CN114032238A - 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
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Claims (2)
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。
2.根据权利要求1所述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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