CN114032238B - gga-miR-146a-5p的抑制物在制备抗J亚群禽白血病病毒感染药物中的应用 - Google Patents
gga-miR-146a-5p的抑制物在制备抗J亚群禽白血病病毒感染药物中的应用 Download PDFInfo
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Abstract
本发明涉及病毒学领域,提供了一种gga‑miR‑146a‑5p的抑制物在制备抗J亚群禽白血病病毒感染药物中的应用,该抑制物为gga‑miR‑146a‑5p的互补序列,其核苷酸序列如SEQ ID NO:1所示,该抑制物可以阻断ALV‑J在细胞中的复制与增殖,可作为ALV‑J复制抑制剂应用,还可以制备成试剂盒和药物等配合净化应用,为抗ALV‑J的研究提供了新的思路和理论基础。
Description
技术领域
本发明涉及病毒学和分子生物学领域,具体涉及gga-miR-146a-5p的抑制物在制备抗J亚群禽白血病病毒感染药物中的应用。
背景技术
J亚群禽白血病病毒(Avian leukosis virus subgroup J,ALV-J),是致癌性逆转录病毒,于1988年由英国科学家Payne及其同事等发现并分离。据报道ALV-J是一外源性白血病病毒与内源性E亚群病毒囊膜基因的重组体,在鸡群中可以通过垂直传播和水平传播。我国于1999年首次分离到ALV-J,近年来ALV-J的宿主范围逐步扩大可以感染我国各品系鸡群,并造成严重危害,已成为我国各种类型鸡群中危害最严重的ALV流行亚群。近年来,甚至在其他禽类体内也检测到了ALV-J阳性感染。由于ALV-J导致鸡的免疫抑制、生长迟缓及多种肿瘤,其传播率也远高于其他ALV亚群,并且动物机体对该病毒表现出免疫耐受,并且目前尚无有效疫苗和药物对其进行防治,从而使其控制和根除变得更加困难,因此需要开发针对ALV-J防治的新措施。
microRNA(miRNA)是一种非编码RNA,长度约为21-23个核苷酸左右,通过直接结合mRNA并影响翻译效率或mRNA丰度来控制蛋白水平。miRNA依靠其成熟体发挥作用。miRNA的成熟需要多个步骤,首先在聚合酶的作用下先转录为pri-microRNA,大多数miRNA的转录是由RNA聚合酶Ⅱ所介导的。pri-microRNA通过两次剪切形成成熟体的miRNA。当miRNA与靶mRNA几乎完全互补时可以导致靶mRNA的降解,此现象常见于植物;而在动物中,miRNA与靶mRNA互补程度较低,可以在不影响靶mRNA的表达下抑制翻译。有大量研究表明,miRNA参与了动植物各种的生理病理过程,包括发育的调节、细胞的增殖分化与免疫活动的调节、病毒感染的发生发展等几乎所有生命活动。
病毒侵入机体后,miRNA在病毒与宿主的互作过程中发挥着重要的调节作用。病毒与宿主都可以编码miRNA,病毒所编码的miRNA有利于帮助病毒规避宿主的免疫反应,而宿主所编码miRNA的则对病毒的入侵有着促进或抑制的作用。由于目前尚无有效疫苗和药物对ALV-J进行防治,因此开发一种可以有效抑制ALV-J复制的miRNA抑制物具有潜在的临床应用价值。
发明内容
本发明的发明人针对现有技术存在的空白之处,提供了一种gga-miR-146a-5p的抑制物在制备抗ALV-J病毒感染药物中的应用,该抑制物为gga-miR-146a-5p的互补序列,其核苷酸序列如SEQ ID NO:1所示,该抑制物可以阻断ALV-J在细胞中的复制与增殖,可作为ALV-J复制抑制剂应用,还可以制备成试剂盒和药物等配合净化应用。为抗ALV-J的研究提供了新的思路和理论基础。
发明人首先研究了ALV-J感染DF-1后细胞中gga-miR-146a-5p的表达情况,结果显示gga-miR-146a-5p的表达显著上调,基于这一发现,发明人设计并合成了gga-miR-146a-5p模拟物和抑制物,并以此为基础研究了其与ALV-J复制的关系:
gga-miR-146a-5p抑制物(inhibitor),其核苷酸序列如SEQ ID NO:1所示(aacccauggaauucaguucuca),
gga-miR-146a-5p模拟物(mimics),其核苷酸序列如SEQ ID NO:2所示(ugagaacugaauuccauggguu);
研究结果发现,将上述gga-miR-146a-5p抑制物(inhibitor)转染至DF-1细胞,转染24h后用ALV-J感染细胞,感染48h后分别用荧光定量PCR或Western blot方法分别在RNA和蛋白水平检测ALV-J的表达量,发现gga-miR-146a-5p抑制物能显著抑制ALV-J在DF-1细胞中的复制和增殖。因此可以用于制备抗ALV-J病毒感染的药物。
附图说明
图1为ALV-J感染后DF-1细胞中gga-miR-146a-5p的定量检测结果,
图中显示ALV-J感染对细胞中gga-miR-146a-5p的调控作用,以MOI为1的ALV-JNX0101株感染DF-1细胞72h后,提取总RNA,进行miRNA反转录,实时荧光定量PCR检测gga-miR-146a-5p的表达,结果表明ALV-J感染显著上调gga-miR-146a-5p的表达;
图2为转染gga-miR-146a-5p抑制物(inhibitor)后DF-1细胞中ALV-J mRNA的定量检测结果,
图中显示gga-miR-146a-5p抑制物(inhibitor)在RNA水平抑制了ALV-J的复制,在DF-1细胞中转染gga-miR-146a-5p inhibitor及其阴性对照,转染24h后以MOI为1接种ALV-JNX0101株,感染48h后提取总RNA进行反转录,实时荧光定量PCR检测ALV-J的表达,结果表明gga-miR-146a-5p inhibitor在RNA水平抑制ALV-J的复制;
图3为转染gga-miR-146a-5p抑制物(inhibitor)后DF-1细胞中ALV-J gp85蛋白的检测结果,
图中显示gga-miR-146a-5p抑制物(inhibitor)在蛋白水平抑制ALV-J的复制,在DF-1细胞中转染gga-miR-146a-5p inhibitor及其阴性对照,转染24h后以MOI为1接种ALV-JNX0101株,感染48h后提取蛋白,Western blot检测ALV-J的表达,结果表明gga-miR-146a-5pinhibitor在蛋白水平抑制ALV-J的复制;
图4为转染gga-miR-146a-5p模拟物(mimics)后DF-1细胞中ALV-J mRNA的定量检测结果,
图中显示gga-miR-146a-5p模拟物(mimics)在RNA水平促进ALV-J的复制,在DF-1细胞中转染gga-miR-146a-5p mimics及其阴性对照,转染24h后以MOI为1接种ALV-JNX0101株,感染48h后提取总RNA进行反转录,实时荧光定量PCR检测ALV-J的表达,结果表明gga-miR-146a-5p mimics在RNA水平促进ALV-J的复制;
图5为转染gga-miR-146a-5p模拟物(mimics)后DF-1细胞中ALV-J gp85蛋白的检测结果,
图中显示gga-miR-146a-5p模拟物(mimics)在蛋白水平促进ALV-J的复制,在DF-1细胞中转染gga-miR-146a-5p mimics及其阴性对照,转染24h后以MOI为1接种ALV-JNX0101株,感染48h后提取提取蛋白,Western blot检测ALV-J的表达,结果表明gga-miR-146a-5pmimics在蛋白水平促进ALV-J的复制。
具体实施方式
为了更清楚地说明本发明,下面结合优选实施例和附图对本发明做进一步的说明。以使本发明的优点和特征能更易于被本领域技术人员理解,从而对本发明的保护范围做出更为清楚明确的界定,下述实施例中所使用的实验方法如无特殊说明,均为常规方法或直接交由基因公司完成;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 ALV-J感染显著上调DF-1细胞中gga-miR-146a-5p的表达
(1)ALV-J感染DF-1细胞
将生长良好的DF-1细胞从细胞瓶中消化下来以1×105均匀的铺板,当细胞生长到汇合度达70%时,将ALV-J(毒株NX0101 GenBank accession number DQ115805.1,本领域常用毒株)病毒液置于冰上融化,用[MOI]=1的ALV-J感染细胞,同时设未接毒的对照(Mock)组,病毒感作1.5h后弃掉病毒液,补充含1%FBS的DMEM维持液维持细胞的生长,于37℃5%CO2条件下培养,于接毒后72h收集细胞。
(2)总RNA提取,miRNA第一链cDNA的合成及荧光定量PCR
使用TRNzol试剂(购自北京TIANGEN公司)提取总RNA,对于DF-1细胞直接在培养板中加入TRNzol Universal试剂裂解细胞,对于组织取50mg组织加入1mL TRNzol Universal试剂,用匀浆仪进行匀浆处理。使用miRcute增强型miRNA cDNA第一链合成试剂盒(购自北京TIANGEN公司)进行miRNA反转录,每20mL体系中加入1ng RNA;反转录程序为42℃60min,95℃3min。将反转录得到的cDNA作为模板,使用miRNA增强型miRNA荧光定量检测试剂盒(SYBR Green)(购自北京TIANGEN公司)通过qPCR检测gga-miR-146a-5p的相对表达量,按照试剂盒说明书配置反应体系和设定反应程序。
实验中的每个样品均设置3个重复,以U6为内参基因,其中U6的上游引物序列见SEQ ID NO:3(ctcgcttcggcagcaca),下游引物序列见SEQ ID NO:4(aacgcttcacgaatttgcgt);gga-miR-146a-5p上游引物序列见SEQ ID NO:5(gtgagaactgaattccatgggtt),下游引物为试剂盒中通用下游引物,用2-ΔΔCT法比较相对表达水平,结果如图1所示ALV-J感染显著上调了DF-1细胞中gga-miR-146a-5p的表达。
为了进一步研究gga-miR-146a-5p模拟物和抑制物与ALV-J复制的关系,接下来的实施例将对gga-miR-146a-5p模拟物和抑制物与ALV-J复制的关系进行研究。
实施例2 gga-miR-146a-5p抑制物和模拟物对ALV-J复制的影响
为了检测gga-miR-146a-5p模拟物转染是否会影响ALV-J的复制,我们合成了gga-miR-146a-5p的模拟物(mimics)和抑制物(inhibitor),
gga-miR-146a-5p抑制物(inhibitor),其核苷酸序列如SEQ ID NO:1所示(aacccauggaauucaguucuca,由吉玛公司合成),
gga-miR-146a-5p模拟物(mimics),其核苷酸序列如SEQ ID NO:2所示(ugagaacugaauuccauggguu,由吉玛公司合成);
DF-1细胞转染inhibitor或mimics 24h后接种ALV-J,病毒感染48h后,收集细胞,提取细胞的蛋白质和RNA,通过Western blot及qRT-PCR的方法分别从蛋白和RNA的水平检测ALV-J的表达量。
(1)gga-miR-146a-5p抑制物和模拟物转染
gga-miR-146a-5p抑制物和模拟物转染前一天,将生长良好的DF-1细胞从细胞瓶中消化下来并通过细胞计数板对细胞悬液进行计数,以每孔1×105均匀的铺板,当细胞的汇合度达到70%时进行转染。按照Lipofectamine 3000(购自美国invitrogen公司)的转染方法,将gga-miR-146a-5p抑制剂或模拟物与Lipofectamine 3000共同孵育制备混合液,将混合液加入细胞含10%FBS(胎牛血清,购自美国Gibico公司)的DMEM培养基(高糖型,购自美国Hyclone公司)中,37℃5%CO2条件下培养24h。
(2)ALV-J感染
将ALV-J(毒株NX0101 GenBank accession number DQ115805.1)病毒液置于冰上融化,DF-1细胞转染24h后,用[MOI]=1的ALV-J感染细胞,感染48h后收集细胞,使用Trizol试剂(购自美国Invitrogen公司)提取细胞总RNA。
(3)ALV-J RNA定量检测
RNA的反转录步骤依据FastKing gDNA Dispelling RT SuperMix试剂盒(购自北京TIANGEN公司)说明书进行,每20mL体系中加入1ng RNA;反转录程序为42℃15min,95℃3min。将反转录得到的cDNA作为模板,使用real-time PCR试剂盒(购自Takara公司)通过qPCR检测ALV-J病毒载量。按照试剂盒说明书配置反应体系和设定反应程序。
实验中的每个样品均设置3个重复,以GAPDH为内参基因,用2-ΔΔCT法比较相对表达水平。GAPDH的上游引物序列见SEQ ID NO:6(gaacatcatcccagcgtcca),下游引物序列见SEQID NO:7(cggcaggtcaggtcaacaac);ALV-J gp85上游引物序上游引物序列见SEQ ID NO:8(tgcgtgcgtggttattatttc),下游引物序列见SEQ ID NO:9(aatggtgaggtcgctgactgt)。
(4)ALV-J蛋白定量检测
利用Western blot方法检测各组细胞内ALV-J囊膜蛋白gp85的表达水平,内参基因选择β-actin,具体步骤如下:
对于细胞样品:细胞用预冷的PBS洗涤三次,在RIPA解液中加入PMSF,使得其终浓度为1mM。在冰上用裂解液裂解细胞约5min,用移液器反复吹打,充分裂解后收集到EP管中;4℃,12000×g离心5min,取上清,BCA法测量蛋白浓度。蛋白变性:取适量的蛋白样品,加入5×的SDS蛋白质上样缓冲液,混匀后于100℃水浴中变性5min;
配制合适浓度的分离胶与浓缩胶,在电泳槽中加入足量1×的电泳缓冲液;每孔加入蛋白样品20g;蛋白Marker加入10L;接通电源,浓缩胶处使用80v电压电泳30min,待染料进入分离胶与浓缩胶的分界线时,调整电压将至110v电泳1.5h左右,至溴酚蓝指示剂至分离胶的底部,关闭电源;
电泳结束后撬开玻璃板,取下凝胶。根据目的蛋白大小,切取适宜的胶块。将PVDF膜剪至如凝胶大小并在在甲醇中活化约1min,然后置于电转缓冲液中浸泡约5min;按三明治法安装转印装置:即负极夹-海绵垫-三层滤纸-凝胶-PVDF膜-三层滤纸-海绵垫-正极夹,确保凝胶与PVDF膜之间没有气泡;将转印装置置于转移槽,使用快速转膜液(购自新赛美公司)400mA恒流转膜30min,将蛋白转至PVDF膜上。
转膜结束后取下PVDF膜,TBST缓冲液漂洗5min,使用含5%脱脂奶粉的TBST溶液37℃封闭2h;封闭结束后,TBST缓冲液洗涤3次,每次10min。用特异性单克隆抗体4℃孵育过夜,TBST缓冲液洗涤3次,每次10min。加入HRP标记的羊抗鼠IgG二抗,37℃孵育1h,TBST缓冲液洗涤3次。
曝光显色:避光条件下配制适量显影液,A液:B液=1:1,用显影液覆盖整张膜,在ECL化学发光显影仪中检测蛋白信号。
结果如下:与转染inhibitor阴性对照组相比在DF-1细胞中转染gga-miR-146a-5pinhibitor后可以在RNA水平显著抑制ALV-J的复制(如图2所示),同时在蛋白水平也显著抑制了ALV-J的复制(如图3所示)。与转染mimics阴性对照组相比在DF-1细胞中转染gga-miR-146a-5p mimics后可以在RNA水平显著促进ALV-J的复制(如图4所示),同时在蛋白水平也显著促进了ALV-J的复制(如图5所示)。说明gga-miR-146a-5p抑制物(inhibitor)有抑制ALV-J复制的功能,可作为ALV-J复制抑制剂应用,还可以制备成试剂盒和药物等配合净化应用,为抗ALV-J的研究提供了新的思路和理论基础。
序列表
<110> 山东农业大学
<120> gga-miR-146a-5p的抑制物在制备抗J亚群禽白血病病毒感染药物中的应用
<160> 9
<170> SIPOSequenceListing 1.0
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<213> 人工序列(人工序列)
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<212> DNA
<213> 人工序列(人工序列)
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ctcgcttcgg cagcaca 17
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<213> 人工序列(人工序列)
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aacgcttcac gaatttgcgt 20
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<212> DNA
<213> 人工序列(人工序列)
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Claims (1)
1.gga-miR-146a-5p的抑制物在制备抗J亚群禽白血病病毒感染药物中的应用,其特征在于:gga-miR-146a-5p的抑制物,其核苷酸序列如SEQ ID NO.1所示。
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AU2013224690A1 (en) * | 2007-06-15 | 2013-09-26 | The Ohio State University Research Foundation | Oncogenic ALL-1 fusion proteins for targeting drosha-mediated microRNA processing |
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