CN117925607A - 一种抑制病毒的miRNA及其应用 - Google Patents
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Abstract
本发明提供了一种抑制病毒的miRNA及其应用,属于生物医药技术领域;本发明从化湿败毒方中提取出序列如SEQ.ID.NO.1~5所示的miRNA;所述miRNA与冠状病毒S蛋白的部分区域完全匹配且具有一定的细胞亲和力,能够抑制冠状病毒的复制,具有抗病毒的能力;所述miRNA在制成病毒抑制剂中具有很好的应用。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种抑制病毒的miRNA及其应用。
背景技术
新型冠状病毒感染(coronavirus disease2019,SARS-COV-2)是由严重急性呼吸系统综合征冠状病毒2型(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)感染导致的急性呼吸道传染病,传染性强、传染途径多、人群普遍易感。冠状病毒属的病毒是具有外套膜包裹的RNA病毒,其直径约100-160nm,遗传物质在所有RNA病毒中最大。SARS-CoV-2感染的前提条件是其进入宿主细胞,并依赖宿主细胞合成新病毒,冠状病毒的正链RNA进入宿主细胞后,可直接作为mRNA链引导蛋白质的合成;也可以通过RNA依赖的RNA聚合酶(RDRP)产生负链,然后以负链为模板,在RDRP的作用下产生正链,达到复制的目的。
miRNA是一类由18-24nt核苷酸构成的非编码内源性小分子RNA,通过7-8个碱基互补配对,与mRNA3’端非编码区结合,抑制或阻碍mRNA翻译,降解mRNA。miRNA与机体代谢、免疫、肿瘤等生理病理过程有密切联系,已经成为人们在生命科学研究中的热点之一。miRNA几乎在所有生物途径中都发挥着重要作用,其表达谱的变化与许多人类疾病相关。miRNA通过抑制翻译和诱导mRNA降解来抑制基因的表达,在病毒的复制或抑制过程中也是如此。
外泌体是指包含了复杂RNA和蛋白质的小膜泡(30-150nm),目前,其特指直径在40-100nm的盘状囊泡。所有培养的细胞在正常及病理状态下均可分泌外泌体,且外泌体天然存在于体液中,包括血液、唾液、尿液、脑脊液和乳汁中。外泌体被视为特异性分泌的膜泡,参与细胞间通讯,其主要来源于细胞内溶酶体微粒内陷形成的多囊泡体,经多囊泡体外膜与细胞膜融合后释放到胞外基质中。
化湿败毒方由生麻黄、苦杏仁、生石膏(先煎)、甘草、藿香(后下)、厚朴、苍术、草果、法半夏、茯苓、生大黄(后下)、生黄芪、葶苈子、赤芍14味中药组成,其可用于重型疫毒闭肺证患者,在临床实践发现其在治疗SARS-COV-2上发挥出了良好的效果。因此,可以从化湿败毒方中寻找一种用于抑制冠状病毒的miRNA。
发明内容
针对现有技术中存在的一些不足,本发明提供了一种抑制病毒的miRNA及其应用;本发明从化湿败毒方中提取出核苷酸序列如SEQ.ID.NO.1~5所示的miRNA;所述miRNA与冠状病毒S蛋白的部分区域完全匹配且具有一定的细胞亲和力,能够抑制冠状病毒的复制,具有抗病毒的能力;所述miRNA在制成病毒抑制剂中具有很好的应用。
为了达到上述目的,本发明采用以下技术方案:
本发明首先提供了一种miRNA,所述miRNA的核苷酸序列如SEQ.ID.NO.1、SEQ.ID.NO.2、SEQ.ID.NO.3、SEQ.ID.NO.4或SEQ.ID.NO.5所示;
其中,SEQ.ID.NO.1:CCGACCUUAGCUCAGUUGGUG;
SEQ.ID.NO.2:CGUUUCACGUCGGGUUCACC;
SEQ.ID.NO.3:GAAACCUGGCUCUGAUACCA;
SEQ.ID.NO.4:GAUCACUCGGUUGUCUGACACAC;
SEQ.ID.NO.5:UUUGGAUUGAAGGGAGCUCUA。
本发明还提供了一种生物安全的载体,所述载体中包含上述miRNA。
优选地,所述载体包括外泌体。
本发明还提供上述miRNA或载体的应用,所述应用包括:
(A)抑制病毒的复制;和/或
(B)制备病毒抑制剂;和/或
(C)制备预防和/或治疗病毒引起的病症及其症状的药物。
优选地,所述病毒包括冠状病毒,更优选为SARS-COV-2病毒。
本发明还提供了一种非治疗目的的抑制病毒复制的方法,所述方法为采用上述miRNA和/或载体抑制病毒复制。
优选地,所述病毒包括冠状病毒,更优选为SARS-COV-2病毒。
本发明还提供了一种病毒抑制剂,所述病毒抑制剂以上述miRNA和/或载体为活性成分。
优选地,所述病毒包括冠状病毒,更优选为SARS-COV-2病毒。
本发明还提供了一种防和/或治疗病毒引起的病症及其症状的药物,所述药物以上述miRNA和/或载体为活性成分。
优选地,所述病毒包括冠状病毒,更优选为SARS-COV-2病毒。
与现有技术相比,本发明的有益效果在于:
本发明从化湿败毒方中筛选获得miRNA,所述miRNA与SARS-COV-2病毒S蛋白的部分区域完全匹配,并且具有一定的亲细胞和力,因此能够一定程度上抑制SARS-COV-2病毒的复制;所述miRNA具有抗病毒能力的效果,能够用于制备病毒抑制剂。
本发明将miRNA导入到生物安全的载体中,例如可以将miRNA转染至HEK293T细胞中,从HEK293T细胞中分离外泌体,借助生物安全的载体,阻断SARS-COV-2病毒S蛋白的合成,抑制SARS-COV-2病毒侵袭能力,快速高效地降低病人体内的病毒拷贝数,从而达到抑制新型冠状病毒的目的。
本发明中以外泌体为载体,所述外泌体被视为特异性分泌的膜泡,参与细胞间通讯。外泌体里含有蛋白质、miRNA等多种生物活性物质,通过膜融合作为细胞间的桥梁,将miRNA和蛋白质等内容物传递给其他细胞。本发明中所述miRNA可以人工合成,所述外泌体可以在低温下储存。并且,所述miRNA和外泌体成本低,可以实现大规模的生产,能够用于抑制病毒的复制、制备病毒抑制剂或制备预防和/或治疗病毒引起的病症及其症状的药物。所述miRNA和外泌体对充分发挥中医药救治新型冠状病毒感染中具有重要意义。此外,本发明为化湿败毒方在治疗新型冠状病毒感染的作用机制上提供了新的思路。
附图说明
图1为化湿败毒方测序后miRNA富集程度的结果图(A)和病毒基因组上miRNA潜在的靶向结合位点数量图(B),其中A1和A2是69个miRNA富集程度的结果图。
图2为细胞培养基收集外泌体的示意图(A)、miRNA转染后透射电镜观察HEK293T细胞外泌体的形态图(B)以及Western Blot实验验证外泌体标志蛋白的结果图(C);其中B1为空白对照,B2~B6是分别含五种miRNA转染的外泌体。
图3为HEK293T细胞外泌体miRNA抗病毒活性的鉴定图(A)和qPCR(n=3)定量检测转染了不同细胞外泌体后细胞中的病毒产量(B),B1~B5是由五种miRNA转染HEK293T细胞后分泌的外泌体抑制SARS-CoV-2复制的实验研究。
具体实施方式
下面结合附图以及具体实施例对本发明作进一步的说明,但本发明的保护范围并不限于此。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。以下实施案例中的方法、设备、材料,如果未进行特别说明,均为本领域常规方法、设备和材料,均可由市场购得。
实施例1:miRNA的获取及测序分析
(1)miRNA的提取:
采用通用植物microRNA提取试剂盒(离心柱型)BioTeke(无锡百泰克生物技术有限公司)化湿败毒方中的miRNA,整个实验中使用无酶无菌的枪头、EP管,从而降低miRNA的降解。通过增大植物提取样品量、在提取过程中增加裂解液剂量和增加氯仿的使用次数,尽可能使核蛋白体分解,以减少植物体蛋白污染。
(2)miRNA测序分析:
委托百迈克生物科技有限公司对步骤(1)中提取到的miRNA进行测序分析,分析步骤如下:
(A)使用Illumina深度测序技术对miRNA片段进行测序,发现富集度高的miRNA;
(B)采用novaseq6000 PE150平台对化湿败毒方中miRNA行测序,平均每个样品产生10M clean reads(单个样品数据量往下浮动小于5%)将比对到参考基因组的reads与miRbase数据库中已知的miRNA的成熟序列及其上游2nt与下游5nt的范围进行比对,比对最多允许一个错配,测序结果如图1所示。
图1为化湿败毒方测序后miRNA富集程度的结果图(A)和病毒基因组上miRNA潜在的靶向结合位点数量图(B)。从图1A中可以看出,化湿败毒方中存在许多miRNA的表达,通过测序中发现了69个miRNA,其中有5个富集程度高的miRNA。
将上述5个富集程度高的miRNA分别输入RNAhybrid软件与冠状病毒SARS-COV-2全基因组序列(NC_045512.2)进行比对分析,获得了针对SARS-COV-2病毒基因组具有靶向能力和结合位点较强的miRNA。通过比对发现,5个miRNA对SARS-COV-2病毒的基因组具有靶向能力,所述miRNA分别为ptc-miR6478、ppt-miR894、aof-miR5139a、pla-miR11607和aof-miR159,其核苷酸序列如下所示:
ptc-miR6478:CCGACCUUAGCUCAGUUGGUG(SEQ.ID.NO.1);
ppt-miR894:CGUUUCACGUCGGGUUCACC(SEQ.ID.NO.2);
aof-miR5139a:GAAACCUGGCUCUGAUACCA(SEQ.ID.NO.3);
pla-miR11607:GAUCACUCGGUUGUCUGACACAC(SEQ.ID.NO.4);
aof-miR159:UUUGGAUUGAAGGGAGCUCUA(SEQ.ID.NO.5)。
为了验证上述5个miRNA抗SARS-COV-2的效果,本实施例中通过生物信息学分析预测了5个miRNA在SARS-CoV-2基因组中的结合位点,预测结果如图1B所示。从图中可以看出,miR6478与SARS-CoV-2基因组有66个结合位点,miR894与SARS-CoV-2基因组有44个结合位点,miRNA5139a与SARS-CoV-2基因组有49个结合位点。miRNA11607与SARS-CoV-2基因组有103个结合位点,miRNA159与SARS-CoV-2基因组有47个结合位点。因此,我们推测上述5个miRNA可能能够抑制SARS-CoV-2蛋白的翻译。
实施例2:miRNA的转染及外泌体的制备
(1)将HEK293T细胞(普诺赛生命科技有限公司)在37℃、5%CO2、10%胎牛血清(南京森贝伽生物科技有限公司)和1%青霉素-链霉素(南京森贝伽生物科技有限公司)的高糖DMEM(Gibco)中培养。待HEK293T细胞复苏、传代后,根据EL转染试剂盒(北京全式金生物技术有限公司)说明,对HEK293T细胞进行转染。将合成5个miRNA或对照非编码RNA(ncRNA)转染至HEK293T细胞中,转染4-6后小时更换培养基,继续培养48后收集上清。
(2)采用如图2A所示的方法外泌体制备,具体步骤为:
根据上述(1)的步骤,分别取ncRNA、miR6478、miR894、miR5139a、miR11607、miR159转染后的细胞上清液,先以4℃、500×g的条件离心10min,去沉淀取上清后,再以4℃、2000×g的条件离心10min,去沉淀取上清后,最后以4℃、10000×g的条件离心20min后取上清液,将得到的上清液使用0.22μm孔径过滤器过滤,然后从收获的上清液在4℃、110000×g的条件离心75min(离心2次)后分离外泌体,弃去沉淀,用PBS洗涤两次,将外泌体重悬浮于PBS中,使用0.22μm孔径过滤器过滤,得到外泌体,-80℃保存,备用。
在透射电镜下观察HEK293T细胞外泌体的形态,观测结果如图2B所示。从图2B中可以看出,转染之后miR6478、miR894、miR5139a、miR11607、miR159这5个miRNA的细胞外泌体个体完整,观察到小囊泡,可见明显完整膜结构,呈茶托样。Western Blot实验验证外泌体标志蛋白的结果如2C所示。
实施例3:miRNA抗病毒成分的验证
将非洲绿猴肾VeroE6细胞系(普诺赛生命科技有限公司)在37℃、5%CO2条件下,在添加10%胎牛血清中培养。SARS-CoV-2毒株(NC_045512.2)在Vero E6细胞中繁殖。所有关于SARS-CoV-2活病毒的实验都是在P3实验室进行。
为了评价上述miRNA对SARS-CoV-2复制的直接影响,采用如图3A所示的鉴定流程来评价HEK293T细胞外泌体miRNA的抗病毒活性,具体方法如下所示:
分别将分离的含miR6478、miR894、miR5139a、miR11607、miR159和ncRNA的细胞外泌体与5×104VeroE6细胞在培养液中孵育8h,孵育结束后更换培养基并感染SARS-CoV-2。在感染后24h,通过实时定量RT-PCR(qRT-PCR)定量检测细胞上清液中病毒的拷贝数来评价疗效,检测结果如图3B所示。
从图3B中可以看出,细胞外泌体miR6478对SARS-CoV-2病毒复制的抑制率为39%(从4.26×109到2.60×109拷贝/mL);细胞外泌体miR894对SARS-CoV-2病毒复制的抑制率为30%(从4.51×109到3.16×109拷贝/mL);细胞外泌体miR5139a对SARS-CoV-2病毒复制的抑制率为23%(从4.03×109到3.11×109拷贝/mL);细胞外泌体miR11607对SARS-CoV-2病毒复制的抑制率为60%(从4.33×109到1.74×109拷贝/mL);细胞外泌体miR159对SARS-CoV-2病毒复制的抑制率为26%(从4.12×109到3.05×109拷贝/mL)。因此,细胞外泌体miR6478、miR894、miR5139a、miR11607和miR159能够不同程度的抑制SARS-CoV-2病毒,这为研究化湿败毒方的作用机制具有重要的意义。
综上所述,本发明提供的5个miRNA与SARS-COV-2病毒S蛋白的部分区域完全匹配,并且具有一定的亲细胞和力,因此能够一定程度上抑制SARS-COV-2病毒的复制;所述miRNA具有抗病毒能力的效果,能够用于制备病毒抑制剂。
所述实施例为本发明的优选的实施方式,但本发明并不限于上述实施方式,在不背离本发明的实质内容的情况下,本领域技术人员能够做出的任何显而易见的改进、替换或变型均属于本发明的保护范围。
Claims (10)
1.一种抑制病毒的miRNA,其特征在于,所述miRNA的核苷酸序列如SEQ.ID.NO.1、SEQ.ID.NO.2、SEQ.ID.NO.3、SEQ.ID.NO.4或SEQ.ID.NO.5所示;
其中,SEQ.ID.NO.1:CCGACCUUAGCUCAGUUGGUG;
SEQ.ID.NO.2:CGUUUCACGUCGGGUUCACC;
SEQ.ID.NO.3:GAAACCUGGCUCUGAUACCA;
SEQ.ID.NO.4:GAUCACUCGGUUGUCUGACACAC;
SEQ.ID.NO.5:UUUGGAUUGAAGGGAGCUCUA。
2.一种载体,其特征在于,所述载体中包含权利要求1所述的miRNA。
3.根据权利要求2所述的载体,其特征在于,所述载体包括外泌体。
4.权利要求1所述的miRNA或权利要求2~3任一项所述的载体的应用,其特征在于,所述应用包括:
(A)抑制病毒的复制;和/或
(B)制备病毒抑制剂;和/或
(C)制备预防和/或治疗病毒引起的病症及其症状的药物。
5.根据权利要求4所述的应用,其特征在于,所述病毒包括冠状病毒,更优选为SARS-COV-2病毒。
6.一种非治疗目的的抑制病毒复制的方法,其特征在于,所述方法为采用权利要求1所述的miRNA和/或权利要求2~3任一项所述的载体抑制病毒复制。
7.根据权利要求6所述的方法,其特征在于,所述病毒包括冠状病毒,更优选为SARS-COV-2病毒。
8.一种病毒抑制剂,其特征在于,所述病毒抑制剂以权利要求1所述的miRNA和/或权利要求2~3任一项所述的载体为活性成分。
9.根据权利要求8所述的病毒抑制剂,其特征在于,所述病毒包括冠状病毒,更优选为SARS-COV-2病毒。
10.一种预防和/或治疗病毒引起的病症及其症状的药物,所述药物以权利要求1所述的miRNA和/或权利要求2~3任一项所述的载体为活性成分。
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