CN117467664A - 一种靶向鸡mbnl1基因的反义寡核苷酸及其应用 - Google Patents
一种靶向鸡mbnl1基因的反义寡核苷酸及其应用 Download PDFInfo
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Abstract
本发明公开了一种靶向鸡MBNL1基因的反义寡核苷酸序列及化学修饰方式,同时本发明还公开了上述反义寡核苷酸在抑制鸡MBNL1基因表达中的应用。所述反义寡核苷酸的序列为5’‑CGGUTATGTAGGAAAUUGGC‑3’。本发明的反义寡核苷酸通过特异性抑制鸡MBNL1基因表达来鉴定基因功能,抑制效率达到50%以上,可应用于优质鸡的遗传育种以及为人类疾病研究提供参考数据,具有很大的经济价值和科研价值。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种通过抑制鸡盲肌样蛋白1(MBNL1)基因表达的反义寡核苷酸(ASO)及其应用。
背景技术
盲肌样蛋白(muscleblind-like protein,MBNL)家族是一类RNA结合蛋白,主要功能是参与前体mRNA的选择性剪接,另有研究表明,MBNL还与mRNA的运输、稳定性以及microRNA的产生过程相关。MBNL1是MBNL蛋白家族研究最为广泛和深入的分子,在组织中广泛表达,在骨骼肌和成肌细胞分化过程中高度表达。人类已有的一些研究,探讨了MBNL1在强直性肌营养不良、血管平滑肌细胞表型转换以及乳腺癌、宫颈癌、前列腺癌等恶性肿瘤中的作用。但在鸡上,对MBNL1的研究还比较少。
在组织中广泛表达的MBNL1有许多转录本异构体,不同异构体的表达具有组织特异性。研究表明,MBNL1 mRNA在鸡不同部位肌肉中的表达与其肌纤维类型组成密切相关,MBNL1 mRNA在胸大肌、髂胫外侧肌等快肌中表达较高,在缝匠肌、腓肠肌内侧头、前背阔肌等慢肌中表达较低;鸡MBNL1蛋白主要表达于细胞核。
反义寡核苷酸(ASO)是一种短的、化学合成的单链寡核苷酸,通过对其骨架和糖基进行修饰,可增加其稳定性、药理特性以及与靶标的结合,并通常具有较小的毒性,常用于细胞和动物体内基因功能研究以及ASO核酸药物的开发等。研究表明,ASO的活性与RNase H水平有关,细胞核中RNase H水平较高,核内靶标mRNAs的降解率要高于胞质,往往比主要在胞浆起作用的siRNA更适用于沉默细胞核定位的RNA。因此,借助ASO技术开展MBNL1基因功能研究,将有助于揭示鸡骨骼肌生长发育和肌纤维类型形成的调控机制,不仅对提升我国家禽产品的市场竞争力具有重要意义,而且对于揭示人类骨骼肌等相关疾病的分子机理也具有很好的借鉴价值。
发明内容
为了揭示鸡骨骼肌生长发育和肌纤维类型形成的调控机制,本发明提供了一种靶向鸡MBNL1基因的反义寡核苷酸及其应用。
第一方面,本发明提供了一种靶向鸡MBNL1基因的反义寡核苷酸,所述反义寡核苷酸的序列为5’-CGGUTATGTAGGAAAUUGGC-3’(SEQ NO.1)。
进一步地,所述反义寡核苷酸的序列全链硫代修饰,并在首尾两端5个碱基的核糖引入2’甲氧基修饰。
第二方面,本发明提供了一种含有编码上述的靶向鸡MBNL1基因的反义寡核苷酸的序列的重组表达载体、转基因细胞系或重组病毒。
第三方面,本发明提供了一种上述的靶向鸡MBNL1基因的反义寡核苷酸的试剂盒。
进一步地,所述试剂盒还包括用于检测鸡MBNL1基因表达的引物组。
第四方面,本发明提供了第一方面所述的反义寡核苷酸,第二方面所述的重组表达载体、转基因细胞系或重组病毒,第三方面所述的试剂盒,在鉴定鸡MBNL1基因功能中的应用。
通过上述技术方案,本发明实现了以下有益效果:
本发明提供了一种能够靶向鸡MBNL1基因的ASO,将其转入原代鸡成肌细胞后可以有效降低鸡MBNL1基因的表达,抑制效率达到50%以上,可见其敲降效率很高。本发明的ASO通过特异性敲降鸡MBNL1基因的表达来鉴定基因功能,可应用于优质鸡的遗传育种以及为人类疾病研究提供参考数据,具有很大的经济价值和科研价值。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式描述中所需要使用的附图作简单地介绍。
图1是用Western blot检测MBNL1在鸡肌肉组织中胞质和胞核表达,泳道1是11胚龄鸡腿肌组织胞浆蛋白,泳道2是11胚龄鸡胸肌组织胞浆蛋白,泳道3是成年鸡腿肌组织胞浆蛋白,泳道4是11胚龄鸡腿肌组织胞核蛋白,泳道5是11胚龄鸡胸肌组织胞核蛋白,泳道6是成年鸡腿肌组织胞核蛋白;
图2是不同ASO在原代鸡成肌细胞干扰鸡MBNL1基因表达的效率,其中,**表示干扰组与NC对照组相比,差异极显著(P<0.01);*表示干扰组与NC对照组相比,差异显著(P<0.05);
图3是不同siRNA在原代鸡成肌细胞干扰鸡MBNL1基因表达的效率,其中,ns表示干扰组与NC对照组相比,差异不显著(P>0.05);
图4是Western blot检测ASO 1在原代鸡成肌细胞分化期干扰鸡MBNL1基因表达的效率,其中,*表示干扰组与NC对照组相比,差异显著(P<0.05);
图5是CCL检测鸡成肌细胞中MBNL1基因表达敲低后对细胞增殖的影响,同一时间点干扰组与NC对照组比较,**表示差异极显著(P<0.01);
图6是实时荧光定量PCR检测鸡成肌细胞中MBNL1基因表达敲低后对细胞增殖分化相关基因表达的影响,其中,**表示干扰组与NC对照组相比,差异极显著(P<0.01)。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式,熟悉此技术的人士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及功效。显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。以下实施例中如无特别说明,均为常规方法,所用的试剂均为市售试剂。
实施例1 Western blot检测鸡MBNL1基因胞质胞核表达定位
用Nuclear Extract Kit(Active Motiff,40010)试剂盒提取肌肉组织样胞质和胞核蛋白,ProStatainTM蛋白质定量试剂盒(Active Motiff,15001)测定蛋白浓度,分别用β-Actin(ERWAN, ER001, 1:10000)和Histone H3(ERWAN, EAB02004, 1:1000)做一抗检测胞质内参蛋白β-Actin和胞核内参蛋白Histone H3,用MBNL1(abcam, ab108519, 1:1000)作一抗检测胞质和胞核中的MBNL1蛋白表达。由图1可见,胞质内参蛋白β-Actin在鸡肌肉组织的胞质和胞核中均表达,胞核内参蛋白Histone H3仅在鸡肌肉组织胞核中表达,MBNL1蛋白主要表达于鸡肌肉组织胞核中。
实施例2靶向鸡MBNL1基因最佳序列筛选
2.1 ASO/siRNA设计与合成
从NCBI在线数据库,获得鸡MBNL1基因共有17个转录本异构体的mRNA序列信息,针对这些转录本的共有CDS序列,设计2个靶向鸡MBNL1基因的ASO序列以及3对siRNA序列(ASO1,ASO 2,siRNA 1,siRNA 2,siRNA 3),由广州锐博生物科技有限公司合成。ASO及siRNA靶向的序列如表1所示。
表1ASO/siRNA靶向的序列
2.2 ASO/siRNA转染原代鸡成肌细胞
从动物组织中分离提取的原代细胞保持了细胞在体内许多重要生物学特征和功能,因此原代细胞不仅被广泛应用于分子、细胞生物学和生物医学基础研究,而且在生物医药领域更具有不可替代性的作用。但相对于细胞系,原代细胞难培养、外源基因转染效率低。本实施例以从11胚龄鸡胚腿肌分离提取的原代鸡成肌细胞为转染细胞,借助转染试剂Lipofectamine 3000将设计合成的靶向鸡MBNL1基因的ASO/siRNA转染入鸡成肌细胞,设ASO阴性对照ASO NC和siRNA阴性对照siRNA NC,每组4个重复孔,转染浓度为100 n mol/L。
2.3 RNA提取和cDNA制备
转染48h后,每孔用PBS清洗2次,选择南京诺唯赞生物科技股份有限公司提取细胞总RNA的试剂RNA isolater Total RNA Extraction Reagent收集和提取转染细胞的总RNA。核酸定量仪测定RNA浓度。按照南京诺唯赞生物科技股份有限公司的HiScript Ⅲ RTSuperMix for qPCR说明书进行cDNA合成。
2.4 实时荧光定量PCR检测ASO/siRNA干扰效率
选择南京诺唯赞生物科技股份有限公司的HiScript Ⅲ RT SuperMix for qPCR(+gDNA wiper)试剂采用SYBR Green Ι法进行实时荧光定量PCR检测ASO/siRNA特异性干扰鸡MBNL1基因表达的效率。实时荧光定量PCR反应的体系如表2所示。每个样品设置3个重复。
表2 实时荧光定量PCR反应的体系
检测鸡MBNL1基因表达的引物对核酸序列如下:
上游引物:5’-ACCCCGTAGGACCTAACGAT-3’(SEQ ID NO.7)
下游引物:5’-CTTGCAGTTCTCCCTCGAAC-3’(SEQ ID NO.8)
检测鸡内参ACTB基因表达的引物对核酸序列如下:
上游引物:5’-TGCTGTGTTCCCATCTATCG-3’(SEQ ID NO.9)
下游引物:5’-TTGGTGACAATACCGTGTTCA-3’(SEQ ID NO.10)
ASO特异性干扰鸡MBNL1基因表达的效率结果见图2,siRNA特异性干扰鸡MBNL1基因表达的效率结果见图3。由图2和图3可见,对MBNL1基因表达抑制,ASO的效果要优于siRNA,ASO 1的抑制效果最佳,达到50%以上,极显著(P<0.01)抑制鸡成肌细胞中鸡MBNL1基因的表达,后续实验选择ASO 1用于研究鸡MBNL1基因功能。
2.5 Western blot检测ASO 1干扰效率
转染72h后,每孔用PBS清洗2次,选择赛默飞RIPA细胞裂解液提取细胞总蛋白,BCA法测定提取的蛋白浓度后,分别用MBNL1(abcam, ab108519, 1:1000)和β-Actin(ERWAN,ER001, 1:10000)作一抗和羊抗兔IgG(Invitrogen, A32732, 1:5000)和羊抗鼠IgG(Invitrogen, A32723, 1:5000)作二抗进行Western blot实验,结果见图4。由图4可见,ASO 1能显著(P<0.05)抑制鸡MBNL1基因蛋白的表达。
实施例3MBNL1基因敲低表达后对鸡成肌细胞增殖的影响
选择南京诺唯赞生物科技股份有限公司的细胞增殖检测试剂Cell Counting-Lite 2.0 Luminescent Cell Viability Assay(CCL)检测鸡成肌细胞中MBNL1基因敲低表达后对细胞增殖能力的影响,结果见图5。由图5可见,在转染 ASO 1后24 h和48 h,MBNL1基因敲低表达组的细胞增殖能力均极显著(P<0.01)低于对照组,提示MBNL1有促进鸡成肌细胞增殖的作用。
实施例4MBNL1基因敲低表达后对细胞增殖、分化相关基因表达的影响
参考实施例2中2.4实时荧光定量PCR法检测鸡MBNL1基因敲低表达后对鸡成肌细胞增殖相关基因PAX7和分化相关基因MYOD1、PGC1α表达的影响。
检测鸡Pax7基因表达的引物对核酸序列如下:
上游引物:5’-TCAGCAACCGACGAGCAAG-3’(SEQ ID NO.11)
下游引物:5’-ATGGTGGATGGTGGCAAGG-3’(SEQ ID NO.12)
检测鸡MyoD1基因表达的引物对核酸序列如下:
上游引物:5’-CAACGCCATCCGCTACAT-3’(SEQ ID NO.13)
下游引物:5’-GTCGAGGCTGGAAACAAC-3’(SEQ ID NO.14)
检测鸡PGC1α基因表达的引物对核酸序列如下:
上游引物:5’-GAAGAGGGAAGAATACCGC-3’(SEQ ID NO.15)
下游引物:5’-CCCACGTAAATCACACGAC-3’(SEQ ID NO.16)
结果见图6。由图6可见,鸡成肌细胞中MBNL1基因敲低表达后,显著抑制PAX7、MYOD1和PGC1α基因的表达,提示MBNL1有促进鸡成肌细胞增殖和分化的作用。
以上结合实施例详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
Claims (6)
1.一种靶向鸡MBNL1基因的反义寡核苷酸,其特征在于,所述反义寡核苷酸的序列为5’-CGGUTATGTAGGAAAUUGGC-3’(SEQ NO.1)。
2.根据权利要求1所述的反义寡核苷酸,其特征在于,所述反义寡核苷酸的序列全链硫代修饰,并在首尾两端5个碱基的核糖引入2’甲氧基修饰。
3.含有编码权利要求1或2所述的靶向鸡MBNL1基因的反义寡核苷酸的序列的重组表达载体、转基因细胞系或重组病毒。
4.含有权利要求1或2所述的靶向鸡MBNL1基因的反义寡核苷酸的试剂盒。
5.根据权利要求4所述的试剂盒,其特征在于,所述试剂盒还包括用于检测鸡MBNL1基因表达的引物组。
6.权利要求1或2所述的反义寡核苷酸,权利要求3所述的重组表达载体、转基因细胞系或重组病毒,权利要求4或5所述的试剂盒,在鉴定鸡MBNL1基因功能中的应用。
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