CN103966212A - A型流感病毒NP基因有干扰作用的siRNA序列的设计及应用 - Google Patents
A型流感病毒NP基因有干扰作用的siRNA序列的设计及应用 Download PDFInfo
- Publication number
- CN103966212A CN103966212A CN201310048338.XA CN201310048338A CN103966212A CN 103966212 A CN103966212 A CN 103966212A CN 201310048338 A CN201310048338 A CN 201310048338A CN 103966212 A CN103966212 A CN 103966212A
- Authority
- CN
- China
- Prior art keywords
- virus
- influenza
- gene
- sirna
- application
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108020004459 Small interfering RNA Proteins 0.000 title claims abstract description 24
- 238000013461 design Methods 0.000 title abstract description 3
- 101900222562 Influenza A virus Nucleoprotein Proteins 0.000 title abstract 3
- 230000000312 effect on influenza Effects 0.000 title abstract 2
- 241000712461 unidentified influenza virus Species 0.000 claims abstract description 14
- 230000000694 effects Effects 0.000 claims abstract description 11
- 206010022000 influenza Diseases 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 101150118742 NP gene Proteins 0.000 claims description 4
- 230000010076 replication Effects 0.000 claims description 3
- 230000030279 gene silencing Effects 0.000 claims description 2
- 238000012226 gene silencing method Methods 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 241000287828 Gallus gallus Species 0.000 abstract description 4
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 4
- 238000000034 method Methods 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 2
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 241000700605 Viruses Species 0.000 description 7
- 230000000890 antigenic effect Effects 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 239000012530 fluid Substances 0.000 description 5
- 230000009368 gene silencing by RNA Effects 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 108091030071 RNAI Proteins 0.000 description 3
- 210000003837 chick embryo Anatomy 0.000 description 3
- 230000003067 hemagglutinative effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 230000001018 virulence Effects 0.000 description 3
- 102000004533 Endonucleases Human genes 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000712464 Orthomyxoviridae Species 0.000 description 1
- 102000000574 RNA-Induced Silencing Complex Human genes 0.000 description 1
- 108010016790 RNA-Induced Silencing Complex Proteins 0.000 description 1
- CWHJIJJSDGEHNS-MYLFLSLOSA-N Senegenin Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(C(O)=O)[C@@H]2CC[C@@]3(C)C(CC[C@]4(CCC(C[C@H]44)(C)C)C(O)=O)=C4[C@@H](CCl)C[C@@H]3[C@]21C CWHJIJJSDGEHNS-MYLFLSLOSA-N 0.000 description 1
- 206010046306 Upper respiratory tract infection Diseases 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 244000144980 herd Species 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 208000033065 inborn errors of immunity Diseases 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 229960003971 influenza vaccine Drugs 0.000 description 1
- 150000004712 monophosphates Chemical class 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- PGZUMBJQJWIWGJ-ONAKXNSWSA-N oseltamivir phosphate Chemical compound OP(O)(O)=O.CCOC(=O)C1=C[C@@H](OC(CC)CC)[C@H](NC(C)=O)[C@@H](N)C1 PGZUMBJQJWIWGJ-ONAKXNSWSA-N 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 208000028529 primary immunodeficiency disease Diseases 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229940061367 tamiflu Drugs 0.000 description 1
- 239000009871 tenuigenin Substances 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明为A型流感病毒NP基因有干扰作用的siRNA序列的设计及应用，公开了四条对A型流感病毒NP基因复制有抑制作用的siRNA；通过鸡胚实验测HA滴度的方法确定了siRNA对流感病毒干扰的有效性。
Description
技术领域
本发明涉及对A型流感病毒NP基因复制有干扰作用的siRNA序列的发现及应用，属于生物技术领域。
背景技术
流行性感冒(Influenza)，在流行性感冒病毒，是一种造成人类及动物患流行性感冒的RNA病毒，在分类学上，流感病毒属于正黏液病毒科，它会造成急性上呼吸道感染，并借由空气迅速的传播，在世界各地常会有周期性的大流行。
流感病毒表面抗原HA和NA的变异有两种形式，即抗原性漂移(antigenic drift)和抗原性转变(antigenic shift)。抗原性漂移是指变异幅度小或连续变异，属于量变，即亚型内变异。一般认为这种变异是由病毒基因点突变和人群免疫力选择所造成的，引起小规模流行。抗原性转变是指变异幅度大，属于质变，即病毒株表面抗原结构发生一种或两种变异，形成新亚型(如H2N2→H3N2)，由于人群对变异病毒株缺少免疫力而容易造成新型流感的大流行。同一时期也可同时流行两个亚型，如1997年甲3型(H3N2)流行时，甲1型(H1N1)同时出现。如果不同型别病毒同时流行，也可发生基因重组而形成新亚型。
尽管由死病毒、减毒毒株或重组表面糖蛋白组成的抗流感疫苗能够有效地保护约70～80％的健康成年人免于患病，但是疫苗只能针对一小部分毒株，对于新的潜在爆发的毒株可能没有作用。而且，对于高风险人群如婴孩、老人、孕妇和其它各种免疫力低下的人群，疫苗的保护作用是非常有限的，保护率低于40％。由于大部分的死亡都是发生在高风险人群中，疫苗对于这些最需要它们的人却不能起到很好的保护作用。正在应用的几种抗流感药物也能够降低流感感染的发生和减轻流感感染时的症状。但是，药物的副作用、病人的承受能力、可能出现的抗药性变异限制了这些药物的大规模使用。所以，对于预防和治疗流感病毒感染的方法仍然有很急切的需要，特别是在高风险人群中和爆发流感时。因此，流感的防治至少在今后50年内仍然是一个严重的问题，寻找更有效的预防和治疗途径是医学研究的重大课题。
RNA干扰(RNA interference，RNAi)既目的RNA与同源的靶目标mRNA结合，阻断病毒翻译蛋白质是解决流感病毒流行的重要手段和方法，其作用原理为：①siRNA形成阶段。RNAi的诱导物在细胞质内被RNaseIII Dicer切割成为21-23nt的小分子干扰RNA(small interfering RNA，siRNA)，siRNA的结构特征是5′端单磷酸，3′端羟基，而且3′端有2～3-nt的碱基突出；②RNA诱导的沉默复合物(RNA-induced Silencing Complex，RISC)的形成阶段。siRNA与RNAi特异性酶(如Ago-2)结合，形成RISC，具有特异性的核酸内切酶活性能特异性地降解与siRNA同源的mRNA；③效应阶段。RISC中siRNA变性，双链解开，卸下正义RNA，反义RNA仍结合在复合物上，并引导RISC与同源的靶目标mRNA结合，在核酸内切酶的作用下，将靶目标mRNA切断(切割位置在siRNA的中央，距离5′端10-nt)，从而阻断了其翻译成蛋白质，表现为基因沉默。
作为一种快速、有效、特异的抑制基因表达的工具，被广泛用于基因功能的研究以及肿瘤、病毒性疾病和遗传性疾病的治疗研究。
发明内容
本发明提供4条对A型流感病毒NP基因复制有抑制作用的siRNA编码序列。如序列表400<1>，400<2>，400<3>，400<4>所示。
本发明所述的四条siRNA载体质粒的动物水平的实验结果表明能效抑制H1N1，H9N2，H2N3的病毒复制。
具体实施方式
实施例1：鸡胚实验验证siRNA载体质粒干扰H9N2流感病毒增殖作用
H9N2病毒毒力ED50108/0.1ml，将H9N2病毒稀释含有100个ED50病毒液，体积0.1ml，注射到9日龄SPF鸡胚尿囊腔中，同时注射SiRNA载体质粒6微克，33℃培养72小时，取尿囊液，测血凝效价。
实施例2：鸡胚实验验证siRNA载体质粒干扰H1N1流感病毒增殖作用
H1N1病毒毒力ED5018.6/0.1ml，将H1N1病毒稀释含有100个ED50病毒液，体积0.1ml，注射到9日龄SPF鸡胚尿囊腔中，同时注射SiRNA载体质粒6微克，33℃培养72小时，取尿囊液，测血凝效价。
实施例3：鸡胚实验验证siRNA载体质粒干扰H3N2流感病毒增殖作用
H3N2病毒毒力ED50107.6/0.1ml，将H1N1病毒稀释含有100个ED50病毒液，体积0.1ml，注射到9日龄SPF鸡胚尿囊腔中，同时注射SiRNA载体质粒6微克，33℃培养72小时，取尿囊液，测血凝效价。
实施例4：尿囊液病毒滴度检测
(1)微量血凝板的每孔中滴加PH7.2PBS100μL，(2)吸取被检尿囊液样品100
μL，倍比稀释12孔，第12孔弃掉100μL。每孔中加入0.1％红细胞悬液100μL。
(2)设阴性对照和流感病毒阳性对照。
(4)置微型振荡器上振荡1min混匀。
(5)放室温下(18～20℃)30min，观察结果。
经三次重复实验，序列表所述的四条序列<400>1，<400>2，<400>3，<400>4对
流感病毒复制的抑制率如下：
序列表
Claims (2)
1.一种对流感病毒NP基因复制有干扰作用的siRNA，其核苷酸序列如序列表400<1>，400<2>，400<3>，400<4>所示。
2.权利要求1、对所述的siRNA作为抑制流感病毒Np基因沉默的靶向基因的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310048338.XA CN103966212A (zh) | 2013-02-06 | 2013-02-06 | A型流感病毒NP基因有干扰作用的siRNA序列的设计及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310048338.XA CN103966212A (zh) | 2013-02-06 | 2013-02-06 | A型流感病毒NP基因有干扰作用的siRNA序列的设计及应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103966212A true CN103966212A (zh) | 2014-08-06 |
Family
ID=51236219
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310048338.XA Pending CN103966212A (zh) | 2013-02-06 | 2013-02-06 | A型流感病毒NP基因有干扰作用的siRNA序列的设计及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103966212A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116814632A (zh) * | 2023-08-23 | 2023-09-29 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | 一种抑制流感病毒的siRNA及其应用 |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1968959A (zh) * | 2002-09-28 | 2007-05-23 | 麻省理工学院 | 流行性感冒治疗 |
| WO2007056861A1 (en) * | 2005-11-18 | 2007-05-24 | Protiva Biotherapeutics, Inc. | Sirna silencing of influenza virus gene expression |
| CN101103111A (zh) * | 2004-11-05 | 2008-01-09 | 因特拉迪格姆公司 | 用于治疗呼吸道病毒感染的组合物及其用途 |
| CN101180395A (zh) * | 2005-03-22 | 2008-05-14 | 麻省理工学院 | 流感治疗剂 |
| CN101184839A (zh) * | 2005-04-08 | 2008-05-21 | 纳斯泰克制药公司 | 用于呼吸道病毒感染的RNAi治疗 |
| CN101254171A (zh) * | 2006-06-13 | 2008-09-03 | 祝加贝 | 含有小分子干扰核糖核酸的喷雾剂 |
| US20080222743A1 (en) * | 2004-08-25 | 2008-09-11 | Avigenics, Inc. | RNA interference and disease resistance in avians |
| WO2008115851A2 (en) * | 2007-03-16 | 2008-09-25 | Mdrna, Inc. | Rnai therapeutic for respiratory virus infection |
| CN101275132A (zh) * | 2008-03-06 | 2008-10-01 | 中山大学 | 对A型流感病毒复制有抑制作用的siRNA及其编码序列 |
| US20090042823A1 (en) * | 2007-08-10 | 2009-02-12 | Mdrna, Inc. | Uses of broad spectrum rnai therapeutics against influenza |
| CN101880677A (zh) * | 2009-11-06 | 2010-11-10 | 中国医学科学院病原生物学研究所 | 针对2009新甲型流感病毒多聚酶基因和核蛋白基因的siRNA序列及其应用 |
| CN101918558A (zh) * | 2007-05-16 | 2010-12-15 | Mat马耳他先进技术有限公司 | 流感的治疗及预防 |
| CN102108354A (zh) * | 2008-03-06 | 2011-06-29 | 中山大学 | 一种对A型流感病毒复制有抑制作用的siRNA及其编码序列 |
-
2013
- 2013-02-06 CN CN201310048338.XA patent/CN103966212A/zh active Pending
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1968959A (zh) * | 2002-09-28 | 2007-05-23 | 麻省理工学院 | 流行性感冒治疗 |
| US20080222743A1 (en) * | 2004-08-25 | 2008-09-11 | Avigenics, Inc. | RNA interference and disease resistance in avians |
| CN101103111A (zh) * | 2004-11-05 | 2008-01-09 | 因特拉迪格姆公司 | 用于治疗呼吸道病毒感染的组合物及其用途 |
| CN101180395A (zh) * | 2005-03-22 | 2008-05-14 | 麻省理工学院 | 流感治疗剂 |
| CN101184839A (zh) * | 2005-04-08 | 2008-05-21 | 纳斯泰克制药公司 | 用于呼吸道病毒感染的RNAi治疗 |
| WO2007056861A1 (en) * | 2005-11-18 | 2007-05-24 | Protiva Biotherapeutics, Inc. | Sirna silencing of influenza virus gene expression |
| CN101254171A (zh) * | 2006-06-13 | 2008-09-03 | 祝加贝 | 含有小分子干扰核糖核酸的喷雾剂 |
| WO2008115851A2 (en) * | 2007-03-16 | 2008-09-25 | Mdrna, Inc. | Rnai therapeutic for respiratory virus infection |
| CN101918558A (zh) * | 2007-05-16 | 2010-12-15 | Mat马耳他先进技术有限公司 | 流感的治疗及预防 |
| US20090042823A1 (en) * | 2007-08-10 | 2009-02-12 | Mdrna, Inc. | Uses of broad spectrum rnai therapeutics against influenza |
| CN101275132A (zh) * | 2008-03-06 | 2008-10-01 | 中山大学 | 对A型流感病毒复制有抑制作用的siRNA及其编码序列 |
| CN102108354A (zh) * | 2008-03-06 | 2011-06-29 | 中山大学 | 一种对A型流感病毒复制有抑制作用的siRNA及其编码序列 |
| CN101880677A (zh) * | 2009-11-06 | 2010-11-10 | 中国医学科学院病原生物学研究所 | 针对2009新甲型流感病毒多聚酶基因和核蛋白基因的siRNA序列及其应用 |
Non-Patent Citations (1)
| Title |
|---|
| JOHANNES MARIO SIMMEL: "TIP-vermittelte Induktion Tetrazyklin-abhängiger Transregulatoren in Saccharomyces cerevisiae und HeLa Zellen", 《ALS DISSERTATION GENEHMIGT VON DER NATURWISSENSCHAFTLICHEN FAKULTÄT DER FRIEDRICH-ALEXANDER-UNIVERSITÄT ERLANGEN-NÜRNBERG》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116814632A (zh) * | 2023-08-23 | 2023-09-29 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | 一种抑制流感病毒的siRNA及其应用 |
| CN116814632B (zh) * | 2023-08-23 | 2023-11-10 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | 一种抑制流感病毒的siRNA及其应用 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wolff et al. | Influenza B, C and D viruses (orthomyxoviridae) | |
| Ma et al. | The pig as a mixing vessel for influenza viruses: Human and veterinary implications | |
| Keshavarz et al. | miRNA-based strategy for modulation of influenza A virus infection | |
| Huang et al. | Pathogenic influenza B virus in the ferret model establishes lower respiratory tract infection | |
| Ichinohe et al. | PolyI: polyC12U adjuvant-combined intranasal vaccine protects mice against highly pathogenic H5N1 influenza virus variants | |
| Pedrazzoli et al. | Vaccination for seasonal influenza in patients with cancer: recommendations of the Italian Society of Medical Oncology (AIOM) | |
| Sivay et al. | Influenza A (H15N4) virus isolation in Western Siberia, Russia | |
| Bamunuarachchi et al. | MicroRNA‐206 inhibits influenza A virus replication by targeting tankyrase 2 | |
| Liu et al. | Avian influenza virus in pregnancy | |
| Shapshak et al. | The influenza pandemic of 2009: lessons and implications | |
| Ma et al. | Antiviral activities of several oral traditional Chinese medicines against influenza viruses | |
| Cui et al. | Cangma Huadu granules, a new drug with great potential to treat coronavirus and influenza infections, exert its efficacy through anti-inflammatory and immune regulation | |
| Kiso et al. | Effect of an asparagine-to-serine mutation at position 294 in neuraminidase on the pathogenicity of highly pathogenic H5N1 influenza A virus | |
| Clark et al. | Recent advances in the influenza virus vaccine landscape: a comprehensive overview of technologies and trials | |
| Stephenson et al. | Influenza: current threat from avian influenza | |
| Tian et al. | Prevalence of neutralizing antibodies to common respiratory viruses in intravenous immunoglobulin and in healthy donors in southern China | |
| Focosi et al. | Passive immunotherapies for the next influenza pandemic | |
| Ashraf et al. | Next-generation vaccines for influenza B virus: advancements and challenges | |
| Wong et al. | Aerosol and nasal delivery of vaccines and antiviral drugs against seasonal and pandemic influenza | |
| CN103966212A (zh) | A型流感病毒NP基因有干扰作用的siRNA序列的设计及应用 | |
| Zhao et al. | Evolutionary analysis of Hemagglutinin and neuraminidase gene variation in H1N1 swine influenza virus from vaccine intervention in China | |
| Zhang et al. | Prediction of the next highly pathogenic avian influenza pandemic that can cause illness in humans | |
| Singh et al. | Swine Flu-A comprehensive view | |
| Zhou et al. | Pandemic (H1N1) 2009 virus in swine herds, People’s Republic of China | |
| Nakayama et al. | Long-term efficacy of an inactivated H5N1 whole-particle influenza vaccine in nonhuman primates |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| DD01 | Delivery of document by public notice |
Addressee: Huo Jin Document name: Notification of Passing Preliminary Examination of the Application for Invention |
|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140806 |