WO2023201836A1 - 一种翘芪复方中药汤剂来源的微小核糖核酸及其制备方法和应用 - Google Patents

一种翘芪复方中药汤剂来源的微小核糖核酸及其制备方法和应用 Download PDF

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WO2023201836A1
WO2023201836A1 PCT/CN2022/094829 CN2022094829W WO2023201836A1 WO 2023201836 A1 WO2023201836 A1 WO 2023201836A1 CN 2022094829 W CN2022094829 W CN 2022094829W WO 2023201836 A1 WO2023201836 A1 WO 2023201836A1
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seq
nucleotide sequence
chinese medicine
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顾晓松
毛苏苏
龚蕾蕾
杨晓明
王星辉
孙华林
徐来
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南通大学
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    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the invention belongs to the technical field of biomedicine, and specifically relates to a microribonucleic acid derived from Qiaoqi compound traditional Chinese medicine decoction and its preparation method and application.
  • Influenza influenza for short
  • influenza virus is an acute respiratory disease commonly suffered by mammals and poultry. It is caused by the influenza virus. It is highly contagious and spreads quickly. It usually leads to simple respiratory infections, including cough, fever, myalgia, Clinical symptoms include chills or sweating, and the discomfort can last from 2 to 8 days and usually develops rapidly. Some patients, especially the elderly, young children and those with other chronic diseases, are prone to viral or secondary pneumonia, and even difficulty breathing and multiple organ failure.
  • the use of anti-influenza drugs is currently a common method of fighting influenza. The prescription is generally determined based on the patient's degree of infection and the patient's physical condition.
  • influenza virus is one of the most intensively studied pathogens, because influenza viruses are highly susceptible to mutation, existing control and treatment options need to be continuously improved.
  • the development of new drugs for viral influenza is not only a major need in today's society, but also a matter of people's death. Significant health needs.
  • the novel coronavirus (referred to as COVID-19) is a new type of virus that has spread widely in the past two years. It is mainly spread through the respiratory route in the form of droplets. Recently, it has also been found that it can be spread through aerosols and contact with objects. It is extremely contagious and spreads very quickly. , the main clinical symptoms are: fever, fatigue, dry cough, anosmia/olfactory disorder, severe pneumonia may be accompanied by difficulty breathing, shortness of breath and hypoxemia, and may cause other complications. Because there is currently no specific treatment for the new coronavirus, it has spread on a large scale around the world. Therefore, in response to the demand for drugs for epidemic prevention and control, the development of new drugs specific for COVID-19 is an important means to respond to COVID-19 and protect people's lives.
  • MicroRNAs are a type of small endogenous non-coding RNA. Normally, miRNA-encoding genes are transcribed by RNA polymerase II and produce primary transcripts, which are processed into small RNAs of approximately 21 nucleotides by RNase III endonucleases Drosha and Dicer. By binding to the coding region or the 3' and 5' untranslated regions of target mRNA, miRNA mediates post-transcriptional gene silencing, plays an important role in a variety of physiological and pathological processes, and has broad application prospects in the medical field.
  • the present invention provides a microribonucleic acid derived from the Qiaoqi compound Chinese medicine decoction and its preparation method and application.
  • Functional plant microribonucleic acid is extracted from the Qiaoqi compound traditional Chinese medicine decoction or contains the microribonucleic acid.
  • Nucleic acid extract, and experiments have verified that the microRNA QQ_159 derived from the Qiaoqi compound traditional Chinese medicine decoction has a certain inhibitory effect and therapeutic effect on viral influenza and pneumonia caused by new coronavirus infection.
  • microribonucleic acid derived from Qiaoqi compound traditional Chinese medicine decoction is selected from novel_mir7, novel_mir33, novel_mir35, novel_mir10, novel_mir20, novel_mir5, novel_mir36, novel_mir28, novel_mir31, ppt-miR894, novel_mir32, novel_mir21, pab-miR3711 , QQ_159 or bdi-miR159a-3p; among them,
  • nucleotide sequence of novel_mir7 is shown in SEQ ID NO.1;
  • nucleotide sequence of novel_mir33 is shown in SEQ ID NO.2;
  • novel_mir35 is shown in SEQ ID NO.3;
  • nucleotide sequence of novel_mir10 is shown in SEQ ID NO.4;
  • nucleotide sequence of novel_mir20 is shown in SEQ ID NO.5;
  • nucleotide sequence of novel_mir5 is shown in SEQ ID NO.6;
  • novel_mir36 is shown in SEQ ID NO.7;
  • nucleotide sequence of novel_mir28 is shown in SEQ ID NO.8;
  • nucleotide sequence of novel_mir31 is shown in SEQ ID NO.9;
  • nucleotide sequence of ppt-miR894 is shown in SEQ ID NO.10;
  • nucleotide sequence of novel_mir32 is shown in SEQ ID NO.11;
  • nucleotide sequence of novel_mir21 is shown in SEQ ID NO.12;
  • the nucleotide sequence of pab-miR3711 is shown in SEQ ID NO.13;
  • the nucleotide sequence of QQ_159 is shown in SEQ ID NO.14;
  • nucleotide sequence of bdi-miR159a-3p is shown in SEQ ID NO.15.
  • the microRNA is QQ_159, including synthetic QQ_159, plant QQ_159, precursor form of QQ_159 or mature form of QQ_159.
  • a preparation method of microribonucleic acid derived from Qiaoqi compound traditional Chinese medicine decoction including the following steps:
  • Step 1) Dispense 300 mL of Qiaoqi compound Chinese medicine decoction into 50 mL enzyme-removing centrifuge tubes, and centrifuge at 2000 rpm to remove impurities; take 10 mL of supernatant from each tube and place it on ice, add 30 mL of TRIzol ls at a ratio of 3 times the volume of the medicinal decoction, After shaking evenly, let it stand at room temperature for 10 minutes, then add 6mL of chloroform, shake evenly and let it stand for 10 minutes;
  • Step 2 Centrifuge at 10,000g, 4°C, for 10 minutes, and collect the supernatant
  • Step 3 Add an equal volume of isopropyl alcohol to precipitate at -20°C for 1 hour, then centrifuge at 12,000g for 15 minutes;
  • Step 4) Carefully discard the supernatant, add 5 mL of 75% ethanol to wash the precipitate, and centrifuge at 12000 g for 5 min at 4°C;
  • Step 5 After centrifugation, carefully discard the ethanol and leave the precipitate. After the precipitate is dry, add 200 ⁇ L of 65°C DEPC water to dissolve it and measure the RNA concentration;
  • Step 6 Use a commercial miRNA isolation kit to purify miRNA, pass 100 ⁇ g RNA through each column, and dissolve each tube with 60 ⁇ L DEPC water;
  • Step 7) Determine the miRNA concentration, freeze-dry for 8 hours, and store at -80°C.
  • microribonucleic acid QQ_159 derived from Qiaoqi compound traditional Chinese medicine decoction in the preparation of drugs that inhibit influenza virus replication.
  • microribonucleic acid QQ_159 derived from Qiaoqi compound traditional Chinese medicine decoction in the preparation of drugs for the treatment of viral influenza.
  • influenza includes influenza B victoria or H5N1 avian influenza.
  • QQ_159 a microRNA derived from Qiaoqi compound traditional Chinese medicine decoction, in the preparation of drugs that inhibit SARS-CoV-2 virus replication.
  • microRNA QQ_159 derived from Qiaoqi compound traditional Chinese medicine decoction in the preparation of drugs for the treatment of viral pneumonia caused by SARS-CoV-2 virus.
  • a pharmaceutical composition for inhibiting influenza virus replication and/or treating influenza including the above-mentioned microRNA QQ_159, and a pharmaceutically acceptable carrier thereof.
  • a pharmaceutical composition for inhibiting SARS-CoV-2 virus replication and/or treating viral pneumonia caused by SARS-CoV-2 virus including the above-mentioned microRNA QQ_159, and a pharmaceutically acceptable carrier thereof.
  • a non-therapeutic method for inhibiting the replication of influenza B virus in vitro contacting the above-mentioned microRNA QQ_159 with cells infected by influenza B virus.
  • the present invention provides an experimental method for extracting microribonucleic acids from traditional Chinese medicine decoction, and uses this method for the first time to extract and identify 15 microribonucleic acids stably present in the Qiaoqi compound traditional Chinese medicine decoction.
  • QQ_159 has been proven to have certain inhibitory and therapeutic effects on viral influenza and pneumonia caused by new coronavirus infection.
  • This invention not only obtains the natural antiviral active ingredients of traditional Chinese medicine, but also provides possible nucleic acid drugs for clinical treatment of influenza or COVID-19.
  • Figure 1 shows the inhibitory effect of QQ_159 on influenza B viral load detected by real-time PCR in Example 2;
  • Figure 2 shows the effect of QQ_159 on the body weight of mice infected with H5N1 avian influenza virus in Example 3;
  • Figure 3 shows the effect of QQ_159 on the survival rate of mice infected with H5N1 avian influenza virus in Example 3;
  • Figure 4 shows the inhibitory effect of QQ_159 on lung inflammation in mice infected with H5N1 avian influenza virus detected by HE staining in Example 3;
  • Figure 5 shows the inhibitory effect of real-time PCR detection of QQ_159 on the viral load in the lungs of mice infected with H5N1 avian influenza virus on the 3rd day (A) and the 5th day (B) in Example 3;
  • Figure 6 shows the inhibitory effect of QQ_159 on lung inflammation in mice infected with SARS-CoV-2 virus detected by HE staining in Example 4.
  • the survival rate of the medium concentration QQ_159 group and the mixed RNA group reached 70%
  • the survival rate of the high concentration QQ_159 group was 50%
  • the infection group had only a 30% survival rate.
  • the HE results showed that compared with the infected control group, lung inflammation in the mixed RNA group and mice with medium concentration QQ_159 was significantly reduced, and the inhibitory effect of medium concentration QQ_159 was better.
  • the medium concentration QQ_159 group, high concentration QQ_159 group and mixed RNA group can effectively inhibit the replication of the virus; as shown in Figure 5B, on the 5th day after infection, the medium concentration QQ_159 group The effect of inhibiting virus replication was significantly weakened, while the high-concentration QQ_159 group and mixed RNA group still had good effects on inhibiting virus replication.
  • SARS-CoV-2 was injected intranasally at 10 5 TCID 50 /mL to infect 6- to 8-week-old hACE2 transgenic mice.
  • mice were sacrificed on the 5th day after infection, and lung tissues were collected.

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Abstract

提供了一种翘芪复方中药汤剂来源的微小核糖核酸及其制备方法，该微小核糖核酸选自核苷酸序列如SEQ ID NO.1～15所示的miRNA。还提供了核苷酸序列如SEQ ID NO.14所示的miRNA QQ_159，包括人工合成的QQ_159、植物QQ_159、QQ_159的前体形式或QQ_159的成熟体形式，及其在制备治疗病毒性流行性感冒和SARS-CoV-2病毒引起的病毒性肺炎的药物中的应用，以及包含miRNA QQ_159和药学上可接受的载体的药物组合物。

Description

一种翘芪复方中药汤剂来源的微小核糖核酸及其制备方法和应用 技术领域
本发明属于生物医药技术领域，具体涉及一种翘芪复方中药汤剂来源的微小核糖核酸及其制备方法和应用。
背景技术
流行性感冒(简称流感)是哺乳动物和家禽常患的一种急性呼吸道疾病，由流感病毒引起，传染性强、传播速度快，通常会导致简单的呼吸道感染，包括咳嗽、发烧、肌痛、发冷或出汗等临床症状，不适感可持续2至8天，通常发病迅速。部分患者，尤其是老年人、幼儿和患有其他慢性疾病的患者，易引发病毒性或继发性肺炎，甚至伴有呼吸困难和多器官衰竭。使用抗流感药物治疗是目前对抗流感的一种常用手段，一般根据患者的感染程度及患者身体状况来决定处方。虽然流感病毒是研究最深入的病原体之一，但由于流感病毒极易变异，现有的控制及治疗方案还需要不断改进，研发针对病毒性流行性感冒的新药既是当今社会的重大需求,也是民生健康的重大需求。
新型冠状病毒(简称新冠)是最近两年广泛传播的一种新型病毒，主要通过飞沫形式的呼吸途径传播，近来还发现可通过气溶胶、物品接触传播，传染性极强，传播速度极快，临床症状主要表现为：发热、疲劳、干咳、嗅觉缺失/嗅觉障碍，严重者或伴有呼吸困难、呼吸急促和低氧血症的严重肺炎并可引发其他并发症。因目前没有针对新冠病毒的特效治疗药物，导致其在全球大规模传播。因此，针对疫情防控的药品需求，研发特异性针对新冠的新药品是应对新冠、保障人民生命安全的重要手段。
微小核糖核酸(microRNAs，简称miRNAs)是一类小的内源性非编码RNA。通常情况下，miRNA编码基因由RNA聚合酶Ⅱ转录并产生初级转录物，由RNaseⅢ核酸内切酶Drosha和Dicer加工成大约21个核苷酸的小RNA。miRNA通过与编码区或靶mRNA的3’和5’非翻译区的结合，介导转录后基因沉默，在多种生理病理过程中发挥着重要作用，在医学领域具有广阔的应用前景。
近年来，植物miRNA相关研究发展迅速，主要通过上调/下调策略研究其生 物学功能，众多研究表明其在光合作用、营养稳态、生长发育、激素信号转导以及胁迫响应等方面都发挥着重要的调控作用；此外，已证实植物miRNA可作为内源性miRNA调节动物基因表达。大量文献报道中药在预防和治疗流感及新冠肺炎过程中发挥了积极作用。但具体是哪种有效成分尚不明确。已有文献报道在中药中含有miRNA，但中药汤剂中存在哪些稳定存在的miRNA，这些miRNA进入动物体内会发挥什么样的功能，人们对其仍不甚了解。
发明内容
针对现有技术的不足，本发明提供一种翘芪复方中药汤剂来源的微小核糖核酸及其制备方法和应用，从翘芪复方中药汤剂中提取功能性植物微小核糖核酸或含该微小核糖核酸提取物，并通过实验验证了该翘芪复方中药汤剂来源的微小核糖核酸QQ_159对病毒性流行性感冒及新型冠状病毒感染导致的肺炎存在一定的抑制效果和治疗作用。
本发明是通过以下技术方案实现的：
一种翘芪复方中药汤剂来源的微小核糖核酸，所述微小核糖核酸选自novel_mir7、novel_mir33、novel_mir35、novel_mir10、novel_mir20、novel_mir5、novel_mir36、novel_mir28、novel_mir31、ppt-miR894、novel_mir32、novel_mir21、pab-miR3711、QQ_159或bdi-miR159a-3p；其中，
所述novel_mir7的核苷酸序列如SEQ ID NO.1所示；
所述novel_mir33的核苷酸序列如SEQ ID NO.2所示；
所述novel_mir35的核苷酸序列如SEQ ID NO.3所示；
所述novel_mir10的核苷酸序列如SEQ ID NO.4所示；
所述novel_mir20的核苷酸序列如SEQ ID NO.5所示；
所述novel_mir5的核苷酸序列如SEQ ID NO.6所示；
所述novel_mir36的核苷酸序列如SEQ ID NO.7所示；
所述novel_mir28的核苷酸序列如SEQ ID NO.8所示；
所述novel_mir31的核苷酸序列如SEQ ID NO.9所示；
所述ppt-miR894的核苷酸序列如SEQ ID NO.10所示；
所述novel_mir32的核苷酸序列如SEQ ID NO.11所示；
所述novel_mir21的核苷酸序列如SEQ ID NO.12所示；
所述pab-miR3711的核苷酸序列如SEQ ID NO.13所示；
所述QQ_159的核苷酸序列如SEQ ID NO.14所示；
所述bdi-miR159a-3p的核苷酸序列如SEQ ID NO.15所示。
优选地，所述微小核糖核酸为QQ_159，包括人工合成的QQ_159、植物QQ_159、QQ_159的前体形式或QQ_159的成熟体形式。
一种翘芪复方中药汤剂来源的微小核糖核酸的制备方法，包括以下步骤：
步骤1)将300mL翘芪复方中药汤剂分装于50mL除酶离心管中，2000rpm离心去除杂质；按每管10mL吸取上清置于冰上，按药汤体积3倍比例加入TRIzol ls 30mL，用力震荡均匀后，室温静置10min，然后加入6mL氯仿，用力震荡均匀，静置10min；
步骤2)离心10000g，4℃，10min，收集上清；
步骤3)加入等体积的异丙醇-20℃沉淀1h，然后离心12000g，15min；
步骤4)小心弃上清，加入5mL的75％的乙醇洗沉淀，4℃，12000g离心5min；
步骤5)离心后，小心弃去乙醇，留沉淀，待沉淀干燥后加200μL 65℃DEPC水溶解，测定RNA浓度；
步骤6)用商用miRNA isolation kit纯化miRNA，每个柱子过100μg RNA，每管用60μL DEPC水溶解；
步骤7)测定miRNA浓度，冷冻干燥8h后，-80℃保存。
一种翘芪复方中药汤剂来源的微小核糖核酸QQ_159在制备抑制流行性感冒病毒复制的药物中的应用。
一种翘芪复方中药汤剂来源的微小核糖核酸QQ_159在制备治疗病毒性流行性感冒的药物中的应用。
优选地，所述流行性感冒包括乙型victoria流行性感冒或H5N1禽流感。
一种翘芪复方中药汤剂来源的微小核糖核酸QQ_159在制备抑制SARS-CoV-2病毒复制的药物中的应用。
一种翘芪复方中药汤剂来源的微小核糖核酸QQ_159在制备治疗SARS-CoV-2病毒引起的病毒性肺炎的药物中的应用。
一种用于抑制流行性感冒病毒复制和/或治疗流行性感冒的药物组合物，包括上述的微小核糖核酸QQ_159，及其药学上可接受的载体。
一种用于抑制SARS-CoV-2病毒复制和/或治疗SARS-CoV-2病毒引起的病毒性肺炎的药物组合物，包括上述的微小核糖核酸QQ_159，及其药学上可接受的载体。
一种体外非治疗目的的抑制乙型victoria流感病毒复制的方法：将上述的微小核糖核酸QQ_159与被乙型victoria流感病毒感染的细胞进行接触。
本发明的有益效果如下：
本发明提供了一种提取中药汤剂中微小核糖核酸的实验方法，并首次用此法提取并鉴定出了15种稳定存在于翘芪复方中药汤剂中的微小核糖核酸。其中，QQ_159被证明对病毒性流行性感冒及新型冠状病毒感染导致的肺炎存在一定的抑制效果和治疗作用。本发明不仅获得了中药抗病毒的天然有效成分，也将为临床治疗流感或新冠肺炎提供可能的核酸药物。
附图说明
图1为实施例2中real-time PCR检测QQ_159对乙型流感病毒载量的抑制效果；
图2为实施例3中QQ_159对感染H5N1禽流感病毒的小鼠体重的影响；
图3为实施例3中QQ_159对感染H5N1禽流感病毒的小鼠存活率的影响；
图4为实施例3中HE染色检测QQ_159对感染H5N1禽流感病毒的小鼠肺部炎症的抑制效果；
图5为实施例3中real-time PCR检测QQ_159对感染H5N1禽流感病毒第3天(A)和第5天(B)的小鼠肺部病毒载量的抑制效果；
图6为实施例4中HE染色检测QQ_159对感染SARS-CoV-2病毒的小鼠肺部炎症的抑制效果。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合附图与具体实施例进行详细描述。
实施例1翘芪复方中药汤剂微小核糖核酸的提取
(1)300mL翘芪复方中药(金银花、连翘、黄芩、青蒿、生黄芪、炒白术、藿香、防风、麦冬、甘草)汤剂分装于50mL除酶离心管中，2000rpm离心去除杂质；按每管10mL吸取上清置于冰上，按药汤体积3倍比例加入TRIzol ls 30mL，用力震荡均匀后，室温静置10min，然后加入6mL氯仿，用力震荡均匀，静置10min。
(2)离心10000g，4℃，10min，收集上清。
(3)加入等体积的异丙醇沉淀1h(-20℃)，然后离心12000g，15min。
(4)小心弃上清，加入5mL的75％的乙醇洗沉淀，4℃，12000g离心5min。
(5)离心后，小心弃去乙醇，留沉淀，待沉淀干燥后加200μL 65℃DEPC水溶解，测定RNA浓度。
(6)用商用miRNA isolation kit纯化miRNA(每个柱子过100μg RNA)，每管用60μL DEPC水溶解。
(7)测定miRNA浓度，冷冻干燥8h后，-80℃保存。
(8)采用BGISEQ-500技术进行微小核糖核酸测序，测序结果如下表1所示。
表1翘芪复方中药汤剂微小核糖核酸列表
Figure PCTCN2022094829-appb-000001
Figure PCTCN2022094829-appb-000002
实施例2测定QQ_159对马丁达比狗肾(MDCK)细胞中乙型流感病毒载量的影响
(1)在24孔培养板内培养MDCK细胞。
(2)分别将10nM、50nM或100nM QQ_159a通过商用转染试剂riboFECTTM CP转染入MDCK细胞，同时将相同浓度的无义序列转染至MDCK细胞作为对照组。
(3)使用乙型Victoria系流感病毒感染上述细胞。
(4)感染病毒24h后，收集上清离心合并细胞，提取RNA，通过real-time PCR检测病毒载量。
结果如图1所示，和对照组相比，100nM QQ_159能显著抑制MDCK细胞中乙型Victoria系流感病毒的病毒载量。
实施例3测定QQ_159对小鼠感染H5N1亚型禽流感病毒的治疗效果
(1)6周龄的BALB/C母鼠感染H5N1亚型禽病毒后2h，腹腔注射药物。动物分组及药物用量见下表2。
表2 QQ_159抗H5N1禽流感动物实验分组及药物处理情况
Figure PCTCN2022094829-appb-000003
表2中：混合RNA为从翘芪复方中药汤剂汇总提取的混合RNA。
(2)连续给药5天，每天观察记录体重变化(若体重下降超过30％，安乐处死，视作死亡)。
(3)分别于攻毒后第3、第5天取肺组织，HE染色检测肺组织炎症情况。
(4)real-time PCR检测肺组织病毒载量。
(5)饲养动物至第10天，观察存活率。
结果：在体重变化方面，如图2所示，中浓度QQ_159(120pmol/只/天，M-QQ_159)、高浓度QQ_159(360pmol/只/天，H-QQ_159)以及混合RNA组在攻毒后的第7天(图2中显示D8)体重变化进入平台期，但感染对照组仍有下降的趋势；高浓度QQ_159组的体重变化率略高于混合RNA组和中浓度QQ_159。在存活率方面，如图3所示，中浓度QQ_159组和混合RNA组的存活率达70％，高浓度QQ_159组的存活率为50％，而感染组仅有30％的存活率。如图4所示，HE结果显示和感染对照组相比，混合RNA组和中浓度QQ_159小鼠肺部炎症明显减轻，且中浓度QQ_159抑制效果更佳。如图5A所示，感染后第3天，中浓度QQ_159组、高浓度QQ_159组和混合RNA组均能有效抑制病毒的复制；如图5B所示，在感染后的第5天，中浓度QQ_159抑制病毒复制效果明显减弱，而高浓度QQ_159组和混合RNA组对抑制病毒复制仍有较好效果。
该实验表明，人工合成的QQ_159及从翘芪复方中药汤剂汇总提取的混合RNA对H5N1禽流感病毒存在一定的治疗作用，表现为体重下降延缓、死亡率的降低以及肺部炎症的减轻。
实施例4研究QQ_159抑制SARS-CoV-2病毒引起的肺炎的效果
(1)SARS-CoV-2以10 5TCID 50/mL滴鼻感染6～8周龄hACE2转基因小鼠。
(2)腹腔注射(ip)给药，给药5天。第一天染毒2h后给药。实验分组和用药剂量见下表3。
表3 hACE2转基因小鼠SARS-CoV-2感染实验
Figure PCTCN2022094829-appb-000004
Figure PCTCN2022094829-appb-000005
(3)在0、1、2、3、4、5天监测体重和症状。
(4)感染后第5天处死小鼠，收集肺组织。
(5)HE染色检测肺组织病理。
结果如图6所示，120pmol/只/天注射QQ_159对感染SARS-CoV-2的hACE2转基因小鼠肺组织炎症具有一定的抑制效果，表明QQ_159对SARS-CoV-2感染引起的小鼠肺炎具有一定抑制效果。
以上所述仅是本发明的优选实施方式，应当指出，对于本领域技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Figure PCTCN2022094829-appb-000006
Figure PCTCN2022094829-appb-000007
Figure PCTCN2022094829-appb-000008

Claims (10)

  1. 一种翘芪复方中药汤剂来源的微小核糖核酸，其特征在于，所述微小核糖核酸选自novel_mir7、novel_mir33、novel_mir35、novel_mir10、novel_mir20、novel_mir5、novel_mir36、novel_mir28、novel_mir31、ppt-miR894、novel_mir32、novel_mir21、pab-miR3711、QQ_159或bdi-miR159a-3p；其中，
    所述novel_mir7的核苷酸序列如SEQ ID NO.1所示；
    所述novel_mir33的核苷酸序列如SEQ ID NO.2所示；
    所述novel_mir35的核苷酸序列如SEQ ID NO.3所示；
    所述novel_mir10的核苷酸序列如SEQ ID NO.4所示；
    所述novel_mir20的核苷酸序列如SEQ ID NO.5所示；
    所述novel_mir5的核苷酸序列如SEQ ID NO.6所示；
    所述novel_mir36的核苷酸序列如SEQ ID NO.7所示；
    所述novel_mir28的核苷酸序列如SEQ ID NO.8所示；
    所述novel_mir31的核苷酸序列如SEQ ID NO.9所示；
    所述ppt-miR894的核苷酸序列如SEQ ID NO.10所示；
    所述novel_mir32的核苷酸序列如SEQ ID NO.11所示；
    所述novel_mir21的核苷酸序列如SEQ ID NO.12所示；
    所述pab-miR3711的核苷酸序列如SEQ ID NO.13所示；
    所述QQ_159的核苷酸序列如SEQ ID NO.14所示；
    所述bdi-miR159a-3p的核苷酸序列如SEQ ID NO.15所示。
  2. 根据权利要求1所述的一种翘芪复方中药汤剂来源的微小核糖核酸，其特征在于，所述微小核糖核酸为QQ_159，包括人工合成的QQ_159、植物QQ_159、QQ_159的前体形式或QQ_159的成熟体形式。
  3. 权利要求1或2所述一种翘芪复方中药汤剂来源的微小核糖核酸的制备方法，其特征在于，包括以下步骤：
    步骤1)将300mL翘芪复方中药汤剂分装于50mL除酶离心管中，2000rpm离心去除杂质；按每管10mL吸取上清置于冰上，按药汤体积3倍比例加入TRIzolls 30mL，用力震荡均匀后，室温静置10min，然后加入6mL氯仿，用力震荡均匀，静置10min；
    步骤2)离心10000g，4℃，10min，收集上清；
    步骤3)加入等体积的异丙醇-20℃沉淀1h，然后离心12000g，15min；
    步骤4)小心弃上清，加入5mL的75％的乙醇洗沉淀，4℃，12000g离心5min；
    步骤5)离心后，小心弃去乙醇，留沉淀，待沉淀干燥后加200μL 65℃DEPC水溶解，测定RNA浓度；
    步骤6)用商用miRNA isolation kit纯化miRNA，每个柱子过100μg RNA，每管用60μL DEPC水溶解；
    步骤7)测定miRNA浓度，冷冻干燥8h后，-80℃保存。
  4. 权利要求2所述一种翘芪复方中药汤剂来源的微小核糖核酸在制备抑制流行性感冒病毒复制的药物中的应用。
  5. 权利要求2所述一种翘芪复方中药汤剂来源的微小核糖核酸在制备治疗病毒性流行性感冒的药物中的应用。
  6. 根据权利要求4或5所述的应用，其特征在于，所述流行性感冒包括乙型victoria流行性感冒或H5N1禽流感。
  7. 权利要求2所述一种翘芪复方中药汤剂来源的微小核糖核酸在制备抑制SARS-CoV-2病毒复制的药物中的应用。
  8. 权利要求2所述一种翘芪复方中药汤剂来源的微小核糖核酸在制备治疗SARS-CoV-2病毒引起的病毒性肺炎的药物中的应用。
  9. 一种用于抑制流行性感冒病毒复制和/或治疗流行性感冒的药物组合物，其特征在于，包括权利要求2所述的微小核糖核酸QQ_159，及其药学上可接受的载体。
  10. 一种用于抑制SARS-CoV-2病毒复制和/或治疗SARS-CoV-2病毒引起的病毒性肺炎的药物组合物，其特征在于，包括权利要求2所述的微小核糖核酸QQ_159，及其药学上可接受的载体。
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