcaccgca gcaggctcct 960
tctcttctct acacgatcag agtgcgatct gaccttcata taatatctgt gtctcaacct 1020
ctgcaggttc cccagttagt accactggaa agagacattg ttgaaaactc tgtggctgtg 1080
cctcttctaa cacatccagg aactgcagca cagataaaag acagaaatac gaatggtcct 1140
ttctttttct gttttcaagg cccttttaca ctttaccact ttctctaaaa tatccaccct 1200
tttttttcag ttggccttat ttgaaaatga tagccacaac tgactttcaa ttgtgtctcc 1260
ttttcagaat gagttatcta tcaacagcac cactagcaac agcaacgact ccccagatgg 1320
cagtgagata ggagagcagg tacttagcga ggatggttac aaaagagatg ggaatggctc 1380
cgagtcaata catgtaggag ggaaggattt tcctactcag cccattttag taaacgaaca 1440
ggggaacact gctgaagaac acaatgacat agaaacatac ggtcatgatg gggtacatgc 1500
gagaggagag aacagcacag caaatggcat caggagccag gtaggcatcg ttgaaaatgc 1560
ggaggaagca gagagcagtg tccacggaca ggctggtcag aatacaaaat ctggaggtgc 1620
tagtgatgta agccagaatg gagatgcgac ccttgtccag gaaaatgagc ctccagaagc 1680
tagcatcaag aatagcacca accatgaggc tggaatacac gggagtgggg ttgctacaca 1740
tgaaacgacg cctcagagag aagggctggg gagtgagaac caggggactg aggtgacacc 1800
aagcatcggg gaagatgcag gtttggatga tactgatggg agtcctagcg ggaacggggt 1860
agaggaggat gaagatacag gctctggtga tggtgagggt gcagaagcag gagatggaag 1920
ggagagccat gatggcacta agggccaggg gggccaatct catgggggaa acactgacca 1980
cagaggtcag agttcagtta gtactgaaga tgatgattct aaagaacaag aaggcttccc 2040
aaatggacac aatggagaca acagcagtga gg 2072
<210> 3
<211> 2072
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gaacctgggg tgttttctca cattagtact ttctccttgt cctagaaaag ggccaaatgt 60
aagaccaaaa tattggggta ctgtggctgt catctttcat cttatgaccc gttttgtggt 120
gttctttgtt ctaaacagac attgattact actcataatg aaaatgaaga taattatata 180
tatatgcatt tgggcaactg cctgggccat tccggtaagt gcatcttccc aatcaagcct 240
tcttactttg ctgtatcttt caacccaatg ttgaaatgta catatttcct tatggtttta 300
cagagaagtt gagtctaaac acttaataga aatgttaaga tttgcattgc agctattatg 360
tgatatcata tggggtctcg atgaaggcaa acacatgcac caatgcatgc tccctccatt 420
cctgttgaaa catcctaatg aaagaatgac cctttttttt taaagtttat ccaaattaat 480
tcagtgctcc aaagtcatga agcttgtctg cttcattcca cacgaattcc actgtaatgt 540
caacacactg tattctgttt gggaaaaaac tgaagaaaga acaggagcta aaagtcagat 600
ctttcaatgt ttcatgtgtg catttgtgtg ttcactgtgg gaaatctgga gcatcagaac 660
aagtacaaag gcagaaacat taagaaagtc gatctgtttg tcatttcatc agctggcttc 720
cacatctaac attgtcacag ggcgtcacat aaccagattc tgggttgttc ctgtacttga 780
gaagttttgt aagcactccg agctcactct tgcagggtga gaattatcag ctaccggggc 840
tgcttctcca gtggtccact ctcatgttgc tttaggggtt tggggctgat cgacaacaac 900
attataaaaa tcctcacttt ctctgcctga aaccccacat aagcaccgca gcaggctcct 960
tctcttctct acacgatcag agtgcgatct gaccttcata taatatctgt gtctcaacct 1020
ctgcaggttc cccagttagt accactggaa agagacattg ttgaaaactc tgtggctgtg 1080
cctcttctaa cacatccagg aactgcagca cagttaaaag acagaaatac gaatggtcct 1140
ttctttttct gttttcaagg cccttttaca ctttaccact ttctctaaaa tatccaccct 1200
tttttttcag ttggccttat ttgaaaatga tagccacaac tgactttcaa ttgtgtctcc 1260
ttttcagaat gagttatcta tcaacagcac cactagcaac agcaacgact ccccagatgg 1320
cagtgagata ggagagcagg tacttagcga ggatggttac aaaagagatg ggaatggctc 1380
cgagtcaata catgtaggag ggaaggattt tcctactcag cccattttag taaacgaaca 1440
ggggaacact gctgaagaac acaatgacat agaaacatac ggtcatgatg gggtacatgc 1500
gagaggagag aacagcacag caaatggcat caggagccag gtaggcatcg ttgaaaatgc 1560
ggaggaagca gagagcagtg tccacggaca ggctggtcag aatacaaaat ctggaggtgc 1620
tagtgatgta agccagaatg gagatgcgac ccttgtccag gaaaatgagc ctccagaagc 1680
tagcatcaag aatagcacca accatgaggc tggaatacac gggagtgggg ttgctacaca 1740
tgaaacgacg cctcagagag aagggctggg gagtgagaac caggggactg aggtgacacc 1800
aagcatcggg gaagatgcag gtttggatga tactgatggg agtcctagcg ggaacggggt 1860
agaggaggat gaagatacag gctctggtga tggtgagggt gcagaagcag gagatggaag 1920
ggagagccat gatggcacta agggccaggg gggccaatct catgggggaa acactgacca 1980
cagaggtcag agttcagtta gtactgaaga tgatgattct aaagaacaag aaggcttccc 2040
aaatggacac aatggagaca acagcagtga gg 2072
<210> 4
<211> 130
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gttccccagt tagtaccact ggaaagagac attgttgaaa actctgtggc tgtgcctctt 60
ctaacacatc caggaactgc agcacaggta aaagacagaa atacgaatgg tcctttcttt 120
ttctgttttc 130
<210> 5
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
acatccagga actgcagcac agg 23
<210> 6
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tacccgggga tcctctagag gaacctgggg tgttttctca 40
<210> 7
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
acagaaaaag aaaggaccat tcgtatttct gtcttttatc tgtgc 45
<210> 8
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gtgcctcttc taacacatcc aggaactgca gcacagataa aa 42
<210> 9
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccaagcttgc atgcctgcag cctcactgct gttgtctcca 40
<210> 10
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tacccgggga tcctctagag gaacctgggg tgttttctca 40
<210> 11
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
acagaaaaag aaaggaccat tcgtatttct gtcttttaac tgtgc 45
<210> 12
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gtgcctcttc taacacatcc aggaactgca gcacagttaa aa 42
<210> 13
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ccaagcttgc atgcctgcag cctcactgct gttgtctcca 40
<210> 14
<211> 2164
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gctcctccct cagtccgaga ctgcatagtg cccgggtaag ggtggggtgt cctttgtcct 60
caggagtgct tgttcagcag caggctctgc aaggtgacct ttgctttgct cagaagacac 120
tgatgatcaa gatgctggcc tgggctccgg gacctgatgc cagtgaggag gaagatgggg 180
tagctaggca acttcaaaac agtgcaatgt gctgccagca tcgagcgagc ggagggtgca 240
caagctgctg ctgtgtgagg aagggagcta aagatgcctt cagaaagctt ctcaggggtg 300
attcttctgc caacccctag gatattctga gctacagagt tattattcca gactgaggaa 360
acaaaagccc aataaagcta ttgaaagtgc ccaagctcag agagcagata gcaggggaag 420
gatttgaatt cagggatctg aaaccaaatc ctgtgttctc tctcctagcc taaactctct 480
cttccttaaa cactgtaaga ggaagatttc ttcctcttac tgggataacg cccaattcta 540
tatagaccag gtgggaaatt acaagtgctt tatcatttaa atctactttt agttaatgat 600
gcttaaagct agcccaggag agacgttacc ctcatggata acagcatagg gccagagcca 660
cgagctatgt actctgtatc ttcatggctg ttgcttccac aggcaggtaa agtcagaagc 720
catgacagtc ctgagcatgc tgaggccccc acataccagg tttattctgg aacctggggt 780
gttttctcac attagtactt tctccttgtc ctagaaaagg gccaaatgta agaccaaaat 840
attggggtac tgtggctgtc atctttcatc ttatgacccg ttttgtggtg ttctttgttc 900
taaacagaca ttgattacta ctcataatga aaatgaagat aattatatat atatgcattt 960
gggcaactgc ctgggccgct accggtgcaa ttccggtaag tgcatcttcc caatcaagcc 1020
ttcttacttt gctgtatctt tcaacccaat gttgaaatgt acatatttcc ttatggtttt 1080
acagagaagt tgagtctaaa cacttaatag aaatgttaag atttgcattg cagctattat 1140
gtgatatcat atggggtctc gatgaaggca aacacatgca ccaatgcatg ctccctccat 1200
tcctgttgaa acatcctaat gaaagaatga cccttttttt ttaaagttta tccaaattaa 1260
ttcagtgctc caaagtcatg aagcttgtct gcttcattcc acacgaattc cactgtaatg 1320
tcaacacact gtattctgtt tgggaaaaaa ctgaagaaag aacaggagct aaaagtcaga 1380
tctttcaatg tttcatgtgt gcatttgtgt gttcactgtg ggaaatctgg agcatcagaa 1440
caagtacaaa ggcagaaaca ttaagaaagt cgatctgttt gtcatttcat cagctggctt 1500
ccacatctaa cattgtcaca gggcgtcaca taaccagatt ctgggttgtt cctgtacttg 1560
agaagttttg taagcactcc gagctcactc ttgcagggtg agaattatca gctaccgggg 1620
ctgcttctcc agtggtccac tctcatgttg ctttaggggt ttggggctga tcgacaacaa 1680
cattataaaa atcctcactt tctctgcctg aaaccccaca taagcaccgc agcaggctcc 1740
ttctcttctc tacacgatca gagtgcgatc tgaccttcat ataatatctg tgtctcaacc 1800
tctgcaggtt ccccagttag taccactgga aagagacatt gttgaaaact ctgtggctgt 1860
gcctcttcta acacatccag gaactgcagc acaggtaaaa gacagaaata cgaatggtcc 1920
tttctttttc tgttttcaag gcccttttac actttaccac tttctctaaa atatccaccc 1980
ttttttttca gttggcctta tttgaaaatg atagccacaa ctgactttca attgtgtctc 2040
cttttcagaa tgagttatct atcaacagca ccactagcaa cagcaacgac tccccagatg 2100
gcagtgagat aggagagcag gtacttagcg aggatggtta caaaagagat gggaatggct 2160
ccga 2164
<210> 15
<211> 2230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gctcctccct cagtccgaga ctgcatagtg cccgggtaag ggtggggtgt cctttgtcct 60
caggagtgct tgttcagcag caggctctgc aaggtgacct ttgctttgct cagaagacac 120
tgatgatcaa gatgctggcc tgggctccgg gacctgatgc cagtgaggag gaagatgggg 180
tagctaggca acttcaaaac agtgcaatgt gctgccagca tcgagcgagc ggagggtgca 240
caagctgctg ctgtgtgagg aagggagcta aagatgcctt cagaaagctt ctcaggggtg 300
attcttctgc caacccctag gatattctga gctacagagt tattattcca gactgaggaa 360
acaaaagccc aataaagcta ttgaaagtgc ccaagctcag agagcagata gcaggggaag 420
gatttgaatt cagggatctg aaaccaaatc ctgtgttctc tctcctagcc taaactctct 480
cttccttaaa cactgtaaga ggaagatttc ttcctcttac tgggataacg cccaattcta 540
tatagaccag gtgggaaatt acaagtgctt tatcatttaa atctactttt agttaatgat 600
gcttaaagct agcccaggag agacgttacc ctcatggata acagcatagg gccagagcca 660
cgagctatgt actctgtatc ttcatggctg ttgcttccac aggcaggtaa agtcagaagc 720
catgacagtc ctgagcatgc tgaggccccc acataccagg tttattctgg aacctggggt 780
gttttctcac attagtactt tctccttgtc ctagaaaagg gccaaatgta agaccaaaat 840
attggggtac tgtggctgtc atctttcatc ttatgacccg ttttgtggtg ttctttgttc 900
taaacagaca ttgattacta ctcataatga aaatgaagat aattatatat atatgcattt 960
gggcaactgc ctgggccgac tacaaagacc atgacggtga ttataaagat catgacatcg 1020
actacaagga tgacgatgac aaggctaccg gtgcaattcc ggtaagtgca tcttcccaat 1080
caagccttct tactttgctg tatctttcaa cccaatgttg aaatgtacat atttccttat 1140
ggttttacag agaagttgag tctaaacact taatagaaat gttaagattt gcattgcagc 1200
tattatgtga tatcatatgg ggtctcgatg aaggcaaaca catgcaccaa tgcatgctcc 1260
ctccattcct gttgaaacat cctaatgaaa gaatgaccct ttttttttaa agtttatcca 1320
aattaattca gtgctccaaa gtcatgaagc ttgtctgctt cattccacac gaattccact 1380
gtaatgtcaa cacactgtat tctgtttggg aaaaaactga agaaagaaca ggagctaaaa 1440
gtcagatctt tcaatgtttc atgtgtgcat ttgtgtgttc actgtgggaa atctggagca 1500
tcagaacaag tacaaaggca gaaacattaa gaaagtcgat ctgtttgtca tttcatcagc 1560
tggcttccac atctaacatt gtcacagggc gtcacataac cagattctgg gttgttcctg 1620
tacttgagaa gttttgtaag cactccgagc tcactcttgc agggtgagaa ttatcagcta 1680
ccggggctgc ttctccagtg gtccactctc atgttgcttt aggggtttgg ggctgatcga 1740
caacaacatt ataaaaatcc tcactttctc tgcctgaaac cccacataag caccgcagca 1800
ggctccttct cttctctaca cgatcagagt gcgatctgac cttcatataa tatctgtgtc 1860
tcaacctctg caggttcccc agttagtacc actggaaaga gacattgttg aaaactctgt 1920
ggctgtgcct cttctaacac atccaggaac tgcagcacag gtaaaagaca gaaatacgaa 1980
tggtcctttc tttttctgtt ttcaaggccc ttttacactt taccactttc tctaaaatat 2040
ccaccctttt ttttcagttg gccttatttg aaaatgatag ccacaactga ctttcaattg 2100
tgtctccttt tcagaatgag ttatctatca acagcaccac tagcaacagc aacgactccc 2160
cagatggcag tgagatagga gagcaggtac ttagcgagga tggttacaaa agagatggga 2220
atggctccga 2230
<210> 16
<211> 2460
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
acaaggacga atctgacagc agtgaccatg acaacagcag tgacagtgag agcaaatcag 60
acagcagtga cagtagtgac gacagcagtg acagcagcga cagtagtgac agcagtgaca 120
gcagtgacag tagtgacagt agtgacagca gcgacagcag tgacagcagc gacagcaaca 180
gtagtagtga cagcagcgac agcagcagta gtagtgacag cagcgacagc agtgacagca 240
gtgacagcag tgacagcagc gatagcagtg acagcagtga cagcagtgac agcagcgata 300
gcagtgacag cagtgacagt agtgacagca gtgacagcag cgacagcagc agtagtagtg 360
acagcagcga cagcagcagt agtagtgaca gcagcgacag cagtgacagc agtgacagca 420
gcgatagcag tgacagcagt gacagcagca gcagcgacag cagcagcagt agcaacagca 480
gtgacagtag tgacagcagt gacagcagca gcagcagcga cagcagcaac agcagcgaca 540
gcagtgacag tagtgacagc agtgacagta gtgacagcag tgacagcagc aacagtagtg 600
acagcagcga cagcagcagt agtagtgaca gcagcgacag cagcagtagt agtgacagca 660
gtgacagtag tgacagtagt gacagcagtg agagcagtga gagcagcgac agcagcaaca 720
gcagtgacag cagcgacagt agtgacagca gtgacagtag cgacagcagc gacagtagtg 780
acagtagcga cagcagtgac agtagcaaca gtagcgacag cagtgacagc agtgacagca 840
gcgacagtag tgacagcagc aacagtagtg acagcagtga cagtagcgac agtagtgaca 900
gcagtgacag cagtgacagc agcgacagta gtgacagcag tgacagtagt gacagcagcg 960
acagtagtga cagcagtgac agcagtgaca gcagtgacag cagcgacagc agcgacagca 1020
gtgacagcag cgacagcagc gacagcagtg acagcagcga cagcagcaac agcagtgaca 1080
gcagtgacag tgacagcaag gatagcagtt ctgacagcag tgatggtgac agcaagtctg 1140
gtaatggcaa cagtgacagc aacagtgaca gcaacagtga cagtgacagt gacagtgaag 1200
gcagtgacag taaccactca accagtgatg attagatcag agagaaccca tgatatcctc 1260
tgtgtgacct cttggtgagt gatgggaagg cagtgaaggt tcctaaccca atgatgacag 1320
gagagatgtg cagactgtgt ggaacccatg gagctcatag ggagtggagc cgagctccag 1380
ctctctcaga gagaatctgg gtgtaccacc tttggtacat gtgtgttaaa atatattcat 1440
gttcagaaaa tatttttaaa aggataaatc taaacaatac tttaacagga actgaagaaa 1500
tcactaagac acatagcttc gatttgaatg gcgggtgctt taaagagcag agctagcaat 1560
gtcacagcct gctgcagcct cctccctcag tgctccgggc accagagagc tagtcttcat 1620
gttgtgcagt gagtaatgct gttctgtgac attcaactca actactctgt catttattta 1680
ttccggggaa aattacattt agggcataat caaaacaccg ctgcaactac tggccctatc 1740
caaggtgctg agataatctt tgtgatgaga caatagctat acattatgaa aattccgaag 1800
aatgaatgag aaaagagccc caaggatggc ttgggcagga tctgacacat gcggttaaat 1860
ttctgcatgg gatggatatg tactaagtcc ccaacccctg cactttgaac agtgtctccc 1920
ttccagcagt ggccctcaaa ccttaaataa acgagcaaca cggatggatg atttcgggag 1980
gtgggatcat attctgagct ctccatgtac cactgtgtta ttagttttct tcgaatcaca 2040
gctcaaacag tttaatcaag agttgtaagg ctgtgcgtga caagagtggg accctgtttg 2100
ggctctaggg ctcctctgaa agcaagagag gtaatgagaa taaaccacac caagacagga 2160
ggtgtgaact gggattgtct caagaaaacc ttaaccctca agccttaagg atatttttga 2220
agatttaggg ttttcctttg tcatttccct atttccccac ataggcagtt atgccaaatt 2280
tgggttaaat agaaactatt aaatacatta taatgataat ctactctatt ctcattttag 2340
gcttatttta cccagagttt cagaagagtt tcttttctca ggtgctcacc tccttttgtg 2400
agagtttctg agttaaggaa tattgctgag gctttcacac gctgctatct gtaaacgcgt 2460
<210> 17
<211> 2499
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
acaaggacga atctgacagc agtgaccatg acaacagcag tgacagtgag agcaaatcag 60
acagcagtga cagtagtgac gacagcagtg acagcagcga cagtagtgac agcagtgaca 120
gcagtgacag tagtgacagt agtgacagca gcgacagcag tgacagcagc gacagcaaca 180
gtagtagtga cagcagcgac agcagcagta gtagtgacag cagcgacagc agtgacagca 240
gtgacagcag tgacagcagc gatagcagtg acagcagtga cagcagtgac agcagcgata 300
gcagtgacag cagtgacagt agtgacagca gtgacagcag cgacagcagc agtagtagtg 360
acagcagcga cagcagcagt agtagtgaca gcagcgacag cagtgacagc agtgacagca 420
gcgatagcag tgacagcagt gacagcagca gcagcgacag cagcagcagt agcaacagca 480
gtgacagtag tgacagcagt gacagcagca gcagcagcga cagcagcaac agcagcgaca 540
gcagtgacag tagtgacagc agtgacagta gtgacagcag tgacagcagc aacagtagtg 600
acagcagcga cagcagcagt agtagtgaca gcagcgacag cagcagtagt agtgacagca 660
gtgacagtag tgacagtagt gacagcagtg agagcagtga gagcagcgac agcagcaaca 720
gcagtgacag cagcgacagt agtgacagca gtgacagtag cgacagcagc gacagtagtg 780
acagtagcga cagcagtgac agtagcaaca gtagcgacag cagtgacagc agtgacagca 840
gcgacagtag tgacagcagc aacagtagtg acagcagtga cagtagcgac agtagtgaca 900
gcagtgacag cagtgacagc agcgacagta gtgacagcag tgacagtagt gacagcagcg 960
acagtagtga cagcagtgac agcagtgaca gcagtgacag cagcgacagc agcgacagca 1020
gtgacagcag cgacagcagc gacagcagtg acagcagcga cagcagcaac agcagtgaca 1080
gcagtgacag tgacagcaag gatagcagtt ctgacagcag tgatggtgac agcaagtctg 1140
gtaatggcaa cagtgacagc aacagtgaca gcaacagtga cagtgacagt gacagtgaag 1200
gcagtgacag taaccactca accagtgatg atgcagggcc cgcttatccg tatgatgtgc 1260
cggattatgc gtagatcaga gagaacccat gatatcctct gtgtgacctc ttggtgagtg 1320
atgggaaggc agtgaaggtt cctaacccaa tgatgacagg agagatgtgc agactgtgtg 1380
gaacccatgg agctcatagg gagtggagcc gagctccagc tctctcagag agaatctggg 1440
tgtaccacct ttggtacatg tgtgttaaaa tatattcatg ttcagaaaat atttttaaaa 1500
ggataaatct aaacaatact ttaacaggaa ctgaagaaat cactaagaca catagcttcg 1560
atttgaatgg cgggtgcttt aaagagcaga gctagcaatg tcacagcctg ctgcagcctc 1620
ctccctcagt gctccgggca ccagagagct agtcttcatg ttgtgcagtg agtaatgctg 1680
ttctgtgaca ttcaactcaa ctactctgtc atttatttat tccggggaaa attacattta 1740
gggcataatc aaaacaccgc tgcaactact ggccctatcc aaggtgctga gataatcttt 1800
gtgatgagac aatagctata cattatgaaa attccgaaga atgaatgaga aaagagcccc 1860
aaggatggct tgggcaggat ctgacacatg cggttaaatt tctgcatggg atggatatgt 1920
actaagtccc caacccctgc actttgaaca gtgtctccct tccagcagtg gccctcaaac 1980
cttaaataaa cgagcaacac ggatggatga tttcgggagg tgggatcata ttctgagctc 2040
tccatgtacc actgtgttat tagttttctt cgaatcacag ctcaaacagt ttaatcaaga 2100
gttgtaaggc tgtgcgtgac aagagtggga ccctgtttgg gctctagggc tcctctgaaa 2160
gcaagagagg taatgagaat aaaccacacc aagacaggag gtgtgaactg ggattgtctc 2220
aagaaaacct taaccctcaa gccttaagga tatttttgaa gatttagggt tttcctttgt 2280
catttcccta tttccccaca taggcagtta tgccaaattt gggttaaata gaaactatta 2340
aatacattat aatgataatc tactctattc tcattttagg cttattttac ccagagtttc 2400
agaagagttt cttttctcag gtgctcacct ccttttgtga gagtttctga gttaaggaat 2460
attgctgagg ctttcacacg ctgctatctg taaacgcgt 2499
<210> 18
<211> 955
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Met Lys Met Lys Ile Ile Ile Tyr Ile Cys Ile Trp Ala Thr Ala Trp
1 5 10 15
Ala Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp
20 25 30
Tyr Lys Asp Asp Asp Asp Lys Ala Thr Gly Ala Ile Pro Asn Glu Leu
35 40 45
Ser Ile Asn Ser Thr Thr Ser Asn Ser Asn Asp Ser Pro Asp Gly Ser
50 55 60
Glu Ile Gly Glu Gln Val Leu Ser Glu Asp Gly Tyr Lys Arg Asp Gly
65 70 75 80
Asn Gly Ser Glu Ser Ile His Val Gly Gly Lys Asp Phe Pro Thr Gln
85 90 95
Pro Ile Leu Val Asn Glu Gln Gly Asn Thr Ala Glu Glu His Asn Asp
100 105 110
Ile Glu Thr Tyr Gly His Asp Gly Val His Ala Arg Gly Glu Asn Ser
115 120 125
Thr Ala Asn Gly Ile Arg Ser Gln Val Gly Ile Val Glu Asn Ala Glu
130 135 140
Glu Ala Glu Ser Ser Val His Gly Gln Ala Gly Gln Asn Thr Lys Ser
145 150 155 160
Gly Gly Ala Ser Asp Val Ser Gln Asn Gly Asp Ala Thr Leu Val Gln
165 170 175
Glu Asn Glu Pro Pro Glu Ala Ser Ile Lys Asn Ser Thr Asn His Glu
180 185 190
Ala Gly Ile His Gly Ser Gly Val Ala Thr His Glu Thr Thr Pro Gln
195 200 205
Arg Glu Gly Leu Gly Ser Glu Asn Gln Gly Thr Glu Val Thr Pro Ser
210 215 220
Ile Gly Glu Asp Ala Gly Leu Asp Asp Thr Asp Gly Ser Pro Ser Gly
225 230 235 240
Asn Gly Val Glu Glu Asp Glu Asp Thr Gly Ser Gly Asp Gly Glu Gly
245 250 255
Ala Glu Ala Gly Asp Gly Arg Glu Ser His Asp Gly Thr Lys Gly Gln
260 265 270
Gly Gly Gln Ser His Gly Gly Asn Thr Asp His Arg Gly Gln Ser Ser
275 280 285
Val Ser Thr Glu Asp Asp Asp Ser Lys Glu Gln Glu Gly Phe Pro Asn
290 295 300
Gly His Asn Gly Asp Asn Ser Ser Glu Glu Asn Gly Val Glu Glu Gly
305 310 315 320
Asp Ser Thr Gln Ala Thr Gln Asp Asn Gln Lys Leu Ser Pro Lys Asp
325 330 335
Thr Arg Asp Ala Glu Gly Gly Ile Ile Ser Gln Ser Glu Ala Cys Pro
340 345 350
Ser Gly Lys Ser Gln Asp Gln Gly Ile Glu Thr Glu Gly Pro Asn Lys
355 360 365
Gly Asn Lys Ser Ile Ile Thr Lys Glu Ser Gly Lys Leu Ser Gly Ser
370 375 380
Lys Asp Ser Asn Gly His Gln Gly Val Glu Leu Asp Lys Arg Asn Ser
385 390 395 400
Pro Lys Gln Gly Glu Ser Asp Lys Pro Gln Gly Thr Ala Glu Lys Ser
405 410 415
Ala Ala His Ser Asn Leu Gly His Ser Arg Ile Gly Ser Ser Ser Asn
420 425 430
Ser Asp Gly His Asp Ser Tyr Glu Phe Asp Asp Glu Ser Met Gln Gly
435 440 445
Asp Asp Pro Lys Ser Ser Asp Glu Ser Asn Gly Ser Asp Glu Ser Asp
450 455 460
Thr Asn Ser Glu Ser Ala Asn Glu Ser Gly Ser Arg Gly Asp Ala Ser
465 470 475 480
Tyr Thr Ser Asp Glu Ser Ser Asp Asp Asp Asn Asp Ser Asp Ser His
485 490 495
Ala Gly Glu Asp Asp Ser Ser Asp Asp Ser Ser Asp Thr Asp Asp Ser
500 505 510
Asp Ser Asn Gly Asp Gly Asp Ser Asp Ser Asn Gly Asp Gly Asp Ser
515 520 525
Glu Ser Glu Asp Lys Asp Glu Ser Asp Ser Ser Asp His Asp Asn Ser
530 535 540
Ser Asp Ser Glu Ser Lys Ser Asp Ser Ser Asp Ser Ser Asp Asp Ser
545 550 555 560
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
565 570 575
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Asn Ser
580 585 590
Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser Asp Ser
595 600 605
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
610 615 620
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
625 630 635 640
Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser Asp Ser
645 650 655
Ser Ser Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
660 665 670
Asp Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser Asp Ser Ser Ser Ser
675 680 685
Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser Ser
690 695 700
Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
705 710 715 720
Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser
725 730 735
Ser Ser Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser
740 745 750
Asp Ser Ser Asp Ser Ser Asp Ser Ser Glu Ser Ser Glu Ser Ser Asp
755 760 765
Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
770 775 780
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
785 790 795 800
Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
805 810 815
Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
820 825 830
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
835 840 845
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
850 855 860
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
865 870 875 880
Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Asp
885 890 895
Ser Lys Asp Ser Ser Ser Asp Ser Ser Asp Gly Asp Ser Lys Ser Gly
900 905 910
Asn Gly Asn Ser Asp Ser Asn Ser Asp Ser Asn Ser Asp Ser Asp Ser
915 920 925
Asp Ser Glu Gly Ser Asp Ser Asn His Ser Thr Ser Asp Asp Ala Gly
930 935 940
Pro Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
945 950 955
<210> 19
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
tgcacttacc ggaatggccc agg 23
<210> 20
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gaattcgagc tcggtacccg gggctcctcc ctcagtccga ga 42
<210> 21
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ccgtcatggt ctttgtagtc ggcccaggca gttgccca 38
<210> 22
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
atgacaaggc taccggtgca attccggtaa gtgcatcttc cc 42
<210> 23
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
caagcttgca tgcctgcagg ctcggagcca ttcccatctc 40
<210> 24
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
tttgggcaac tgcctgggcc gactacaaag accatgacgg tg 42
<210> 25
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gaagatgcac ttaccggaat tgcaccggta gccttgtcat cgtcatcctt gtag 54
<210> 26
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ctctctgatc taatcatcac tgg 23
<210> 27
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gaattcgagc tcggtacccg ggacaaggac gaatctgaca gca 43
<210> 28
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
tccggcacat catacggata agcgggccct gcatcatcac tggttgagtg gttac 55
<210> 29
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
tatccgtatg atgtgccgga ttatgcgtag atcagagaga acccatgata t 51
<210> 30
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
caagcttgca tgcctgcagg acgcgtttac agatagcagc 40
<210> 31
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
ctccgagctc actcttgcag 20
<210> 32
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
atctcactgc catctgggga 20
<210> 33
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
tctcagcctg gaaagagaga 20
<210> 34
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
tggcttacat cactagcacc tc 22
<210> 35
<211> 945
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Met Lys Met Lys Ile Ile Ile Tyr Ile Cys Ile Trp Ala Thr Ala Trp
1 5 10 15
Ala Ile Pro Val Pro Gln Leu Val Pro Leu Glu Arg Asp Ile Val Glu
20 25 30
Asn Ser Val Ala Val Pro Leu Leu Thr His Pro Gly Thr Ala Ala Gln
35 40 45
Asn Glu Leu Ser Ile Asn Ser Thr Thr Ser Asn Ser Asn Asp Ser Pro
50 55 60
Asp Gly Ser Glu Ile Gly Glu Gln Val Leu Ser Glu Asp Gly Tyr Lys
65 70 75 80
Arg Asp Gly Asn Gly Ser Glu Ser Ile His Val Gly Gly Lys Asp Phe
85 90 95
Pro Thr Gln Pro Ile Leu Val Asn Glu Gln Gly Asn Thr Ala Glu Glu
100 105 110
His Asn Asp Ile Glu Thr Tyr Gly His Asp Gly Val His Ala Arg Gly
115 120 125
Glu Asn Ser Thr Ala Asn Gly Ile Arg Ser Gln Val Gly Ile Val Glu
130 135 140
Asn Ala Glu Glu Ala Glu Ser Ser Val His Gly Gln Ala Gly Gln Asn
145 150 155 160
Thr Lys Ser Gly Gly Ala Ser Asp Val Ser Gln Asn Gly Asp Ala Thr
165 170 175
Leu Val Gln Glu Asn Glu Pro Pro Glu Ala Ser Ile Lys Asn Ser Thr
180 185 190
Asn His Glu Ala Gly Ile His Gly Ser Gly Val Ala Thr His Glu Thr
195 200 205
Thr Pro Gln Arg Glu Gly Leu Gly Ser Glu Asn Gln Gly Thr Glu Val
210 215 220
Thr Pro Ser Ile Gly Glu Asp Ala Gly Leu Asp Asp Thr Asp Gly Ser
225 230 235 240
Pro Ser Gly Asn Gly Val Glu Glu Asp Glu Asp Thr Gly Ser Gly Asp
245 250 255
Gly Glu Gly Ala Glu Ala Gly Asp Gly Arg Glu Ser His Asp Gly Thr
260 265 270
Lys Gly Gln Gly Gly Gln Ser His Gly Gly Asn Thr Asp His Arg Gly
275 280 285
Gln Ser Ser Val Ser Thr Glu Asp Asp Asp Ser Lys Glu Gln Glu Gly
290 295 300
Phe Pro Asn Gly His Asn Gly Asp Asn Ser Ser Glu Glu Asn Gly Val
305 310 315 320
Glu Glu Gly Asp Ser Thr Gln Ala Thr Gln Asp Asn Gln Lys Leu Ser
325 330 335
Pro Lys Asp Thr Arg Asp Ala Glu Gly Gly Ile Ile Ser Gln Ser Glu
340 345 350
Ala Cys Pro Ser Gly Lys Ser Gln Asp Gln Gly Ile Glu Thr Glu Gly
355 360 365
Pro Asn Lys Gly Asn Lys Ser Ile Ile Thr Lys Glu Ser Gly Lys Leu
370 375 380
Ser Gly Ser Lys Asp Ser Asn Gly His Gln Gly Val Glu Leu Asp Lys
385 390 395 400
Arg Asn Ser Pro Lys Gln Gly Glu Ser Asp Lys Pro Gln Gly Thr Ala
405 410 415
Glu Lys Ser Ala Ala His Ser Asn Leu Gly His Ser Arg Ile Gly Ser
420 425 430
Ser Ser Asn Ser Asp Gly His Asp Ser Tyr Glu Phe Asp Asp Glu Ser
435 440 445
Met Gln Gly Asp Asp Pro Lys Ser Ser Asp Glu Ser Asn Gly Ser Asp
450 455 460
Glu Ser Asp Thr Asn Ser Glu Ser Ala Asn Glu Ser Gly Ser Arg Gly
465 470 475 480
Asp Ala Ser Tyr Thr Ser Asp Glu Ser Ser Asp Asp Asp Asn Asp Ser
485 490 495
Asp Ser His Ala Gly Glu Asp Asp Ser Ser Asp Asp Ser Ser Asp Thr
500 505 510
Asp Asp Ser Asp Ser Asn Gly Asp Gly Asp Ser Asp Ser Asn Gly Asp
515 520 525
Gly Asp Ser Glu Ser Glu Asp Lys Asp Glu Ser Asp Ser Ser Asp His
530 535 540
Asp Asn Ser Ser Asp Ser Glu Ser Lys Ser Asp Ser Ser Asp Ser Ser
545 550 555 560
Asp Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
565 570 575
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
580 585 590
Ser Asn Ser Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser Ser Asp Ser
595 600 605
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
610 615 620
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
625 630 635 640
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser Ser Asp Ser
645 650 655
Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
660 665 670
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser Asp Ser
675 680 685
Ser Ser Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
690 695 700
Ser Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp
705 710 715 720
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser
725 730 735
Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser Ser
740 745 750
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Glu Ser Ser Glu
755 760 765
Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
770 775 780
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
785 790 795 800
Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
805 810 815
Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
820 825 830
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
835 840 845
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
850 855 860
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
865 870 875 880
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser
885 890 895
Asp Ser Asp Ser Lys Asp Ser Ser Ser Asp Ser Ser Asp Gly Asp Ser
900 905 910
Lys Ser Gly Asn Gly Asn Ser Asp Ser Asn Ser Asp Ser Asn Ser Asp
915 920 925
Ser Asp Ser Asp Ser Glu Gly Ser Asp Ser Asn His Ser Thr Ser Asp
930 935 940
Asp
945
<210> 36
<211> 984
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Met Lys Met Lys Ile Ile Ile Tyr Ile Cys Ile Trp Ala Thr Ala Trp
1 5 10 15
Ala Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp
20 25 30
Tyr Lys Asp Asp Asp Asp Lys Ala Thr Gly Ala Ile Pro Val Pro Gln
35 40 45
Leu Val Pro Leu Glu Arg Asp Ile Val Glu Asn Ser Val Ala Val Pro
50 55 60
Leu Leu Thr His Pro Gly Thr Ala Ala Gln Asn Glu Leu Ser Ile Asn
65 70 75 80
Ser Thr Thr Ser Asn Ser Asn Asp Ser Pro Asp Gly Ser Glu Ile Gly
85 90 95
Glu Gln Val Leu Ser Glu Asp Gly Tyr Lys Arg Asp Gly Asn Gly Ser
100 105 110
Glu Ser Ile His Val Gly Gly Lys Asp Phe Pro Thr Gln Pro Ile Leu
115 120 125
Val Asn Glu Gln Gly Asn Thr Ala Glu Glu His Asn Asp Ile Glu Thr
130 135 140
Tyr Gly His Asp Gly Val His Ala Arg Gly Glu Asn Ser Thr Ala Asn
145 150 155 160
Gly Ile Arg Ser Gln Val Gly Ile Val Glu Asn Ala Glu Glu Ala Glu
165 170 175
Ser Ser Val His Gly Gln Ala Gly Gln Asn Thr Lys Ser Gly Gly Ala
180 185 190
Ser Asp Val Ser Gln Asn Gly Asp Ala Thr Leu Val Gln Glu Asn Glu
195 200 205
Pro Pro Glu Ala Ser Ile Lys Asn Ser Thr Asn His Glu Ala Gly Ile
210 215 220
His Gly Ser Gly Val Ala Thr His Glu Thr Thr Pro Gln Arg Glu Gly
225 230 235 240
Leu Gly Ser Glu Asn Gln Gly Thr Glu Val Thr Pro Ser Ile Gly Glu
245 250 255
Asp Ala Gly Leu Asp Asp Thr Asp Gly Ser Pro Ser Gly Asn Gly Val
260 265 270
Glu Glu Asp Glu Asp Thr Gly Ser Gly Asp Gly Glu Gly Ala Glu Ala
275 280 285
Gly Asp Gly Arg Glu Ser His Asp Gly Thr Lys Gly Gln Gly Gly Gln
290 295 300
Ser His Gly Gly Asn Thr Asp His Arg Gly Gln Ser Ser Val Ser Thr
305 310 315 320
Glu Asp Asp Asp Ser Lys Glu Gln Glu Gly Phe Pro Asn Gly His Asn
325 330 335
Gly Asp Asn Ser Ser Glu Glu Asn Gly Val Glu Glu Gly Asp Ser Thr
340 345 350
Gln Ala Thr Gln Asp Asn Gln Lys Leu Ser Pro Lys Asp Thr Arg Asp
355 360 365
Ala Glu Gly Gly Ile Ile Ser Gln Ser Glu Ala Cys Pro Ser Gly Lys
370 375 380
Ser Gln Asp Gln Gly Ile Glu Thr Glu Gly Pro Asn Lys Gly Asn Lys
385 390 395 400
Ser Ile Ile Thr Lys Glu Ser Gly Lys Leu Ser Gly Ser Lys Asp Ser
405 410 415
Asn Gly His Gln Gly Val Glu Leu Asp Lys Arg Asn Ser Pro Lys Gln
420 425 430
Gly Glu Ser Asp Lys Pro Gln Gly Thr Ala Glu Lys Ser Ala Ala His
435 440 445
Ser Asn Leu Gly His Ser Arg Ile Gly Ser Ser Ser Asn Ser Asp Gly
450 455 460
His Asp Ser Tyr Glu Phe Asp Asp Glu Ser Met Gln Gly Asp Asp Pro
465 470 475 480
Lys Ser Ser Asp Glu Ser Asn Gly Ser Asp Glu Ser Asp Thr Asn Ser
485 490 495
Glu Ser Ala Asn Glu Ser Gly Ser Arg Gly Asp Ala Ser Tyr Thr Ser
500 505 510
Asp Glu Ser Ser Asp Asp Asp Asn Asp Ser Asp Ser His Ala Gly Glu
515 520 525
Asp Asp Ser Ser Asp Asp Ser Ser Asp Thr Asp Asp Ser Asp Ser Asn
530 535 540
Gly Asp Gly Asp Ser Asp Ser Asn Gly Asp Gly Asp Ser Glu Ser Glu
545 550 555 560
Asp Lys Asp Glu Ser Asp Ser Ser Asp His Asp Asn Ser Ser Asp Ser
565 570 575
Glu Ser Lys Ser Asp Ser Ser Asp Ser Ser Asp Asp Ser Ser Asp Ser
580 585 590
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
595 600 605
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Asn Ser Ser Ser Asp
610 615 620
Ser Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
625 630 635 640
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
645 650 655
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
660 665 670
Ser Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser
675 680 685
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
690 695 700
Asp Ser Ser Asp Ser Ser Ser Ser Asp Ser Ser Ser Ser Ser Asn Ser
705 710 715 720
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser
725 730 735
Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
740 745 750
Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Ser Ser
755 760 765
Ser Asp Ser Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser Asp Ser Ser
770 775 780
Asp Ser Ser Asp Ser Ser Glu Ser Ser Glu Ser Ser Asp Ser Ser Asn
785 790 795 800
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
805 810 815
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser
820 825 830
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn
835 840 845
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
850 855 860
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
865 870 875 880
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
885 890 895
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
900 905 910
Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Asp Ser Lys Asp
915 920 925
Ser Ser Ser Asp Ser Ser Asp Gly Asp Ser Lys Ser Gly Asn Gly Asn
930 935 940
Ser Asp Ser Asn Ser Asp Ser Asn Ser Asp Ser Asp Ser Asp Ser Glu
945 950 955 960
Gly Ser Asp Ser Asn His Ser Thr Ser Asp Asp Ala Gly Pro Ala Tyr
965 970 975
Pro Tyr Asp Val Pro Asp Tyr Ala
980
Claims (10)
1.一种DGI疾病动物模型的构建方法，其特征在于，所述方法包括以下步骤：
1)将具有DSPP基因c.144+1G>A或c.144+1G>T点突变的孤雄单倍体胚胎干细胞与卵细胞结合获得细胞胚胎；
2)培育所述细胞胚胎即获得DGI疾病动物模型。
2.根据权利要求1所述的构建方法，其特征在于，所述DSPP基因c.144+1G>A或c.144+1G>T点突变后编码的蛋白质氨基酸序列如SEQ ID NO.1所示。
3.根据权利要求1所述的构建方法，其特征在于，所述孤雄单倍体胚胎干细胞为DKO-AG-haESCs。
4.根据权利要求1所述的构建方法，其特征在于，所述孤雄单倍体胚胎干细胞来源于哺乳动物；优选的，所述孤雄单倍体胚胎干细胞来源于啮齿动物；更优选的，所述孤雄单倍体胚胎干细胞来源于小鼠。
5.根据权利要求1所述的构建方法，其特征在于，所述构建方法还包括以下特征中的一项或几项：
1)所述卵细胞与所述孤雄单倍体胚胎干细胞来源于同种动物；
2)所述卵细胞与所述孤雄单倍体胚胎干细胞结合采用卵胞浆注射法；
3)培育所述细胞胚胎是指将细胞胚胎移入到母体中，母鼠怀孕后生产获得点突变杂合鼠即为DGI疾病动物模型。
6.根据权利要求1-5任一所述的构建方法，其特征在于，所述DGI疾病动物模型的构建方法还包括以下特征：在c.144+1G>A或c.144+1G>T点突变的DSPP基因组上插入有标签。
7.根据权利要求6所述的构建方法，其特征在于，在c.144+1G>A或c.144+1G>T点突变的DSPP基因组的5’端和3’端插入有标签；优选的，5’端和3’端的标签不同。
8.根据权利要求7所述的构建方法，其特征在于，c.144+1G>A或c.144+1G>T点突变的DSPP基因组5’端插入标签后的核苷酸序列如SEQ ID NO.15所示，3’端插入标签后的核苷酸序列如SEQ ID NO.17所示。
9.一种DGI疾病动物模型，其特征在于，所述DGI疾病动物模型由权利要求1-8任一权利要求所述的构建方法制备获得。
10.权利要求9所述的DGI疾病动物模型在研究DSPP蛋白的时空表达谱式、切割谱式、蛋白定位以及DGI疾病发生发展机制中的用途。
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