CN114196650A - E3泛素连接酶hrd1在调控初级纤毛发生中的应用 - Google Patents
E3泛素连接酶hrd1在调控初级纤毛发生中的应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域,具体涉及E3泛素连接酶HRD1在调控初级纤毛发生中的应用。更具体地,本发明提供了HRD1在促进纤毛发生、制备纤毛相关疾病的药物中的应用;以及,HRD1抑制剂在抑制纤毛发生、构建胚胎发育缺陷的动物模型中的应用。
Description
技术领域
本发明涉及生物技术领域,具体涉及E3泛素连接酶HRD1在调控初级纤毛 发生中的应用。
背景技术
初级纤毛是从中心体的母中心粒发出、从细胞表面延伸出的棒状结构。正常 情况下,每个细胞仅有一根初级纤毛。它是一种在进化上非常保守的细胞器,在 低等动物斑马鱼和果蝇体内均有分布,更是广泛存在于哺乳动物的各个组织。初 级纤毛曾经被认为是一种退化的没有功能的细胞器,但近年来研究发现初级纤毛 上富集了多种信号转导通路的受体蛋白,如Hedgehog通路受体、Wnt通路受体、 Notch通路受体和PDGF受体等。纤毛如同细胞天线一样能够接收并整合胞外信 号,在控制细胞生长分化和调节组织器官发育成熟等方面具有重要功能。
越来越多的研究表明,初级纤毛相关基因的突变常常会导致其结构缺陷或功 能紊乱,并引发一系列人类遗传性疾病,统称为纤毛相关疾病(ciliopathy),包 括肾消耗病、Joubert综合征、Bardet-Biedl综合征、Meckel综合征和综 合征等。这类疾病可能出现的临床症状有神经系统发育不全、视力减退、耳聋、 智力低下、生殖缺陷、肾脏肝脏多囊性病变、多趾、肥胖及糖尿病等。很多纤毛 疾病严重时可导致胎儿在胚胎期死亡,或在婴幼儿时期出现器官畸形、功能缺陷 等病征。此外,纤毛发生异常与肿瘤发生密切相关,有研究表明纤毛的丢失是细 胞发生癌变的前提。因此,深入探索纤毛的发生机制和生理、病理调节功能,将 为纤毛相关疾病和肿瘤的诊治提供新的理论依据。
HRD1/SYVN1是介导内质网应激(ER stress)过程中非折叠或错误蛋白降解 途径(ERAD)不可缺少的E3泛素连接酶,发挥着重要的保护功能。它参与识 别内质网中损伤蛋白并对其进行K48位泛素化,从而保证损伤蛋白被转运出ER, 进入蛋白酶体降解,减少对ER的损伤。除了在ER stress过程中扮演了重要角 色,HRD1蛋白还在其它生理过程中发挥重要功能,比如调控P53依赖的凋亡、 PGC-1b依赖的线粒体代谢和DC细胞中BLIMP1依赖的MHC-II表达等。
发明内容
在探索纤毛组装机制过程中,我们发现在细胞中敲低HRD1显著抑制了初 级纤毛发生,并且,在HRD1敲低的细胞中回转HRD1基因,可以逆转HRD1 敲低导致的纤毛缺陷表型。
本发明所提供的研究成果能帮助人们加深对于初级纤毛组装过程的认识和 纤毛缺陷相关疾病的理解,同时也可能为纤毛疾病的诊治提供新的策略。
HRD1在促进纤毛发生、治疗纤毛相关疾病中的应用
一方面,本发明提供了一种促进纤毛发生、治疗纤毛相关疾病的方法,所述 方法包括使用表达HRD1/促进HRD1表达的生物材料。
本发明所述“纤毛”在结构和功能上分为动纤毛和静纤毛,静纤毛在后续的 研究中又被称之为初级纤毛。
优选地,所述纤毛是初级纤毛。
优选地,所述纤毛相关疾病包括初级纤毛相关疾病包括:Bardet-Biedl综合 症(BBS)、青少年消耗性肾病(NPHP)、Senior-Loken综合症(SLNS)、Alstrom 综合症(ALMS)、Meckel-Grubber综合症(MKS)、Joubert综合症(JBTS)、1型 口面指综合症(OFD1)、埃利伟氏综合症(EVC)及先天性利伯氏黑矇(LCA)、 多囊肾病(PKD)等。
优选地,所述生物材料包括编码基因、表达盒、重组载体、细胞(重组细胞)、 蛋白质(蛋白)。
优选地,所述使用促进HRD1表达的生物材料包括HRD1促进剂。
如本领域所熟知,所述“编码基因”是在基因组上的能够被转录成RNA的 区域,所述RNA具有调节功能、催化功能,和/或编码蛋白。
本发明所述编码基因可以是包含或不包含内含子的编码序列,凡可编码生成HRD1的mRNA或蛋白的核酸序列皆包含在所述编码基因的范围内。
优选地,所述HRD1的编码基因还可以与基因工程中常用的调控元件连接, 从而构建成表达盒或重组载体。
优选地,所述常用的调控元件包括但不限于增强子、启动子、内部核糖体进 入位点(IRES)和其他表达控制元件(例如转录终止信号,或者多腺苷酸化信号 和多聚U序列等)。
优选地,所述载体是表达载体,表达载体可以通过转化,转导或者转染导入 宿主细胞,使其携带的遗传物质元件在宿主细胞中获得表达。所述表达载体包括 成品表达载体或根据使用目的自行构建的表达载体,所述成品载体示例性的包括 pVAX1,pBudCE4.1,pTracer CMV2,pcDNA3.1等。
优选地,所述启动子可以根据使用目的选择无特异性启动子(组成型启动子)、 或特异性启动子。所述特异性启动子还可以包括细胞特异性启动子、组织特异性 启动子、器官特异性启动子;可以根据需要HRD1表达的具体环境进行选择。
本发明所述“特异性启动子”所启动的HRD1中仅在需要的部位特异表达。 从而克服了无特异性启动子启动的外源基因在受体中非特异、持续、高效表达所 造成的浪费,增加促进基因表达的效果。示例性的举例如:骨γ-羧基谷氨酸蛋白 2(OG-2)启动子(成骨细胞表达)、3-酮酸CoA转移酶2B(Oxct2B)启动子(单 倍体精细胞表达)、表面活化蛋白B(SP-B)启动子(肺细胞表达)、突触蛋白启 动子(神经细胞表达)、Wiskott-Aldrich综合征蛋白(WASP)启动子(造血细胞 表达)等。
示例性的无特异性的启动子包括但不限于巨细胞病毒(CMV)极早期启动 子,牛痘病毒的H5、P7.5和Pll启动子,延长因子l-α(EFla)启动子,泛激素 C启动子(UBC),磷酸甘油酸激酶-1(PGK)启动子等。
优选地,本文中使用的术语“重组细胞”,是指含有前述HRD1编码基因或 者其中导入了前述表达盒、重组载体,并且支持HRD1编码基因、前述表达盒、 重组载体的复制和/或表达的细胞。所述重组细胞可以是原核细胞例如大肠杆菌, 或者是真核细胞例如真菌、酵母、昆虫、两栖动物、线虫、哺乳动物细胞等。
所述哺乳动物细胞包括人类细胞,具体可以包括取自于人体的细胞或市售的 成品细胞系:例如RPE1细胞、SW480细胞、u87MG细胞、HOS细胞、C8166 细胞、MT-4细胞、Molt-4细胞、HeLa细胞、HT1080细胞、293细胞、TE671细 胞等。
另一方面,本发明提供了一种促进纤毛发生、治疗纤毛相关疾病的产品,所 述产品包括表达HRD1、促进HRD1表达的生物材料。
优选地,所述产品包括药物、药物组合物、试剂盒。
另一方面,本发明提供了HRD1在促进纤毛发生、治疗纤毛相关疾病中的应 用。
优选地,所述HRD1是表达HRD1、促进HRD1表达的生物材料、或前述产 品。
抑制纤毛发生、构建胚胎发育缺陷的动物模型
另一方面,本发明提供了HRD1抑制剂在抑制纤毛发生、构建胚胎发育缺陷 的动物模型中的应用。
优选地,所述抑制剂包括siRNA干扰、CRISPR/cas9方法、同源重组、基因 敲除、基因置换、基因沉默、点突变实、化学药物、Morpholinos方法所使用的 试剂。
优选地,所述抑制剂是siRNA干扰方法、Morpholinos方法中所使用的试剂; 更具体地,如本发明具体实施例所述。所述Morpholinos(反义吗啉环寡核苷酸) 方法是斑马鱼中最为常见的一种基因敲降技术,其基本原理是把核苷酸上的五碳 糖用吗啉环取代,并对原有的磷酸基团做一定的改变。
优选地,所述应用是体外发生的,例如抑制具体细胞系的纤毛发生;更具体 地,如本发明具体实施例所述。
优选地,所述动物模型包括任何模式生物,例如线虫、果蝇、斑马鱼、小鼠 等;具体地,如本发明具体实施例所述,所述动物模型是斑马鱼。
优选地,所述胚胎发育缺陷的动物模型还具有KV泡纤毛发生缺陷、左右不 对称性缺陷的特征。
另一方面,本发明提供了构建纤毛缺失的动物模型的方法,以及该方法所构 建的动物模型;所述方法包括敲低受试动物的HRD1的表达。
检测HRD1在判断纤毛状态、诊断胚胎致死中的应用
另一方面,本发明提供了判断纤毛状态、胚胎发育状态的方法,所述方法包 括通过HRD1表达量的检测结果进行判断。
另一方面,本发明提供了一种判断纤毛状态、胚胎发育状态的试剂盒,所述 试剂盒包括检测样品中HRD1的表达量所使用的试剂。
另一方面,本发明检测HRD1的表达量的试剂在判断纤毛状态、胚胎发育状 态中的应用;
所述HRD1在纤毛状态不正常、胚胎发育状态不正常的样品中表达下调。
所述“纤毛状态不正常”包括纤毛缺失,所述“胚胎发育状态不正常”包括 胎儿在胚胎期死亡,或在婴幼儿时期出现器官畸形、功能缺陷等病征。
优选地,所述样品包括待检测的细胞、胚胎、组织等。
优选地,所述检测表达量的试剂包括检测蛋白表达量和/或mRNA表达量的 试剂。
优选地,所述检测HRD1蛋白表达量的试剂可以是以下任意方法中所使用 的试剂:蛋白质印迹(Western Blot法)、酶联免疫吸附测定(ELISA)、放射性免 疫测定(RIA)、夹心测定、免疫组织化学染色、质谱检测法、免疫沉淀分析法、 补体结合分析法、流式细胞荧光分析技术和蛋白质芯片法。
优选地,所述检测HRD1 mRNA表达量的试剂可以是以下任意方法中使用 到的试剂:基于PCR的检测方法、Southern杂交方法、Northern杂交方法、点杂 交方法、荧光原位杂交方法、DNA微阵列方法、ASO法、高通量测序平台方法。
优选地,所述检测HRD1表达量是检测HRD1蛋白表达量的试剂。
优选地,所述检测HRD1蛋白表达量的试剂包括HRD1抗体。
优选地,当HRD1检测试剂检测到HRD1表达时,显示出可检测标记。
如本文中所使用的,术语“可检测的标记”是指,可通过荧光、光谱、光化 学、生物化学、免疫学、电学、光学或化学手段检测的任何组合物。在本发明中, 特别优选的是,此类标记能够适用于免疫学检测(例如,酶联免疫测定法、放射 免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。这类标记是本领域熟 知的,包括但不限于,酶、放射性核素、荧光染料(例如,异硫氰酸荧光素、荧 光素、异硫氰酸四甲基罗丹明、藻红蛋白、德克萨斯红、罗丹明、量子点或花菁 染料衍生物)、吖啶酯类化合物、磁珠、测热标记物(例如胶体金、有色玻璃、 塑料珠)、以及用于结合上述标记物修饰的亲和素(例如,链霉亲和素)的生物 素。
本发明所提供的方法或应用的施用对象、以及上述受试动物,都指动物受试 者,特别是脊椎动物受试者,落在本发明的范围内的合适的脊椎动物包括但不限 于脊索动物亚门的任何成员,包括灵长目动物(包括人)、鱼(包括斑马鱼)、啮 齿动物(例如,小鼠、大鼠、豚鼠)、兔形目动物(例如,家兔、野兔)、牛科动 物(例如,牛)、绵羊类动物(例如,绵羊)、山羊类动物(例如,山羊)、猪类动 物(例如,猪)、马科动物(例如,马)、犬科动物(例如,狗)、猫科动物(例 如,猫)、鸟类动物、爬行动物等。
本发明的优点和有益效果:
(1)本发明提供了一种全新的初级纤毛缺失的动物模型,所述动物模型中 HRD1低表达,可以用于研究治疗纤毛相关疾病的发病机制、研究和测试新药, 提供了良好的可视化的动物模型;
(2)本发明阐明了HRD1和纤毛发生之间的关系,而纤毛相关基因的突变 和蛋白功能的缺失与多种复杂疾病相关,很多纤毛相关疾病严重时可导致胎儿在 胚胎期死亡,或在婴幼儿时期出现器官畸形、功能缺陷等病征,因此通过检测 HRD1的表达量可以在及时的检测胚胎发育状态,为纤毛相关疾病的诊断提供新 的策略;
(3)同时本发明在具体实施例中证明了过表达HRD1可以改变纤毛缺失的 状态,为纤毛相关疾病的治疗提供新的策略。
附图说明
图1是初级纤毛检测结果图,图1A是荧光下显微镜下结果图,图1B是细胞数 量统计结果图,图1C是HRD1的表达检测结果图。
图2是回转HRD1后对初级纤毛进行检测的结果图,图2A是荧光下显微镜下结 果图,图2B是细胞数量统计结果图。
图3是斑马鱼胚胎发育情况的检测结果图,图3A是体式镜下结果图,图3B是 斑马鱼胚胎正常、脊柱弯曲、心脏肿大表型统计结果图,图3C是hrd1-sMO抑 制剪切核酸大小检测结果,图3D是hrd1-aMO抑制翻译的表达检测结果图。
图4是斑马鱼对称性的检测结果图,图4A是显微镜下结果图,图4B是cmlc2 左侧分布、居中分布、右侧分布斑马鱼胚胎数统计结果图。
图5是斑马鱼KV泡纤毛的检测结果图,图5A是荧光下显微镜下结果图,图5B 是KV泡内纤毛数量统计结果图,图5C是KV泡内纤毛长度统计结果图。
具体实施方式
下面结合实施例对本发明做进一步的说明,以下所述,仅是对本发明的较佳 实施例而已,并非对本发明做其他形式的限制,任何熟悉本专业的技术人员可能 利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明 方案内容,依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化, 均落在本发明的保护范围内。
实验方法
1.细胞培养和转染
RPE1细胞在含有2mM L-谷氨酸盐、10%胎牛血清和青链霉素双抗的 DMEM/F12完全培养基中培养,均置于37℃恒温和5%CO2气体的饱和湿度培 养箱中。
种细胞12h后开始转染,更换新鲜培养基,过表达质粒用Thermo Scientific公 司LTX with PlusTMReagent,干涉转染试剂为Invitrogen公司的LipofectamineTMRNAiMAX。轻柔混匀,静置15min后加入细胞培养基中,6h后 换液。
在做mcherry-HRD1逆转HRD1敲低导致的纤毛缺陷时,要求细胞密度转染 的时候在20%左右,更换新鲜培养基siRNA用量为50mM先干涉,6h后换成完 全培养基;第二天更换新鲜培养基每个24孔板每孔0.5μg质粒过表达,6h后换 成完全培养基继续培养。
2.免疫荧光染色
(1)将细胞接种在有盖玻片的24孔板中。
(2)将血清饥饿或者正常培养的细胞置于冰上处理10min,废除细胞内一些 不稳定的微管。
(3)固定:纤毛染色或者中心体定位蛋白染色,采用冰甲醇-20℃固定打孔5-10min;过表达HRD1质粒染色直接用4%多聚甲醛室温固定10min(冰甲醇固定 法很容易导致外源表达质粒丢失);转染GFP-centrin2的细胞,为了取得更好的 中心体定位效果,采用冰甲醇-20℃固定打孔2min后,再用4%多聚甲醛室温固 定10min。
(4)打孔:PBS洗去多聚甲醛后,加入含0.1%Triton X-100的PBS在冰上 孵育10min(冰甲醇固定法不需要此步)。
(5)封闭:用封闭液(PBS+3%NGS+0.1%Trinton)室温封闭1h。
(6)一抗:对应抗体采用合适比例稀释于封闭液中,将玻片抠出来置于预先 准备好的包有封口膜的玻璃板上,每孔30微升,4℃过夜或室温3h孵育。
(7)将玻片转移置24孔板中,用PBS洗三次,每次3min。
(8)二抗:标记488、546或者647的相应种属二抗1:400稀释于封闭液 中,将玻片抠出来转移到预先准备好的包有封口膜的玻璃板上,每孔30微升, 室温避光保湿孵育1h。
(9)将玻片重新转移置24孔板中,PBS洗三次,每次5min。
(10)染核:DNA染料Hoechst 1:500配于PBS中,每孔400微升,室温避 光染核10min。
(11)封片:用封片剂封片,室温静置至干燥固定。
3.Western
Blot蛋白印记杂交
(1)由于RPE1细胞特别薄,收取蛋白的时候蛋白量特别少,我们一般准 备一个六孔板的细胞用100微升M2 buffer裂解,混匀后冰上裂解30min,12000 rpm离心10min后吸取上清。
(2)采用Bio-Rad蛋白定量测定蛋白浓度后,添加对应体积M2 buffer时每 组蛋白浓度一致,在加入相应体积5*loading,使蛋白充分变性。
(3)将蛋白样品煮沸10min后转移至冰上待冷却到室温后,用进样针加入 到SDS-PAGE胶加样孔内。
(4)进行SDS-PAGE常规电泳,浓缩胶电压为80V约30min跑完,分离胶 电压为120V,约2-3h,分离时间与需要检测蛋白分子量有关系。
(5)使用PVDF膜转印,恒流400mA。
(6)用5%的牛血清白蛋白(BSA)或者脱脂奶粉室温封闭1h。
(7)稀释相应一抗于封闭液中,室温孵育1h或4℃过夜,具体时间与抗体 效价有关系。
(8)TBST洗膜3次,每次5分钟。
(9)稀释相应二抗于封闭液中。常规二抗1:5000使用,室温孵育1h。
(10)TBST洗膜3次,每次5分钟。
(11)孵育ECL发光液暗室压片,压片时间与抗体效价有关系,常规1min- 3min,经过显影和定影后观察目的蛋白的条带。
4.斑马鱼原位杂交
4.1合成探针
(1)PCR引物设计特定针对斑马鱼hrd1序列的引物,靶向序列在700bp左 右,从斑马鱼cDNA文库中将目标序列用设计引物PCR扩增出来,凝胶回收目 标条带。
(2)连接、转化,将目标片段连接到T载体上。3.5微升回收目的片段+0.5 微升切好的T载体,采用蓝白斑筛选挑取白斑。
(3)挑菌,采用T7+hrd1-F与T7-hrd1-R两对引物菌液PCR鉴定,确定 hrd1转录方向。
(4)摇菌,提取质粒,选择合适的内切酶,37℃酶切充分,凝胶回收有明 显滞后的条带。
(5)酶切产物回收:(此步开始无RNase操作)
a.加ddH2O补齐至100微升,加入十分之一体积3M醋酸钠10微升,再加 入2倍体积无水乙醇200微升,-20℃沉淀1-2小时。
b.4℃,13200rpm离心15min,弃上清。
c.加入75%乙醇200微升,4℃,13200rpm离心15min,弃上清,晾干。
d.加入7微升RNase-free water,用RNase-free枪尖不断吹洗侧壁上的沉淀, 转移至RNase-free的EP管中,-20℃保存。
(6)探针合成:(10微升体系)
模板DNA5.5微升+DIG 1微升+DTT1微升+转录缓冲液2微升+RNase inhibitor 0.2微升+T7 or Sp6 polymerase 0.5微升
超净台冰上配置上述体系,37℃孵育1.5小时,160V电泳检测RNA质量, 合成后加入1微升DNase I,37℃消化模板DNA15min。
(7)纯化RNA
a.加入RNase-freewater补齐至100微升,再加入10微升3M NaAc,最后 加入200微升无水乙醇,混匀,至于-20冰箱,沉淀2小时或者过夜。
b.4℃,13200rpm离心15min,弃上清。
c.加入75%乙醇,4℃,13200离心15min,弃上清。
d.吸干上清,不宜晾干,加入30微升RNase-freewater溶解即可。
e.最后加入270微升Hybe+(杂交液),-20℃储存,工作时1:200使用。
4.2原位杂交第一天
(1)收取14h胚胎,用4%多聚甲醛,4℃固定过夜。
(2)换成PBST,人工手动剥壳,用注射器枪尖,小心不要伤到胚胎。
(3)换成无水乙醇,-20℃脱水5h以上,时间越长约好。
(4)复水,一次用75%甲醇,50%甲醇,25%甲醇,PBST洗涤胚胎,每次 五分钟,躺着放,让胚胎与溶液充分接触。
(5)Permeablization,室温,加入proteinase K(1ug/ml),处理胚胎,16h前 的胚胎不需要该步,24h的胚胎该步处理6min。
(6)终止反应,加入PBST置换3次,每次5min。
(7)采用4%PFA室温固定20min,平放桌上即可,再用PBST洗五次,每 次5min,最后用Hybe-/PBS泡5min。
(8)预杂交,65℃,用Hybe+孵育固定后胚胎1h。
(9)杂交,吸去上步用Hybe+,加入1:200稀释好的探针,每管100微升, 65℃过夜,至少12h。
4.3原位杂交第二天
(1)回收探针,放-20℃储存,可反复利用多次。
(2)采用预热到65℃的Hybe-与SSC溶液洗涤胚胎,最终梯度置换成浓度 为0.2SSC的溶液。
(3)采用常温的0.2SSC与MAB tw溶液洗涤胚胎,最终梯度置换成100% MABtw。
(4)加入MAB block,常温震动1h。
(5)弃掉封闭液,加入抗体稀释液,保鲜膜包好,震动一会儿,4℃过夜。
4.4原位杂交第三天
(1)室温震动1h,回收抗体,置于4℃保存,可再用两次。
(2)采用MABtw室温洗涤胚胎八次,每次15min。
(3)采用BCL室温洗涤胚胎三次,每次15min。
(4)采用BM purpleTM:BCL buffer=1:1,避光,倾斜,时间长短,因样 品而异。
(5)染好后,用PBST洗三次,每次5min。
(6)采用脱色液脱色,10-15min。
(7)用PBST室温震荡3次,每次5min。
(8)4%PFA室温至少固定20min。
(9)最后置换成90%丙三醇,4℃保存,前往拍摄。
实施例1、E3酶HRD1定位于中心体并调控初级纤毛发生
siRNA干涉(RNA interference)实验中具体选用5’- UUCUCCGAACGUGUCACGUAA-3’为对照(附图中标记为siCtrl,Seq ID NO.:1); 5’-GCCAAGAGACUGCCCUGCAACCACA-3’(附图中标记为siHRD1-1#,Seq ID NO.:2)和5’-GAUACUUGUCUGGCCUUCACCGUUU-3’(附图中标记为siHRD1- 2#,Seq ID NO.:3)为实验组对HRD1进行敲低。按照通用方法进行siRNA干涉 实验后,结果显示:HRD1敲低抑制RPE1细胞初级纤毛发生,图1A是荧光下 显微镜下结果图,图1B是对显微镜下有初级纤毛的细胞数量进行了统计的统计 结果图,图1C是western blot实验验证了siRNA干涉实验使HRD1的表达降低。 Ac-tubulin、γ-tubulin分别是初级纤毛和中心体的标志物。
随后进行回转HRD1实验,过表达质粒上携带编码对siHRD1-1#干涉具有抵 抗效应的全长基因(野生型HRD1质粒,突变siHRD1-1#靶向序列部分核苷酸, 在氨基酸编码不影响的前提下,对siHRD-1具有抵抗效应)。
结果显示:回转HRD1可以逆转HRD1敲低导致的纤毛缺陷,如图2:图2A 是荧光下显微镜下结果图,图2B是对显微镜下有初级纤毛的细胞数量进行了统 计的统计结果图。RPE细胞在正常培养环境下,及含有10%胎牛血清的培养基中 培养(+Serum组),长有少量初级纤毛;在饥饿的环境下,即无血清的培养基中 培养(-Serum组),RPE1细胞出现生长周期阻滞长出大量纤毛。可以看出不管 是正常组(+Serum)还是饥饿组(-Serum),干涉HRD1均能抑制初级纤毛的发 生。
实施例2、hrd1调控斑马鱼胚胎发育和初级纤毛发生
斑马鱼作为广泛应用的模式生物,是研究初级纤毛生理功能非常好的模型。 由于初级纤毛与早期胚胎发育密切相关,所以我们接下来想检测敲低hrd1是否 会导致斑马鱼胚胎发育过程中出现纤毛缺陷相关表型。
斑马鱼培养及Morpholinos注射在上海生化所斑马鱼平台完成。
(1)Morpholino由Gene Tools公司设计并合成。
hrd1-aMO:5’-TTACAAGAGCGGCTCGCACCATC-3’(Seq ID NO.:4)
hrd1-sMO:5’-TGGGTCTGTGTGATGTACCTGTAAA-3’(Seq ID NO.:5)
hrd1-misMO:5’-TTACTAGTGCCGCTGGCTCCATC-3’(Seq ID NO.:6)
(2)参考斑马鱼HRD1基因组,设计验证sMO抑制剪切效果的测序引物如 下,HRD1-sMO-F:5’-GCGATCCTGATGACTATGGTCCTCACAA-3’(Seq ID NO.:7);HRD1-sMO-R:5’-TGCACACATTGTCGGTGGCTTGCAAGTC-3’(Seq ID NO.:8)。引物在英俊公司合成。
(3)参考斑马鱼Cmlc2基因组,设计引物制作原位杂交探针,在英俊公司合 成,序列如下,Cmlc2-F:5’-AGTTATCAGGGCTCCTGTATTTAG-3’(Seq ID NO.:9);
Cmlc2-R:5’-TAATACGACTCACTATACCAATAGACGTGGCTGGAAATAT -3’(Seq ID NO.:10)。
我们设计了两种不同的morpholinos,通过显微注射的方法来敲低hrd1。一 种是hrd1-aMO负责结合在hrd1 mRNA的起始密码子atg附近,进而抑制hrd1 的翻译。另一种是hrd1-sMO负责结合在hrd1剪切位点,进而影响hrd1剪切过 程导致错误的mRNA的剪切。hrd1-misMO是在hrd1-aMO的基础上做了五个核 苷酸突变,失去靶向hrd1起始密码子附近能力,作为对照组morpholinos。
如图3所示hrd1敲低导致斑马鱼胚胎发育缺陷;斑马鱼胚胎发育缺陷包括 脊柱弯曲、心脏肿大等表型,图3B对3A进行了统计,说明两种不同的morpholinos 敲低hrd1均能导致斑马鱼出现脊柱弯曲、心脏肿大等胚胎发育缺陷。
进一步地,hrd1敲低导致斑马鱼左右不对称性缺陷(如图4);hrd1敲低导 致斑马鱼KV泡(Kupffer’s vesicle,KV)中纤毛的数量和长度显著减少(图5)。
以上数据表明,hrd1敲低导致的纤毛缺陷对于斑马鱼早期胚胎发育过程至 关重要。
序列表
<110> 中国人民解放军军事科学院军事医学研究院
<120> E3泛素连接酶HRD1在调控初级纤毛发生中的应用
<141> 2021-12-19
<160> 10
<170> SIPOSequenceListing 1.0
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ttacaagagc ggctcgcacc atc 23
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Claims (10)
1.一种促进纤毛发生的方法,所述方法包括使用表达HRD1/促进HRD1表达的生物材料;
优选地,所述生物材料包括编码基因、表达盒、重组载体、细胞、蛋白质;
优选地,所述纤毛是初级纤毛。
2.一种促进纤毛发生、治疗纤毛相关疾病的产品,所述产品包含表达HRD1/促进HRD1表达的生物材料;
优选地,所述纤毛是初级纤毛;
优选地,所述生物材料包括编码基因、表达盒、重组载体、细胞、蛋白质;
优选地,所述产品包括药物组合物、试剂盒。
3.HRD1在促进纤毛发生、制备纤毛相关疾病的药物中的应用;
优选地,所述纤毛是初级纤毛;
优选地,所述HRD1是表达HRD1/促进HRD1表达的生物材料、或权利要求2所述的产品;
优选地,所述纤毛相关疾病包括初级纤毛相关疾病包括:Bardet-Biedl综合症、青少年消耗性肾病、Senior-Loken综合症、Alstrom综合症、Meckel-Grubber综合症、Joubert综合症、1型口面指综合症、埃利伟氏综合症、先天性利伯氏黑矇、多囊肾病。
4.HRD1抑制剂在抑制纤毛发生、构建胚胎发育缺陷的动物模型中的应用;
优选地,所述抑制剂包括siRNA干扰、CRISPR/cas9方法、同源重组、基因敲除、基因置换、基因沉默、点突变实、化学药物、Morpholinos方法所使用的试剂;
优选地,所述抑制剂是siRNA干扰方法、Morpholinos方法中所使用的试剂;
优选地,所述胚胎发育缺陷的动物模型具有KV泡纤毛发生缺陷、左右不对称性缺陷的特征。
5.一种判断纤毛状态的方法,所述方法包括通过HRD1表达量的检测结果进行判断;
优选地,所述表达量是蛋白表达量。
6.一种判断纤毛状态、胚胎发育状态的试剂盒,所述试剂盒中包含检测样品中HRD1的表达量所使用的试剂。
7.检测HRD1的表达量的试剂在判断纤毛状态、制备判断胚胎发育状态的产品中的应用。
8.如权利要求7所述的应用,其特征在于,所述检测表达量的试剂包括检测蛋白表达量和/或mRNA表达量的试剂;
优选地,所述检测HRD1蛋白表达量的试剂包括以下任意方法中所使用的试剂:蛋白质印迹法、酶联免疫吸附测定、放射性免疫测定、夹心测定、免疫组织化学染色、质谱检测法、免疫沉淀分析法、补体结合分析法、流式细胞荧光分析技术、蛋白质芯片法;
优选地,所述检测HRD1 mRNA表达量的试剂包括以下任意方法中使用到的试剂:基于PCR的检测方法、Southern杂交方法、Northern杂交方法、点杂交方法、荧光原位杂交方法、DNA微阵列方法、ASO法、高通量测序平台方法。
9.一种构建胚胎发育缺陷的动物模型的方法,所述方法包括敲低受试动物的HRD1的表达。
10.权利要求9所述的方法所构建的动物模型。
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