CN114081952B - 初级纤毛生成相关标志物及其应用 - Google Patents
初级纤毛生成相关标志物及其应用 Download PDFInfo
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- CN114081952B CN114081952B CN202111399728.2A CN202111399728A CN114081952B CN 114081952 B CN114081952 B CN 114081952B CN 202111399728 A CN202111399728 A CN 202111399728A CN 114081952 B CN114081952 B CN 114081952B
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Abstract
本发明公开了初级纤毛生成相关标志物及其应用，所述标志物为HOIP、HOIL‑1L和/或SHARPIN。本发明首次发现了HOIP、HOIL‑1L和/或SHARPIN的异常表达引起纤毛缺陷，回补相关标志物相关缺陷获得恢复，提示所述标志物可应用于相关疾病的诊断和治疗。
Description
技术领域
本发明属于生物医药领域，涉及初级纤毛生成相关标志物及其应用。
背景技术
纤毛是一种以细胞微管为主形成的突出于细胞表面的结构，分布于哺乳动物体内的大多数细胞。纤毛结构大体上可以分为三部分：轴丝、纤毛基质和纤毛膜。轴丝是由从基体(basal body)开始组装的9排环形排列的双联体微管束及其附属蛋白构成的,环形的中心常常存在一对中心微管；纤毛膜是细胞膜的延伸(成分与细胞膜不太一致)，均匀地包裹着轴丝；纤毛基质填充在轴丝和纤毛膜之间。
根据纤毛是否具有运动性和是否具有中心微管，将其分为4种类型：“9+2”运动型：这类纤毛轴丝包括外围的9组双联体微管和一对中心微管，轴丝微管束上面附着辐条蛋白、连接蛋白和内外动力蛋白臂；“9+0”不动型：这类纤毛轴丝缺少中心微管，而且微管束之间缺少动力蛋白臂；“9+2”不动型和“9+0”运动型属于不常见的类型。
纤毛具有极高的复杂性并且行使很多重要的功能，能够调控繁多的细胞生理活动和复杂的动物发育过程。研究发现，纤毛的缺失会导致多种发育缺陷性疾病，包括多囊肾疾病，Nephronophthisis疾病(NPHP)、Joubert综合症(JBTS)、Meckel-Gruber综合症(MKS)、Bardet Biedl综合症(BBS)等，这些疾病的症状主要包括内脏转位，多指(趾)，生殖能力降低，视觉、嗅觉、听觉的减退,呼吸系统感染，肝肾的多囊性增生等，这些症状出现的比率不一。
截止到目前为止，关于纤毛的研究仍处于初步阶段，临床上并没有较好的产前诊断标志物来判定胎儿是否患有纤毛相关疾病，因此研究与纤毛相关性疾病相关的基因，为未来纤毛疾病的分子病理学研究和纤毛缺陷疾病以及发育缺陷疾病的临床治疗提供有力的靶点。
发明内容
为了弥补现有技术的不足，本发明的目的在于提供一种预测纤毛缺陷性疾病的分子标志物以及治疗纤毛缺陷性疾病的分子靶标。
为了实现上述目的，本发明采用如下技术方案：
本发明第一方面提供了一种药物组合物，所述药物组合物包括HOIP、HOIL-1L和/或SHARPIN功能性表达的促进剂，和/或药学上可接受的载体。
进一步，所述促进剂为含有能编码功能性HOIP、HOIL-1L和/或SHARPIN蛋白的核酸的试剂、HOIP、HOIL-1L和/或SHARPIN蛋白的激活剂、含有HOIP、HOIL-1L和/或SHARPIN蛋白质的试剂。
进一步，所述试剂为含有能编码功能性HOIP、HOIL-1L和/或SHARPIN蛋白的核酸的试剂。
进一步，所述试剂为含有HOIP、HOIL-1L和/或SHARPIN核酸的表达载体。
本发明第二方面提供了一种诊断产品，所述产品包括检测HOIP、HOIL-1L和/或SHARPIN的试剂。
进一步，所述试剂包括通过数字成像技术、蛋白免疫技术、染料技术、核酸测序技术、核酸杂交技术、色谱技术、质谱技术检测HOIP、HOIL-1L和/或SHARPIN表达水平的试剂；
进一步，所述试剂包括针对HOIP、HOIL-1L和/或SHARPIN的引物、探针、抗体。
本发明第三方面提供了一种筛选治疗纤毛缺陷性疾病的候选药物的方法，步骤包括：
测试组中，在培养体系中添加测试化合物，并观察所述测试组中HOIP、HOIL-1L和/或SHARPIN的表达量和/或活性；在对照组中，在相同的培养体系中不添加测试化合物，并观察对照组中HOIP、HOIL-1L和/或SHARPIN的表达量和/或活性；
其中，如果测试组中HOIP、HOIL-1L和/或SHARPIN的表达量和/或活性高于对照组，就表明该测试化合物是治疗纤毛缺陷性疾病的候选药物。
在本发明中，所培养述体系包括(但不限于)：细胞体系、亚细胞体系、溶液体系、组织体系、器官体系或动物体系。
在本发明中，所述的步骤还包括：对获得的候选药物进行进一步的细胞实验和/或动物试验，以从候选药物中进一步选择可以治疗纤毛缺陷性疾病的物质。
本发明第四方面提供了一种筛选治疗胚胎发育缺陷性疾病的候选药物的方法，步骤包括：
测试组中，在培养体系中添加测试化合物，并观察所述测试组中HOIP、HOIL-1L和/或SHARPIN的表达量和/或活性；在对照组中，在相同的培养体系中不添加测试化合物，并观察对照组中HOIP、HOIL-1L和/或SHARPIN的表达量和/或活性；
其中，如果测试组中HOIP、HOIL-1L和/或SHARPIN的表达量和/或活性高于对照组，就表明该测试化合物是治疗胚胎发育缺陷性疾病的候选药物。
在本发明中，所培养述体系包括(但不限于)：细胞体系、亚细胞体系、溶液体系、组织体系、器官体系或动物体系。
在本发明中，所述的步骤还包括：对获得的候选药物进行进一步的细胞实验和/或动物试验，以从候选药物中进一步选择可以治疗胚胎发育缺陷性疾病的物质。
本发明第五方面提供了如下任一项所述的应用：
1)本发明第一方面所述的药物组合物在制备治疗纤毛缺陷性疾病的药物中的应用；
2)本发明第一方面所述的药物组合在制备治疗胚胎发育缺陷性疾病的药物中的应用；
3)本发明第一方面所述的药物组合物在制备治疗缺陷性左右不对称/脊柱侧弯疾病的药物中的应用；
4)本发明第一方面所述的药物组合物在制备促进纤毛生成的产品中的应用；
5)HOIP、HOIL-1L和/或SHARPIN的抑制剂在制备抑制纤毛生成的产品中的应用；
6)本发明第二方面所述的诊断产品在制备诊断纤毛缺陷性疾病的工具中的应用；
7)本发明第二方面所述的诊断产品在制备诊断胚胎发育缺陷性疾病的工具中的应用；
8)HOIP、HOIL-1L和/或SHARPIN在构建纤毛缺陷性疾病模型或胚胎发育缺陷性疾病模型中的应用；
9)本发明第三方面所述的方法在筛选治疗纤毛缺陷性疾病的候选药物中的应用；
10)本发明第四方面所述的方法在筛选治疗胚胎发育缺陷性疾病的候选药物中的应用。
进一步，所述的纤毛缺陷性疾病包括多囊肾病、Nephronophthisis疾病、Joubert综合症、Meckel-Gruber综合症、Bardet Biedl综合症。
进一步，纤毛缺陷性疾病为HOIP、HOIL-1L和/或SHARPIN缺失导致的纤毛缺陷性疾病。
进一步，相比正常对照，HOIP、HOIL-1L和/或SHARPIN在纤毛缺陷性疾病中表达水平或者表达活性下调。
进一步，5)中所述的抑制剂包括核酸抑制剂，蛋白抑制剂，蛋白水解酶，蛋白结合分子。
进一步，所述核酸抑制剂包括shRNA、小干扰RNA、dsRNA、微小RNA、反义核酸及其构建物。
进一步，所述核酸抑制剂选自反义核酸。
进一步，所述反义核酸选自吗啉代反义寡核苷酸。
进一步，所述吗啉代反义寡核苷酸的序列如SEQ ID NO：5和6所示。
进一步，所述核酸抑制剂选自小干扰RNA。
进一步，所述小干扰RNA的序列如SEQ ID NO：2-4所示。
进一步，6)中通过施用HOIP、HOIL-1L和/或SHARPIN的抑制剂构建纤毛缺陷性疾病模型或胚胎发育缺陷性疾病模型。
进一步，所述抑制剂包括核酸抑制剂，蛋白抑制剂，蛋白水解酶，蛋白结合分子；
进一步，所述核酸抑制剂包括shRNA、小干扰RNA、dsRNA、微小RNA、反义核酸或其构建物。
进一步，所述核酸抑制剂选自反义核酸或其构建物。
进一步，所述反义核酸选自吗啉代反义寡核苷酸。
进一步，所述吗啉代反义寡核苷酸的序列如SEQ ID NO：5和6所示。
进一步，所述模型为动物模型。
进一步，所述动物模型为斑马鱼模型。
进一步，所述核酸抑制剂选自小干扰RNA；
进一步，所述小干扰RNA的序列如SEQ ID NO：2-4所示。
进一步，所述模型为细胞模型。
进一步，所述细胞模型为RPE-1细胞模型。
附图说明
图1是HOIP、HOIL-1L、SHARPIN对初级纤毛的影响图，其中，图a是HOIP，或HOIL-1L+SHARPIN的敲低效果图；图b是抑制初级纤毛的免疫荧光图；图c是抑制初级纤毛的统计图；图d是蛋白表达图；图e是初级纤毛发生比率图。
图2是HOIP对初级纤毛的影响图，其中，图a是aMO的敲除效率图；图b是sMO的敲除效率图；图c是斑马鱼KV泡纤毛染色图；图d是每个KV泡的纤毛数量图；图e是斑马鱼发育图，图f是斑马鱼发育统计图；图g是rescue实验斑马鱼发育统计图；图h是斑马鱼左右对称图；图i是斑马鱼左右对称统计图；图j是rescue实验斑马鱼左右对称统计图。
具体实施方式
本发明经过广泛而深入的研究，发现HOIP、HOIL-1L、SHARPIN与纤毛的生成相关，在培养的细胞中抑制HOIP、HOIL-1L、SHARPIN会抑制纤毛的发生过程，进行斑马鱼实验，发现抑制HOIP会影响斑马鱼的发育，出现纤毛功能异常导致的左右对称缺陷，以及出现脊柱侧弯，而过表达则会回补HOIP敲低所引起的缺陷。说明HOIP、HOIL-1L、SHARPIN可能导致纤毛异常相关疾病，其可作为相关疾病诊断和治疗的靶标，在此基础上完成了本发明。
标志物
在本发明中，HOIP基因(gene ID为5072)，HOIL-1L(gene ID：10616)；SHARPIN(gene ID:81858)包括野生型、突变型或其片段。本发明的HOIP、HOIL-1L、SHARPIN核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。
促进剂和药物组合物
基于本发明的发现，本发明提供了一种药物组合物，所述药物组合物包含HOIP、HOIL-1L和/或SHARPIN功能性表达的促进剂。
所述的HOIP、HOIL-1L和/或SHARPIN的促进剂是指任何可提高HOIP、HOIL-1L和/或SHARPIN基因或表达产物稳定性、上调HOIP、HOIL-1L和/或SHARPIN的表达、增加HOIP、HOIL-1L和/或SHARPIN的有效作用时间或促进HOIP、HOIL-1L和/或SHARPIN基因的转录的物质，这些物质均可用于本发明，作为对于上调HOIP、HOIL-1L和/或SHARPIN基因的表达有用的物质，从而可用于促进初级纤毛的发生。
在本发明中，“功能性表达”意指功能性基因产物的转录和/或翻译。“功能性表达”可以在至少两个水平上失调。第一，在DNA水平上，例如通过基因的缺失或破坏，或者是没有转录发生(在两种情况下都阻止相关基因产物的合成)。转录的缺失可以例如由表观遗传的变化(例如DNA甲基化)或由功能缺失性突变导致。如本文中所使用的“功能缺失”或“LOF”突变是，相对于赋予蛋白质增强的或新的活性的功能获得性突变而言，阻止、减少或消除基因产物的功能的突变。功能性缺失可由广泛的突变类型引起，包括但不限于整个基因或基因部分的删除、剪接位点突变、由小的插入和删除引起的移码突变、无义突变、取代了必需氨基酸的错义突变、和阻止产物的正确细胞定位的突变。该定义也包括HOIP、HOIL-1L和/或SHARPIN基因的启动子或调控区域的突变，如果这些突变干扰了基因的功能。无效突变是完全破坏基因产物功能的LOF突变。一个等位基因中的无效突变通常将使表达水平降低50％但可能对基因产物的功能具有严重影响。值得注意的是，功能性表达也可以因为功能获得性突变而失调：通过赋予蛋白质新的活性，蛋白质的正常功能失调，表达的功能活性蛋白减少。反之亦然，功能性表达可以例如通过基因复制或通过缺乏DNA甲基化而增加。功能性表达也可以因为功能获得性突变而失调：通过赋予蛋白质新的活性，蛋白质的正常功能失调，表达的功能活性蛋白减少。反之亦然，功能性表达可以例如通过基因复制或通过缺乏DNA甲基化而增加。第二，在RNA水平上，例如通过缺乏有效的翻译，例如因为mRNA的不稳定(例如通过UTR变体)，可以导致在转录物翻译之前mRNA被降解。或者通过缺乏有效的转录，例如因为突变诱导了新的剪接变体。
作为本发明的一种优选方式，所述的HOIP、HOIL-1L和/或SHARPIN的促进剂是一种含有HOIP、HOIL-1L和/或SHARPIN的表达载体。所述的表达载体通常还含有启动子、复制起点和/或标记基因等。
本领域的技术人员熟知的方法能用于构建本发明所需的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如卡拉霉素、庆大霉素、潮霉素、氨苄青霉素抗性。
在本发明中，是本领域已知的各种载体，如市售的载体、包括质粒、粘粒、噬菌体、病毒等。表达载体向宿主细胞中的导入可以使用电穿孔法、磷酸钙法、脂质体法、DEAE葡聚糖法、显微注射、病毒感染、脂质体转染、与细胞膜透过性肽的结合等周知的方法。
术语“宿主细胞”包括原核细胞和真核细胞。常用的原核宿主细胞的例子包括大肠杆菌、枯草杆菌等。常用的真核宿主细胞包括酵母细胞、昆虫细胞和哺乳动物细胞。较佳地，该宿主细胞是真核细胞，如CHO细胞、COS细胞等。
在本发明中，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。
在本发明中，药学上可接受的载体实例是软化水或蒸馏水；盐水溶液；植物油类，如花生油、红花油、橄榄油、棉籽油、玉米油，麻油类，如花生油(peanutoil)、红花油、橄榄油、棉籽油、玉米油、麻油、花生油(arachisoil)或椰子油；硅油类，包括聚硅氧烷类，如甲基聚硅氧烷、苯基聚硅氧烷和甲基苯基聚硅氧烷；挥发性硅酮类；矿物油类，如液体石蜡、软石蜡或角鲨烷；纤维素衍生物，如甲基纤维素、乙基纤维素、羧甲基纤维素、羧甲基纤维素钠或羟丙基甲基纤维素；低级烷醇类，例如乙醇或异丙醇；低级芳烷醇类(aralkanols)；低级聚烷撑二醇类或低级烷撑二醇类，例如聚乙二醇、聚丙二醇、乙二醇、丙二醇、1，3-丁二醇或甘油；脂肪酸酯类，如棕榈酸异丙酯、肉豆蔻酸异丙酯或油酸乙酯；聚乙烯吡咯烷酮；琼脂；黄耆树胶或阿拉伯胶和凡士林。一般载体或多种载体形成组合物的10wt％至99.9wt％。
组合物可以是适合注射给药的剂型、适合口服摄取的剂型(如胶囊、片剂、囊片、酏剂)、适合局部给药的油膏、乳膏或洗剂形式、适合用作滴眼剂的递送剂型、适合通过吸入给药的气雾剂形式(如通过鼻内吸入或口吸入)、适合胃肠外给药的剂型，即皮下、肌肉内或静脉内注射。
对于可注射溶液或悬液的给药，无毒的胃肠外可接受的稀释剂或载体可包括林格氏溶液、等渗盐水、磷酸盐缓冲盐水、乙醇和1，2丙二醇。
用于口服的合适载体、稀释剂、赋形剂和佐剂的一些实例包括花生油、液体石蜡、羧甲基纤维素钠、甲基纤维素、海藻酸钠、阿拉伯胶、黄耆树胶、右旋糖、蔗糖、山梨醇、甘露醇、明胶和卵磷脂。另外，这些口服剂型可含有合适的调味剂和着色剂。当以胶囊剂型使用时，胶囊可用能延缓崩解的化合物包被，如单硬脂酸甘油酯或二硬脂酸甘油酯。
佐剂典型地包括润滑剂、乳化剂、增稠剂、防腐剂、杀菌剂和缓冲剂。
口服给药的固体剂型可包含在人和兽医药学实践中可接受的粘合剂、增甜剂、崩解剂、稀释剂、调味剂、包衣剂、防腐剂、润滑剂和/或延时剂(timedelayagent)。合适的粘合剂包括阿拉伯胶、明胶、玉米淀粉、黄耆树胶、海藻酸钠、羧甲基纤维素或聚乙二醇。合适的增甜剂包括蔗糖、乳糖、葡萄糖、阿斯巴特或糖精。合适的崩解剂包括玉米淀粉、甲基纤维素、聚乙烯吡咯烷酮、瓜尔胶、黄原胶、膨润土、藻酸或琼脂。合适的稀释剂包括乳糖、山梨醇、甘露醇、右旋糖、高岭土、纤维素、碳酸钙、硅酸钙或磷酸二钙。合适的调味剂包括薄荷油、冬青油、樱桃、柑橘或树莓调味剂。合适的包衣剂包括丙烯酸和/或甲基丙烯酸和/或它们的酯类的聚合物或共聚物、蜡、脂肪醇、玉米蛋白、虫胶或麸质。合适的防腐剂包括苯甲酸钠、维生素E、α-生育酚、抗坏血酸、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯或亚硫酸氢钠。合适的润滑剂包括硬脂酸镁、硬脂酸、油酸钠、氯化钠或滑石。合适的延时剂包括单硬脂酸甘油酯或二硬脂酸甘油酯。
用于口服给药的液体剂型除了上述药物以外，还可包含液体载体。合适的液体载体包括水、油类如橄榄油、花生油(peanutoil)、麻油、向日葵油、红花油、花生油(arachisoil)、椰子油、液体石蜡、乙二醇、丙二醇、聚乙二醇、乙醇、丙醇、异丙醇、甘油、脂肪醇、甘油三酯或其混合物。
用于口服给药的悬液可进一步包括分散剂和/或悬浮剂。合适的悬浮剂包括羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、聚乙稀吡咯烷酮、海藻酸钠或乙酰乙醇。合适的分散剂包括卵磷脂、如硬脂酸之类的脂肪酸的聚氧乙烯酯类、聚氧乙烯山梨醇单-或二-油酸酯、-硬脂酸酯或-月桂酸酯、聚氧乙烯脱水山梨糖醇单-或二-油酸酯、-硬脂酸酯或-月桂酸酯和类似物。
用于口服给药的乳剂可进一步包括一种或多种乳化剂。合适的乳化剂包括上面所例举的分散剂或天然树胶，如瓜尔胶、阿拉伯胶或黄耆树胶。
本发明的药物导入组织或者细胞的方式可以分为体外或者体内的方式。体外方式包括将含有HOIP、HOIL-1L和/或SHARPIN基因，或HOIP、HOIL-1L和/或SHARPIN蛋白质促进剂的药物导入细胞中，再将细胞移植或回输到体内。体内方式包括直接将含有HOIP、HOIL-1L和/或SHARPIN基因，或者HOIP、HOIL-1L和/或SHARPIN蛋白质抑制物的药物注入体内组织中。
作为本发明的一种可选择的实施方式，还可以将通过本发明的筛选治疗纤毛缺陷性疾病的候选药物方法分离的化合物作为药物施用于人或其它哺乳动物，其包括但不限于小鼠、大鼠、豚鼠、兔、猫、犬、羊、猪、牛、猴子、狒狒、黑猩猩时，分离的化合物可以直接施用，或者可以利用已知的药物制备方法配制成各种剂型。例如，根据需要，所述药物可以作为糖衣片剂、胶囊剂、酏剂和微胶囊口服施用；或者用水或任何其它药物可接受的液体配制成无菌溶液或悬浮液，以注射剂的形式非口服施用。例如，可以将化合物以一般接受的药物施用方式所需的单位剂型(unit dose)、与药学可接受的载体或介质混合在一起，所述载体或介质包括但不限于无菌水、生理盐水、植物油、乳化剂、悬浮剂、表面活性剂、稳定剂、调味剂、赋形剂(excipient)、媒介物(vehicle)、防腐剂、粘合剂等。根据这些制剂中有效成分的含量，可以获得在指定范围内的合适的给药量。
诊断产品及方法
在本发明中，诊断纤毛缺陷性疾病的产品可以是任何形式，包括(但不限于)芯片、制剂、试剂盒，只要其能够检测HOIP、HOIL-1L和/或SHARPIN基因或其表达产物的表达水平即可。
作为一种可选择的实施方式，本发明中的芯片包括：固相载体；以及有序固定在所述固相载体上的寡核苷酸探针，所述的寡核苷酸探针特异性地对应于HOIP、HOIL-1L和/或SHARPIN所示的部分或全部序列。
具体地，可根据本发明所述的基因，设计出适合的探针，固定在固相载体上，形成“寡核苷酸阵列”。所述的“寡核苷酸阵列”是指具有可寻址位置(即以区别性的，可访问的地址为特征的位置)的阵列，每个可寻址位置均含有一个与其相连的特征性寡核苷酸。根据需要，可将寡核苷酸阵列分成多个亚阵。
术语“探针”指能与另一分子的特定序列或亚序列或其它部分结合的分子。除非另有指出，术语“探针”通常指能通过互补碱基配对与另一多核苷酸(往往称为“靶多核苷酸”)结合的多核苷酸探针。根据杂交条件的严格性，探针能和与该探针缺乏完全序列互补性的靶多核苷酸结合。探针可作直接或间接的标记，其范围包括引物。杂交方式，包括，但不限于：溶液相、固相、混合相或原位杂交测定法。
所述固相载体可采用基因芯片领域的各种常用材料，例如包括但不限于塑料制品、微颗粒、膜载体等。所述塑料制品可通过非共价或物理吸附机制与抗体或蛋白抗原相结合，最常用的塑料制品为聚苯乙烯制成的小试管、小珠和微量反应板；所述微颗粒是由高分子单体聚合成的微球或颗粒，其直径多为微米，由于带有能与蛋白质结合的功能团，易与抗体(抗原)形成化学偶联，结合容量大；所述膜载体包括硝酸纤维素膜、玻璃纤维素膜及尼龙膜等微孔滤膜。
本发明还提供了一种检测纤毛相关疾病的方法，在一个优选例中，包括步骤：分别检测待测样本和对照样本HOIP、HOIL-1L和/或SHARPIN的表达水平，如果与对照样本相比，待测样本的HOIP、HOIL-1L和/或SHARPIN的表达水平存在异常降低，则是潜在的纤毛异常相关疾病患病样本。
在另一优选例中，所述异常的降低是指：与对照样本相比，所述HOIP、HOIL-1L和/或SHARPIN基因或其表达产物的表达水平的降低幅度≥10％，较佳地≥20％，较佳地≥50％，更佳地≥80％，最佳地≥100％。
本发明提供了HOIP、HOIL-1L和/或SHARPIN在制备纤毛缺陷性疾病的模型中的应用。
作为本发明的一种优选方式，通过施用HOIP、HOIL-1L和/或SHARPIN的抑制剂来制备纤毛缺陷性疾病的模型。
作为本发明的更为优选的实施方式，所述的HOIP、HOIL-1L和/或SHARPIN的抑制剂是一种特异性与HOIP、HOIL-1L和/或SHARPIN结合的抗体。所述特异性抗体包括单克隆抗体、多克隆抗体；本发明不仅包括完整的抗体分子，也包括抗体的任何片段或修饰，例如，嵌合抗体、scFv、Fab、F(ab’)2、Fv等。只要所述片段能够保留与HOIP、HOIL-1L和/或SHARPIN蛋白的结合能力即可。用于蛋白质水平的抗体的制备时本领域技术人员公知的，并且本发明可以使用任何方法来制备所述抗体。
作为本发明的一种优选方式，所述HOIP、HOIL-1L和/或SHARPIN的抑制剂是一种HOIP、HOIL-1L和/或SHARPIN特异性的小干扰RNA分子。如本文所用，所述的“小干扰RNA”是指一种短片段双链RNA分子，能够以同源互补序列的mRNA为靶目标降解特定的mRNA，这个过程就是RNA干扰(RNA interference)过程。小干扰RNA可以制备成双链核酸的形式，它含有一个正义链和一个反义链，这两条链仅在杂交的条件下形成双链。一个双链RNA复合物可以由相互分离的正义链和反义链来制备。因此，举例来讲，互补的正义链和反义链是化学合成的，其后可通过退火杂交，产生合成的双链RNA复合物。
本发明的核酸抑制剂如siRNA可以化学合成，也可以通过一个重组核酸结构里的表达盒转录成单链RNA之后进行制备。siRNA等核酸抑制物，可通过采用适当的转染试剂被输送到细胞内，或还可采用本领域已知的多种技术被输送到细胞内。
作为本发明的一种优选地实施方式，所述HOIP、HOIL-1L和/或SHARPIN的抑制剂是反义寡核苷酸。所述反义寡核苷酸具有与目的序列互补的序列，并且通过所述互补序列，可以实现目的基因的抑制，所述反义寡核苷酸是核糖核酸或DNA。作为一种优选地实施方式，反义寡核苷酸包含至少一个化学改性。反义寡核苷酸能包含一个或一个以上的锁核酸(LNAs，Locked nucleic acids)。锁核酸为改性核糖核酸，在核糖部位的2’至4’碳之间包含额外的桥键，来具有锁定(locked)形态，并以此具有锁核酸的寡核苷酸具有改善的热稳定性。择一性地，反义寡核苷酸可包含肽核酸(PNA，peptide nucleic acids)，并且反义寡核苷酸包含基于肽的主链来代替糖-磷酸主链。反义寡核苷酸能包含的其他化学改性包括：如2’-O-烷基(例如，2’-O-甲基，2’-O-甲氧基乙基)、2’-氟及4’-硫氧改性一样的糖改性；硫代磷酸、吗啉代或磷羧酸键之类的主链改性。反义寡核苷酸的长度为7-50核苷酸，优选为10-40核苷酸，更优选为15-30核苷酸，最优选为20-25核苷酸。
作为本发明的一种可选方式，所述的HOIP、HOIL-1L和/或SHARPIN的抑制剂也可以是一种“小发夹RNA(Small hairpin RNA，shRNA)”，其是能够形成发夹结构的非编码小RNA分子，小发夹RNA能够通过RNA干扰途径来抑制基因的表达。如上述，shRNA可以由双链DNA模板来表达。双链DNA模板被插进一个载体，例如质粒或病毒载体，然后在体外或体内连接到一个启动子进行表达。shRNA在真核细胞内DICER酶的作用下，可被切割成小干扰RNA分子，从而进入RNAi途径。“shRNA表达载体”是指一些本领域常规用于构建shRNA结构的质粒，通常该质粒上存在“间隔序列”以及位于“间隔序列”两边的多克隆位点或供替换序列，从而人们可以将shRNA(或类似物)相应的DNA序列通过正向和反向的方式插入多克隆位点或替换其上的供替换序列，该DNA序列转录后的RNA可形成shRNA(Short Hairpin)结构。所述的“shRNA表达载体”目前已经完全可以通过商购的途径购买获得，例如一些病毒载体。
下面结合具体的实施例进一步说明本发明，本发明的实施例仅用于解释本发明，并不意味着限制本发明的保护范围。
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。
实施例1 细胞检测HOIP、HOIL-1L、SHARPIN对初级纤毛发生的影响
实验原理：利用RNA干扰技术(RNAi)对细胞内源的HOIP、HOIL-1L、SHARPIN进行48h敲低，随后血清饥饿诱导初级纤毛的发生，并通过免疫荧光染色对细胞的初级纤毛进行检测，从而评价HOIP的敲低对初级纤毛的影响。
实验细胞系：人视网膜上皮细胞(RPE-1)(由中科院朱学良老师赠送)
实验材料：Ac-tubulin抗体：sigma公司，货号T6793,γ-tubulin抗体：sigma公司，货号T6557。
实验方法：
1.细胞培养：用advanced DMEM/F12培养基在37℃，5％CO2的培养箱里培养RPE-1细胞。传一代后进行下一步的实验。
2.种细胞：前一天晚上以3×104cells/ml细胞密度分别种在有爬片的24孔板和6孔板上，12小时后视密度合适继续进行下一步实验。
3.干扰和rescue实验
针对HOIP、HOIL-1L、SHARPIN，设计干扰序列，购置于Thermo fisher公司，干扰序列如下：
siCtrl：5′-UUCUCCGAACGUGUCACGUAA-3′(SEQ ID NO：1)
siHOIP：5′-CACCACCCUCGAGACUGCCUCUUCU-3′(SEQ ID NO：2)
siHOIL-1L：5′-CCGCUGCAGGGUAAAUGGGAUUCCU-3′(SEQ ID NO：3)
siSHARPIN：5′-UGCCUGAGCGCAGCCUUGCCUCUUA-3′(SEQ ID NO：4)
配制干扰体系，HOIP的干扰序列终浓度为50nM，HOIL-1L和SHARPIN共干扰时干扰序列浓度各为一半，即25nM，转染试剂为RNAiMAX(Thermo fisher)，体系如下：
24孔板：(0.75μl siRNA+50μlopti-MEM)+(0.75μlRNAiMAX+50μl opti-MEM)
6孔板：(2.5μl siRNA+50μlopti-MEM)+(2.5μl RNAiMAX+50μlopti-MEM)
见上述配好的体系静置15min后，滴加入孔板中，充分混匀，6小时后换正常培养基进行培养。
针对HOIP，构建野生型HOIP和C885S突变的HOIP的质粒，6小时后，进行质粒转染，24孔板换正常培养液200μl，6孔板换正常培养液900μl，转染试剂为LipofectamineTM LTXReagent with PLUSTM Reagent，转染体比例为plasmid：LTX:plus＝1：2.5：1。体系为：
24孔板：(0.05μg plasmids+0.05μl plus+25μl opti-MEM)+(0.125μl LTX+25μlopti-MEM)
6孔板：(0.25μg plasmids+0.25μl plus+50μl opti-MEM)+(0.625μl LTX+50μlopti-MEM)
上述体系配置好后静置5min，然后分别加入细胞中。
4.收集细胞，干扰48小时后，目标蛋白被siRNAs敲低，将6孔板中的细胞用M2buffer冰上裂解30分钟(M2 buffer里加入常用的蛋白抑制剂cocktail)，12000rpm离心15min，测量蛋白浓度，根据蛋白浓度标准曲线将所有蛋白裂解液的浓度归一化；最后免疫印迹检测敲低效果。
5.免疫印迹实验
(1)制胶：配制10％的SDS-PAGE蛋白凝胶。待浓缩胶凝固后，小心拔下梳子。
(2)上样：将凝胶固定于电泳装置上，加入足量的1×Tris-甘氨酸电泳缓冲液，在加样孔中分别加入各样品40μL(40ug总蛋白)。
(3)电泳：首先在80V恒压的下进行电泳分离，待loading跑至分离胶后将电压调至120V，直至loading跑出胶底。
(4)转印：PVDF膜转印，400mA下转印2.5h。
(5)封闭：配制5％的脱脂牛奶(以TBST buffer为封闭稀释液)，封闭1h。
(6)一抗孵育：anti-sheep-HOIP，anti-sheep-HOIL-1L以及anti-rabbit-SHARPIN在4℃孵育过夜。
(7)二抗孵育：用sheep或rabbit二抗在室温状态下孵育1h。
(8)显影：采用底物催化的方式。将A、B液以1:1混合后滴于转印膜上，涂匀，置于暗盒中进行显影。
6.24孔板里的细胞在正常培养基条件下培养48h后，换Opti-MEM减血清培养基培养48h。收集24孔板里的爬片，免疫荧光染色检测初级纤毛的发生情况。免疫荧光实验步骤如下：
(1)将24孔板置于冰上10分钟，之后弃掉培养基，然后用PBS洗涤3次。
(2)固定与透化：-20℃冰甲醇固定细胞(300ul)5分钟，PBS洗涤3次。
(3)封闭：按0.25％PBST+3％NGS(羊血清)配置封闭液，每孔300ul，封闭1h。
(4)一抗孵育：以封闭液为抗体稀释液，将AC-tubulin抗体以1:400的比例以及γ-tubulin以1:200的比例稀释到封闭液中，每个爬片30ul。室温孵育1h。
(5)PBS洗涤三次，每次10分钟。
(6)二抗孵育：以封闭液为二抗稀释液，将Alexa Fluor-488和Alexa Fluor-546二抗以1：400的比例稀释到封闭液中，每个爬片30ul。室温孵育1h。
(7)PBS洗涤3次。每次10分钟。
(8)复染：Hoechst 10分钟。
(9)封片：Sigma mounting medium封片剂封闭爬片。
(10)置于荧光镜下镜检。
7.统计学方法
采用Prism 6.0(GraphPad Software,Inc.)软件对数据进行统计学分析，对于两组数据之间比较采用Student's t test，对于非正态分布的数据进行比较使用Mann-Whitney rank-sum test；大于两组数据的多组间进行比较采用one-way ANOVA,P<0.05认为存在显著性差异。*P<0.05；**P<0.01；***P<0.001；NS,not significant。
实验结果：
通过RNAi技术将人视网膜上皮细胞(RPE-1)中的内源HOIP、HOIL-1L、SHARPIN蛋白水平敲低(图1a)，随后利用血清饥饿培养方式诱导初级纤毛的发生，Ac-tubulin指示初级纤毛，γ-tubulin指示中心体，结果发现，在人视网膜上皮细胞(RPE-1)中敲低HOIP、HOIL-1L和SHARPIN能够强烈地抑制血清饥饿诱导的初级纤毛的发生(见图1b和c)。rescue实验将结果显示，干涉HOIP后再回补的外源HOIP-WT或C885S的蛋白水平和内源的HOIP保持一致(图1d)，当回补HOIP-WT时，初级纤毛能有效地得到恢复(图1e)。
实施例2 斑马鱼实验检测HOIP对初级纤毛发生的影响
实验原理：利用靶向HOIP的Morpholino，包括翻译抑制型(aMO)和剪切抑制型(sMO)敲低斑马鱼中HOIP的蛋白表达水平，再利用免疫荧光染色检测HOIP敲低对斑马鱼KV泡纤毛发生以及斑马鱼发育的影响。
实验材料：斑马鱼，由中科院上海生命科学研究院斑马鱼资源中心提供。Ac-tubulin抗体：sigma公司，货号T6793。
HOIP的靶向序列：
sMO:5′-GCTGAACATGATGATTTTACCTGTT-3′(SEQ ID NO：5)；
aMO:5′-ATCAGTGAGAGAGGCCATAGCGC-3′(SEQ ID NO：6)，
Ctrl-MO:5′-CCTCTTACCTCAGTT-3′(SEQ ID NO：7)。
实验方法：
一、检测aMO以及sMO对HOIP的敲低效果
1.为了检测aMO的敲低效果，将带有aMO靶序列的pCS2-UTR-rnf31-EGFP质粒与aMO或control-MO共注入斑马鱼胚胎的单细胞阶段。胚胎培养7小时后检测斑马鱼中EGFP的表达情况。
2.为了检测sMO对HOIP mRNA的抑制作用，收取对照组和注射sMO组斑马鱼，用trizol法提取斑马鱼的总RNA，RNA经过逆转录后进行PCR扩增，利用测序及凝胶电泳法检测HOIP mRNA的完整性。
PCR扩增引物：
E5-F:5′-GCACCCATCCTCATCCAG-3′(SEQ ID NO：8)；
E7-R:5′-CTGAGTCGCTTTGTGGGT-3′(SEQ ID NO：9)；
测序引物：
sMO-F:5′-GTACTTAACCTGTACGGCTACACC-3′(SEQ ID NO：10)；
sMO-R:5′-TTTACACTGCCACTCAG ATATTGT-3′(SEQ ID NO：11)。
二、斑马鱼免疫荧光染色检测KV纤毛
1.在斑马鱼胚胎(一个细胞水平)注射对照组和实验组的morpholino(分别aMO和sMO)后，在28℃恒温水浴中培养到10体节时期，然后用吸管转移到1.5ml EP管中(每管可以固定100个左右的胚胎,吸走多余的液体，加入配置好的固定液，在混悬仪上室温固定2小时。
2.用1ml的枪尖吸走固定液，用配好的PBST溶液(PBS中加0.1％吐温)清洗三次，每次在旋转仪混悬5分钟，注意斑马鱼胚胎比较小，很脆弱，每一次的步骤都需要小心。
3.在斑马鱼中加入胚胎封闭液(PBS中加2％BSA,0.5％NGS,1％DMSO,0.5％Triton-X100)，室温封闭1-2小时。
4. 500μl封闭液中加入AC-tubulin抗体(1：600)，4℃混悬过夜。
5.用1ml含0.5％TritonX-100/PBS洗三次，每次10分钟。
6.封闭液中加入二抗Alexa Fluor-546(1：400，)，室温混悬孵育2小时，然后用1ml含0.5％TritonX-100/PBS洗三次，每次10分钟。
10.在PBST中加入Hoechst染料(1：1000)室温混悬染色5分钟，然后用PBST溶液洗三次，每次5分钟。
12.利用Zeiss LSM880共聚焦显微镜对斑马鱼KV泡的纤毛进行拍摄。
三、rescue实验
使用mMESSAGE mMACHINETM SP6试剂盒(ThermoFisher Scientific)的pCS2-aMO-resistant-rnf31质粒转录斑马鱼pCS2-aMO-resistant全长mRNA，并与rnf31-aMOs一起注射到单细胞时期的斑马鱼胚胎中。观察不同时期的表型并收取胚胎。
四、斑马鱼左右不对称分析
通过原位杂交染色的方式检测斑马鱼的左右不对称性，收取分别注射了control-MO，sMO以及aMO的斑马鱼(受精后26小时，26hpf)，在4％的多聚甲醛中固定，随后在65℃条件下用dig标记的cmlc2探针进行杂交。
五、统计学方法
采用Prism 6.0(GraphPad Software,Inc.)软件对数据进行统计学分析，对于两组数据之间比较采用Student's t test，对于非正态分布的数据进行比较使用Mann-Whitney rank-sum test；大于两组数据的多组间进行比较采用one-way ANOVA,P<0.05认为存在显著性差异。*P<0.05；**P<0.01；***P<0.001；NS,not significant。
实验结果：
通过定量PCR，基因测序，以及荧光表达实验分别证明了aMO和sMO在斑马鱼中敲低了HOIP的表达水平(见图2a和2b)。随后的免疫荧光染色检测斑马鱼发育10体节时期的KV泡纤毛的发生情况，结果发现，HOIP的敲低强烈地抑制了斑马鱼中KV泡纤毛的发生(见图2c和d)。HOIP的敲低引起躯体弯曲和心包水肿(图2e和图2f)，回补HOIP躯体弯曲和心包水肿现有效的得到恢复(图2g)。同时，斑马鱼中HOIP的敲低引起缺陷的左右不对称发育(图2h和图2i)，回补HOIP左右不对称有效的得到恢复(图2j)。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。
序列表
<110> 中国人民解放军军事科学院军事医学研究院
<120> 初级纤毛生成相关标志物及其应用
<141> 2021-11-19
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 1
uucuccgaac gugucacgua a 21
<210> 2
<211> 25
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 2
caccacccuc gagacugccu cuucu 25
<210> 3
<211> 25
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccgcugcagg guaaauggga uuccu 25
<210> 4
<211> 25
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 4
ugccugagcg cagccuugcc ucuua 25
<210> 5
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gctgaacatg atgattttac ctgtt 25
<210> 6
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atcagtgaga gaggccatag cgc 23
<210> 7
<211> 15
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cctcttacct cagtt 15
<210> 8
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gcacccatcc tcatccag 18
<210> 9
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ctgagtcgct ttgtgggt 18
<210> 10
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gtacttaacc tgtacggcta cacc 24
<210> 11
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
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Claims (24)
1.一种筛选治疗纤毛缺陷性疾病的候选药物的方法，其特征在于，步骤包括：
测试组中，在培养体系中添加测试化合物，并观察所述测试组中HOIP的表达量和/或活性；在对照组中，在相同的培养体系中不添加测试化合物，并观察对照组中HOIP的表达量和/或活性；
其中，如果测试组中HOIP的表达量和/或活性高于对照组，就表明该测试化合物是治疗纤毛缺陷性疾病的候选药物。
2.一种筛选治疗胚胎发育缺陷性疾病的候选药物的方法，其特征在于，步骤包括：
用待筛选物质处理表达或含有HOIP基因或其编码的蛋白的体系；和
检测所述体系中HOIP基因或其编码的蛋白的表达或活性；
其中，若候选物质可增加HOIP的表达或活性，则表明该候选物质是治疗胚胎发育缺陷性疾病的候选药物。
3.如下任一项应用：
1）药物组合物在制备治疗纤毛缺陷性疾病的药物中的应用，所述药物组合物包括HOIP功能性表达的促进剂；
2）药物组合在制备治疗胚胎发育缺陷性疾病的药物中的应用，所述药物组合物包括HOIP功能性表达的促进剂；
3）药物组合物在制备治疗缺陷性左右不对称/脊柱侧弯疾病的药物中的应用，所述药物组合物包括HOIP功能性表达的促进剂；
4）药物组合物在制备促进纤毛生成的产品中的应用，所述药物组合物包括HOIP功能性表达的促进剂；
5）HOIP抑制剂或HOIL-1L和SHARPIN的抑制剂在制备抑制纤毛生成的产品中的应用；
6）诊断产品在制备诊断纤毛缺陷性疾病的工具中的应用，所述产品包括检测HOIP的试剂或检测HOIL-1L和 SHARPIN的试剂，其中HOIP或HOIL-1L和SHARPIN在纤毛缺陷性疾病中表达下调；
7）诊断产品在制备诊断胚胎发育缺陷性疾病的工具中的应用，所述产品包括检测HOIP的试剂，其中HOIP在胚胎发育缺陷性疾病中表达下调；
8）HOIP在构建纤毛缺陷性疾病模型或胚胎发育缺陷性疾病模型中的应用，其中，通过施用HOIP的抑制剂构建纤毛缺陷性疾病模型或胚胎发育缺陷性疾病模型；
9）HOIL-1L和SHARPIN在构建纤毛缺陷性疾病模型中的应用，其中通过施用HOIL-1L和SHARPIN的抑制剂构建纤毛缺陷性疾病模型。
4.根据权利要求3所述的应用，其特征在于， 1)-4)中的药物组合物还包括药学上可接受的载体。
5.根据权利要求3所述的应用，其特征在于，1）-4）中的促进剂为含有能编码功能性HOIP蛋白的核酸的试剂、HOIP蛋白的激活剂、含有HOIP蛋白质的试剂。
6.根据权利要求5所述的应用，其特征在于，所述促进剂为含有能编码功能性HOIP蛋白的核酸的试剂。
7.根据权利要求6所述的应用，其特征在于，所述促进剂为含有HOIP核酸的表达载体。
8.根据权利要求3所述的应用，其特征在于，所述6）中所述的试剂包括通过数字成像技术、蛋白免疫技术、染料技术、核酸测序技术、核酸杂交技术、色谱技术、质谱技术检测HOIP表达水平的试剂或检测HOIL-1L和SHARPIN表达水平的试剂。
9.根据权利要求3所述的应用，其特征在于，所述7）中所述的试剂包括通过数字成像技术、蛋白免疫技术、染料技术、核酸测序技术、核酸杂交技术、色谱技术、质谱技术检测HOIP表达水平的试剂。
10.根据权利要求8所述的应用，其特征在于，所述试剂包括针对HOIP的引物、探针、抗体或针对HOIL-1L和SHARPIN的引物、探针、抗体。
11.根据权利要求9所述的应用，其特征在于，所述试剂包括针对HOIP的引物、探针、抗体。
12.根据权利要求3所述的应用，其特征在于，所述纤毛缺陷性疾病包括Nephronophthisis疾病、Joubert 综合症、Meckel-Gruber 综合症、Bardet Biedl 综合症。
13.根据权利要求12所述的应用，其特征在于，相比正常对照，HOIP在纤毛缺陷性疾病中表达水平或者表达活性下调或HOIL-1L和SHARPIN在纤毛缺陷性疾病中表达水平或者表达活性下调。
14.根据权利要求3所述的应用，其特征在于，5）、8）或9）中所述的抑制剂包括核酸抑制剂，蛋白抑制剂，蛋白水解酶，蛋白结合分子。
15.根据权利要求14所述的应用，其特征在于，所述核酸抑制剂包括shRNA、小干扰RNA、微小RNA、反义核酸及其构建物。
16.根据权利要求15所述的应用，其特征在于，所述核酸抑制剂选自反义核酸。
17.根据权利要求16所述的应用，其特征在于，所述反义核酸选自吗啉代反义寡核苷酸。
18.根据权利要求17所述的应用，其特征在于，所述吗啉代反义寡核苷酸的序列如SEQID NO：5和6所示。
19.根据权利要求15所述的应用，其特征在于，所述核酸抑制剂选自小干扰RNA。
20.根据权利要求19所述的应用，其特征在于，所述小干扰RNA的序列如SEQ ID NO：2-4所示。
21.根据权利要求3所述的应用，其特征在于，8）或9）中的模型为动物模型。
22.根据权利要求21所述的应用，其特征在于，所述动物模型为斑马鱼模型。
23.根据权利要求3所述的应用，其特征在于，8）或9）中的模型为细胞模型。
24.根据权利要求23所述的应用，其特征在于，所述细胞模型为RPE-1细胞模型。
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