CN112062827A - Cep55蛋白在调控纤毛去组装和制备纤毛相关疾病模型中的应用 - Google Patents

Cep55蛋白在调控纤毛去组装和制备纤毛相关疾病模型中的应用 Download PDF

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CN112062827A
CN112062827A CN202010971546.7A CN202010971546A CN112062827A CN 112062827 A CN112062827 A CN 112062827A CN 202010971546 A CN202010971546 A CN 202010971546A CN 112062827 A CN112062827 A CN 112062827A
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cep55
cilia
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李慧艳
李爱玲
周涛
张宇程
吴敏
涂海情
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Academy of Military Medical Sciences AMMS of PLA
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Abstract

本发明公开了CEP55蛋白在调控纤毛去组装和制备纤毛相关疾病模型中的应用。本发明提供了CEP55蛋白或其相关生物材料在如下任一中的应用：(A1)调控纤毛去组装；(A2)制备用于调控纤毛去组装的产品；所述相关生物材料为能够促进所述CEP55蛋白表达和/或使其活性增加的物质或能够抑制所述CEP55蛋白表达和/或使其活性降低的物质。本发明揭示了CEP55蛋白参与调控纤毛的去组装，并评价Cep55基因敲除小鼠表型，为全面阐明CEP55在体内功能奠定了基础，发现其组织学特征与临床上纤毛相关疾病诊断特征一致，本发明为产前诊断提供了可能标志物。

Description

CEP55蛋白在调控纤毛去组装和制备纤毛相关疾病模型中的 应用
技术领域
本发明涉及生物技术领域，具体涉及CEP55蛋白在调控纤毛去组装和制备纤毛相关疾病模型中的应用。
背景技术
初级纤毛是由特化的微管为结构基础的细胞器，存在于大多数哺乳动物细胞表面。这种细胞器如同细胞天线，负责接收并整合胞外信号，对维持胚胎的正常发育至关重要。纤毛根据运动性分为运动纤毛和非运动纤毛：运动纤毛存在于多个器官中，包括大脑、肺、中耳和生殖器官，被认为具有有节奏的摆动能力和驱动细胞外液体的流动能力；而非运动的初级纤毛存在于机体绝大多数细胞和组织中，充当着细胞外物理和化学信号的传感器，参与大量生物学过程。多项研究报道，纤毛的结构缺陷或功能紊乱会导致全身多器官受累的人类疾病，统称为纤毛相关疾病。该类疾病临床症状有脑发育不全、认知障碍、肾脏肝脏多囊性病变、视网膜变性和生殖缺陷等。纤毛的组装(微管延伸)、去组装(微管解聚)及纤毛信号转导异常，都可能诱发纤毛相关疾病。因此，对这类疾病发病机制的揭示很大程度上为临床疾病诊治提供依据。
CEP55蛋白在细胞中具有中间体和中心体双重定位，已知报道其调控有丝分裂中间体的分离，但其中心体上的功能研究知之甚少。目前，尚未有CEP55蛋白参与纤毛去组装，以及用于制备纤毛相关疾病模型中的相关报道。
发明内容
本发明的目的是提供CEP55蛋白在调控纤毛去组装和制备纤毛相关疾病模型中的应用。
第一方面，本发明要求保护CEP55蛋白或其相关生物材料在如下任一中的应用：
(A1)调控纤毛去组装；
(A2)制备用于调控纤毛去组装的产品；
所述相关生物材料可为能够促进所述CEP55蛋白表达和/或使其活性增加的物质或能够抑制所述CEP55蛋白表达和/或使其活性降低的物质。
在所述应用中，所述动物细胞内或所述动物体内所述CEP55蛋白表达量和/或活性降低，所述动物细胞或所述动物体中纤毛的长度增长和/或密度增加；和/或，所述动物细胞内或所述动物体内所述CEP55蛋白表达量和/或活性提高，所述动物细胞或所述动物体中纤毛的长度减短和/或密度降低。
所述应用为非疾病诊断治疗性应用。
第二方面，本发明要求保护能够抑制CEP55蛋白表达和/或使其活性降低的物质在如下任一中的应用：
(B1)抑制动物细胞或动物体中纤毛去组装，或制备用于抑制动物细胞或动物体中纤毛去组装的产品；
(B2)促进动物细胞或动物体中纤毛发生，或制备用于促进动物细胞或动物体中纤毛发生的产品；
(B3)制备纤毛相关疾病动物模型；
(B4)制备具有如下症状中全部或部分的动物模型：脑小症、侧脑室和/或第3脑室扩张、脑积水、肾小球和/或肾小管微囊肿、纤毛长度增加、形成纤毛细胞比例增加。
所述应用为非疾病诊断治疗性应用。
第三方面，本发明要求保护能够促进CEP55蛋白表达和/或使其活性增加的物质在如下任一中的应用：
(C1)促进动物细胞或动物体中纤毛去组装，或制备用于促进动物细胞或动物体中纤毛去组装的产品；
(C2)抑制动物细胞或动物体中纤毛发生，或制备用于抑制动物细胞或动物体中纤毛发生的产品。
在上述各方面中，所述能够促进CEP55蛋白表达和/或使其活性增加的物质可为能够表达所述CEP55蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系或者CEP55蛋白的促进剂。
在上述各方面中，所述能够抑制所述CEP55蛋白表达和/或使其活性降低的物质可为能够实现对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行抑制表达或敲除的物质。
进一步地，能够实现对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行抑制表达的物质如能够抑制编码所述CEP55蛋白的核酸分子表达的siRNA；能够实现对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行敲除的物质如采用CRISPR/Cas9技术对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行敲除时所用的sgRNA或能够转录得到所述sgRNA的DNA分子或含有所述DNA分子的表达盒或表达载体，当然还可包括Cas9蛋白或用于表达Cas9蛋白的表达载体，和/或用于同源重组的donor载体。
在本发明的具体实施方式中，能够实现对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行抑制表达的物质具体为核苷酸序列如SEQ ID No.1或SEQ IDNo.2或SEQ ID No.3所示的siRNA。采用CRISPR/Cas9技术对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行敲除时所用的sgRNA的编码序列中的特异性识别序列(对应于spacer)如SEQ ID No.6或SEQ ID No.7所示。
所述应用为非疾病诊断治疗性应用。
第四方面，本发明要求保护一种调控动物细胞或动物体中纤毛去组装的方法。
本发明所要求保护的调控动物细胞或动物体中纤毛去组装的方法，可为如下任一：
方法A1：一种抑制动物细胞或动物体中纤毛去组装的方法，可包括如下步骤：降低动物细胞内或动物体内CEP55蛋白的表达量和/或活性。
方法A2：一种促进动物细胞或动物体中纤毛去组装的方法，可包括如下步骤：提高动物细胞内或动物体内CEP55蛋白的表达量和/或活性。
所述方法为非疾病诊断治疗性方法。
第五方面，本发明要求保护一种制备纤毛相关疾病动物模型的方法。
本发明要求保护的制备纤毛相关疾病动物模型的方法，可包括如下步骤：降低动物体内CEP55蛋白的表达量和/或活性。
第六方面，本发明要求保护一种制备具有如下症状中全部或部分的动物模型的方法，可包括降低动物体内CEP55蛋白的表达量和/或活性的步骤：脑小症、侧脑室和/或第3脑室扩张、脑积水、肾小球和/或肾小管微囊肿、纤毛长度增加、形成纤毛细胞比例增加。
第七方面，本发明要求保护胞内CEP55蛋白的表达量和/或活性降低的动物细胞模型或体内CEP55蛋白的表达量和/或活性降低的动物模型如下任一中的应用：
(D1)筛选用于预防和/或治疗纤毛相关疾病的药物；
(D2)筛选能够改善如下症状中全部或部分的药物中的应用：脑小症、侧脑室和/或第3脑室扩张、脑积水、肾小球和/或肾小管微囊肿、纤毛长度增加、形成纤毛细胞比例增加。
在上述各方面中，降低所述动物细胞内或所述动物体内所述CEP55蛋白的表达量和/或活性可通过对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行抑制表达或敲除实现。
进一步地，对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行抑制表达可通过向所述动物细胞或所述动物导入能够抑制编码所述CEP55蛋白的核酸分子表达的siRNA实现。
更进一步地，所述siRNA的核苷酸序列具体如SEQ ID No.1或SEQ ID No.2或SEQID No.3所示。
进一步地，对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行敲除可通过CRISPR/Cas9技术实现。
更进一步地，通过CRISPR/Cas9技术对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行敲除时，采用的sgRNA的编码序列中的特异性识别序列(对应于spacer)如SEQ ID No.6或SEQ ID No.7所示。
第八方面，本发明要求保护如下任一应用：
(E1)CPE55蛋白或编码所述CEP55蛋白的核酸分子作为标记物在制备用于纤毛相关疾病产前诊断的产品中的应用。
(E2)用于检测CPE55蛋白或编码所述CEP55蛋白的核酸分子的物质在制备用于纤毛相关疾病产前诊断的产品中的应用。
(E3)CEP55基因在制备用于治疗由于CEP55基因缺陷或异常引起的纤毛相关疾病的产品中的应用。
其中(E3)涉及基因疗法。基因治疗(gene therapy)是指将外源正常基因导入靶细胞，以纠正或补偿缺陷和异常基因引起的疾病。具体到本发明，为将外源正常的CEP55基因导入靶细胞，以纠正或补偿由于CEP55基因缺陷或异常引起的疾病。
在上述各方面中，所述纤毛可为初级纤毛。
在上述各方面中，所述动物细胞可为哺乳动物细胞；所述动物可为哺乳动物。
进一步地，所述哺乳动物细胞可为肾小管上皮细胞或胚胎成纤维细胞或人视网膜色素上皮细胞(如RPE-1细胞)。
进一步地，所述哺乳动物可为人或小鼠。
在上述各方面中，所述CEP55蛋白的氨基酸序列可如SEQ ID No.4(小鼠)或SEQ IDNo.8(人源)所示。相应的，编码所述CEP55蛋白的核酸分子的序列可如SEQ ID No.5(小鼠)或SEQ ID No.9(人源)所示。
在上述各方面中，所述纤毛相关疾病具体可为具有如下症状中全部或部分的纤毛相关疾病：脑小症、侧脑室和/或第3脑室扩张、脑积水、肾小球和/或肾小管微囊肿、纤毛长度增加、形成纤毛细胞比例增加。
本发明揭示了CEP55蛋白参与调控纤毛的去组装，并评价Cep55基因敲除小鼠表型，为全面阐明CEP55在体内功能奠定了基础，发现其组织学特征与临床上纤毛相关疾病诊断特征一致，本发明为产前诊断提供了可能标志物。并且，有望采用基因疗法正常的利用CEP55基因治疗由于CEP55基因缺陷或异常引起的纤毛相关疾病。
附图说明
图1为胚胎18.5天Cep55-/-纯合敲除小鼠显示纤毛相关疾病病例特征——脑发育异常、多囊肾。a为Cep55+/+与Cep55-/-小鼠胚胎脑部外观代表图；b为Cep55+/+与Cep55-/-小鼠胚胎脑部冠状面及矢状面核磁共振成像图；黄色箭头指示Cep55-/-胎脑脑积水部位；c为Cep55+/+与Cep55-/-小鼠胚胎脑部冠状切片苏木精-伊红染色图；d为Cep55+/+与Cep55-/-小鼠胚胎肾脏冠状切片苏木精-伊红染色图，虚线框放大部分为肾小球。
图2为Cep55-/-纯合敲除小鼠脑室、肾脏等细胞纤毛过度生长。a为Cep55+/+与Cep55-/-小鼠脑室内脉络膜上皮细胞纤毛形成情况；b为对图a中细胞纤毛长度统计结果；c为Cep55+/+与Cep55-/-小鼠肾小管上皮细胞纤毛形成情况，虚线内指示肾小管；d为对图c中细胞纤毛长度统计结果；e为Cep55+/+与Cep55-/-原代小鼠成纤维细胞初级纤毛形成情况(含血清培养)；f为对图d中细胞纤毛发生比例统计结果；g为Cep55+/+与Cep55-/-原代小鼠成纤维细胞初级纤毛形成情况(去除血清培养)；h为对图g中细胞纤毛长度统计结果。
图3为CEP55介导纤毛去组装。a为含血清培养的视网膜上皮细胞中，siRNA敲低CEP55后细胞纤毛形成情况；b对图a中细胞纤毛发生比例统计结果(-表示对照组)；c为去除血清培养的视网膜上皮细胞中，siRNA敲低CEP55后细胞纤毛长度情况(-表示对照组)；d对图c中细胞纤毛长度统计结果；e为细胞水平诱导初级纤毛组装与去组装流程；f为siRNA敲低CEP55抑制血清诱导的纤毛去组装效应；g为图f的代表性图片(-表示对照组)。
各图的定量结果中，*表示p<0.05；**表示p<0.01；***表示p<0.001。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中所涉及的CEP55蛋白的氨基酸序列如SEQ ID No.4(小鼠)或SEQ IDNo.8(人源)所示，其编码核苷酸序列如SEQ ID No.5(小鼠，cDNA)、SEQ ID No.10(小鼠，基因组)或SEQ ID No.9(人源)所示。
人视网膜色素上皮细胞(RPE-1)：为中科院朱学良老师赠送。记载于“Tu HQ,QinXH,Liu ZB,et al.Microtubule asters anchored by FSD1 control axoneme assemblyand ciliogenesis.Nat Commun.2018Dec 11；9(1):5277.”一文中，公众可从申请人处获得，尽可用于重复本发明实验使用，不得他用。
实施例1、CEP55蛋白在调控纤毛去组装和制备纤毛相关疾病模型中的应用
一、实验材料
抗体：Ac-Tubulin抗体，Sigma公司，货号T6793；γ-Tubulin抗体：Sigma公司，货号T6557；GT335抗体，Adipogen公司，货号AG-20B-0020B-C100。
siRNA购自Thermo Fisher Scientific公司：
siCEP55-1#：5’-GGAACAACAGAUGCAGGCAUGUACU-3’(SEQ ID No.1)；
siCEP55-2#：5’-GAGGGAGCAGGUGUUGAAAGCCUUA-3’(SEQ ID No.2)；
siCEP55-3#：5’-ACGAAUUGCUGAACUUGAAAGCAAA-3’(SEQ ID No.3)；
Control siRNA：5’-UUCUCCGAACGUGUCAGGUTT-3’。
其他试剂：Advanced DMEM/F12(1:1)培养基(12634-010)、DMEM培养基(11995-065)、Opti-MEM培养基(31985-070)、磷酸盐缓冲液PBS(C10010500BT)购自Gibco公司；胎牛血清(FND500)购自澳洲ExCell Bio公司；4％多聚甲醛(P1110)购自Macgene公司；组织包埋剂OCT购自日本SAKURA公司；牛血清白蛋白BSA(VA10411)购自GenStar公司；DNA染料Hoechst(H3570)购自Invitrogen公司；封片剂Mounting Medium(ZLI-9556)购自中杉金桥公司；Alexa Fluor 488及546免疫荧光二抗、转染试剂RNAiMAX Transfection Reagent(13778)购自Thermo Fisher Scientific公司。
二、实验方法
1、Cep55loxP/loxP条件敲除小鼠的构建
Cep55loxP/loxP条件敲除小鼠是利用CRISPR/Cas9基因编辑技术，由申请人团队订制，委托南京大学-南京生物医药研究院制备获得。具体策略为靶向Cep55基因的3号至5号外显子进行敲除，将导致移码突变，使Cep55蛋白翻译提前终止。
首先，构建sgRNA。
5’-序列：5’-TAGAGGTGATGGGGTCATGAGGG-3’(SEQ ID No.6)；
3’-序列：5’-GGAATGAGCACTTTGAGAAAGGG-3’(SEQ ID No.7)。
本发明所选sgRNA与Cep55内含子反向互补，5’sgRNA在2号外显子下游，3’sgRNA在5号外显子下游。
体外检测sgRNA切割效率良好后，设计带有同源臂，loxp位点及限制性酶切位点的donor克隆载体，将其与两个sgRNA及Cas9体系共同注射入C57BL/6J小鼠受精卵中，再移植入代孕雌鼠体内，得到F0代小鼠。测序验证Cep55loxP/wt阳性F0代小鼠，将其与野生C57BL/6J小鼠回交，测序验证得到Cep55loxP/wt阳性F1代小鼠。进一步，将获得的Cep55loxP/loxP小鼠和诱导全身敲除的EIIa-Cre工具小鼠(003724，Jackson Laboratory)进行杂交，得到Cep55+/-小鼠，继而通过Cep55+/-小鼠杂交以获得Cep55-/-等位基因敲除小鼠。
2、小鼠基因型鉴定PCR
A.将小鼠组织用裂解液进行裂解，利用两对PCR引物扩增，鉴定小鼠基因型：
F1：5’-AGCATGCCTGCAAACTGCTTC-3’；
R1：5’-GTCTCAAACACTCAGTTCTCC-3’；
F2：5’-TGTCTGGTAAGGATCTTCAAGCTG-3’；
R2：5’-TGGAAGTTAGCTTGGGCTACG-3’。
B.将每组两对引物PCR产物用2％琼脂糖凝胶跑样；
C.Cep55+/+野生小鼠经引物F1/R1扩增后仅在245bp处出现阳性条带(引物F2/R2扩增无条带)，Cep55-/-纯合敲除小鼠经引物F2/R2扩增后仅在约300bp处出现阳性条带(引物F1/R1扩增无条带)，Cep55+/-杂合小鼠则在两处均出现阳性条带(引物F1/R1扩增在245bp处出现阳性条带，引物F2/R2扩增后在约300bp处出现阳性条带)。
3、小鼠胚胎的分离与取材技术
A.取妊娠期18.5天Cep55+/-孕鼠，颈椎脱臼法将其处死，取得胚胎放入冰PBS中；
B.分离各个胚胎丢弃胎盘，逐个放入盛有PBS的6孔板中；
C.顺序编号，PCR鉴定胎鼠基因型；
D.体视镜下分离胎鼠脑、肾等组织，拍照或固定。
4、小鼠组织石蜡切片制作
A.固定：将分离后组织置于4％多聚甲醛中，4℃过夜；
B.水洗：去离子水或PBS洗涤，2遍；
C.脱水：75％、80％、85％、95％、100％、100％梯度酒精各30分钟；
D.透明：二甲苯40分钟，3遍；
E.浸蜡：65℃石蜡30分钟，3遍；
F.包埋：迅速取出置于预热好的组织槽中，进行石蜡包埋；
G.切片：石蜡切片机，厚度3-5μm，置于玻片盒中室温冷处储藏。
5、石蜡切片苏木精-伊红染色(HE)
A.烤片：65℃烤箱1小时；
B.脱蜡：二甲苯15分钟，2遍；
C.脱二甲苯：100％、100％、95％、95％、90％、80％梯度酒精各5分钟；
D.复水：蒸馏水洗2分钟，2遍；
E.染核：苏木素中染5-10分钟后，自来水冲洗3次至水清；
F.分化：395ml无水乙醇+5ml浓盐酸+100ml蒸馏水中迅速涮2次；
G.温水返蓝：在温水中涮3次；
H.脱水：80％、90％、95％、95％、100％、100％梯度酒精各2分钟；
I.二甲苯2分钟，2遍；
J.封片：向切片样本处滴加1-2滴中性树脂，加盖玻片。
K.将切片通风橱内晾干过夜，用NanoZoomer扫描切片。
6、小鼠头部核磁共振成像(MRI)
(1)小鼠胚胎的分离与取材：
A.取妊娠期18.5天Cep55+/-孕鼠，将其处死后取得胚胎放入冰PBS中；
B.分离胚胎并进行基因型鉴定；
C.配制3％琼脂糖凝胶，将胚胎密封其中，待到MRI成像时取出。
(2)MRI成像：
A.将胚胎从琼脂糖凝胶中取出，固定于小动物MRI设备；
B.用Paravision 6.0.1软件平台记录成像信息；
C.粗拍确定位置后，调整最佳参数，进行拍摄；
D.将胚胎依次至于相同条件下拍摄。
参数为：TR/TE:4500/100ms，FOV 20×20cm；
图像大小：256×256，拍摄厚度：0.7mm，20averages。
7、小鼠组织冰冻切片制作
A.固定：将分离后组织置于4％多聚甲醛中，4℃过夜；
B.洗涤：PBS洗涤2次；
C.脱水：30％蔗糖(PBS配制)4℃过夜或至组织沉入底部；
D.包埋：选取合适组织大小的组织包埋盒，加入OCT，缓慢倒入液氮，待其冷凝为固体状，置于-80℃冰箱保存；
E.切片：按冰冻切片标准流程操作进行切片，厚度10-12μm，-80℃冰箱保存。
8、细胞培养
RPE-1细胞培养于Advanced DMEM/F-12(1:1)，培养时添加10％胎牛血清及100U/mL双抗(青霉素、链霉素)。原代小鼠成纤维细胞(MEFs)由E13.5胚胎分离获得，培养于DMEM培养基中，添加15％胎牛血清及100U/mL双抗。纤毛组装实验，将细胞培养基更换为Opti-MEM培养基中去除血清培养48小时；纤毛去组装实验，去除血清培养48小时后，将细胞培养基更换为含血清培养基，进行血清回刺激0，2，18，24小时。上述细胞均置于5％CO2、37℃的培养箱中培养。
9、原代MEFs分离
A.取妊娠期13.5天Cep55+/-孕鼠，颈椎脱臼法将其处死，取得胚胎放入冰PBS中；
B.分离各个胚胎丢弃胎盘，逐个放入盛有冰PBS的6孔板中；
C.顺序编号，取小段尾巴做基因型鉴定；
D.细胞分离：将胚胎的头、内脏及四肢去除干净，剩余组织放入胰酶中置于冰上，全部胚胎分离完后统一用剪刀剪碎，于37℃摇床上孵育15分钟；
E.加入完全培养基(15％胎牛血清的DMEM培养基)进行中和，离心弃上清；
F.将沉淀用新鲜完全培养基重悬吹散后，依照密度种入培养皿中；
G.细胞铺平后6小时后或过夜进行换液或补液。
10、转染siRNA敲低基因
(1)将RPE-1细胞种植于24孔板中，以干涉时细胞密度约20％为宜，转染前更换新鲜培养基至一半总体积量。
(2)转染：siRNA终浓度为20nM，步骤如下(以6孔板为例)：
A.取20-50nM siRNA混于100μL Opti-MEM中；
B.取2μL RNAiMax转染试剂混于100μL Opti-MEM中，静置5分钟；
C.将A加入B中混合均匀，静置15分钟；
D.向细胞内逐滴加入转染体系，200μL/孔，终体积为1mL。
(3)转染6小时后更换培养基。
11、免疫荧光
(1)冰冻切片的免疫荧光
A.溶解OCT：切片取出，迅速置于冰PBS中，室温放置10-15分钟；
B.画圈：在切片上组织外围，用疏水笔画好疏水圈；
C.固定：圈内滴加4％多聚甲醛，固定10-15分钟；
D.洗涤：PBS洗两次，每次5分钟；
E.封闭：3％BSA(0.1％PBST配制)室温1小时或4℃过夜；
F.一抗：弃封闭液，滴加一抗(如GT335/Ac-tubulin/γ-tubulin)4℃过夜；
G.洗涤：PBS洗三次，每次5分钟；
H.二抗：滴加二抗(如Alexa Fluor 488/546)室温1小时，避光；
I.洗涤：PBS洗三次，每次5分钟，避光；
J.染核：Hoechst配制浓度为1：1000，PBS配制，避光；
K.PBS洗三次，每次5分钟，避光；
L.封片：滴取封片剂，将盖玻片盖于组织上，避光晾干，4℃留存。
M.待组织切片晾干后，用Zeiss LSM 880显微镜拍摄图像。
(2)细胞玻片的免疫荧光(冰甲醇固定法)
A.解聚微管：取出孔板置于冰上5分钟后，弃去培养基；
B.洗涤：PBS洗两次；
C.固定及透化：加入冰甲醇置于-20℃冰箱5-10分钟；
D-L.步骤同免疫荧光(1)中D-L；
M.待细胞爬片晾干后，用DeltaVision显微镜拍摄图像，进行形成纤毛细胞比例及长度统计。
三、实验结果与分析
1、Cep55纯合敲除小鼠围产期致死情况
统计Cep55基因敲除小鼠各基因型围产期致死情况，如表1。
表1 Cep55纯合敲除小鼠围产期致死
Figure BDA0002684252360000091
注：[n*]代表死亡幼仔数目；％为该基因型幼仔数与该时期总幼仔数的比值。
为确定Cep55纯合敲除小鼠致死时间，本发明分别取不同时间Cep55杂合孕鼠，观察并记录其幼仔基因型及死亡情况。由表1分析可知，Cep55杂合孕鼠杂交产生后代符合孟德尔遗传定律，Cep55纯合敲除小鼠在出生P0-P2天死亡，具有纤毛相关疾病围产期死亡的表型。以上结果提示，Cep55对小鼠胚胎发育有重要的作用。
2、Cep55纯合敲除小鼠显示纤毛相关疾病病例特征——脑发育异常、多囊肾
首先，本发明对E18.5胚胎进行脑部外观评价。与野生型相比，Cep55纯合敲除胎鼠具有脑小症表型，呈现大脑皮层变薄等缺陷(图1中a)。其次，本发现利用核磁共振成像技术对小鼠进行脑积水评价，发现Cep55纯合敲除胎脑具有脑积水表型(图1中b)。进一步，本发现对E18.5胎鼠的大脑与肾脏进行HE染色，从组织形态学分析得到，Cep55纯合敲除胎鼠具有纤毛相关疾病临床特征，如脑发育异常，表现为脑小症、侧脑室及第3脑室扩张，海马发育不全等(图1中c)，以及肾小球及肾小管微囊肿(图1中d)。
3、Cep55纯合敲除胎鼠脑室、肾脏等细胞纤毛过度生长
为了检测Cep55纯合敲除鼠出现的纤毛相关疾病表型是否由于纤毛异常导致，本发明以GT335/Ac-tubulin抗体标记初级纤毛，利用小鼠脑与肾的冰冻切片，进行免疫荧光染色，以评价P0.5天小鼠脑室脉络膜上皮细胞(CPECs)及E18.5天胎鼠肾小管上皮细胞(RTECs)纤毛发生情况。结果显示Cep55纯合敲除胚胎中纤毛较野生型相比长度增加(图2中a-d)。进一步，本发明分离小鼠胚胎成纤维细胞(MEFs)，并进行免疫荧光染色，评价其纤毛形成情况。结果显示Cep55纯合敲除MEFs纤毛发生比例及长度均增加(图2中e-h)。这些数据说明Cep55纯合敲除小鼠大脑和肾脏发育异常可能由于其体内纤毛过度生长。
4、CEP55参与调控纤毛去组装
为了进一步验证CEP55调控纤毛形成的功能，本发明在人视网膜色素上皮细胞(RPE-1)建立了纤毛评价实验体系。本发明在含血清培养的RPE-1细胞中利用siRNA敲低CEP55，发现敲低CEP55直接诱导纤毛形成(图3中a和b)。并且，在去除血清培养诱导纤毛形成的RPE-1细胞中，敲低CEP55增加纤毛长度(图3中c和d)。进一步，本发明在RPE-1细胞中敲低CEP55，再利用上述纤毛去组装体系，发现敲低CEP55抑制血清诱导的纤毛去组装(图3中e-g)。这些数据表明CEP55通过介导纤毛去组装，负向调控纤毛发生，对纤毛动态平衡至关重要。
以上结果说明，Cep55敲除小鼠存在与人类纤毛相关疾病一致病理特征，CEP55具有调控纤毛去组装的功能。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
<110> 军事科学院军事医学研究院生物医学分析中心
<120> CEP55蛋白在调控纤毛去组装和制备纤毛相关疾病模型中的应用
<130> GNCLN201052
<160> 10
<170> PatentIn version 3.5
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ccagggagaa acgtttgtgg tagcaggttc ctaaaacacg ttaactgtgc atttgcctcg 1080
gcagattgta ctagcaattt tacttggtat ttgtcaagat agtgggcaaa accaggttga 1140
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tatgactatt agtattaatt acagtttaag aatatgtgaa tctccttttc ttttgttgta 1320
ttgctgttgc ctgctacact gttctgaaag cttcctagca tccgagactc tatgttgttt 1380
cttcttcttt ctgttgcttt agccagctta tatccagtcc cgattaggtt acctggtaaa 1440
aaggctttgt agtaatagtc tgtgatttgt ttaaacagtt ttgtgcagct aggctgcctt 1500
cagtctatgc aaatgaggct ggccgtgaac tcctgcccat cctgcctcta cttcctgagt 1560
gctaggatta ttagcctgta tcaccatagc tttgtagtaa cagttgaatg aaggaaggtc 1620
ttatcttcct ttcaattcca tgatcacctt gccttcattt ttatctcagg acatatgaga 1680
aaaatggttt tattaaatct cttaatggag agctattttt tacattattt atatatacag 1740
tgtattttac tacacatctg cctgcctaga gtttgtattc tacatttgct tctaaactct 1800
catctagtac tattagcaga tgtgaaactt ggcctaactt catttgccca ttcatttctt 1860
tcaccccccc ccaatctctg agaattacac ctctttttct ataaatgtct tgtgttggat 1920
tttctcactt cttcctgcca tacgctctaa aacgaataag atagagttct gtagcctccc 1980
ccagccttga agcaggctga ttcagacttt gctgccactg aatatgaggt cacctggggt 2040
gtagccttcg acttagatgg ctatattatg tcaactacag atgctcttct ctgagactct 2100
gctacctgct gagagaacct tgagacatcc cggagacaca tcctatcctg catgtcgcct 2160
acttgtcttg gcttcatcat tcttttctca aagtctaagt ctctttcagc cccaccctgc 2220
cttccagaaa tacccggttc atgtaggcct cgggcgactt acatcagagt tcaactacac 2280
ataccttcta aagcagagag cacacgttga gttcatgact ataaactcag aattagagtt 2340
tttattcttg agtatgatat caccttgaaa gtggaaggcc tggggaccct tccactgatt 2400
gtaaaccaaa actttctgtt aatttacagg tgcctgtaaa cctataacct gggaggctga 2460
agcaggaggc ctgtgagttc ggtgccaccc aagttacgta gtgagttcca gactctcctg 2520
ggctgccttg agagatctgt tcgcaaatat cagcaaaaac aagtatgcaa attatatttc 2580
tagaaggtag atatttccct tgtatctacc acctaagagt tttagattca taataacacc 2640
ctaaaaagct tactcaattt tctttccgga ccccactgat ttggataaat cacttctatt 2700
tagagcagag tacattataa ggcctctgcc aagcctgaga tatgaaaatt taaactcaga 2760
cttcaaaagc agttgaattt ttaaaaatca ttttttgtat tttcacagat cattgcaacg 2820
atgtcttcaa gaagtcccaa agatttaatt aaaagcaaat ggggatcaag gcctagtagc 2880
tccaagtcag acactgcatt agagaaattt aagggagaaa ttgcagcctt taaaacatcc 2940
ctggatgaaa tcacctctgg gaaaggaaaa atggccgaaa aaggcagaag cagactcttg 3000
gaggtaaatg gtccttgaat cctttaaggt gaatacactc tagaaagcgg tgtgtgtggc 3060
tttctgatgc tacagtcttc ttcttcccgt tgcccgcgct tgacctgatc tctttaggtc 3120
tagaaagccg atcttgaatt tggaatctta aattcctgct ttgatttttc tgacctttag 3180
acatacttta gttcttgcta aatctctctt ttaaaatgca attgagtatg tgttttgggg 3240
ttaccaggca ttaagttgtt tcagaaataa agacatgatt aatagttttg tatcttatat 3300
aatataatgt gctatgatct ggtttttttc ccctcacttt taaaagattt attttttgtg 3360
tatgagtgtt ttgccaacat gtatgtgtgc acatcatatg catgcctggc acctgcagag 3420
gtcaggagaa ggcatagggt cccctggaac tggagtttgg gtgattatta gctaccatgt 3480
gggtgctggg aattaaatac gattcctttg cgaaagcaac aagtgctctt aactactgaa 3540
ccaacagtcc actcccagac cctcacattt tttgttgttt ttgttttgtt ttgctctttc 3600
aggtagggtc tcatgtaata caggttggtc ttgaacttga actgaggatg agcctaaatt 3660
tctgattccc cttgcaccca cttttaagtt ctatgattat gtgttgtaac atttatttca 3720
acatttcaaa tattgaaaac aaaccaataa gcttggcatt catcatgtaa tttttttgat 3780
aatttatctt ttaaaaaata tttatttact tatttttatt tcatgtgtat tggtgttttg 3840
cctgcatgta tgcccatgtg agggtgttgg tgttttgcct gcatatatgc acatgtgagg 3900
gtgttgagtc tcctggaact accattacag ttatgagctg ctgtgtgtgt gctgagaatt 3960
gaatttatat cctctggaag aacagcttgt gctcttaacc actgagccat tctctctagc 4020
tcatttatct tagttatatt aatattattg atacaaattt gttatataat atcagtcttt 4080
tgtattgtta caacacagtg ctatctatga gtaatttgag tagggagaaa gagaaatatt 4140
tagctgctaa gtccaaaagc aaagtatgtc tggtaaggat cttcaagctg tgttatcaca 4200
taagagaagg ccaggcaagc atatgaggta tatggaggac caggcctgaa ttcattttaa 4260
ctgataagtg acccctgaca accacatgag tctactcaag agctctgtgc cctcatgacc 4320
ccatcacctc taacactgtt gcattgaagg atcatattca aaccatagca tgctgtgttt 4380
aagattgtaa aacatactag cttactgcat gtattgtcat tttcattggc tctataactt 4440
ggaaacgctc tgtttccatg atttggggga tgccagaaag atactgtgcc attctctacc 4500
tacttccctc ccctttaata tttactcagc tgttaacatg atttggtttt tgttttttgc 4560
tagttcacca gatatataaa aacaaaaaca aaacaatcaa acttatgcac ttaaaatcgc 4620
caaattgttt atccacttca aagtgttagt gttagggttc cttctaactg ttcagaatcc 4680
atcttatcgt tcctgttggc tgccctgtta gcctttcctt actgacagtt gtttttatca 4740
tctttctctt ggttctttcc tgtttctcca cagctatgtg acaagcattg gtttagcctc 4800
ctaccgccat tgatccagat gttttttcct ttctctctct ctttctctct gtctgtctgt 4860
ctgtctgtct atttggtgtg tatacatgtt aactagaaca cagatggcgt tgatgaaagg 4920
gcttgtgtgt ctgtgagctg tctaagcagg ggactgagct tcagcttgta ttttcagtcc 4980
tgatctcgta aaatccaagt tgatatgtaa taaatacttc ttaaacactt gttgcaatat 5040
actgtttagc tggatgtttg acctggctta gttctaagat tcttcttagt tcctatctag 5100
aaaatccaag ttcttgaagc cgagagagaa aagaatgttt attacctcct ggagaaggac 5160
aaagaaatac agagactcaa agaccaccta aggtcaagat atagctcttc ttcattgttc 5220
gagcagctgg aagagaaaac aaaggaatgt gagaagaagc agcagttgct ggagtcccta 5280
tccaaagaga cagatgtcct gaaaaaccag ctgtccgcca cgacgaaaag actttctgaa 5340
cttgaaagca aagccagtac actccacctg tcacaggtgc taagctttct ttaaactgag 5400
atctttgccc gtctggtaca acttataatt cctgtgagga gtgagttttc tacggtttgt 5460
gaaatgatgc aacaccttag ttagtgcacc tgcagatcac ttaccttctt attgtttggt 5520
tttgttttta gttgagtccc tcttccataa gtaaaaatag ttcatttggt tccctgcact 5580
ccccacacag tgttcctccc agatgaatag tcagaagaca gtctgtgctg tagctacaat 5640
aatcgagatt ttgctgagat tttatttcaa tagtttgcac tacataaacg ttatgttgac 5700
gtttttgatt cgttttcata tttgagataa acagttatcc caatgtgttt ttttctaact 5760
gcacatacat ttttttcttt ccctcttgtc tgagaggaac aatgtctcag gaagtacaag 5820
gatctgtgta aaaatcgtct gggtttaaat cttgattctg ccacttgata gcatgtaact 5880
ctgagcagac aaaccccttg cttctctggc tggagtttgt cagtcttcct tttataagcc 5940
ttgccttcat atcagtaaag ctgggccaag agcactgatg attaattatg ataatgatgg 6000
caagctgcct tgcagaaagc ttggaatata ctaagtgctt agttaatgat caaacctccg 6060
gttcttttga catctgaata ttactaacaa ttagtaatag tacccataga aaaataagtt 6120
cagttgtgat ttctcagtgg tgtgatgcgg tatgggttac ttttcctgtg gctgtgttaa 6180
aatactctga tgagagcagt ttaagggagg aagggtttgt tctggtttct agtccttggg 6240
gatcattcca tcttggctgg gaaggaggcc tggcagcaag aacaagatgg tcacattgct 6300
ctggcactca gcaaccaaag ggagaacaag tgaagccctg cctataaagc ttcaaggcct 6360
gaccccagtg acccccttcc tccagcaaga ctccatccct acaggttctg tagacttccc 6420
aaacaaccct agtatccaca caagcctttc cttacagata caccaagatc acccaaccct 6480
gaaaagtagg cttttcagtt tgctggctag cctgtgatga tagccacctg gttttctcta 6540
tgtgagaatg aagaaaggaa agccattatg gaattatttt ttgaattctg tgaaaactat 6600
acaaatagtg ttactattta gtattgttat agtctcccaa atctgaaact tttcaaatgt 6660
ttacaagtag aaaaattcac acctgacctc acatagctat cacagcaaga aactgaacac 6720
attaaacaat ttgtggacag gctatgtgta tatgtgaaac aaatggccaa gtggtggtat 6780
tgcatgcctt taatcccagt acttgtgagg cagaggcagg cagatctctg agtttgaagc 6840
cagcctggtc tacagagcaa gttctaggga aactagggct atatagagaa accctgtctt 6900
ggaaaaaaaa atgaatttca tatttagagt tgagtctcat cccaaatata tctcattatg 6960
tatctaaata tttcaaactc caaaaaagta cagtatgaaa cattttggtt ctggtaagtt 7020
tgggaatcct cagcctatgc tgatataaaa tgtaactcta gaaaacactt tatgtctttg 7080
atagtttggt tcgtttattt gaaatagcct agtttgactt tttttgatga aaactcagtg 7140
ggctatttgt aggggttgaa ggttctggat attggggggt ctaggaaatc acagtaaaat 7200
tagatctgaa gtctcggggg gcgggggtcc tagattgctt aggacagtgg ttttctacct 7260
ttcaaatgct gggactcttt aatacagtcc tcaggatgtg gtgaccccaa ccataaaatt 7320
atttcattgc tacttcataa ctctaatgtt gctactggta tgaatcataa tgaaagtatt 7380
tttggaaata gaggataacc aagggtggtg cgacccacag gttgagaact actggtatag 7440
gagactatcc agggaattta gttctggtgg tagtcccctg ataattctca cctggcttgg 7500
tggttcacca gtagtagagg tgtggtttca ttgtcttgag aaatgttttg tatttttatg 7560
gaaatataat gaaatttgta ttgttaagtt gatgatgtca gctactattg gcttatttac 7620
agagcatgcc tgcaaactgc ttcaactcct caatgaatag tattcatgaa aaggagatgc 7680
agctgaaaga tgtaagttgt tttgttgttg ttggtggtgg tggtggtggt taaacaatgg 7740
ataaagaaga caaactcgta tgttctttaa attttataga attggaagca ttaatgttat 7800
gactttaaaa aaaaaaaaaa aaaaaaacaa gtctaatgaa gagtcaggag aactgagtgt 7860
ttgagaccat ggtgtagaaa ctctgtagat tgcaacgccc tggaactaag cttccagctg 7920
ccttagcagc attcttttcc acctcagtct aactcgccca aattgttgct gttatggtgc 7980
aatttaattt atgcttttct ttttgtttat ctatttgata tgtatttatt gagtgaaaaa 8040
cagaaataaa aaggaaagga agggacatgc ctgctcctag gtatggtgga ggagaaagtt 8100
tattataggt aaaagggaaa gccagaagca gggggacatc tgggagtcca gagtggtctt 8160
gaccctgagc cacgtgagga cagatgggga gagagggtag ccaggtacag catccaggag 8220
gccaaaggta ttaaatgaat gaatgaatga atgaatgaat aaataaataa ataaataaat 8280
aaggtgggta acctataatg tctggattat attgggaaga acctcttggg gaagggcagc 8340
ccagccctgg atttaagtgt tcagggtagg ggtggggttc gtcagcctta ttctgtaacc 8400
tgtttggact ctgggggaac ctggaagcct gatcagcttt gatatgttaa gtaggcacct 8460
gagctgtttg tctgggattt gaaacctaac tttccttatc ttccagtctt tctccaagag 8520
ttgggaagga aatgtggtct atgtatgcgc agtagttctc cagaattaga catgaggtta 8580
ctttagatgg accggaaagc tttgctttat tatttctaac ttttataagt ggataagtaa 8640
gttttgttgg aaattgaata aaatgaggtc acaggactgt ctgcatcttt tcaagtttta 8700
gatgaaaata tatgaagtat aaagttgttg atataattat tagatcacca ggtagctgtt 8760
tatattacag aaacatggtg tgtttacctt gatgcctgat aagtctaccc ttaagatttc 8820
tgttttattt aatctagcaa gtgtcttttg aagtctattt gattttcaaa agaagaattt 8880
aaatatcttg agtatctata caggatggtt tggtactttt atcaaagtat ttacctggag 8940
gtaattgaaa taaacataat tttaaaatat gttacatatt taaatatgct agacataagt 9000
tacaaaatag agtatttcgg tatcatcttt tacagagaat tttgtccatc tgtgtatact 9060
ggtacctacc aattagatgc ctgtatttag gtcatttacc ttacatgcct tccattttct 9120
cctcctttgc ttcagtcttg ctggatttcc aaccattcct taaaaggaaa accctggttt 9180
cctgggtggg cccctctgtt tacataagaa aaactctagt gcagtaaaat ctaaaccaat 9240
tcacctgcaa gctaaatcta acctgagcct agactttttg agtccagagg attttcttga 9300
aatgtagatt ttggatttag taggtctggg gtggggcttc gattgtgcat ttcctgaatg 9360
ctgttgctgc tgatacaagg gcacccttgg attattccta ccttgaagag cttctagcta 9420
ctcagctctt ctgggttctt gaagatctcc tcatctgttt gaggaggagg aagaagagaa 9480
ttacccttgg gagacttgga gagcaagaaa gagccattca ttcactcctc agtggacgct 9540
gcgtgtttgc cttgtgagag cctggaaact ggttccatct cggttcccag gttacggcct 9600
caggaggctg gtccagaaga actagtattt gttccatgta aaataagctc taggaatgca 9660
tggatgtgtt tcctacagta ttcagtcacc atttaggtct gtcagacatt ttgaaataat 9720
tacctggtaa taattcttag gtttcttagg catctaaaag agacataaag gcagaaagag 9780
aagtgcatct gtccttagga aggagtgttt attggtggac agggttgtgt atgttgtatg 9840
tgggatgatc tcacccatgt ggatgggtgc atcttctact cctgtcactt tagattcaaa 9900
gaggccctgg ggccagttaa aggttcctag agcttttggc tataggcaag ttgaaaattc 9960
caaaagttct cagccagtct tagctagtgg agagctcttg gaatctttta ctgatatgga 10020
aatagccaag ggatcctgtg aaccccagac ctaatcgggc gcctctaatg gattaccttg 10080
tcccagcatt ttgctctgct gcatggtgtt tggagagggc atggacacac ccctgtggat 10140
ggttttgcca tatagcccac agcatgcagg cagacgagat tcccatggaa aaggtggctc 10200
agcctttctt agcgggagaa agtggctatc gtacctatgt tacattcctt cccgcatcct 10260
tcccttctgg agttgtcctg cttggctgca aaggaagctg ggaagtatag tctttctgaa 10320
ggaaactctg gataatgcca taaatgttcc cagatatcag tcttatcatt tctgtgcaaa 10380
ctatactgac cataaccagc ggaaggttgg cagaccaaga catggtctta atgttacaaa 10440
attggatttt taaacagctt agaggagata agtaacctta attttttttt taatttagaa 10500
gatacaaata gaaaagttct aagaatgaga atgatacagg tatctctttt tatattcttt 10560
acctaaattt aaccattcat gacagtttta agcccatttt tgtactattc ttataccttt 10620
cacctacaca aatatacaaa gtattttttc ctaagccatt caagagggag ctgcatatat 10680
ctgtaattat ttaaccctgc ctacatatgc atttactata ccttaagcag gaacttagta 10740
aatttaacat gaacacaata cttttcgtta acctaccatc cctaatccag ttttaccaat 10800
gaactcacat tttttaatta ttctactaca gctcacattt ttacctattt taaaaatatt 10860
gtccacagag tacttagcac atccatggcc aaacgtagag gaacaccacc cttccacagt 10920
acttcatttt ttttttaact ttagggtttt cattgtaatt tagatgggca tgtcgagtgc 10980
atgcctcctg tgtatataga tgaacagaaa gccagaagga atagctgatg tgacgatgtc 11040
tccataaatt taagcccaat atcaatgttt attgagagca tcctagtctg accattgatg 11100
gttttagaga agaaagggta gatgcatatg aattggagag aagagcaatg ttggcttagc 11160
tcagcattgt gacatagact tgtaatatta gctgcttgag aggctgaggc aggaggatca 11220
taagttcaag gcctgcttag gctacagttc tgtgtttgtg accagcaagg cagaggtaga 11280
ggctagaaga gcttactttg ttcagcatta ttgacattgt gtggctctta aatcttttct 11340
ttaaaaagat ttttctctcc caacttttgt tacttacaaa tcatataact aaagtctaat 11400
gagtctattc agtgttgctc ctgtgaatat gtttttagtg ttgaccactt ggcattagat 11460
aaccaggctg ggaaagtctg gtttcttctc cccagcctcc aacagccatt atgtggctgt 11520
aaacttaaat atatttttag ttcatgcact tcagtaaagt atcaacagat gcatttgaaa 11580
aacattgatg accatctgat gtagacttaa attatttgca ttgccacttc tttttttttt 11640
taagatgtat gtatgtatgt atgtatgtat gtatgtatgt atttatttat ttattacatg 11700
taagtacact gtagctgtca tcagacactg cagaagaggg agtcagatct cattacgggt 11760
ggttgtgagc cactacgtgg ttgctgggat ttgaacttcg gacctttgga agagcagtcg 11820
ggtgctctta cccactgagc catctcacca gtccgcattg ccacttctta aagataggca 11880
gatataaagt ttgatagaaa attagatagc tttaatattt aatagtgtaa tctgtaggta 11940
attagatata cacatgtgct ctttgggtca cacagtgttc ttctgttttg ttttgtttgt 12000
ttgtttgttt gagatagagt ttctctgtat aacagccctg gctgtcttgg aacttgctct 12060
gaagagcagg ctggcctcga actcacaaag atcctcttgc ctctgtttcc caagtgctgg 12120
gattaaaggc ttgcctcact aggcccagtg acaacagtgg gttttagaac tgaatttgaa 12180
catgaactag aagtcctcat gaggactcaa tccagaggag cttggccagt gtgaaggaat 12240
gttaggctaa ggcatacatc atcacatatg tgtttcttgg aacccttcac ccaaaacatt 12300
taacatgcac atgatatcag gttcggacgt gcataactat ttttcacaag taattgttaa 12360
aacttatttc ctggcctctc aggatcatag aacaagaggg gacaactgtg tcctattagc 12420
ctgacaagta ggttgggcag gatttgtaat ctcattgcat attcgtgttg tgttcctatt 12480
tctaggcctt cttccctccc cattaacagc agcttagtag tcttttcatt tatttatttt 12540
tatttttgag acagtgtctc agtatatagc cttggccggt cttaaactca gagatccttc 12600
tgcctctgtc tccccactac taagattaaa agtgggtgcc accacaccga gctatgtagt 12660
tctttagaga caaagtccca ccacatagcc cacgtgggtc tttaactcat ggtcttagct 12720
gattgccgca gaccctctgg gcccctttgt ctgcgtggaa cgggtctcta gtctcgggtg 12780
gacaaagtgt aggatgagtg acaaacagac gcgcacagga gagttcgtgt ggaatctgag 12840
tgtaattttt caaatcgaac atcagacttt ttatgcagaa gacaataagg aagttgggtg 12900
acatacccgc aaggtacaaa tgaggcaacc ggatgcttaa tgactcttac acagaacaga 12960
ggaatgaaaa cgcaaagact ggcaggaact gggcaataaa ataactgaga caaagtcagc 13020
tctatctaag gtcagctata gtcttagaag ccaggtgtga ggacttttca ctcctagggc 13080
aagggctttc acacccaagt catggttcta actagggagt tccgttctag ctaaccatct 13140
catgaataat gcaatactct aaaaccacag ctggatctac ttcctaaacc attgtaaatt 13200
cctgtatatg ggagtgactt ggcttttgat agtgtagcac cagaggctat tctgaatgtc 13260
actgaatagg caacactctt actgaattcc aagcccatgg tcttgctcaa ggacattcta 13320
ggactgttgg aacactggcg gaaggcttta gctatgtcag aattcaatct taaaaggcac 13380
ttatgatagg aaaatattga aagagagcac gtggatccat acactagact aaccgtctca 13440
tggaaatgga actcgggaat gaaaagttaa tgtatgggta gagaacacta gactccagga 13500
ggagagcttc cttgaaactc ttttgcctca tgagtgactt ttaagccttt aggcttgtcc 13560
agctgactcg accagagagc gtagccgctg atgtagagct ggggctatag gttccagcct 13620
aacccttctg taggttttca cttgaaaatt ccttattttc agtaacaaaa tggcctacct 13680
tccctagcaa aattgggtga aagaaagcaa ggaggagagg catgtgtctg tcttctagga 13740
atggctggtc tgcctgacag actccttttg tattgatttg gttaagtttc taagagtcaa 13800
ccagtaaagg atggtgagtg ttagtaccag agctctttct agaagtaaga ttttgaacag 13860
cttatttctg tgcctgtccc aggggcattc tagctaacag acaaaattag ttctctggaa 13920
actgactgtc atgtattcta aagggatgta tgtagcatgc agtgactgcc tgtaagtgac 13980
ttataagcca taagattttc tatgtattag aaaagacaat gttatggaga cacacacccc 14040
cttacatatt cttagttctg ggctttttac tatgctgttc atttttcatt cactataagg 14100
ctctggagaa aaaccagcaa tggcttgtgt atgatcagca gcgagaggcc tacgttaaag 14160
ggcttttagc aaagatcttc gagctggaaa agagaacaga gacagccgct gcttccctca 14220
cacagcagat gaagaagatt gaatcagaag gtactgagct atacaagtgg tcttttggga 14280
taagcctgac atttcccaac aggagtgatt catcaaattt agaccttttt ttaatatata 14340
tacatatgtg tcagaaagta aaatcacatc atgtctttta agcttcaagt aagaaatagt 14400
ctgtttcctt tccccttgtt ttgagattaa aagaggacac aagccacata ggtgagacgg 14460
aattatgaat acctttcccc tttctcaaag tgctcattcc ctccagagct gacctgggta 14520
ccagtttcct tttgcggggg gggggggggg ggggggggga gtggatgacg gattgagatg 14580
aagtcttgct agatcatagt ccaggctagc ctggtactta ccacgtagcc caagctaact 14640
tccaattttt ttgttacagg ttatcttcaa gtggagaagc aaaaatatga tcatctcttg 14700
gaaaatgcca aaaaagatct cgaagttgag cgacaagccg tgactcagtt gcgtttagaa 14760
ctcgatgaat ttcgaaggaa atacgaagaa gctcgaaaag aagttgaaga cttaaatcag 14820
ctgttgagct ctcagcgaaa ggcagacatc cagcacctgg aggaggacaa gcagaagacg 14880
gagaggatcc aaaagctgag ggaagagagc agtatcttca agggaaagct ggaagaggag 14940
aggaagaggt cggaggagct cttatcccag gttagccagc ctgaggtctc cgaacttcag 15000
aaatcaacca aggctaggga agcccggagc acgggcgaac ctgcaagggg cagaagcagt 15060
ctctactatg gaggcagacc gtgctttagt atctaaatag atggataagc tcaggaagat 15120
aaagcaggat ggtgtgaaaa gagagaacgg gcgcatggcg gcctcgcaga agggatggta 15180
ttagacagag gattgatggt gggaatctgc agtgaggctg gggattcccc atcagtggag 15240
gagatcacac ttctaaggct tgagggaaga gtagagcatg tgggaagcca gaggaaagca 15300
gtaagggaat ccagtcactc ccaggctgca ctgggagtgc gctggtgttg tgctccaacg 15360
aatgacatag caccttcatg atttgagaca gcatggtttg ggcgtgttag aattgaatac 15420
tactgtgaca gaaagtcatt aatgtaactt ttgcttggca acatggagga ctagaaagaa 15480
cagtgaagtc cgtacctacc ttttctgaca cacagcattc actatcaaat gcaagatatc 15540
ctctacttgc ctcctaagac gactgaggag taaagtgaaa gcactttgaa aactaagtag 15600
atgccaatgg cggttatggc tactagaata gaatgtttaa catccctcgg aagttgagag 15660
ctgaggaaca gtcagtaatc ctaacatttg cttcctccct cttcagtctg cttactttga 15720
ctgataggtg tgagtaccaa tatttaggaa attaggaaag gttaactaga tacaagtaaa 15780
aatgtccctg tcactagtgt aagagatgag gcacatcctt ctgttatcct gctctgaaat 15840
gatggctaac accaaagcct ccaggaacag gggcatggtc acacagagct gtacccatca 15900
ctatgtggct gctcagggga ctcaggcagg atgctgctgc taatcttgct taaatagaaa 15960
gcctcagata atgagctggg atgagtgtca cgcttagcag gtgttgtcct tgttactggg 16020
atgagtgatg tgggttcttt gtctggggag gagcaggtga gataatgcag gctgtcattg 16080
tatcctccag gtccgaattc tctacgattc tctgttaaag caccaagagg agcaggccag 16140
ggtagctcta ctggaacagc aggtatttac tgtggatgct ccaaaggcca ttcagctggt 16200
aaaaaccaga actccctctg gtatttatac tgttctatgg acaactactt gaaagttgta 16260
aaccaactct acaccattca tgtatctgta cctgtcctta ggccaaaccg agtcttagag 16320
gtgtggtggc tgttgtcctg acaggttgct cttcagtggc tgtagttcct acttctagcc 16380
taacaggatc tttttctagg ccagctcgta agttcaagca gagcagacac catctattct 16440
cacaatgtag acagaaattc ttaatatatt tcaaaccagg gaatagccat ctttcccctc 16500
ttttccacac ccaccaaaaa tgccaagaac gtgaaggaat aaccccaacc agcaacttct 16560
taagctgcct tctgctcatc cataccttag gccttggctt tgctggttca taaagcagtg 16620
tttgagccat aaacgtcact tactaacaat ctcactgtgc tcacatgagc tcttgataag 16680
aaggcccggc aagtgttctt gtgctgacaa agatggtaaa catatagtga agagaactgg 16740
gcaccatctg ccagctctaa ctgtctcgtc ctaacatatt ccagatgcag gcatgtactc 16800
tagactttga aaatgaaaaa ctcgaccgcc agaatatgca gcatcaactc tatgtgattc 16860
ttaaggaact ccgaaaagca aaaagtcaaa taacacagtt ggaatccttg gtaagctttg 16920
aatggctact gtaaacctta ttcttcatgc atggatttgg atttatggca ggctcatctc 16980
cctgggcctc tcatagtgtc ccatgctaga gcaaattgtg gctcctaacc attgcccagc 17040
ctccgtgctg gcactgaaat gggtcacact gtgagaagga aaaaaagaaa aaaggaaaaa 17100
cttattcttc attctttttt tgctaccttc ccatttgtgt cagctcagtt tccaaaccag 17160
ttacatagct taacctaacc ttacagcatc aattcagaac tcggaaggag tggtgccgcg 17220
gccctcttac tcataggtac tttactgatg tctgggacca tccctaccca ggttgggtgc 17280
caccattcta gtagttgttt gcagagaggt gagaaggtag aaaagaggac agcatgacat 17340
tctttttttt gtatttctaa tgaatctatg tgcatctatg tccccacatg ccccgttagt 17400
gcacattcat gtcttgtctt agaactacag aatatttgaa agtagagaaa tattataaat 17460
ataactccta gttcctaagg gagaggaaaa tattaactac taagtaactg aaaatgttta 17520
tattaataaa ataactatct tcataatgta tatttttaag gtttcctaac tattttaaat 17580
tcttttttta aaaagcaaag tctcaaagct gtataatgtc ctgctcatct gtgtgtgtga 17640
cttgtctgag ctactgaaag tcttggctgc tctcagtttc gcatagggtt ttgttgctgc 17700
tctgttttga tttatgcttg tgctgcccag ggttggtctg gggtctttgt acatctttat 17760
tccttagctt acttctgttt tacaaagctg gcaaacagat tcacattcct gaaaatgtca 17820
tgataaatcc cattattttg tgcagtgaat ttaagaaaag ggaaaggagt ggtgtcttcc 17880
ctctacagtc aactctgtgc ctcttagcaa agcctacata catgagcact aattcattac 17940
gtccctgagt aatgaattag tcacaccatc caagtgattc atgactacca catatgttac 18000
ccttaaaata ttgatgaaca gctgagcagt ggtggcacat gcttttaatc ccagcccttg 18060
ggaggcagag ataggcagat ctctgagttg gaggccagcc tggtctacaa agtgtattca 18120
agatagccag ggctccacag agaaactctt gttttaaaaa acaaaacaac gataacaaca 18180
aaaatattga tgagcagaaa tccaacacaa actgtctgta gggctgaagc acgcacgcag 18240
aggatgtggg agcagttcac actgctgagt gctcaggctt atgttcaaat ggtgttcatt 18300
tgccgtttgt tttgcagaag cagctacatg ggtttaccat cacagagcag ccattcccac 18360
tccagagaga acctgaaagc agagtgaaag ctacctcacc caaaagtccc agtgctgcac 18420
tcaatgacag cctagtggaa tgtcccaagt gcagcgtaca gtacccagcc accgagcatc 18480
gagacctgct tgtccatgtc gaatactgca tgaagtagca gaaccattct tgcctttgtg 18540
ctgaaaaagt ccacactgta ttctatgttt ctttataggc actttaaacc acagatgaca 18600
tcttttatac aagaaactcc atctgccctt ggcactctgg catatgaggg catccttgta 18660
tttttctggc cactttgcat ttccttggta gccattcctc cctgagttgg ttcgtcatca 18720
ggctgcagtg acagattgtg gtgagcagtg gctatccaaa cttgtagcac ttcacgctgt 18780
tgtaatgact agcatttcac acggttcttc tacttgataa acagagctat ttgggttttt 18840
ggttatggct taaagctact gagcagtaag caaacatttt atgctttggg ggttttaaga 18900
gaatcagaag gtaattaacc atggatctta ctgttttgtt ttttttaaat aagtgctttg 18960
tatactgaca cttccatttg atacctcttg ctattgtaga acggttacta gcaaagttgg 19020
gaagttttgt aatgttgagt gctgttaact gttctaagca caaagcaggt tttgtttgaa 19080
gaatgccttt aagtggctgt atatttttac tcctattacc tggccataac tttgacttaa 19140
atttcattag tacaggtcag tttcctaaaa tgccaaaatg aacaaaactt tagcaaaact 19200
atcttgcctc atttgatttg cagggatttt attatatcat tttgttttgt tttgtttttt 19260
agcactctta aagatggttg ggcagtgtaa ttatcagatg tttgttttct aaatagatat 19320
ttttatatat gctttatgta aaccaaaatg tgttttattc cttcagattt tctaacatgc 19380
ttacacttgg gctactattt agcaatatga aggaggaaat aataaaatta tttaataagt 19440
tttac 19445

Claims (10)

1.CEP55蛋白或其相关生物材料在如下任一中的应用：
(A1)调控纤毛去组装；
(A2)制备用于调控纤毛去组装的产品；
所述相关生物材料为能够促进所述CEP55蛋白表达和/或使其活性增加的物质或能够抑制所述CEP55蛋白表达和/或使其活性降低的物质。
2.能够抑制CEP55蛋白表达和/或使其活性降低的物质在如下任一中的应用：
(B1)抑制动物细胞或动物体中纤毛去组装，或制备用于抑制动物细胞或动物体中纤毛去组装的产品；
(B2)促进动物细胞或动物体中纤毛发生，或制备用于促进动物细胞或动物体中纤毛发生的产品；
(B3)制备纤毛相关疾病动物模型；
(B4)制备具有如下症状中全部或部分的动物模型：脑小症、侧脑室和/或第3脑室扩张、脑积水、肾小球和/或肾小管微囊肿、纤毛长度增加、形成纤毛细胞比例增加。
3.能够促进CEP55蛋白表达和/或使其活性增加的物质在如下任一中的应用：
(C1)促进动物细胞或动物体中纤毛去组装，或制备用于促进动物细胞或动物体中纤毛去组装的产品；
(C2)抑制动物细胞或动物体中纤毛发生，或制备用于抑制动物细胞或动物体中纤毛发生的产品。
4.一种调控动物细胞或动物体中纤毛去组装的方法，为如下任一：
方法A1：一种抑制动物细胞或动物体中纤毛去组装的方法，包括如下步骤：降低动物细胞内或动物体内CEP55蛋白的表达量和/或活性；
方法A2：一种促进动物细胞或动物体中纤毛去组装的方法，包括如下步骤：提高动物细胞内或动物体内CEP55蛋白的表达量和/或活性。
5.一种制备纤毛相关疾病动物模型的方法，包括如下步骤：降低动物体内CEP55蛋白的表达量和/或活性。
6.一种制备具有如下症状中全部或部分的动物模型的方法，包括降低动物体内CEP55蛋白的表达量和/或活性的步骤；
脑小症、侧脑室和/或第3脑室扩张、脑积水、肾小球和/或肾小管微囊肿、纤毛长度增加、形成纤毛细胞比例增加。
7.胞内CEP55蛋白的表达量和/或活性降低的动物细胞模型或体内CEP55蛋白的表达量和/或活性降低的动物模型如下任一中的应用：
(D1)筛选用于预防和/或治疗纤毛相关疾病的药物；
(D2)筛选能够改善如下症状中全部或部分的药物中的应用：脑小症、侧脑室和/或第3脑室扩张、脑积水、肾小球和/或肾小管微囊肿、纤毛长度增加、形成纤毛细胞比例增加。
8.根据权利要求4-7中任一所述的方法或应用，其特征在于：降低所述动物细胞内或所述动物体内所述CEP55蛋白的表达量和/或活性通过对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行抑制表达或敲除实现；
和/或
对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行抑制表达通过向所述动物细胞或所述动物导入能够抑制编码所述CEP55蛋白的核酸分子表达的siRNA实现；
进一步地，所述siRNA的核苷酸序列如SEQ ID No.1或SEQ ID No.2或SEQ ID No.3所示；
和/或
对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行敲除通过CRISPR/Cas9技术实现；
进一步地，通过CRISPR/Cas9技术对所述动物细胞内或所述动物体内编码所述CEP55蛋白的核酸分子进行敲除时，采用的sgRNA的编码序列中的特异性识别序列如SEQ ID No.6或SEQ ID No.7所示。
9.CPE55蛋白或编码所述CEP55蛋白的核酸分子作为标记物在制备用于纤毛相关疾病产前诊断的产品中的应用；
或，用于检测CPE55蛋白或编码所述CEP55蛋白的核酸分子的物质在制备用于纤毛相关疾病产前诊断的产品中的应用；
或，CEP55基因在制备用于治疗由于CEP55基因缺陷或异常引起的纤毛相关疾病的产品中的应用。
10.根据权利要求1-9中任一所述的应用或方法，其特征在于：所述纤毛为初级纤毛；
和/或
所述动物细胞为哺乳动物细胞；所述动物为哺乳动物；
进一步地，所述哺乳动物细胞为肾小管上皮细胞或胚胎成纤维细胞或人视网膜色素上皮细胞；
进一步地，所述哺乳动物为人或小鼠；
和/或
所述CEP55蛋白的氨基酸序列如SEQ ID No.4所示；
进一步地，编码所述CEP55蛋白的核酸分子的序列如SEQ ID No.5所示。
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