CN1847256B - Antiviral prepn containing both CpG single-strand deoxyoligonucleotide and ribavirin - Google Patents

Antiviral prepn containing both CpG single-strand deoxyoligonucleotide and ribavirin Download PDF

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CN1847256B
CN1847256B CN 200510064537 CN200510064537A CN1847256B CN 1847256 B CN1847256 B CN 1847256B CN 200510064537 CN200510064537 CN 200510064537 CN 200510064537 A CN200510064537 A CN 200510064537A CN 1847256 B CN1847256 B CN 1847256B
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ribavirin
cpg odn
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于永利
包木胜
王丽颖
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长春华普生物技术有限公司
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    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02A50/38Medical treatment of vector-borne diseases characterised by the agent
    • Y02A50/381Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a virus
    • Y02A50/384Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a virus of the genus Flavivirus
    • Y02A50/385Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a virus of the genus Flavivirus the disease being Dengue
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y02A50/38Medical treatment of vector-borne diseases characterised by the agent
    • Y02A50/381Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a virus
    • Y02A50/384Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a virus of the genus Flavivirus
    • Y02A50/389Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a virus of the genus Flavivirus the disease being Japanese encephalitis

Abstract

The present invention provides single strand deoxidized oligonucleotides containing one or several CpG dinucleotides. The single strand deoxidized oligonucleotides containing CpG dinucleotide may be joint applied with ribavirin to produce obvious antiviral effect, especially RNA viral resisting effect. The combination of the single strand deoxidized oligonucleotides containing CpG dinucleotide and ribavirin may be applied in preventing and treating viral infection and viral infection caused diseases.

Description

含CpG单链脱氧寡核苷酸和利巴韦林联合应用产生的抗病 CpG-containing single-stranded oligodeoxynucleotides antiviral nucleotides and ribavirin produced

毒作用 Cytotoxicity

[0001] 发明领域 [0001] Field of the Invention

[0002] 本发明涉及一种含CpG 二核苷酸的单链脱氧核苷酸和利巴韦林,特别是涉及含CpG 二核苷酸的单链脱氧核苷酸和利巴韦林联合应用,本发明所涉及的含CpG 二核苷酸的单链脱氧核苷酸和利巴韦林联合应用时可用于病毒感染疾病及病毒感染相关疾病的治疗和预防。 [0002] The present invention relates to a dinucleotide CpG-containing single-stranded deoxynucleotides and ribavirin, in particular, it is concerned with a single-stranded CpG dinucleotides deoxynucleotides and ribavirin when dinucleotide CpG-containing single-stranded oligodeoxynucleotides according to the present invention and ribavirin can be used for viral infections and viral infections related disease treatment and prevention. 本发明所指的病毒包括但不限于流感病毒、口蹄疫病毒、登革热病毒、日本脑炎病毒、丙型肝炎病毒、乙型肝炎病毒、人类免疫缺陷性病毒和乳头瘤病毒。 Viruses of the invention include, but are not limited to the meaning of influenza virus, foot and mouth disease virus, dengue virus, Japanese encephalitis virus, hepatitis C virus, hepatitis B virus, human immunodeficiency virus and papillomavirus. 本发明还提供了含CpG 二核苷酸的单链脱氧核苷酸的序列。 The present invention further provides a dinucleotide sequence CpG-containing single-stranded deoxynucleotides.

[0003] 发明背景 [0003] Background of the Invention

[0004] CpG ODN是人工合成的含一个或多个CpG 二核苷酸的脱氧寡核苷酸单链DNA, 其中的CpG是由胞嘧啶和鸟嘌呤通过磷酸连接成的二核苷酸,C代表胞嘧啶,G代表鸟嘌呤,P代表磷酸,胞嘧啶位于5'端。 [0004] CpG ODN is a synthetic single-stranded oligodeoxynucleotides containing one or more CpG dinucleotides in DNA, which are connected by CpG cytosine and guanine through phosphoric acid into dinucleotide, C cytosine, G indicates guanine, P Representative phosphate, cytosine at the 5 'end. 由于序列尤其是CpG两侧的序列的不同,CpG ODN可有多种多样的形式。 Since different sequences are particular sequences of both CpG, CpG ODN may have a wide variety of forms. 有些CpG ODN具有明确的免疫调节作用,表现出较好的临床应用价值(ffeiner GJ. The immunobiology and clinical potential of immunostimulatory CpGoligodeoxynucleotides. J Leukoc Biol 2000 Oct ;68 (4) :455-63)。 Some CpG ODN have a clear role in immune regulation, showed better clinical value (ffeiner GJ The immunobiology and clinical potential of immunostimulatory CpGoligodeoxynucleotides J Leukoc Biol 2000 Oct; 68 (4):.. 455-63). 有些CpG ODN 的作用具有种属特异性,即对一种动物表现生物学作用的CpG 0DN,在另一种动物则未必表现出同样性质或强度的生物学活性(Gunther Hartmann, et al. Delineation of a CpGPhosphorοthioate Oligodeoxynucleotide for Activating Primate Immune ResponsesIn Vitro and In Vivol The Journal of Immunology,2000,164 :1617-1624)。 Some of the role of CpG ODN species specific, i.e. the biological effect on the performance of an animal CpG 0DN, in another animal is not necessarily exhibit the same biological activity or strength properties (Gunther Hartmann, et al. Delineation of a CpGPhosphorοthioate oligodeoxynucleotide for Activating Primate Immune ResponsesIn Vitro and In Vivol The Journal of Immunology, 2000,164: 1617-1624).

[0005] 根据功能的特点,CpG ODN被分为三种类型:A型CpG ODN (CpG-A 0DN)、B型CpG ODN(CpG-B 0DN)和C型CpG ODN(CpG-C 0DN)。 [0005] The characteristic feature, CpG ODN are divided into three types: A type CpG ODN (CpG-A 0DN), B-type CpG ODN (CpG-B 0DN) and type C CpG ODN (CpG-C 0DN). 主要表现为激活树突状细胞、天然杀伤细胞、 刺激树突状细胞分泌干扰素活性的CpG ODN被归类为A型CpG ODN0 A型CpG ODN被期望用于肿瘤、病毒感染性疾病和其它用干扰素治疗的疾病的治疗。 Mainly for the activation of dendritic cells, natural killer cells, dendritic cells CpG ODN stimulate secretion of interferon activity is classified as Type A CpG ODN0 A type CpG ODN is intended for cancer, viral and other infectious diseases with interferon therapy treatment of disease. 主要表现为激活B淋巴细胞,增强体液免疫应答活性的CpG ODN被归类为B型CpG ODN0 B型CpG ODN是一类分子佐剂,可增强疫苗的免疫效果。 Mainly activated B lymphocytes, enhanced humoral immune response activity of CpG ODN are classified as Type B CpG ODN0 B-type CpG ODN adjuvant is a class of molecules, can enhance the immune effect of the vaccine. 兼备A型CpG ODN和B型CpG ODN主要活性的CpGODN被归类为C 型CpG ODN0 Both type A and type B CpG ODN CpG ODN CpGODN main activity is classified as type C CpG ODN0

[0006] 实验表明,CpG ODN具有抗病毒的生物学活性。 [0006] Experiments show that, CpG ODN biologically active antiviral. 经阴道应用CpG ODN可使小鼠或豚鼠提高抵抗II型单纯疱疹病毒(HSV-2)的能力(Richard B. Pyles et al. Use ofImmunostimulatory sequence-Containing Oligonucleotides as Topical Therapy forGenital Herpes Simplex Virus Type 2 Infection.Journal of Virology, November 2002,Vol. 76,No. 22 p. 11387-11396)。 CpG ODN vaginal application can improve the ability of mouse or guinea pig (Richard B. Pyles et al resistant herpes simplex virus type II (HSV-2) a. Use ofImmunostimulatory sequence-Containing Oligonucleotides as Topical Therapy forGenital Herpes Simplex Virus Type 2 Infection. Journal of Virology, November 2002, Vol. 76, No. 22 p. 11387-11396). 给小鼠应用CpG ODN 可减少其呼吸道合胞病毒(RSV)的荷量(Cho JY, et al. Immunostimulatory DNA sequences inhibit respiratorysyncytial viral load, airway inflammation, and mucus secretion. J Allergy ClinImmunol 2001 Nov ; 108 (5) :697-702)。 Application of CpG ODN to mice can reduce the amount of charge (Cho JY, et al respiratory syncytial virus (RSV) is Immunostimulatory DNA sequences inhibit respiratorysyncytial viral load, airway inflammation, and mucus secretion J Allergy ClinImmunol 2001 Nov;.. 108 (5 ): 697-702). 注射CpG ODN 的小鼠在经致死量的HSV-2攻击后,阴道分泌物中病毒滴度明显降低(Harandi AM, Eriksson K, Holmgren J. A protectiverole of locally administered immunostimulatory CpGoligodeoxynucleotide in amouse model of genital herpes infection. J Virol 2003 Jan ;77 (2) :953-62)。 CpG ODN injected mice after a lethal dose of HSV-2 attacks, vaginal secretions decreased viral titers (Harandi AM, Eriksson K, Holmgren J. A protectiverole of locally administered immunostimulatory CpGoligodeoxynucleotide in amouse model of genital herpes infection . J Virol 2003 Jan; 77 (2): 953-62).

[0007] 禾0 E1 , # (l-β -D-ribofuranosyl-1,2,4-triazol e-3-carboxamide, Rikivirin),也称Virazole,是一种核苷类似物,具有广谱的抗病毒活性(Gilbert,B Eand Knight V. Biochemistry and clinical applications of ribavirin. Antimicrob. Agents Chemother. 1986,30(2) :201-205)(Streeter DG, Witkowski JT, Khare GP, Sidwell Rff, Bauer RJ, Robins RK, Simon LN. Mechanism of action of 1—D-ribofuranosyl-1,2, 4-triazole-3_carboxamide(Virazole), a newbroad-spectrum antiviral agent.Proc Natl Acad Sci US A. 1973 Apr ;70 (4) :1174-8.),被用于多种病毒感染性疾病的治疗(Gilbert, BE and Knight V. Biochemistry andclinical applications of ribavirin. Antimicrob. Agents Chemother. 1986,30(2) :201-205)。 [0007] Wo 0 E1, # (l-β -D-ribofuranosyl-1,2,4-triazol e-3-carboxamide, Rikivirin), Virazole, also known, is a nucleoside analog with a broad spectrum of anti- viral activity (Gilbert, B Eand Knight V. Biochemistry and clinical applications of ribavirin Antimicrob Agents Chemother 1986,30 (2):... 201-205) (Streeter DG, Witkowski JT, Khare GP, Sidwell Rff, Bauer RJ, Robins . RK, Simon LN Mechanism of action of 1-D-ribofuranosyl-1,2, 4-triazole-3_carboxamide (Virazole), a newbroad-spectrum antiviral agent.Proc Natl Acad Sci US A. 1973 Apr; 70 (4): . 1174-8), it is used to treat a variety of viral infectious diseases (Gilbert, bE and Knight V. Biochemistry andclinical applications of ribavirin Antimicrob Agents Chemother 1986,30 (2...): 201-205). 利巴韦林和α 干扰素联合应用是治疗由丙型肝炎病毒感染引起的慢性的丙型肝炎的有效方法(Davis GL, Esteban-Mur R, Rustgi V,Hoefs J,Gordon SC,TrepoC,Shiffman ML,Zeuzem S,Craxi A,Ling MH,Albrecht J0 Interferon alfa-2b aloneor in combination with ribavirin for the treatment of relapse of chronic hepatitisC.International Hepatitis Interventional Therapy Group N Engl J Med. 1998 Novl9 ;339 (21) :1493-9.)。 Interferon α and ribavirin combination is an effective method (Davis GL treatment of chronic hepatitis C caused by the hepatitis C virus infection, Esteban-Mur R, Rustgi V, Hoefs J, Gordon SC, TrepoC, Shiffman ML , Zeuzem S, Craxi A, Ling MH, Albrecht J0 Interferon alfa-2b aloneor in combination with ribavirin for the treatment of relapse of chronic hepatitisC.International Hepatitis Interventional Therapy Group N Engl J Med 1998 Novl9; 339 (21):. 1493- 9.). 利巴韦林和长效α 干扰素(pegylated interferon, peginterferon)联合应用对治疗丙型肝炎有明显的疗效(Torriani FJ, Rodriguez-Torres Μ, RockstrohJK, Lissen Ε, Gonzalez-Garcia J, Lazzarin A, Carosi G, Sasadeusz J, Katlama C, Montaner J, Sette H Jr, Passe S, De Pamphilis J, Duff F, Schrenk UM, DieterichDT ;APRICOT Study Group. Peginterferon Alfa-2a plus ribavirin for chronichepatitis C virus infection in HIV-infected patients. N Engl J Med. 2004 Jul 29 ;351 (5) :438-50)。 Long-acting interferon α and ribavirin (pegylated interferon, peginterferon) combined treatment of hepatitis C has a significant effect (Torriani FJ, Rodriguez-Torres Μ, RockstrohJK, Lissen Ε, Gonzalez-Garcia J, Lazzarin A, Carosi G, Sasadeusz J, Katlama C, Montaner J, Sette H Jr, Passe S, De Pamphilis J, Duff F, Schrenk UM, DieterichDT;. APRICOT Study Group Peginterferon Alfa-2a plus ribavirin for chronichepatitis C virus infection in HIV-infected patients . N Engl J Med 2004 Jul 29; 351 (5):. 438-50).

[0008] 流感病毒是引起人流感的病原体。 [0008] Influenza virus is human influenza caused by a pathogen. 在1918-1919年,全世界有两千万人死于流感病毒的流行(Patterson,KD & Pyle,GF (1991)Bull. Hist. Med. 65,4-213)。 In the 1918-1919 year 20 million people worldwide died of epidemic influenza virus (Patterson, KD & Pyle, GF (1991) Bull. Hist. Med. 65,4-213). 接种疫苗和应用抗病毒药物是防治流感病毒的两类主要方法(Bridges,CB,Fukuda, K.,Cox, NJ & Singleton, J. Α. (2001)Morbid. Mortal, ffkly. Rep. 50,1-44),但事实证明,这两类方法均不能有效地控制流感的流行(Webby, RJ& Webster, RG (2001)Philos. Trans. R. Soc. London 356,1817-18¾)。 Vaccination and antiviral drugs are two main types of method (Bridges, CB, Fukuda, K., Cox, NJ & Singleton, J. Α prevention and treatment of influenza virus. (2001) Morbid. Mortal, ffkly. Rep. 50,1 -44), but the fact that these two methods can not effectively control the flu epidemic (Webby, RJ & Webster, RG (2001) Philos. Trans. R. Soc. London 356,1817-18¾). 在易感人群,接种疫苗的短期(半年)保护率约为39% (Fukuda, K. , NJ (1999)Morbid. Mortal, ffkly. Rep. 48,1-28, Castle, SC Clinical relevance of age-related immune dysfunction. ClinInfect Dis. 2000 Aug ;31 (2) :578-85. Epub 2000 Sep 14. Review) 0由于流感病毒抗原[血细胞凝集素(HA)和神经酰胺酶(NA)]性的不断变化,现行的疫苗几乎每年都要重新改建结构。 In the short term vulnerable populations, vaccinations (six months) protection rate of about 39% (Fukuda, K., NJ (1999) Morbid. Mortal, ffkly. Rep. 48,1-28, Castle, SC Clinical relevance of age- . related immune dysfunction ClinInfect Dis 2000 Aug; 31 (2):. 578-85 2000 Sep 14. Review) 0 Since the influenza virus antigen [hemagglutinin (HA) and neuraminidase (NA)] of changing Epub. , the current vaccine almost every year remodeled structure. 由于副作用较大和新的抗药流感病毒株不断出现,一些获得批准的抗流感病毒药物的效果也不理想(Luscher-Mattli, Μ.Influenza chemotherapy :a review of thepresent state of art and of new drugs in development. Arch Virol. 2000 ;145 (11) :2233-48. Review.)。 Because of side effects and new drug-resistant strains of influenza viruses emerging, some of the effects of anti-influenza virus drugs approved is not ideal (Luscher-Mattli, Μ.Influenza chemotherapy: a review of thepresent state of art and of new drugs in development .. Arch Virol 2000; 145 (11): 2233-48 Review)...

[0009] 丙型肝炎病毒(HCV)是引起丙型肝炎的病原体,在全世界感染了大约1亿七千多万人。 [0009] Hepatitis C virus (HCV) Hepatitis C is caused by pathogens in the world infected about 100 million more than seven thousand people. 肝癌和肝硬化是HCV感染引起的两个严重的并发症。 Liver cancer and cirrhosis of the liver are two serious complications of HCV infection. 在世界范围内,采用α-干扰素和利巴韦林联合应用是治疗丙型肝炎病毒感染的方法,其有效率约为40% [Jon Cohen. Science, 1999, 285 :26-30]。 Worldwide, the use of α- interferon and ribavirin is the method of treating a hepatitis C viral infection, the efficiency is about 40% [Jon Cohen Science, 1999, 285:. 26-30]. 丙型肝炎病毒是一种有包膜的单股正链RNA病毒,属黄病毒科病毒。 Hepatitis C virus is an enveloped single-strand RNA virus, genus Flaviviridae. 黄病毒科的另外两种病毒,登革热病毒(Wang WK, Lin SR, Lee CM, King CC, Chang SC. Dengue type 3 virus in plasma is a population of closely related genomes : quasispecies. J Virol. 2002 May ;76 (9) :4662-5.)和日本脑炎病毒(Yun Si,Kim SY,Rice CM,Lee YM. Development and application of a reverse genetics system forJapanese encephalitis virus. J Virol. 2003 Jun ;77 (11) :6450-65.)。 . Flaviviridae other two viruses, dengue virus (Wang WK, Lin SR, Lee CM, King CC, Chang SC Dengue type 3 virus in plasma is a population of closely related genomes: quasispecies J Virol 2002 May; 76.. (9): 4662-5) and Japanese encephalitis virus (Yun Si, Kim SY, Rice CM, Lee YM Development and application of a reverse genetics system forJapanese encephalitis virus J Virol 2003 Jun; 77 (11...).: 6450-65.). 登革热病毒、日本脑炎病毒也是单股正链的RNA病毒。 Dengue virus, Japanese encephalitis virus is a positive strand RNA viruses. 由于尚无研究丙型肝炎病毒的细胞和动物模型,采用登革热病毒、日本脑炎病毒做实验获得的结果可在相当的程度上代表用丙型肝炎病毒做细胞实验获得的结果。 Because no studies of hepatitis C virus in cell and animal models, using dengue virus, Japanese encephalitis virus do experimental results obtained can be made on behalf of cells results obtained from experiments with Hepatitis C virus in a considerable extent.

[0010] 口蹄疫(foot-and-mouthdisease,FMD)是由口蹄疫病毒引起的烈性传染病。 [0010] deadly infectious FMD (foot-and-mouthdisease, FMD) is caused by foot and mouth disease virus. 猪、 牛、羊都可感染口蹄疫病毒而发生口蹄疫,轻者在口、舌、唇、蹄、乳房等部位出现水泡、破溃及烂斑等病变,重者发生死亡。 Pigs, cattle, sheep can be infected with foot and mouth disease and foot and mouth disease virus, the light blistering, ulceration and rotten spots and other lesions in the mouth, tongue, lips, shoes, and other parts of the breast, severe cases, death. 口蹄疫的发生和流行会造成巨大的直接和间接的经济损失[江鹏斐,赵启祖,谢庆阁,口蹄疫研究进展中国农业科学1999,32 (6) :93〜100]。 And foot and mouth disease epidemic will cause huge direct and indirect economic losses [Jiang Pengfei, Zhao Qizu, Xie Qing Court, foot and mouth disease research progress China Agricultural Science 1999,32 (6): 93~100]. 国际兽疫局(OIE)将口蹄疫列为A类家畜传染病。 OIE (OIE) as the foot and mouth disease of livestock category A diseases. 世界各国对预防和控制口蹄疫都十分重视。 The world for the prevention and control of foot and mouth disease very seriously. 除屠杀感染口蹄疫的家畜外,接种疫苗是预防和控制口蹄疫的主要措施[M. Woolhouse, 2001]。 In addition to the livestock slaughter infected with foot and mouth disease, vaccination is the primary measure to prevent and control FMD [M. Woolhouse, 2001]. 传统的口蹄疫疫苗由灭活的初步纯化的口蹄疫病毒加油佐剂乳化制成的灭活苗,对易感家畜有相当的保护作用,但存在灭活不充分而引发口蹄疫的可能。 FMD vaccine by the conventional fuel preliminarily purified inactivated vaccine made from inactivated FMD virus adjuvant emulsion, a significant protective effect against susceptible livestock, but there are not sufficient to inactivate FMD may be caused. 此外,在生产这种疫苗过程中动用的有活力的口蹄疫病毒是一种不可忽视的潜在传染源。 In addition, the use of the vaccine in the production process of dynamic foot and mouth disease virus is a potential source of infection can not be ignored.

[0011] 二十世纪80年代初以来,许多国家投入了大量人力、物力和财力研制新型口蹄疫疫苗,如重组蛋白疫苗,合成肽疫苗,活载体疫苗和DNA疫苗等[Broekhuigsen MP,1987 ; Morgan D 0,1990 ;Ward, G. , Rieder, Ε. & Mason, PW Plasmid DNA encodingrepIieating foot-and-mouth disease virus genomes induces antiviral immuneresponses in swine. Journal of Virology 1997,71,7442-7447. ;Berinstein A, TamiC, Taboga 0, Smitsaart E,Carrillo E. Protective immunity against foot-and-mouthdisease virus induced by a recombinant vaccinia virus. Vaccine. 2000 Apr28 ; 18 (21) :2231-8.]。 [0011] Since the early 80s of the twentieth century, many countries have invested a lot of manpower, material and financial resources to develop new FMD vaccines, such as recombinant protein vaccines, synthetic peptide vaccine, live vector vaccines and DNA vaccines [Broekhuigsen MP, 1987; Morgan D 0,1990; Ward, G., Rieder, Ε & Mason, PW Plasmid DNA encodingrepIieating foot-and-mouth disease virus genomes induces antiviral immuneresponses in swine Journal of Virology 1997,71,7442-7447;... Berinstein A, TamiC , Taboga 0, Smitsaart E, Carrillo E. Protective immunity against foot-and-mouthdisease virus induced by a recombinant vaccinia virus Vaccine 2000 Apr28; 18 (21):... 2231-8]. 一些疫苗显示了较好的效果,但大部分仍在实验室的探索之中。 Some vaccines show good results, but most are still exploring laboratories into.

[0012] 口蹄疫病毒(foot-and-mouth disease virus,FMDV)是口蹄疫的病原体,是小RNA病毒科(Picornaviridae) 口蹄疫病毒属(Aphthovirus)的成员,现已发现了7 个血清型,即0、A、C型(称欧洲型),SATU SAT2、SAT3型(南非1、2、3型,也称非洲型)和AsiaI型(亚洲I型,称亚洲型)。 [0012] FMDV (foot-and-mouth disease virus, FMDV) is foot and mouth disease pathogens, is a small RNA virus family member (Picornaviridae) FMD virus genus (Aphthovirus), has discovered seven serotypes, namely 0, A, C type (called European type), SATU SAT2, SAT3 type (type 1, 2, South Africa, also known as the African type) and AsiaI type (Asia type I, said the Asian type). 0型口蹄疫病毒是近年来世界范围内流行较广的血清型。 0 type FMD virus is prevalent in the world in recent years a wide range of serotypes. 研究表明,针对的体液免疫在肌体抗口蹄疫病毒感染中起主要作用。 Studies have shown that humoral immune response plays a major role in the body's anti-FMD virus infection. 在家畜体内,口蹄疫病毒中和抗体的水平与其抵抗口蹄疫病毒攻击的能力呈明显的正相关关系[Pay TW, Hingley PJ. Correlation of 140S antigen dose with the serum neutralizing antibody responseand the level of protection induced in cattle by foot-and-mouth diseasevaccines. Vaccine.1987 Mar ;5 (1) :60-4.]。 In animal body, foot and mouth disease virus neutralizing antibody levels of resistance to FMD virus attacks and their was a significant positive correlation between [Pay TW, Hingley PJ. Correlation of 140S antigen dose with the serum neutralizing antibody responseand the level of protection induced in cattle by . foot-and-mouth diseasevaccines Vaccine.1987 Mar; 5 (1): 60-4].. 基于这禾中观察,许多实验室做了大量实验,在口蹄疫病毒上寻找能剌激机体产生病毒中和抗体的靶点,发现在口蹄疫病毒的外壳蛋白(VP)存在五个侯选的结构[Crowther JR,Farias S,Carpenter WC, Samuel AR. Identification of a fifth neutralizable site on type 0 foot-and-mouth disease virusfollowing characterization of single and quintuple monoclonal antibody escapemutants. J Gen Virol.1993 Aug ;74 (Pt 8):1547-53. ;Kitson JD, McCahon D,BelshamGJ. Sequence analysis of monoclonal antibody resistant mutants of type O foot andmouth disease virus -evidence for the involvement of the three surface exposedcapsid proteins in four antigenic sites. Virology. 1990 Nov ; 179(1) J6-34.],其中的三个位于外壳蛋白1 (Vpl)中。 Based on these Wo observed in many laboratory experiments done a lot to find the virus can stimulate the body to produce antibodies and targets on foot and mouth disease virus, found that the structure of the five candidates in the presence of foot and mouth disease virus coat protein (VP) [ . Crowther JR, Farias S, Carpenter WC, Samuel AR Identification of a fifth neutralizable site on type 0 foot-and-mouth disease virusfollowing characterization of single and quintuple monoclonal antibody escapemutants J Gen Virol.1993 Aug; 74 (Pt 8).: 1547-53;... Kitson JD, McCahon D, BelshamGJ Sequence analysis of monoclonal antibody resistant mutants of type O foot andmouth disease virus -evidence for the involvement of the three surface exposedcapsid proteins in four antigenic sites Virology 1990 Nov;. 179 ( 1) J6-34.], wherein the housing is located three protein 1 (Vpl) in. 实验证明,从口蹄疫病毒中分离出的Vpl蛋白具有良好的免疫原性Dachrach HL, Moore DM, McKercher PD, Polatnick J. Immune andantibody responses to an isolated capsid protein of foot-and-mouth disease virus. JImmunol.1975 Dec ;115(6) :1636-41.; Kleid DG,Yansura D,Small B,Dowbenko D,MooreDM,Grubman MJ,McKercher PD,Morgan DO, Robertson BH, Bachrach HL Cloned viralprotein vaccine for foot-and-mouth disease -responses in cattle and swine. Science. 1981 Dec 4 ;214(4525) :1125-9.; Strohmaier K,1982 ;Rodriguez A,Saiz JC,NovellalS,Andreu D,Sobrino F. Antigenic specificity of porcine T cell response againstfoot-and-mouth disease virus structural proteins !identification of T helperepitopes in VPl. Virology. 1994Nov 15 ;205(1) :24-33.] ο用Vpl蛋白的肽段(141-160和200-213)免疫豚鼠和牛产生的中和性抗体能保护动物免于感染口蹄疫病毒Dittle几,Houghten RA,Alexmder H, Shinnick TM, Sutcliffe JG, Lerner RA, Rowl Experiments show that, separated from the foot and mouth disease virus protein Vpl good immunogenicity Dachrach HL, Moore DM, McKercher PD, Polatnick J. Immune andantibody responses to an isolated capsid protein of foot-and-mouth disease virus. JImmunol.1975 Dec; 115 (6): 1636-41 .; Kleid DG, Yansura D, Small B, Dowbenko D, MooreDM, Grubman MJ, McKercher PD, Morgan DO, Robertson BH, Bachrach HL Cloned viralprotein vaccine for foot-and-mouth disease -responses in cattle and swine Science 1981 Dec 4; 214 (4525):.. 1125-9 .; Strohmaier K, 1982; Rodriguez A, Saiz JC, NovellalS, Andreu D, Sobrino F. Antigenic specificity of porcine T cell response againstfoot ! -and-mouth disease virus structural proteins identification of T helperepitopes in VPl Virology 1994Nov 15; 205 (1):... 24-33] ο Vpl protein with peptides (141-160 and 200-213) immunized guinea pigs and cattle neutralizing antibodies can protect animals from infection of FMD virus Dittle few, Houghten RA, Alexmder H, Shinnick TM, Sutcliffe JG, Lerner RA, Rowl ands DJ, BrownF. Protection against foot-and-mouth disease by immunization with a chemicalIysynthesized peptide predicted from the viral nucleotide sequence. Nature. 1982 Jull ;298 (5869) :30-3.; Pfaff EiMussgay Μ,Bohm HO,Schulz GE,Schaller H. Antibodiesagainst a preselected peptide recognize and neutralize foot and mouth diseasevirus. EMBO J. 1982 ;1(7): 869-74. ;DiMarchi R, Brooke G, Gale C, Cracknell V, DoelT, Mowat N. Protection of cattle against foot-and-mouth disease by a syntheticpeptide. Science.1986 May 2 ;232 (4750) :639-41. ]。 .. Ands DJ, BrownF Protection against foot-and-mouth disease by immunization with a chemicalIysynthesized peptide predicted from the viral nucleotide sequence Nature 1982 Jull; 298 (5869):. 30-3 .; Pfaff EiMussgay Μ, Bohm HO, Schulz GE , Schaller H. Antibodiesagainst a preselected peptide recognize and neutralize foot and mouth diseasevirus EMBO J. 1982; 1 (7):.. 869-74; DiMarchi R, Brooke G, Gale C, Cracknell V, DoelT, Mowat N. Protection of . cattle against foot-and-mouth disease by a syntheticpeptide Science.1986 May 2; 232 (4750): 639-41].. VPl 140-160氨基酸构成的“环”状结构暴露在病毒表面,其中的RGD序列在口蹄疫病毒高度保守,是口蹄疫病毒结合细胞表面受体的关键结构,可能介导口蹄疫病毒进入被感染的细胞[Jackson Τ, 2002 ;Almeida MR,1998]。 VPl 140-160 amino acid "ring" structure composed of a surface exposed to the virus, wherein the RGD sequence is highly conserved in foot and mouth disease virus, foot and mouth disease virus binding structure is a key cell surface receptor may mediate FMD virus infected cells into the [ jackson Τ, 2002; Almeida MR, 1998]. 依据VPl环状结构合成的肽段能诱导产生高水平的中和抗体。 Peptides synthesized according VPl cyclic structure can induce high levels of neutralizing antibodies. 也有研究表明,用从口蹄疫病毒分离出来的或基因工程方法得到的Vpl蛋白免疫动物,可以产生部分保护作用。 Studies have also shown that, Vpl animals immunized with the protein isolated from foot and mouth disease virus or genetic engineering methods to get, some protective effect can be produced. 通过基因工程手段把Vpl蛋白与某些来源于其他微生物的蛋白融合在一起可以提高Vpl蛋白的免疫原性[Clarke BE,Newton SE,Carroll AR,Francis MJ,Appleyard G,Syred AD,Highfield PE, Rowlands DJ, Brown F. Improved immunogenicityof a peptide epitope after fusion to hepatitis B core protein. Nature. 1987 Nov26_Dec 2 ;330 (6146) :381-4. ;Corchero JL,Villaverde A. Antigenicity of a viralpeptide displayed on beta—galactosidase fusion proteins is influenced by thepresence of the homologous partner protein. FEMS Microbiol Lett. 1996 Nov15 ;145(1) :77-82.]。 By means of genetic engineering the protein Vpl protein derived from certain other microorganisms can together increase the immunogenicity of protein Vpl [Clarke BE, Newton SE, Carroll AR, Francis MJ, Appleyard G, Syred AD, Highfield PE, Rowlands . DJ, Brown F. Improved immunogenicityof a peptide epitope after fusion to hepatitis B core protein Nature 1987 Nov26_Dec 2; 330 (6146):.. 381-4; Corchero JL, Villaverde A. antigenicity of a viralpeptide displayed on beta-galactosidase fusion proteins is influenced by thepresence of the homologous partner protein FEMS Microbiol Lett 1996 Nov15; 145 (1):.. 77-82]..

[0013] 日本脑炎是由黄病毒属的日本脑炎病毒引起的。 [0013] Japanese encephalitis flaviviruses by the Japanese encephalitis virus. 大多数人感染日本脑炎病毒后都不会发病。 Most people infected with the Japanese encephalitis virus will not develop the disease. 多数发病者临床症状较轻,常见发烧、头痛、恶心、倦怠、腹痛或出现意识障碍及精神症状。 Most of the onset of clinical symptoms were mild, common fever, headache, nausea, fatigue, abdominal pain or disturbance of consciousness and mental symptoms. 重症病例多以突发高烧、头痛、呕吐开始,接着出现脑脊膜刺激现象,3-5日出现痉挛、异常行为、肌强直,意识明显变化甚至昏迷、死亡。 Severe cases and many more sudden high fever, headache, vomiting began, then appear meningeal irritation, cramps 3-5 days, abnormal behavior, muscle rigidity, significant changes in consciousness and even coma and death.

[0014] 日本脑炎在亚洲许多国家,仍然是严重威胁健康的流行病。 [0014] Japanese encephalitis in many countries in Asia, remains a serious threat to the health epidemic. 由于预防注射的实施,日本、南韩及台湾的病例已减少很多,但其邻近的许多国家,包括中国大陆、菲律宾、印尼、 马来西亚、印度、尼泊尔等国仍有许多日本脑炎患者,偶有该病的流行。 Since the implementation of vaccination, Japan, South Korea and Taiwan have been reduced a lot of cases, but many of its neighboring countries, including mainland China, the Philippines, Indonesia, Malaysia, India, Nepal and other countries there are still many Japanese encephalitis patients, sometimes the epidemic disease.

[0015] 乙型肝炎病毒是一种DNA病毒。 [0015] Hepatitis B virus is a DNA virus. 完整的乙型肝炎病毒颗粒直径为42nm,可分为包膜与核心两部分。 Complete HBV particle diameter of 42nm, the coated core can be divided into two parts. 包膜上的蛋白质称为乙型肝炎表面抗原(HBsAg),它在肝细胞内合成,然后释出入血液循环中,本身并无传染性。 Envelope protein known as the hepatitis B surface antigen (of HBsAg), it is synthesized in the liver cells, and then released out of the blood circulation in itself is not infectious. 核心部分含有环状双股的DNA、DNA聚合酶、核心抗原(HBcAg)和e抗原(HBeAg),是病毒复制的主体,并有传染性。 The core portion contains a cyclic DNA double strand, DNA polymerase, core antigen (of HBcAg) and the e antigen (HBeAg-), viral replication is the body, and infectious.

[0016] 艾滋病是由于感染了人类免疫缺陷病毒(简称HIV)后引起的一种致死性传染病。 [0016] AIDS is a fatal infectious disease due to infection with the human immunodeficiency virus after (referred to as HIV). HIV主要破坏人体的免疫系统,使机体逐渐丧失防卫能力而不能抵抗外界的各种病原体,因此极易感染一般健康人所不易患的感染性疾病和肿瘤,最终导致死亡。 The main HIV attacks the body's immune system, so that the body gradually lose the ability to defend against a variety of pathogens and not the outside world, and therefore vulnerable to infection generally healthy people do not susceptible to infectious diseases and cancer, eventually leading to death. 2000年我国艾滋病疫情呈平稳上升趋势,与上一年相比,报告HIV感染者人数增加11.2%,艾滋病病例增加1.3%。 AIDS epidemic in China in 2000 showed a steady upward trend, compared with the previous year, an increase of 11.2% in the number of reported HIV infection, AIDS cases increased by 1.3%. 截止2000年底,全国31个省、自治区、直辖市(不包括台湾省、香港和澳门特别行政区)累积报告HIV感染者22517例,其中艾滋病病例880例。 By the end of 2000, 31 provinces, autonomous regions and municipalities (excluding Taiwan Province, Hong Kong and Macao Special Administrative Regions) cumulative report 22,517 cases of HIV infection, AIDS cases among 880 cases. 报告死亡病例496例(AIDS 死亡466例)。 496 cases of reported deaths (AIDS deaths 466 cases). 2000年报告艾滋病死亡病例110例,HIV感染者死亡17例。 2000 report of 110 cases of AIDS deaths, HIV infection 17 patients died.

[0017] 艾滋病病毒(HIV)是带有包膜的RNA逆转录病毒,分类上属于逆转录病毒科。 [0017] AIDS virus (HIV) is a retrovirus with RNA enveloped retrovirus belonging to families on classification. HIV 呈球形或卵形,直径100-130nm,由包膜和核心两个部分组成。 HIV spherical or ovoid shape, the diameter of 100-130nm, the envelope and the core of two parts. 包膜蛋白包括外膜糖蛋白(gpl20)和跨膜糖蛋白(gp41)。 Outer membrane proteins include envelope glycoprotein (gpl20) and transmembrane glycoprotein (gp41). 核心部分由核壳蛋白、两个相同拷贝的核酸基因组RNA和酶类组成。 The core part of the nucleocapsid protein, two identical copies of a nucleic acid consisting of genomic RNA and enzymes.

[0018] 目前世界已研制了一些能够有效抑制艾滋病病毒的药物,这些药物已能在某种程度上缓解艾滋病病人的症状,延长患者的生命和提高其生活质量。 [0018] The world has developed some capable of effectively inhibiting HIV drugs, these drugs have been able to alleviate the symptoms of AIDS patients in a way to extend the lives of patients and improve their quality of life. 但是,这些药物十分昂贵,且有很大的副作用,而且有些药物长时间使用后对艾滋病病毒就没有效果了。 However, these drugs are very expensive, and there is a lot of side effects, and some drugs after prolonged use of the AIDS virus would be no effects. 目前,我们国家还没有正式进口或生产这些药物。 At present, our country has not officially import or production of these drugs. 至今为止,世界上还没有研制出能彻底治愈艾滋病的药物和有效预防艾滋病病毒的疫苗。 So far, the world has not developed can be completely cured of AIDS drugs and effective in preventing HIV vaccine.

[0019] 乳头瘤病毒属于乳多空病毒科(Papovaviridae)的乳头瘤病毒属,它包括多种动物的乳头瘤病毒和人乳头瘤病毒(Human papillomavirus,HPV)。 [0019] papillomavirus belonging Papovaviridae (Papovaviridae) of papillomavirus genus, which includes a variety of animal and human papilloma virus HPV (Human papillomavirus, HPV). HPV是一种小的DNA病毒,直径45〜55nm,衣壳呈二十面体立体对称,含72个壳微粒,没有囊膜。 HPV is a small DNA virus, diameter 45~55nm, icosahedral capsid symmetry perspective, shell particles containing 72, no capsule. HPV基因组是一闭环双股DNA,分子量5X106道尔顿。 HPV genome is a closed double-stranded DNA, a molecular weight of 5X106 Daltons. 按功能可分为早期区(E区)、晚期区(L区)和非编码区(NCR)三个区域。 Divide into the early region (E regions), late region (L region) and a non-coding region (NCR) three regions. E区分为El〜E7开放阅读框架,主要编码与病毒复制、转录、调控和细胞转化有关的蛋白。 E divided into El~E7 open reading frame encoding the major viral replication, transcription, and cell transformation related to the regulation of protein. L区分Ll和L2,分别编码主要衣壳蛋白和次要衣壳蛋白。 L and Ll distinguish L2, respectively, encoding the major capsid protein and the minor capsid protein. NCR是E 区与L区间-6. 4〜1. Obp的DNA片段,可负责转录和复制的调控。 NCR is the E region and the L section -6. 4~1. Obp DNA fragment, may be responsible for regulation of transcription and replication.

[0020] 据流行病学调查资料显示:全球乳头瘤病毒的感染率在9-13%左右,即每年大约有6亿3千万人感染乳头瘤病毒。 [0020] According to epidemiological survey data show: Global HPV infection rate of about 9-13%, that every year about 630 million people are infected with HPV. 乳头瘤病毒与很多的皮肤粘膜疾病密切相关。 HPV is closely related to many diseases of skin and mucous membranes. 乳头瘤病毒的感染可导致皮肤的扁平疣,生殖器的尖锐湿疣,更为严重的是乳头瘤病毒的感染与宫颈癌、阴茎癌和肛门的癌症密切相关。 HPV infection can cause genital warts skin warts, genital more serious is papillomavirus infection and cervical cancer, penile cancer and anal cancer are closely related. 美国每年有大约的性生活活跃的成年人患尖锐湿疣,另外还有至少15%的亚临床感染者。 United States each year about the sexually active adults suffering from genital warts, in addition to at least 15% of subclinical those. (L Koutsky. Epidemiology of genital human papillomavirus infection. Am JMed,May 5,1997 ; 102 (5A) :3-8) „ 据世界卫生组织(WHO) 报道,全世界每年新发生的宫颈癌患者4. 5万人,其80%的病人位于发展中国家,宫颈癌是仅次于乳腺癌的女性第二杀手。其中,我国每年新发病例13. 5万,约占世界的三分之一。近年来由于人乳头状瘤病毒(HPV)感染增多,使宫颈癌发病率明显上升且趋于年轻化。(博生肿瘤咨询)全球每年死于宫颈癌的患者有232000人,其中发展中国家192000人[0021] 另有研究证实,单纯疱疹II型病毒和人乳头瘤病毒,是导致宫颈癌的重要生物因子,是生殖系统感染最为常见的病原体。被宫颈单纯疮疹II型病毒感染的妇女宫颈癌的发病率高于健康妇女8倍。人乳头瘤病毒导致宫颈癌的危险比单纯疱疹II型病毒更大。 (L Koutsky Epidemiology of genital human papillomavirus infection Am JMed, May 5,1997; 102 (5A):.. 3-8) cervical cancer patients, "according to World Health Organization (WHO) reported that the new worldwide occur each year 4.5 million people, 80% of patients in the developing world, cervical cancer is second only to breast cancer killer of women. among them, China's new cases each year 135,000, about one-third of the world. in recent years, As the human papilloma virus (HPV) infection increases the incidence of cervical cancer increased significantly and tend to be younger. (Berson tumor consulting) worldwide die each year from cervical cancer patients have 232000 people, including 192,000 people in developing countries [ 0021] another study demonstrated that type II herpes simplex virus and human papilloma virus, is an important biological factors lead to cervical cancer, reproductive system infection is most common pathogens. infected cervical herpes simplex virus type II cervical cancer in women a high incidence in healthy women eight times the risk of HPV lead to cervical cancer more than herpes simplex virus type II.

[0022] 目前鉴定出的人乳头瘤病毒(HPV)已超过100个型别,根据各型病毒与其致病危害性的不同,将HPV分为低危型、高危型。 [0022] identified human papillomavirus (HPV) has more than 100 types, in accordance with various harmful pathogenic virus and its different type of HPV is divided into low-risk, high-risk type. 低危型,主要引起尖锐湿疣等良性病变,其中HPV 6 型感染占70% 以上、其次是HPVll 型(BrownDRJchroeder JM, Fife KH,etal. Detection of muItiplehuman papillomavirus types in condylomata acuminata lesions from otherwise healthy andimmunosuppressed patients. J Clin Microhiol,1999,37(10): 3316-3322);高危型,主要与宫颈癌、外阴癌、阴茎癌等恶性肿瘤的发生有关,以HPV16型为主。 Low-risk, the main cause benign lesions such as genital warts, HPV 6 infection where more than 70%, followed by HPVll type (BrownDRJchroeder JM, Fife KH, etal. Detection of muItiplehuman papillomavirus types in condylomata acuminata lesions from otherwise healthy andimmunosuppressed patients. J Clin Microhiol, 1999,37 (10): 3316-3322); high risk, primary malignancy with cervical cancer, vulvar cancer, penile cancer and the like related to the main type HPV16. (人乳头瘤病毒分子生物学研究进展邓燕飞2000年4月国外医学•生理、病理科学与临床分册,第20卷,第2期。) (Human papillomavirus molecular biology research progress Dengyan Fei April 2000 • International Journal of physiology, pathology and clinical science volumes, Volume 20, Issue 2.)

[0023] 采取有效的疫苗,预防和治疗乳头瘤病毒感染是预防和治疗宫颈癌和性病并发症的理想途径。 [0023] to take effective vaccine, prevention and treatment of papillomavirus infections is the ideal way to prevent and treat cervical cancer and sexually transmitted diseases complications. 科学家一直在进行不懈的探索,试图寻找到一种行之有效的疫苗,如DNA疫苗、病毒载体疫苗和合成肽疫苗等。 Scientists have been making unremitting exploration, trying to find an effective vaccine, such as DNA vaccines, viral vectors, vaccines and synthetic peptide vaccines. 迄今为止,在世界上尚未研制出有效的乳头瘤病毒疫苗。 So far, the world has not yet developed an effective HPV vaccine.

发明内容 SUMMARY

[0024] 本发明的目的之一是提供人工合成的含CpG单链脱氧核苷酸(CpG 0DN),它们由含一个或多个CpG的寡核苷酸单链DNA分子构成。 [0024] One object of the present invention is to provide a synthetic CpG-containing single-stranded oligodeoxynucleotides (CpG 0DN), which consists of a single-stranded oligonucleotide DNA molecule containing one or more of CpG. CpG ODN的磷酸二酯键可以是非硫化的, 部分硫化的,也可以是完全硫化的。 CpG ODN of phosphodiester linkages may be non-sulfurized, partially sulfurized, also be completely cured. 人工合成的含CpG单链脱氧核苷酸可以是经化学修饰的。 Synthetic CpG-containing single-stranded oligodeoxynucleotides may be chemically modified.

[0025] 本发明的目的之二是提供CpG ODN的抗病毒的作用以及CpG ODN和利巴韦林联合应用产生的抗病毒的作用。 [0025] The object of the present invention is to provide a two CpG ODN antiviral effect and antiviral effect of CpG ODN and ribavirin produced.

[0026] 本发明的目的之三是提供用CpG ODN和利巴韦林联合应用治疗和预防病毒感染及病毒感引起的相关疾病的方法和设想。 [0026] another object of the present invention is to provide a method and contemplated by CpG ODN and related diseases ribavirin treatment and prophylaxis of viral infections and viral flu caused.

[0027] 本发明的目的之四是提供用CpG ODN和利巴韦林联合应用治疗和预防包括但不限于由流感病毒、口蹄疫病毒、登革热病毒、日本脑炎病毒、丙型肝炎病毒、乙型肝炎病毒、人类免疫缺陷性病毒和乳头瘤病毒感染引起的疾病的方法。 [0027] The object of the present invention is to provide a four-CpG ODN and ribavirin for the treatment and prophylaxis include, but are not limited to the influenza virus, foot and mouth disease virus, dengue virus, Japanese encephalitis virus, hepatitis B virus hepatitis virus, human immunodeficiency virus and disease caused by HPV infection method.

[0028] 另外,需要指出的是,在本申请的上下文的公开内容的基础上,本发明的其它具有实质性特点的方面和创造性的有益效果对本领域的普通技术人员来说是可以直接推知的。 [0028] Further, to be noted that, on the basis of the present application, the disclosure content and the context of the other substantive features aspects and inventive advantages of the present invention, one of ordinary skill in the art that can be directly inferred .

具体实施方式 Detailed ways

[0029] 在本发明的上下文中,所使用的术语除非另外说明,一般具有本领域的普通技术人员通常理解的含义。 [0029] In the present context, the term is used unless stated otherwise, meaning having ordinary skill in the art generally understood. 特别地,下列术语具有如下的含义: In particular, the following terms have the following meanings:

[0030] 优选地,本发明的CpG ODN具有如下所示的序列: [0030] Preferably, CpG ODN of the present invention has the sequence shown below:

[0031] CpG ODN 1 :5,_ggggacgatatcgatgggggg_3, [0031] CpG ODN 1: 5, _ggggacgatatcgatgggggg_3,

[0032] CpG ODN 2 :5,_ggggtcgttcgtcgttgggggg_3, [0032] CpG ODN 2: 5, _ggggtcgttcgtcgttgggggg_3,

[0033] CpG ODN 3 :5,_ggggtgcaacgttcagggggg_3, [0033] CpG ODN 3: 5, _ggggtgcaacgttcagggggg_3,

[0034] CpG ODN 4 :5,_tcgtgcgacgatcgtcgcagggggg_3,[0035] CpG ODN 5 : 5, -tcgagcgatcgctcgagggggg-3, [0034] CpG ODN 4: 5, _tcgtgcgacgatcgtcgcagggggg_3, [0035] CpG ODN 5: 5, -tcgagcgatcgctcgagggggg-3,

[0036] CpG ODN 6 : 5, -ggggtgctggccgtcgttgggggg-3, [0036] CpG ODN 6: 5, -ggggtgctggccgtcgttgggggg-3,

[0037] CpG ODN 7 : 5, -tcgaaacgtttcgggggg-3, [0037] CpG ODN 7: 5, -tcgaaacgtttcgggggg-3,

[0038] CpG ODN 8 : 5, -tcgtcgggtgcgacgtcgcagggggg-3, [0038] CpG ODN 8: 5, -tcgtcgggtgcgacgtcgcagggggg-3,

[0039] CpG ODN 9 : 5, -tcggacgatcgtcgggggg-3, [0039] CpG ODN 9: 5, -tcggacgatcgtcgggggg-3,

[0040] CpG ODN 10 :5: '-tcgtcgggtgcgatcgcagggggg-3' [0040] CpG ODN 10: 5: '-tcgtcgggtgcgatcgcagggggg-3'

[0041] CpG ODN 11 :5: '-tcgtgcgacgtcgcagatgat-3' [0041] CpG ODN 11: 5: '-tcgtgcgacgtcgcagatgat-3'

[0042] CpG ODN 12 :5: '-tcgtatgcatcgatgcatagggagg-3' [0042] CpG ODN 12: 5: '-tcgtatgcatcgatgcatagggagg-3'

[0043] CpG ODN 13 :5: ' -tcgtcgtttcgtcgttgggg-3' [0043] CpG ODN 13: 5: '-tcgtcgtttcgtcgttgggg-3'

[0044] CpG ODN 14 :5 '-tcgtcgggtgcatcgatgcagggggg-3' [0044] CpG ODN 14: 5 '-tcgtcgggtgcatcgatgcagggggg-3'

[0045] CpG ODN 15 :5: '-tcgtcgcagaacgttctgggggg-3' [0045] CpG ODN 15: 5: '-tcgtcgcagaacgttctgggggg-3'

[0046] CpG ODN 16 :5: '-tcgtcgggtgcgacgtcgca-3' [0046] CpG ODN 16: 5: '-tcgtcgggtgcgacgtcgca-3'

[0047] CpG ODN 17 :5: '-gtcgttttcgtcgacgaattgggggggg-3, [0047] CpG ODN 17: 5: '-gtcgttttcgtcgacgaattgggggggg-3,

[0048] CpG ODN 18 :5: '-tcgtcgtcgactcgtggtcggggg-3' [0048] CpG ODN 18: 5: '-tcgtcgtcgactcgtggtcggggg-3'

[0049] CpG ODN 19 :5 '-tcggggacgatcgtcgggggg-3 ' [0049] CpG ODN 19: 5 '-tcggggacgatcgtcgggggg-3'

[0050] CpG ODN 20 :5: '-gggggcgtcgttttcgtcgacgaatt-3 ' [0050] CpG ODN 20: 5: '-gggggcgtcgttttcgtcgacgaatt-3'

[0051] 其中的磷酸 二! 指键可以是部分硫化的,全部硫化的,也可以是未硫化的 [0051] wherein phosphodiester! Refers to a bond may be partially cured, the entire sulfurized, and may be an unvulcanized

[0052] 本发明的CpG ODN可通过已知的方法生产,例如采用固相亚磷酰胺三酯法进行生产。 [0052] CpG ODN of the present invention can be produced by a known method, for example, produced using the solid phase phosphoramidite triester method. 以下的实施例详细地例举了一种生产本发明的人工合成的含CpG单链脱氧寡核苷酸的方法。 The following examples illustrate a process for producing the present invention synthesized CpG-containing single-stranded oligodeoxynucleotides detail.

[0053] 在预防和治疗病毒感染和病毒感染引发的疾病时,这些CpG ODN的一次用药剂量为1-5000微克。 [0053] in the prevention and treatment of viral infections and diseases caused by viral infection, once these CpG ODN dose of 1 to 5000 micrograms. 利巴韦林的用量为300-1000毫克。 Ribavirin in an amount of 300-1000 mg.

[0054] CpG ODN的应用方式包括粘膜表面(包括呼吸道、消化道和泌尿生殖道黏膜)应用,皮下、肌肉注射应用,胃肠应用,腹腔应用,静脉注射等方式应用。 [0054] CpG ODN application methods include mucosal surfaces (including the respiratory, gastrointestinal and urogenital tract) applications, subcutaneous, intramuscular application, parenteral application, intraperitoneal application, intravenous injection, etc. applications.

[0055] 利巴韦林的应用方式包括粘膜表面(包括呼吸道、消化道和泌尿生殖道黏膜)应用,皮下、肌肉注射应用,胃肠应用,腹腔应用,静脉注射等方式应用。 [0055] Ribavirin application methods include mucosal surfaces (including the respiratory, gastrointestinal and urogenital tract) applications, subcutaneous, intramuscular application, parenteral application, intraperitoneal application, intravenous injection, etc. applications.

[0056] 下面结合具体的制备实施例和生物学效果实施例,并参照附图进一步详细地描述本发明。 [0056] In particular embodiments in conjunction with the following Preparation Examples and biological effects, and the present invention is described in further detail with reference to the drawings. 应理解,这些实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。 It should be understood that these examples are merely to illustrate the invention without limiting the scope of the invention in any manner.

[0057] 实施例 [0057] Example

[0058] 在如下实施例中,未详细描述的各种过程和方法是本领域中公知的常规方法,例如合成采用固相亚磷酰胺三酯法。 [0058] In the following examples, various processes and methods not described in detail are conventional methods well known in the art, for example, synthesized by the solid phase phosphoramidite triester method.

[0059] 在如下实施例中,所用试剂的来源、商品名和/或有必要列出其组成成分者,均只标明一次。 [0059] In the following examples, the reagent sources, trade names and / or which are necessary components listed only once are marked. 在其后所用相同试剂如无特殊说明,不在赘述上述内容。 Thereafter the same reagents Unless otherwise specified, the above is not repeated.

[0060] 实施例1 CpG ODN的制备 Preparation Example 1 CpG ODN of the [0060] embodiment

[0061 ] 采用固相亚磷酰胺三酯法合成CpG ODN [0061] The solid phase phosphoramidite triester Synthesis of CpG ODN

[0062] 1、试剂和材料: [0062] 1. Reagents and Materials:

[0063]三氯乙酸(Trichloroacetic Acid, TCA)、可控多孔玻璃(Controlled Pore Glass)、DMT (二甲氧基三苯甲基)、四氮唑活化剂、乙酸酐、N-甲基咪唑、A、T、C、G四种核苷酸单体、ABI DNA合成仪、高效液相色谱层析仪等 [0063] TCA (Trichloroacetic Acid, TCA), controlled pore glass (Controlled Pore Glass), DMT (dimethoxytrityl), tetrazolium activator, acetic anhydride, N- methylimidazole, A, four nucleotide monomers T, C, G, ABI DNA synthesizer, HPLC chromatograph, etc.

[0064] 2、方法: [0064] 2. Method:

[0065] 1)脱保护基 [0065] 1) Deprotection

[0066] 用三氯乙酸(Trichloroacetic Acid,TCA)脱去连结在可控多孔玻璃(Controlled Pore Glass)上的核苷酸的保护基团二甲氧基三苯甲基(DMT),获得游离的5' -羟基端,以供下一步缩合反应。 [0066] off coupled to the controlled pore glass (Controlled Pore Glass) nucleotide protective dimethoxytrityl group (DMT) with trichloroacetic acid (Trichloroacetic Acid, TCA), to give the free 5 '- hydroxyl ends, for the next condensation reaction.

[0067] 2)活化 [0067] 2) activation

[0068] 将亚磷酰胺保护的核苷酸单体与四氮唑活化剂混合并进入合成柱,形成亚磷酰胺四唑活性中间体(其3'-端已被活化,但5'-端仍受DMT保护),此中间体将与可控多孔玻璃上的已脱保护基的核苷酸发生缩合反应。 [0068] The protected nucleotide phosphoramidite monomer with a tetrazolium agent and activator mixed into the synthesis column tetrazole forming phosphoramidite reactive intermediate (3'-end thereof has been activated, but the 5'-end DMT still under protection), the condensation reaction of this intermediate with the deprotected nucleotide has on the controlled pore glass.

[0069] 3)连接 [0069] 3) connected

[0070] 亚磷酰胺四唑活性中间体遇到可控多孔玻璃上已脱保护基的核苷酸时,将与其5' -羟基发生亲合反应,缩合并脱去四唑,此时合成的寡核苷酸链向前延长一个碱基。 When [0070] tetrazolyl phosphoramidite reactive intermediate is deprotected encountered on controlled pore glass nucleotides, 5 to its' - hydroxyl groups of the affinity reaction, tetrazole condense off, then synthetic a forward oligonucleotide chain extension base.

[0071] 4)封闭 [0071] 4) is closed

[0072] 缩合反应后为了防止连在可控多孔玻璃上的未参与反应的5' -羟基在随后的循环反应中被延伸,常通过乙酰化来封闭此端羟基,一般乙酰化试剂是用乙酸酐和N-甲基咪唑等混合形成的。 [0072] After the condensation reaction is not involved in the reaction in order to prevent even on a controlled pore glass 5 '- hydroxyl group is extended in a subsequent reaction cycle, this normally closed terminal hydroxyl group by acetylation, generally acetylating agent is acetic a mixed acid anhydride and N- methylimidazole formed.

[0073] 5)氧化 [0073] 5) oxide

[0074] 缩合反应时核苷酸单体是通过亚磷酯键与连在可控多孔玻璃上的寡核苷酸连接, 而亚磷酯键不稳定,易被酸、碱水解,此时常用碘的四氢呋喃溶液将亚磷酰转化为磷酸三酯,得到稳定的寡核苷酸。 [0074] The nucleotide phosphoramidite monomer by ester linkage to the oligonucleotide attached to the controlled pore glass when the condensation reaction is connected, and the phosphorous ester bond labile and susceptible to acid, alkaline solution, typically at a tetrahydrofuran solution of iodine is converted to the phosphoramidite triester phosphate, a stable oligonucleotide.

[0075] 经过以上五个步骤后,一个脱氧核苷酸就被连到可控多孔玻璃的核苷酸上,同样再用三氯乙酸脱去新连上的脱氧核苷酸5'-羟基上的保护基团DMT后,重复以上的活化、连接、封闭、氧化过程即可得到一DNA片段粗品。 [0075] After the above five steps, a deoxynucleotide was attached to controlled pore glass nucleotide, also trichloroacetic acid and then removed deoxy-5'-nucleotides on the newly connected hydroxy after the DMT protecting group, repeat the above activation, connection closing, to oxidation to give a crude DNA fragment. 最后对其进行切割、脱保护基(一般对A、C 碱基采用苯甲酰基保护;G碱基用异丁酰基保护;T碱基不必保护;亚磷酸用腈乙基保护)、 纯化(常用的有HAP,PAGE, HPLC,C18,OPC等方法)、定量等合成后处理即可得到符合实验要求的寡核苷酸片段。 Finally, its cutting, deprotection (typically for A, C using benzoyl protecting bases; G nucleotide protection isobutyryl; T bases do not have protection; nitrile ethyl phosphite protection), purified (common there HAP, PAGE, HPLC, C18, OPC or the like), quantification can be obtained by post-synthesis treatment with the experimental oligonucleotides requirements.

[0076] 未硫化的CpG ODN在ABI 3900 DNA合成仪上合成;全硫化及部分硫化CpG单链脱氧寡核苷酸的合成采用置换法,在ABI 394 DNA合成仪上合成。 [0076] CpG ODN unvulcanized synthesized on a ABI 3900 DNA Synthesizer; partially cured fully cured and a single-stranded CpG oligodeoxynucleotides synthesized by metathesis, synthesized on ABI 394 DNA synthesizer.

[0077] 实施例2、实施例3、实施例4、实施例5、实施例6中使用的CpG ODN序列均如下所示: [0077] Example 2, Example 3, Example 4, Example 5, CpG ODN sequence in Example 6 were used in the examples are as follows:

[0078] CpG ODN 1 5, -ggggacgatatcgatgggggg-3, [0078] CpG ODN 1 5, -ggggacgatatcgatgggggg-3,

[0079] CpG ODN 2 5, -ggggtcgttcgtcgttgggggg-3, [0079] CpG ODN 2 5, -ggggtcgttcgtcgttgggggg-3,

[0080] CpG ODN 3 5, -ggggtgcaacgttcagggggg-3, [0080] CpG ODN 3 5, -ggggtgcaacgttcagggggg-3,

[0081] CpG ODN 4 5, -tcgtgcgacgatcgtcgcagggggg-3, [0081] CpG ODN 4 5, -tcgtgcgacgatcgtcgcagggggg-3,

[0082] CpG ODN 5 5, -tcgagcgatcgctcgagggggg-3, [0082] CpG ODN 5 5, -tcgagcgatcgctcgagggggg-3,

[0083] CpG ODN 6 5, -ggggtgctggccgtcgttgggggg-3, [0083] CpG ODN 6 5, -ggggtgctggccgtcgttgggggg-3,

[0084] CpG ODN 7 5, -tcgaaacgtttcgggggg-3, [0084] CpG ODN 7 5, -tcgaaacgtttcgggggg-3,

[0085] CpG ODN 8 5, -tcgtcgggtgcgacgtcgcagggggg-3[0086] CpG ODN 9 : 5'. -tcggacgatcgtcgggggg-3, [0085] CpG ODN 8 5, -tcgtcgggtgcgacgtcgcagggggg-3 [0086] CpG ODN 9: 5 '-tcggacgatcgtcgggggg-3,.

[0087] CpG ODN 10 :5, -tcgtcgggtgcgatcgcagggggg-3, [0087] CpG ODN 10: 5, -tcgtcgggtgcgatcgcagggggg-3,

[0088] CpG ODN 11 :5, -tcgtgcgacgtcgcagatgat-3, [0088] CpG ODN 11: 5, -tcgtgcgacgtcgcagatgat-3,

[0089] CpG ODN 12 :5, -tcgtatgcatcgatgcatagggagg-3, [0089] CpG ODN 12: 5, -tcgtatgcatcgatgcatagggagg-3,

[0090] CpG ODN 13 :5, -tcgtcgtttcgtcgttgggg-3' [0090] CpG ODN 13: 5, -tcgtcgtttcgtcgttgggg-3 '

[0091] CpG ODN 14 :5' -tcgtcgggtgcatcgatgcagggggg-3' [0091] CpG ODN 14: 5 '-tcgtcgggtgcatcgatgcagggggg-3'

[0092] CpG ODN 15 :5, -tcgtcgcagaacgttctgggggg-3, [0092] CpG ODN 15: 5, -tcgtcgcagaacgttctgggggg-3,

[0093] CpG ODN 16 :5, -tcgtcgggtgcgacgtcgca-3, [0093] CpG ODN 16: 5, -tcgtcgggtgcgacgtcgca-3,

[0094] CpG ODN 17 :5, -gtcgttttcgtcgacgaattgggggggg-3' [0094] CpG ODN 17: 5, -gtcgttttcgtcgacgaattgggggggg-3 '

[0095] CpG ODN 18 :5, -tcgtcgtcgactcgtggtcggggg-3, [0095] CpG ODN 18: 5, -tcgtcgtcgactcgtggtcggggg-3,

[0096] CpG ODN 19 :5' -tcggggacgatcgtcgggggg-3' [0096] CpG ODN 19: 5 '-tcggggacgatcgtcgggggg-3'

[0097] CpG ODN 20 :5, -gggggcgtcgttttcgtcgacgaatt-3, [0097] CpG ODN 20: 5, -gggggcgtcgttttcgtcgacgaatt-3,

[0098] 实施例2 CpG ODN和CpG ODN与利巴韦林联合应用的抗滤泡口炎病毒作用比较 Comparison of anti-virus effect of follicular stomatitis [0098] Example 2 CpG ODN and with CpG ODN of ribavirin

[0099] 1、滤泡口炎病毒(VSV)的扩增 [0099] 1, follicular stomatitis virus (VSV) Amplification

[0100] 1)仪器设备和材料:低温冰箱、二氧化碳孵箱、超净工作台、倒置显微镜、液氮罐、 蒸馏水器、真空泵、细胞培养瓶、各种规格的吸管、加样器、滴管等。 [0100] 1) Materials and equipment: cryogenic refrigerator, carbon dioxide incubator, clean bench, inverted microscope, a liquid nitrogen tank, distilled water, a vacuum pump, cell culture flask, a variety pipette, pipette, dropper Wait.

[0101] 2) RPMI1640 培养基:含L-谷氨酰胺的RPMI1640 (GIBC0BRL) 10. 4 克,2. 0 克碳酸氢钠,10单位庆大霉素,加三蒸水至体积1000毫升。 [0101] 2) RPMI1640 medium: RPMI1640 (GIBC0BRL) containing 10.4 g of L- glutamine, 20 g of sodium bicarbonate, 10 units of gentamicin, plus three to a volume of 1,000 ml distilled water. 0. 22微米的滤膜真空泵抽滤除菌、分装。 0.22 micron filter sterilized vacuum filtration, packaging.

[0102] 3)方法:用含10%小牛血清anvitrogen,经56°C 30分钟处理)的RPMI1640培养基培养Vero E6细胞。 [0102] 3) Method: containing 10% calf serum anvitrogen, by 56 ° C 30 min Process) in Vero E6 cells cultured in RPMI1640 medium. 在IOOml培养瓶中长成单层后,更换培养液。 After IOOml grown monolayer culture flasks, medium was changed. 加入含滤泡口炎病毒(VSV)的培养液,其含量为10TCID50。 Add broth containing follicular stomatitis virus (VSV), and an amount of 10TCID50. 设无病毒对照。 Set no virus control. 约对-48小时后,感染VSV的Vero E6细胞全部发生病变,收集病变细胞和上清,将其置-20°C过夜。 After about 48 hours, Vero E6 cells infected with VSV of all lesions, diseased cells, and the supernatant was collected, it was placed overnight at -20 ° C. 次晨,将其融解,用吸管猛烈吹打,离心收取上清。 The next morning, it was thawed, fierce wind and percussion with a straw, centrifuged supernatant was collected. 在测定TCID50后,将上清贮存于-20°C备用。 After the measurement TCID50, and the supernatant was stored at -20 ° C for use.

[0103] 2、滤泡口炎病毒(VSV) TCID50的测定 [0103] 2, was measured follicular stomatitis virus (VSV) TCID50 of

[0104] 1)仪器设备和材料:低温冰箱、二氧化碳孵箱、超净工作台、倒置显微镜、液氮罐、 蒸馏水器、真空泵、细胞培养瓶、各种规格的吸管、加样器、滴管等。 [0104] 1) Materials and equipment: cryogenic refrigerator, carbon dioxide incubator, clean bench, inverted microscope, a liquid nitrogen tank, distilled water, a vacuum pump, cell culture flask, a variety pipette, pipette, dropper Wait.

[0105] 2) RPMI1640 培养基:含L-谷氨酰胺的RPMI1640 (GIBC0BRL) 10. 4 克,2. 0 克碳酸氢钠,10单位庆大霉素,加三蒸水至体积1000毫升。 [0105] 2) RPMI1640 medium: RPMI1640 (GIBC0BRL) containing 10.4 g of L- glutamine, 20 g of sodium bicarbonate, 10 units of gentamicin, plus three to a volume of 1,000 ml distilled water. 用0. 22微米的滤膜滤过除菌、分装。 With a 0.22 micron membrane filtration sterilization, packaging.

[0106] 3)含10%小牛血清的RPMI1640培养基:10毫升小牛血清Qnvitrogen,经56°C 30 分钟处理),90毫升RPMI1640培养基。 [0106] 3) RPMI1640 medium containing 10% fetal bovine serum: 10 ml calf serum Qnvitrogen, by 56 ° C 30 min Process), 90 ml RPMI1640 medium.

[0107] 4)0. 5%结晶紫染液:结晶紫0.5克,NaCl 0. 85克,溶于50毫升无水乙醚。 [0107] 4) 05% crystal violet dye solution: 0.5 g crystal violet, NaCl 0. 85 g, dissolved in 50 ml of anhydrous ether. 加3 毫升甲醛,47毫升蒸馏水。 Add 3 ml of formalin, 47 ml of distilled water.

[0108] 5)结晶紫脱色液:50毫升乙二醇单甲醚与50毫升蒸馏水混合。 [0108] 5) crystal violet destaining solution: 50 ml of ethylene glycol monomethyl ether mixed with 50 ml of distilled water.

[0109] 6)0. 25%胰蛋白酶(Trypsin) [0109] 6) 0.25% trypsin (Trypsin)

[0110] 7)方法:用0.25%胰蛋白酶消化生长良好的Vero E6细胞,3_5分钟。 [0110] 7) Method: with 0.25% trypsin in Vero E6 cells grow well, 3_5 minutes. 离心(1000rpm,5分钟)洗涤细胞。 Cells were washed by centrifugation (1000rpm, 5 minutes). 用10%小牛血清的RPMI1640培养基调细胞浓度为4X 105个/毫升。 10% fetal calf serum RPMI1640 culture with a cell concentration of 4X 105 tone / ml. 加细胞悬液于96孔平底培养板(Costar),每孔100微升,设三个复孔。 The cell suspension was added to 96 well flat bottom culture plates (Costar), 100 microliters per well, three wells provided. 37°C CO2 孵箱培养M小时后,细胞形成单层。 After the 37 ° C CO2 incubator M-hour culture, the cells formed a monolayer. 更换培养液,其中含倍比稀释的待滴定VSV(长春生物制品所)。 The medium was changed, wherein the dilution containing the VSV be titrated (Changchun Vaccine). 设无VSV对照。 No VSV control setting. M小时后,用结晶紫染色的方法判定细胞病变的程度。 After M hour, determined by the degree of cytopathic crystal violet staining method. 吸弃培养液,每孔加200μ1 0.5%结晶紫染液,37°C,15分钟。 Aspirating the medium, each well was added 200μ1 0.5% crystal violet dye, 37 ° C, 15 min. 流水冲掉结晶紫染液。 Flush water crystal violet dye. 每孔加入200 μ 1结晶紫脱色液,振荡器振荡,使染料完全从细胞内脱出,用分光光度计在MOnm波长处比色(Cytokines. A practical approach, second edition, edited by FR BALKffILLThe practical approach series.)。 Per well was added 200 μ 1 crystal violet solution bleaching, oscillator, the dye is completely released from the cell, a colorimetric spectrophotometer (Cytokines. A practical approach, second edition, edited by FR BALKffILLThe practical approach series at a wavelength MOnm .). 以出现50%细胞病变的病毒稀释度的倒数XlO为此病毒液的TCID50/毫升数值。 Appears at 50% cytopathic XlO reciprocal of virus dilution TCID50 / ml virus solution for this value.

[0111] 3、人外周血单个核细胞的分离 [0111] 3, human peripheral blood mononuclear cells isolated

[0112] 1)仪器设备和器材:低温冰箱、二氧化碳孵箱、超净工作台、倒置显微镜、液氮罐、 蒸馏水器、真空泵、细胞培养瓶、滤菌器、滤过瓶、各种规格的吸管、加样器、滴管、血球计数板、水平式离心机等。 [0112] 1) The equipment and materials: deep freezer, carbon dioxide incubator, clean bench, inverted microscope, a liquid nitrogen tank, distilled water, a vacuum pump, cell culture flask, a bacteria filter, filtration bottles, a variety of pipette, pipette, dropper, hemacytometer, centrifuge horizontal.

[0113] 2)试剂和材料:肝素抗凝的人全血购自长春市中心血站。 [0113] 2) Reagents and Materials: heparinized human whole blood was purchased from Changchun Blood Center. 聚蔗糖-泛影葡胺: 比重1.077士0.001,购自北京鼎国生物技术有限公司。 Ficoll - hypaque: specific gravity 1.077 0.001 disabilities, purchased from Beijing Ding States Biological Technology Co., Ltd. RPMI1640培养液:L_谷氨酰胺的RPMI1640 (GIBC0BRL) 10. 4克,碳酸氢钠2. 0克,庆大霉素10万单位,加三蒸水至1000毫升。 RPMI1640 medium: L_ glutamine RPMI1640 (GIBC0BRL) 10. 4 g, 2.0 g of sodium bicarbonate, gentamicin 100,000 units, was added to 1000 ml triple-distilled water. 0. 22微米的滤膜抽滤除菌、分装。 0.22 micron filter filtration sterilization, packaging.

[0114] 3)方法:用聚蔗糖-泛影葡胺淋巴细胞分层液分离人外周血的单个核细胞。 [0114] 3) Methods: Ficoll - Hypaque Ficoll was isolated from human peripheral blood mononuclear cells. 分层液与肝素抗凝外周血的体积比约为2 : 1。 And the volume of heparinized peripheral blood was stratified ratio of about 2: 1. 水平离心(l,000Xg,20min)。 Centrifuge (l, 000Xg, 20min). 用吸管吸取含单个核细胞的液带,置入另离心管中。 Pipet-containing solution with mononuclear cells, into another centrifuge tube. 加入等体积的无血清培养液。 Serum-free medium was added an equal volume. l,000g离心15min,弃上清。 l, 000g centrifugation 15min, the supernatant was discarded. 重复洗涤两次。 Washing was repeated twice. 弃上清,用2ml培养液重悬细胞,并进行细胞计数。 The supernatant was discarded, a 2ml culture was resuspended cells, and the cells counted.

[0115] 4、CpG ODN和CpG ODN和利巴韦林联合应用抗滤泡口炎病毒的作用的测定 [0115] 4, CpG ODN and CpG ODN and ribavirin measuring the Resistance of follicular stomatitis virus application

[0116] 用含10%小牛血清的RPMI1640培养基调人外周血单个核细胞的终浓度为3 X IO6 个/毫升。 [0116] RPMI1640 culture tone people with 10% calf serum-containing peripheral blood mononuclear cells at a final concentration of 3 X IO6 cells / ml. 加细胞悬液于12孔培养板,每孔anl。 The cell suspension was added to 12-well culture plate, each well anl. 加CpG ODN(1-20)至终浓度6yg/ml。 Plus CpG ODN (1-20) to a final concentration 6yg / ml. 37°C,5%二氧化碳孵箱培养48小时,收集上清。 37 ° C, 5% carbon dioxide incubator for 48 hours, the supernatant was collected.

[0117] 设立CpG组、利巴韦林组、CpG+利巴韦林组、IMDM组。 [0117] CpG established group, ribavirin group, CpG group + ribavirin, IMDM group. 将生长状态良好的Vero E6细胞接种于96孔培养板,每孔1. 3X IO4个细胞,37° ,5% CO2培养M小时后,CpG组加入1 : 100稀释的CpG ODN刺激的人PBMC培养上清,利巴韦林组加入利巴韦林(至终浓度10 μ mol/1),CpG+利巴韦林组加入1 : 100稀释的CpG ODN刺激的人PBMC培养上清和利巴韦林(浙江诚意药业有限公司),IMDM组只加入IMDM。 The good state of growth of Vero E6 cells were seeded in 96-well culture plate, each hole 1. 3X IO4 cells, 37 °, 5% CO2 M h after the culture, the group of CpG was added 1: 100 diluted CpG ODN-stimulated human PBMC cultures the supernatant, ribavirin ribavirin group added (to a final concentration of 10 μ mol / 1), CpG + ribavirin was added group 1: 100 dilution of CpG ODN to stimulate human PBMC culture supernatants and ribavirin ( Zhejiang sincerity Pharmaceutical Co., Ltd.), IMDM group only added IMDM. 继续培养1. 5小时后,加入250TCID50/ml的VSV病毒,同时设正常VERO细胞对照组,培养44小时。 After cultured for 1.5 hours, addition of 250TCID50 / ml of VSV virus There was also a control group of VERO cells cultured for 44 hours. 用结晶紫染色的方法判定细胞病变的程度。 Stained with crystal violet method determines the degree of cytopathic. 吸弃培养液。 Culture medium was aspirated. 每孔加200μ1 0.5%结晶紫染液,37°C,孵育15分钟。 Each well was added 200μ1 0.5% crystal violet dye, 37 ° C, incubated for 15 minutes. 流水冲掉结晶紫染液。 Flush water crystal violet dye. 每孔加入200微升结晶紫脱色液,振荡器振荡,使染料完全从细胞内脱出,用分光光度计在MOnm波长测定分光光度值。 200 microliters per well of crystal violet solution bleaching, oscillator, the dye is completely released from the cell, was measured with a spectrophotometer MOnm wavelength spectrophotometric values.

[0118] 5、结果:CpG ODN组和CpG ODN与利巴韦林联合应用组相比较,CpG ODN与利巴韦林联合应用组的OD值显著地高于CpG ODN组,这一结果说明,CpG ODN具有明显的抗病毒作用,CpG ODN和利巴韦林联合应用具有更显著的抗病毒作用。 [0118] 5. Results: Group CpG ODN and with CpG ODN compared ribavirin group, the OD value of CpG ODN in combination with ribavirin group is significantly higher than that CpG ODN group, the results indicate, CpG ODN have significant antiviral activity, CpG ODN and ribavirin have a more significant antiviral activity. 两组间的OD值比较见表1。 OD values ​​between the two groups is shown in table 1.

[0119] 表1 CpG ODN和CpG ODN与利巴韦林联合应用的抗滤泡口炎病毒作用比较 Comparison of anti-virus effect of follicular stomatitis [0119] Table. 1 CpG ODN and with CpG ODN of ribavirin

[0120] 实施例3 CpG ODN和CpG ODNN与利巴韦林联合应用的抗流感病毒作用比较 Comparison of anti-influenza virus effect [0120] Example 3 CpG ODN and with CpG ODNN of ribavirin

[0121] 1、人外周血单个核细胞的分离 [0121] 1, human peripheral blood mononuclear cells

[0122] 1)仪器设备和器材:低温冰箱、二氧化碳孵箱、超净工作台、倒置显微镜、液氮罐、 蒸馏水器、真空泵、细胞培养瓶、滤菌器、滤过瓶、各种规格的吸管、加样器、滴管、血球计数板、水平式离心机等。 [0122] 1) The equipment and materials: deep freezer, carbon dioxide incubator, clean bench, inverted microscope, a liquid nitrogen tank, distilled water, a vacuum pump, cell culture flask, a bacteria filter, filtration bottles, a variety of pipette, pipette, dropper, hemacytometer, centrifuge horizontal. [0123] 2)试剂和材料:肝素抗凝的人全血购自长春市中心血站。 [0123] 2) Reagents and Materials: heparinized human whole blood was purchased from Changchun Blood Center. 聚蔗糖-泛影葡胺:比重1. 077士0. 001,购自北京鼎国生物技术有限公司。 Ficoll - hypaque: specific gravity 1.077 0.001 disabilities, purchased from Beijing Ding States Biological Technology Co., Ltd. DMEM培养液:1000ml含庆大霉素10万单位。 DMEM medium: 1000ml containing gentamicin 100,000 units. 0.22微米的滤膜抽滤除菌、分装。 0.22 micron filter filtration sterilization, packaging.

[0124] 3)方法:用聚蔗糖-泛影葡胺淋巴细胞分层液分离人外周血的单个核细胞。 [0124] 3) Methods: Ficoll - Hypaque Ficoll was isolated from human peripheral blood mononuclear cells. 分层液与肝素抗凝外周血的体积比约为2 : 1。 And the volume of heparinized peripheral blood was stratified ratio of about 2: 1. 水平离心(l,000Xg,20min)。 Centrifuge (l, 000Xg, 20min). 用吸管吸取含单个核细胞的液带,置入另离心管中。 Pipet-containing solution with mononuclear cells, into another centrifuge tube. 加入等体积的无血清培养液。 Serum-free medium was added an equal volume. l,000g离心15min,弃上清。 l, 000g centrifugation 15min, the supernatant was discarded. 重复洗涤两次。 Washing was repeated twice. 弃上清,用2ml培养液重悬细胞,并进行细胞计数。 The supernatant was discarded, a 2ml culture was resuspended cells, and the cells counted.

[0125] 2、CpG ODN和CpG ODN与利巴韦林联合应用的抗流感病毒作用测定 [0125] 2, anti-influenza virus effect of CpG ODN and with CpG ODN ribavirin measured

[0126] 用DMEM培养液调人外周血单个核细胞的终浓度为3X IO6个/毫升。 [0126] DMEM medium adjusted with human peripheral blood mononuclear cells at a final concentration 3X IO6 cells / ml. 加此细胞悬液于12孔培养板,每孔anl。 This cell suspension was added to 12-well culture plate, each well anl. 加CpG ODN至终浓度eyg/ml。 CpG ODN added to a final concentration eyg / ml. 37 °C,5% 二氧化碳孵箱培养48小时,收集上清,检测其抗流感病毒的活性。 37 ° C, 5% carbon dioxide incubator for 48 hours, the supernatant was collected, detected anti-influenza virus activity.

[0127] 设立CpG组、利巴韦林组、CpG+利巴韦林组、DMEM组。 [0127] CpG established group, ribavirin group, CpG group + ribavirin, DMEM group. 用0. 3% BSA DMEM调节生长状态良好的Vero E6细胞浓度至3X IO5个/ml。 Good growth state adjusted with 0. 3% BSA DMEM Vero E6 cell concentration to 3X IO5 cells / ml. 将细胞接种于96孔平底培养板,每孔1. 3X104个细胞,37° and 5% CO2培养M小时后,CpG组加入1 : 100稀释的CpG ODN刺激的人PBMC培养上清,利巴韦林组加入利巴韦林(至终浓度ΙΟμπιοΙ/l),CpG+利巴韦林组加入1 : 100稀释的CpG ODN刺激的人PBMC培养上清和利巴韦林(浙江诚意药业有限公司),DMEM组只加入DMEM。 Cells were seeded in 96-well flat bottom plates, 1. 3X104 cells per well, 37 ° and 5% CO2 M h after the culture, the group of CpG was added 1: 100 dilution of CpG ODN to stimulate human PBMC culture supernatants, ribavirin Lin group ribavirin was added (to a final concentration ΙΟμπιοΙ / l), CpG + ribavirin was added group 1: 100 dilution of CpG ODN to stimulate human PBMC culture supernatants and ribavirin (Zhejiang We Pharmaceutical Co., Ltd.), DMEM group added only DMEM. 继续培养1. 5小时后,每孔加入0. 3% BSA DMEM(4 μ g/ml胰酶)稀释的流感病毒液(长春生物制品研究所)200 μ 1,同时设正常VERO细胞对照。 After cultured for 1.5 hours, each well was 0. 3% BSA DMEM (4 μ g / ml trypsin) was diluted with influenza virus (Changchun Institute of Biological Products) 200 μ 1, VERO cells while the normal control is provided. 37°C、 5%C02孵箱培养44小时,用结晶紫染色的方法判定细胞病变的程度。 37 ° C, 5% C02 incubator for 44 hours culture, the degree of cytopathic determining the crystal violet staining method. 吸弃培养液。 Culture medium was aspirated. 每孔加200μ1 0.5%结晶紫染液,37°C,15分钟。 Each well was added 200μ1 0.5% crystal violet dye, 37 ° C, 15 min. 流水冲掉结晶紫染液。 Flush water crystal violet dye. 每孔加入200微升结晶紫脱色液,振荡器振荡,使染料完全从细胞内脱出,用分光光度计在MOnM波长测定分光光度值。 200 microliters per well of crystal violet solution bleaching, oscillator, the dye is completely released from the cell, was measured with a spectrophotometer MOnM wavelength spectrophotometric values.

[0128] 3结果:CpG ODN组和CpG ODN与利巴韦林联合应用组相比较,CpG ODN与利巴韦林联合应用组的OD值显著地高于CpG ODN组,这一结果说明,CpG ODN具有明显的抗流感病毒作用,CpG ODN和利巴韦林联合应用具有更显著的抗流感病毒作用。 [0128] Results 3: CpG ODN group and CpG ODN compared to ribavirin group, the OD value of CpG ODN in combination with ribavirin group is significantly higher than that CpG ODN group, the results indicate that, of CpG ODN has significant anti-influenza virus effect, CpG ODN and ribavirin have a more significant role in the anti-influenza virus. 两组间的OD值比较见表2。 OD values ​​between the two groups is shown in table 2.

[0129] 表2 CpG ODN和CpG ODNN与利巴韦林联合应用的抗流感病毒作用比较 [0129] Anti-influenza virus effect Table 2 CpG ODN and with CpG ODNN ribavirin Comparison

[0130] 实施例4 CpG和利巴韦林抗流感病毒的小鼠体内实验 In vivo experiments in mice [0130] Example 4 CpG and Ribavirin against influenza virus

[0131] 1材料:雌性昆明鼠,14-16克,流感病毒Hmi型肺适应株(FMl)购自长春生物制品所。 [0131] 1 Materials: female Kunming mice, 14-16 grams, influenza virus Hmi pulmonary adapted strains (FML) are purchased from Changchun Institute of Biological Products.

[0132] 2方法:设立正常小鼠对照、模型对照组(阴性对照组)、利巴韦林组、CpG组、利巴韦林加CpG组。 [0132] Method 2: establishment of normal mouse control, model control group (negative control group), ribavirin group, CpG group, ribavirin plus CpG group. CpG组小鼠皮下注射CpG ODN 80 μ g(CpG ODN 1_20),48小时后,将流感病毒用0. 3% BSA DMEM稀释至IOLD5tl,除正常小鼠对照组外,其它各组小鼠经乙醚麻醉后,鼻腔给予20μ 1的IOLD5tl的流感病毒。 CpG ODN CpG mice were injected subcutaneously 80 μ g (CpG ODN 1_20), 48 hours later, influenza virus diluted to 0. 3% BSA DMEM IOLD5tl, in addition to the normal mice in the control group, other groups of mice with ether after anesthesia, intranasal administration of IOLD5tl of 20μ 1 influenza virus. 2小时后,采用灌胃的方法给予利巴韦林组小鼠,50mg 利巴韦林/kg体重,每只小鼠0.2-0.細1,每日3次。 After 2 hours, using the method of intragastric administration of ribavirin mice, 50mg ribavirin / kg body weight, each mouse 0.2-0. 1 Fine, 3 times a day. 采用皮下注射的方式给予CpG组小鼠80 μ gCpG 0DN,每两天注射一次。 Administered by subcutaneous injection using CpG mice 80 μ gCpG 0DN, injected once every two days. 利巴韦林加CpG组,给予利巴韦林和CpG,给药剂量和给药方式同利巴韦林组和CpG组。 Ribavirin plus CpG group, and CpG ribavirin, dose and mode of administration of ribavirin with CpG group, and groups. 4天后,解剖小鼠,取肺,称重,判定肺部病变指数。 After 4 days, mice were dissected, lung, weighed, determining lung disease index. 肺部病变指数(Biochemical and Biophysical Research Communications 279,158-161 (2000). Inhibitory Effects of an Antisense Oligonucleotide in an ExperimentallyInfectedMouse Model of Influenza A Virus.)判定指标: Pulmonary lesions index (.. Biochemical and Biophysical Research Communications 279,158-161 (2000) Inhibitory Effects of an Antisense Oligonucleotide in an ExperimentallyInfectedMouse Model of Influenza A Virus) determining indicators:

[0133] -未有明显病变 [0133] - No obvious lesions

[0134] +25%肺组织发生病变 [0134] + 25% lung lesions

[0135] ++25-50%肺组织发生病变 [0135] ++ 25-50% lung lesions

[0136] +++50-75%肺组织发生病变 [0136] +++ 50-75% lung lesions

[0137] ++++ > 75 %肺组织发生病变 [0137] ++++> 75% lung lesions

[0138] 3结果:CpG+利巴韦林组小鼠的肺部病变指数显著低于CpG组和利巴韦林组。 [0138] 3. Results: CpG + ribavirin lung disease index was significantly lower than mice CpG group and ribavirin group. 说明CpG和利巴韦林联合应用具有比CpG和利巴韦林单独应用时更好的效果。 DESCRIPTION CpG and ribavirin better than Ribavirin and CpG alone effect. 各组间肺病变指数的比较见表3。 Lung lesion index of each group is shown in table 3.

[0139] 4结论:CpG ODN具有明显的抗流感病毒作用,CpG ODN和利巴韦林联合应用具有更显著的抗流感病毒作用。 [0139] 4 Conclusion: CpG ODN have significant anti-influenza virus effect, CpG ODN and ribavirin have a more significant role in the anti-influenza virus.

[0140] 实施例5 CpGODN和CpGODNN与利巴韦林联合应用的抗口蹄疫病毒作用比较 Example 5 CpGODN anti-FMD virus activity in combination with ribavirin and CpGODNN Application [0140] Comparative embodiment

[0141] 1、人外周血单个核细胞的分离 [0141] 1, human peripheral blood mononuclear cells

[0142] 1)仪器设备和器材: [0142] 1) The equipment and materials:

[0143] 低温冰箱、二氧化碳孵箱、超净工作台、倒置显微镜、液氮罐、蒸馏水器、真空泵、细胞培养瓶、滤菌器、滤过瓶、各种规格的吸管、加样器、滴管、血球计数板、水平式离心机等。 [0143] cryogenic refrigerator, carbon dioxide incubator, clean bench, inverted microscope, a liquid nitrogen tank, distilled water, a vacuum pump, cell culture flask, a bacteria filter, filtration bottles, a variety pipette, pipette, dropwise tube, hemacytometer, centrifuge horizontal.

[0144] 2)试剂和材料:0型口蹄疫病毒由内蒙古生物制药厂提供,抗病毒实验在内蒙古生物制药厂进行。 [0144] 2) Reagents and Materials: 0 by Inner Mongolia FMDV bio-pharmaceutical antiviral experiments in Inner Mongolia biological laboratories. 肝素抗凝的人全血购自长春市中心血站。 Heparinized human whole blood was purchased from Changchun city blood bank. 聚蔗糖-泛影葡胺:比重1.077 士0.001,购自北京鼎国生物技术有限公司。 Ficoll - hypaque: specific gravity 1.077 0.001 disabilities, purchased from Beijing Ding States Biological Technology Co., Ltd. IMDM培养液:1000ml含庆大霉素10万单位。 IMDM medium: 1000ml containing gentamicin 100,000 units. 0.22微米的滤膜抽滤除菌、分装。 0.22 micron filter filtration sterilization, packaging.

[0145] 3)方法:用聚蔗糖-泛影葡胺淋巴细胞分层液分离人外周血的单个核细胞。 [0145] 3) Methods: Ficoll - Hypaque Ficoll was isolated from human peripheral blood mononuclear cells. 分层液与肝素抗凝外周血的体积比约为2 : 1。 And the volume of heparinized peripheral blood was stratified ratio of about 2: 1. 水平离心(l,000Xg,20min)。 Centrifuge (l, 000Xg, 20min). 用吸管吸取含单个核细胞的液带,置入另离心管中。 Pipet-containing solution with mononuclear cells, into another centrifuge tube. 加入等体积的无血清培养基。 Serum-free medium was added in equal volume. l,000g离心15min,弃上清。 l, 000g centrifugation 15min, the supernatant was discarded. 重复洗涤两次。 Washing was repeated twice. 弃上清,用2ml培养基重悬细胞并进行细胞计数。 The supernatant was discarded, the cells were resuspended with 2ml medium and cell counts.

[0146] 2、CpG ODN和CpG ODN与利巴韦林联合应用的抗口蹄疫病毒作用测定 [0146] 2, anti-FMD virus activity CpG ODN and with CpG ODN ribavirin measured

[0147] 用含10%胎牛血清的IMDM培养液调人外周血单个核细胞的终浓度为3X106个/ 毫升。 [0147] with 10% fetal calf serum-containing culture medium IMDM adjusted final concentration of human peripheral blood 3X106 / ml mononuclear cells. 加此细胞悬液于12孔培养板,每孔anl。 This cell suspension was added to 12-well culture plate, each well anl. 加CpG ODN至终浓度eyg/ml。 CpG ODN added to a final concentration eyg / ml. 37°C,5% 二氧化碳孵箱培养48小时,收集上清,检测其抗口蹄疫病毒的活性。 37 ° C, 5% carbon dioxide incubator for 48 hours, the supernatant was collected, detected anti-FMD virus activity.

[0148] 设立CpG组、利巴韦林组、CpG+利巴韦林组、IMDM组。 [0148] CpG established group, ribavirin group, CpG group + ribavirin, IMDM group. 用5%胎牛血清IMDM调节生长状态良好的BHK细胞浓度至3 X IO5个/ml。 Good growth state adjusted with 5% fetal calf serum IMDM BHK cell concentration to 3 X IO5 cells / ml. 将细胞接种于96孔平底培养板,每孔1. 3 X IO4 个细胞,37°、5% (X)2培养M小时后,CpG组加入1 : 100稀释的CpG ODN刺激的人PBMC 培养上清,利巴韦林组加入利巴韦林(至终浓度10 μ mol/1),CpG+利巴韦林组加入1 : 100 稀释的CpG ODN刺激的人PBMC培养上清和利巴韦林(浙江诚意药业有限公司),IMDM组只加入IMDM。 Cells were seeded in 96-well flat bottom culture plate, each well of 1. 3 X IO4 cells, 37 °, 5% (X) 2 M h after culturing, group of CpG was added 1: 100 diluted CpG ODN-stimulated human PBMC cultures on Qing, ribavirin ribavirin group is added (to a final concentration of 10 μ mol / 1), CpG + ribavirin was added group 1: 100 dilution of CpG ODN to stimulate human PBMC culture supernatants and ribavirin (Zhejiang sincerity Pharmaceutical Co., Ltd.), IMDM group only added IMDM. 继续培养1. 5小时后,每孔加入2% FBS IMDM稀释的口蹄疫病毒液(内蒙生物制药厂)200 μ 1,设正常Vero细胞对照。 After cultured for 1.5 hours, dilution was added per well of 2% FBS IMDM FMD virus solution (Inner biological laboratories) 200 μ 1, disposed normal Vero cell control. 37°C、5% C02孵箱培养44小时,用结晶紫染色的方法判定细胞病变的程度。 37 ° C, 5% C02 incubator for 44 hours culture, the degree of cytopathic determining the crystal violet staining method. 吸弃培养液。 Culture medium was aspirated. 每孔加200μ1 0.5%结晶紫染液,37°C,15分钟。 Each well was added 200μ1 0.5% crystal violet dye, 37 ° C, 15 min. 流水冲掉结晶紫染液。 Flush water crystal violet dye. 每孔加入200微升结晶紫脱色液,振荡器振荡,使染料完全从细胞内脱出,用分光光度计在MOnm波长测定分光光度值。 200 microliters per well of crystal violet solution bleaching, oscillator, the dye is completely released from the cell, was measured with a spectrophotometer MOnm wavelength spectrophotometric values.

[0149] 3结果:CpG ODN组和CpG ODN与利巴韦林联合应用组相比较,CpG ODN与利巴韦林联合应用组的OD值显著地高于CpG ODN组,这一结果说明,CpG ODN具有明显的抗口蹄疫病毒作用,CpG ODN和利巴韦林联合应用具有更显著的抗口蹄疫病毒作用。 [0149] Results 3: CpG ODN group and CpG ODN compared to ribavirin group, the OD value of CpG ODN in combination with ribavirin group is significantly higher than that CpG ODN group, the results indicate that, of CpG ODN has significant anti-FMD virus activity, CpG ODN and ribavirin have a more significant role in the anti-FMD virus. 两组间的OD 值比较见表4。 OD values ​​between the two groups is shown in table 4.

[0150] 表4 CpGODN和CpGODNN与利巴韦林联合应用的抗口蹄疫病毒作用比较 Comparison of anti-FMD virus activity [0150] Table 4 CpGODN and CpGODNN combination with ribavirin applications

[0151 ] 实施例6 CpG ODN和CpG ODNN与利巴韦林联合应用抗单股正链RNA日本脑炎病 [0151] Example 6 CpG ODN and with CpG ODNN ribavirin embodiment of combined use of single strand RNA Japanese encephalitis

毒作用比较 Comparison of cytotoxicity

[0152] 1、人外周血单个核细胞的分离同实施例2。 [0152] 1, human peripheral blood mononuclear cells isolated in Example 2.

[0153] 2、获取CpG ODN (6 μ g/ml)刺激人外周血PBMC培养上清同实施例2。 [0153] 2, acquires CpG ODN (6 μ g / ml) stimulation of human PBMC in culture supernatants in Example 2.

[0154] 3、CpG ODN和CpG ODN与利巴韦林联合应用的抗单股正链RNA日本脑炎病毒作用测定 [0154] 3, anti- an RNA positive stranded CpG ODN and with CpG ODN ribavirin Japanese encephalitis virus activity assay

[0155] 1)材料:日本脑炎病毒购自长春生物制品所。 [0155] 1) Material: Japanese encephalitis virus purchased from Changchun Vaccine. 10% FCSRPMI1640完全培养液:同实施例2。 10% FCSRPMI1640 complete medium: same as Example 2.

[0156] 2)方法: [0156] 2) Method:

[0157] 用含10%小牛血清的RPMI1640培养基调人外周血单个核细胞的终浓度为3 X IO6 个/毫升。 [0157] RPMI1640 culture tone people with 10% calf serum-containing peripheral blood mononuclear cells at a final concentration of 3 X IO6 cells / ml. 加细胞悬液于12孔培养板,每孔anl。 The cell suspension was added to 12-well culture plate, each well anl. 加CpG ODN至终浓度6 μ g/ml。 CpG ODN added to a final concentration of 6 μ g / ml. 37°C,5% 二氧化碳孵箱培养48小时,收集上清。 37 ° C, 5% carbon dioxide incubator for 48 hours, the supernatant was collected.

[0158] 设立CpG组、利巴韦林组、CpG+利巴韦林组、IMDM组。 [0158] CpG established group, ribavirin group, CpG group + ribavirin, IMDM group. 将生长状态良好的BHK细胞接种于96孔培养板,每孔1.3X104个细胞,37°、5% CO2培养对小时后,CpG组加入1 : 100 稀释的CpG ODN刺激的人PBMC培养上清,利巴韦林组加入利巴韦林(至终浓度10 μ mol/ 1),CpG+利巴韦林组加入1 : 100稀释的CpG ODN刺激的人PBMC培养上清和利巴韦林(浙江诚意药业有限公司),IMDM组只加入IMDM。 The well-grown BHK cells were seeded in 96-well culture plate, 1.3X104 cells per well, 37 °, 5% CO2 after hours of culture, the group of CpG was added 1: CpG ODN to stimulate human PBMC 100 dilution of culture supernatant, ribavirin ribavirin group is added (to a final concentration of 10 μ mol / 1), CpG + ribavirin was added group 1: 100 dilution of CpG ODN to stimulate human PBMC culture supernatants and ribavirin (Zhejiang We drug Co., Ltd.), IMDM group only added IMDM. 继续培养1. 5小时后,加入100TCID50/ml的日本脑炎病毒,同时设正常BHK细胞对照,培养44小时。 After cultured for 1.5 hours, addition of 100TCID50 / ml of Japanese encephalitis virus, There was also a control BHK cells, cultured for 44 hours. 在显微镜下观察细胞病变(CPE), 出现25%的细胞病变记“ + ”,出现沈-50%的细胞病变记“++”,出现51-75%的细胞病变记“+++”,出现76-100%的细胞病变记“++++”。 Cytopathic (CPE) under the microscope, 25% of cell lesions in mind a "+" appears Shen -50% of cell lesions in mind, "++", a 51-75% cytopathic mind "+++" 76-100% of the cells appear diseased mind, "++++."

[0159] 3)结果(CPE法):CpG ODN组和CpG ODN与利巴韦林联合应用组相比较,CpG ODN 与利巴韦林联合应用组的细胞病变显著地低于CpG ODN组,这一结果说明,CpG ODN具有明显的抗日本脑炎病毒作用,CpG ODN和利巴韦林联合应用具有更显著的抗日本脑炎病毒作用。 [0159] 3) Results (CPE method): CpG ODN group and CpG ODN compared to ribavirin group, cytopathic CpG ODN in combination with ribavirin group is significantly lower than the CpG ODN group, which a result explained, CpG ODN has a significant role in the anti-Japanese encephalitis virus, CpG ODN and ribavirin have a more significant role in the anti-Japanese encephalitis virus. 两组间的细胞病变的比较见表5。 Table 5. Comparison between the two groups of cytopathic.

[0160] 表5 CpG ODN和CpG ODNN与利巴韦林联合应用抗单股正链RNA日本脑炎病毒作用比较 [0160] Table 5 CpG ODN and CpG ODNN and ribavirin anti-single-strand RNA virus of Japanese encephalitis COMPARISON

[0161] 上述的实验结果表明,本发明的CpG ODN具有明显的抗日本脑炎病毒的作用,CpG ODN和利巴韦林联合应用有更显著的抗日本脑炎病毒的作用。 [0161] The experimental results show that, CpG ODN of the present invention has a significant role in the anti-Japanese encephalitis virus, CpG ODN and ribavirin have more significant anti-Japanese encephalitis virus. 丙型肝炎病毒和登革热病毒、 日本脑炎病毒一样同是单股正链的RNA病毒,因此,CpG ODN和利巴韦林联合应用同样可以具有抗丙型肝炎病毒的作用。 Hepatitis C virus and dengue virus, Japanese encephalitis virus is the same as positive strand RNA viruses, therefore, CpG ODN and ribavirin may also have anti-hepatitis C virus.

[0162] 附表部分 [0162] Schedule section

[0163] 表1 CpG ODN和CpG ODN与利巴韦林联合应用的抗滤泡口炎病毒作用比较 Comparison of anti-virus effect of follicular stomatitis [0163] Table. 1 CpG ODN and with CpG ODN of ribavirin

Figure CN1847256BD00161

[0164] IMDM 组0. 213 士0. 07 [0164] IMDM groups 0.213 persons 0.07

[0165]利巴韦林组 0. 398 士0. 09 [0165] ribavirin group of 0.398 persons 0.09

[0166]正常 VERO 细胞:1. 625 士0. 11 [0166] VERO cells Normal: 1625 persons 0.11

[0167] 表2 CpG ODN和CpG ODNN与利巴韦林联合应用的抗流感病毒作用比较 [0167] Anti-influenza virus effect Table 2 CpG ODN and with CpG ODNN ribavirin Comparison

Figure CN1847256BD00171

[0168] DMEM 组0. 245 士0. 08 [0168] DMEM group of 0.245 persons 0.08

[0169]利巴韦林组 0. 423 士0. 09 [0169] ribavirin group of 0.423 persons 0.09

[0170]正常 VERO 细胞对照1. 3¾ 士0. 13 [0170] VERO cells of normal persons control 1. 3¾ 0.13

[0171] 表3 CpG ODN和CpG ODN与利巴韦林联合应用抗流感病毒的小鼠体内实验 [0171] Table. 3 CpG ODN and with CpG ODN ribavirin in vivo in mice against influenza virus

Figure CN1847256BD00181

[0172] 模型组(阴性对照)++++ [0172] model group (negative control) ++++

[0173] 正常细胞组- [0173] Normal cells group -

[0174] 利巴韦林组+++ [0174] Ribavirin group +++

[0175] 表4 CpGODN和CpGODNN与利巴韦林联合应用的抗口蹄疫病毒作用比较 Comparison of anti-FMD virus activity [0175] Table 4 CpGODN and CpGODNN combination with ribavirin applications

Figure CN1847256BD00191

[0176] IMDM 组0. 267 士0. 05 [0176] IMDM groups 0.267 persons 0.05

[0177]利巴韦林组 0. 561 士0. 09 [0177] ribavirin group of 0.561 persons 0.09

[0178]正常 BHK 细胞1. 782 士0. 14 [0178] BHK cells of normal persons 0.14 1.782

[0179] 表5 CpG ODN和CpG ODNN与利巴韦林联合应用抗单股正链RNA日本脑炎病毒作 [0179] Table 5 CpG ODN and CpG ODNN and ribavirin anti-single-strand RNA virus as Japanese encephalitis

Figure CN1847256BD00201

[0180] IMDM 组++++ [0180] IMDM group ++++

[0181] 利巴韦林组++ [0181] ribavirin group ++

[0182] 正常BHK细胞- [0182] BHK cells Normal -

[0183] CpG 序列 [0183] CpG sequence

[0184] 设计序列如下: [0184] Design of the following sequence:

[0185] (G) η (L) nXlX2CGYlY2 (M) η (G) η [0185] (G) η (L) nXlX2CGYlY2 (M) η (G) η

[0186] Xl = Α, Τ, G ;Χ2 = A, T ;Yl = A, T ;Y2 = Α, Τ, C ;L, M = A,T,C,G ;n 为0-6 [0186] Xl = Α, Τ, G; Χ2 = A, T; Yl = A, T; Y2 = Α, Τ, C; L, M = A, T, C, G; n is 0-6

[0187] 5,-ggggTCgTTCgTCgTTgggggg-3,(SEQ ID NO :1) [121] [0187] 5, -ggggTCgTTCgTCgTTgggggg-3, (SEQ ID NO: 1) [121]

[0188] 5,-ggggATAACgTTgCgggggg-3,(SEQ ID NO :2) [143]0189] 5, -ggggTgCAACgTTCAgggggg-3,(SEQ ID NO :3) [402] [0188] 5, -ggggATAACgTTgCgggggg-3, (SEQ ID NO: 2) [143] 0189] 5, -ggggTgCAACgTTCAgggggg-3, (SEQ ID NO: 3) [402]

0190] 5, -ggggTCCTACgTAggAgggggg-3,(SEQ ID NO :4) [123] 0190] 5, -ggggTCCTACgTAggAgggggg-3, (SEQ ID NO: 4) [123]

0191] 5, -ggggTCCATgACgTTCCTgAAgggggg-3,(SEQ ID NO :5) [603] 0191] 5, -ggggTCCATgACgTTCCTgAAgggggg-3, (SEQ ID NO: 5) [603]

0192] 5, -gggggACgTCgCCggggggg-3,(SEQ ID NO :6) [118] 0192] 5, -gggggACgTCgCCggggggg-3, (SEQ ID NO: 6) [118]

0193] 5, -ggATCCgTACgCATgggggg-3,(SEQ ID NO :7) [320] 0193] 5, -ggATCCgTACgCATgggggg-3, (SEQ ID NO: 7) [320]

0194] 5' -gggggAATCgATTCgggggg-3' (SEQ ID NO :8) [154] 0194] 5 '-gggggAATCgATTCgggggg-3' (SEQ ID NO: 8) [154]

0195] 5' -gggATgCATCgATgCATCgggggg-3' (SEQ ID NO :9) [464] 0195] 5 '-gggATgCATCgATgCATCgggggg-3' (SEQ ID NO: 9) [464]

0196] 5' -ggTgCgACgTCgCAgggggg-3' (SEQ ID NO :10) [471] 0196] 5 '-ggTgCgACgTCgCAgggggg-3' (SEQ ID NO: 10) [471]

0197] 5' -gggACgTACgTCgggggg-3' (SEQ ID NO :11) [390] 0197] 5 '-gggACgTACgTCgggggg-3' (SEQ ID NO: 11) [390]

0198] 5' -gggggATCgACgTCgATCgggggg-3,(SEQ ID NO :12) [322] 0198] 5 '-gggggATCgACgTCgATCgggggg-3, (SEQ ID NO: 12) [322]

0199] 5' -ggCgATCgATCgATCggggggg-3' (SEQ ID NO :13) [333] 0199] 5 '-ggCgATCgATCgATCggggggg-3' (SEQ ID NO: 13) [333]

0200] 5, -ggggTCgATCgATCgAgggggg-3' (SEQ ID NO :14) [113] 0200] 5, -ggggTCgATCgATCgAgggggg-3 '(SEQ ID NO: 14) [113]

0201] 5, -ggTCgCgATCgCgAgggggg-3,(SEQ ID NO :15) [307] 0201] 5, -ggTCgCgATCgCgAgggggg-3, (SEQ ID NO: 15) [307]

0202] 5, -ggGGTCAACGTTGAgggggG-3,(SEQ ID NO :16) [156] 0202] 5, -ggGGTCAACGTTGAgggggG-3, (SEQ ID NO: 16) [156]

0203] 5, -gTCgTTTTCgTCgACgAATTgggggggg-3,(SEQ ID NO :17)[222] 0203] 5, -gTCgTTTTCgTCgACgAATTgggggggg-3, (SEQ ID NO: 17) [222]

0204] 5, -gTCgTTATCgTTTTTTCgTAgggggg-3,(SEQ ID NO :18) [151] 0204] 5, -gTCgTTATCgTTTTTTCgTAgggggg-3, (SEQ ID NO: 18) [151]

0205] 5, -ggCgTTAACgACgggggg-3,(SEQ ID NO :19) [288] 0205] 5, -ggCgTTAACgACgggggg-3, (SEQ ID NO: 19) [288]

0206] 5, -gTCggCACgCgACgggggg-3,(SEQ ID NO :20) [157] 0206] 5, -gTCggCACgCgACgggggg-3, (SEQ ID NO: 20) [157]

0207] 5, -ggTgCgACgTCgCAgggggg-3,(SEQ ID NO :21) [312] 0207] 5, -ggTgCgACgTCgCAgggggg-3, (SEQ ID NO: 21) [312]

0208] 5, -gTCTATTTTgTACgTACgTgggg-3,(SEQ ID NO :22) [360] 0208] 5, -gTCTATTTTgTACgTACgTgggg-3, (SEQ ID NO: 22) [360]

0209] 5, -gACgTCgACgTCgACgTCAggggg-3,(SEQ ID NO :23) [209] 0209] 5, -gACgTCgACgTCgACgTCAggggg-3, (SEQ ID NO: 23) [209]

0210] 5, -ggggTCgATCgTTgCTAgCgggggg-3,(SEQ ID NO :24) [399] 0210] 5, -ggggTCgATCgTTgCTAgCgggggg-3, (SEQ ID NO: 24) [399]

0211] 5, -gggggACgTTATCgTATTggggggg-3,(SEQ ID NO :25) [600] 0211] 5, -gggggACgTTATCgTATTggggggg-3, (SEQ ID NO: 25) [600]

0212] 5, -ggggTCgTCgTTTgTCgTgTgTCgTTgggggg-3,(SEQ ID NO :26) [408] 0212] 5, -ggggTCgTCgTTTgTCgTgTgTCgTTgggggg-3, (SEQ ID NO: 26) [408]

0213] 5, -ACgATCgATCgATCgggggg-3,(SEQ ID NO :27) [304] 0213] 5, -ACgATCgATCgATCgggggg-3, (SEQ ID NO: 27) [304]

0214] 5, -AgACgTCTAACgTCggggg-3,(SEQ ID NO :28) [301] 0214] 5, -AgACgTCTAACgTCggggg-3, (SEQ ID NO: 28) [301]

0215] 5, -ggggTgCTggCCgTCgTTgggggg-3,(SEQ ID NO :29) [266] 0215] 5, -ggggTgCTggCCgTCgTTgggggg-3, (SEQ ID NO: 29) [266]

0216] 5, -ggggTCgTTgCCgTCgggggg-3,(SEQ ID NO :30) [248] 0216] 5, -ggggTCgTTgCCgTCgggggg-3, (SEQ ID NO: 30) [248]

0217] 5, -ACCggTATCgATgCCggTgggggg-3,(SEQ ID NO :31) [389] 0217] 5, -ACCggTATCgATgCCggTgggggg-3, (SEQ ID NO: 31) [389]

0218] 5, -TTCgTTgCATCgATgCATCgTTgggggg-3,(SEQ ID NO :32) [287] 0218] 5, -TTCgTTgCATCgATgCATCgTTgggggg-3, (SEQ ID NO: 32) [287]

0219] (G) η (L) nCG (XY) nCG (M) η (G) η 0219] (G) η (L) nCG (XY) nCG (M) η (G) η

0220] X =A, T ;Y = A, T ;L, M = A, T, C,G ;n 为0—6 0220] X = A, T; Y = A, T; L, M = A, T, C, G; n is 0-6

0221] 5, -ggggACgATACgTCggggggg-3,(SEQ ID NO :33) [546] 0221] 5, -ggggACgATACgTCggggggg-3, (SEQ ID NO: 33) [546]

0222] 5, -ggggACgATATCgATgggggg-3,(SEQ ID NO :34) [1007] 0222] 5, -ggggACgATATCgATgggggg-3, (SEQ ID NO: 34) [1007]

0223] 5' -ggACgATCgATCgTgggggg-3' (SEQ ID NO :35) [521] 0223] 5 '-ggACgATCgATCgTgggggg-3' (SEQ ID NO: 35) [521]

0224] 5' -TCggggACgATCgTCgggggg-3' (SEQ ID NO :36) [677] 0224] 5 '-TCggggACgATCgTCgggggg-3' (SEQ ID NO: 36) [677]

0225] 5' -gggggATCgATATCgATCgggggg-3' (SEQ ID NO :37) [576] 0225] 5 '-gggggATCgATATCgATCgggggg-3' (SEQ ID NO: 37) [576]

0226] 5' -ggATCgATCgATCgATgggggg-3' (SEQ ID NO :38) [268] 0226] 5 '-ggATCgATCgATCgATgggggg-3' (SEQ ID NO: 38) [268]

0227] 5' -ggTgCATCgATCgATgCAgggggg-3' (SEQ ID NO :39) [101][0228] 5' -ggTgCATCgTACgATgCAgggggg-3' (SEQ ID NO :40) [100] 0227] 5 '-ggTgCATCgATCgATgCAgggggg-3' (SEQ ID NO: 39) [101] [0228] 5 '-ggTgCATCgTACgATgCAgggggg-3' (SEQ ID NO: 40) [100]

[0229] 5' -ggTgCgATCgATCgCAgggggg-3' (SEQ ID NO :41) [134] [0229] 5 '-ggTgCgATCgATCgCAgggggg-3' (SEQ ID NO: 41) [134]

[0230] 5,-gggggggTCgATCgATgggggg-3' (SEQ ID NO :42) [519] [0230] 5, -gggggggTCgATCgATgggggg-3 '(SEQ ID NO: 42) [519]

[0231] 5,-ggggTCgTCgAACgTTgggggg-3,(SEQ ID NO :43)[350] [0231] 5, -ggggTCgTCgAACgTTgggggg-3, (SEQ ID NO: 43) [350]

[0232] 5,-TgTCgTTCCTTgTCgTT-3,(SEQ ID NO :44) [387] [0232] 5, -TgTCgTTCCTTgTCgTT-3, (SEQ ID NO: 44) [387]

[0233] 5,-TTCgCTTCgCTTTTCgCTTCgCTT-3,—3,(SEQ ID NO :45) [212] [0233] 5, -TTCgCTTCgCTTTTCgCTTCgCTT-3, -3, (SEQ ID NO: 45) [212]

[0234] 5' -ACCgCCAAggAgAAgCCgCAggAggg-3,(SEQ ID NO :46) [166] [0234] 5 '-ACCgCCAAggAgAAgCCgCAggAggg-3, (SEQ ID NO: 46) [166]

[0235] 5,-TACAACggCgAggAATACC-3,(SEQ ID NO :47) [176] [0235] 5, -TACAACggCgAggAATACC-3, (SEQ ID NO: 47) [176]

[0236] 5,-gTACAACggCgAggAATACCT-3,(SEQ ID NO :48) [523] [0236] 5, -gTACAACggCgAggAATACCT-3, (SEQ ID NO: 48) [523]

[0237] 5,-ACCgTCgTTgCCgTCggCCC-3,(SEQ ID NO :49)[230] [0237] 5, -ACCgTCgTTgCCgTCggCCC-3, (SEQ ID NO: 49) [230]

[0238] 5,-TgCTggCCgTCgTT-3,(SEQ ID NO :50) [435] [0238] 5, -TgCTggCCgTCgTT-3, (SEQ ID NO: 50) [435]

[0239] 5,-gTCggCACgCgACg-3,(SEQ ID NO :51) [325] [0239] 5, -gTCggCACgCgACg-3, (SEQ ID NO: 51) [325]

[0240] 5,-gTCggCACgCgACgCCCCCC-3,(SEQ ID NO :52) [523] [0240] 5, -gTCggCACgCgACgCCCCCC-3, (SEQ ID NO: 52) [523]

[0241] 5,-TCCCgCTggACgTT-3,(SEQ ID NO :53) [188] [0241] 5, -TCCCgCTggACgTT-3, (SEQ ID NO: 53) [188]

[0242] 5,-TTACCggTTAACgTTggCCggCC-3,(SEQ ID NO :54) [403] [0242] 5, -TTACCggTTAACgTTggCCggCC-3, (SEQ ID NO: 54) [403]

[0243] 5,-ACCggTTAACgTTgTCCCCgggg-3,(SEQ ID NO :55) [420] [0243] 5, -ACCggTTAACgTTgTCCCCgggg-3, (SEQ ID NO: 55) [420]

[0244] 5,-CgTTgACgATCgTCCCATggCggg-3,(SEQ ID NO :56) [104] [0244] 5, -CgTTgACgATCgTCCCATggCggg-3, (SEQ ID NO: 56) [104]

[0245] 5,-TCTgCggCCTTCgTCg-3,(SEQ ID NO :57) [257] [0245] 5, -TCTgCggCCTTCgTCg-3, (SEQ ID NO: 57) [257]

[0246] 5,-TAgTAACCggTCCggCgCCCCC-3,(SEQ ID NO :58) [221] [0246] 5, -TAgTAACCggTCCggCgCCCCC-3, (SEQ ID NO: 58) [221]

[0247] 5,-TTgCAgCgCTgCCggTggg-3,(SEQ ID NO :59) [611] [0247] 5, -TTgCAgCgCTgCCggTggg-3, (SEQ ID NO: 59) [611]

[0248] 5,-CggCCCATCgAgggCgACggC-3,(SEQ ID NO :60) [378] [0248] 5, -CggCCCATCgAgggCgACggC-3, (SEQ ID NO: 60) [378]

[0249] 5,-TCATCgACTCTCgAgCgTTC-3,(SEQ ID NO :61) [599] [0249] 5, -TCATCgACTCTCgAgCgTTC-3, (SEQ ID NO: 61) [599]

[0250] 5,-ATCgTCgACTCTCgTgTTCTC-3,(SEQ ID NO :62) [201] [0250] 5, -ATCgTCgACTCTCgTgTTCTC-3, (SEQ ID NO: 62) [201]

[0251] 5,-TgCAgCTTgCTgCTTgCTgCTTC-3,(SEQ ID NO :63) [153] [0251] 5, -TgCAgCTTgCTgCTTgCTgCTTC-3, (SEQ ID NO: 63) [153]

[0252] 5,-ggTgCgACgTCgCAgATgAT-3,(SEQ ID NO :64) [116] [0252] 5, -ggTgCgACgTCgCAgATgAT-3, (SEQ ID NO: 64) [116]

[0253] 5,-ggTCgAACgTTCgAgATgAT-3,(SEQ ID NO :65) [133] [0253] 5, -ggTCgAACgTTCgAgATgAT-3, (SEQ ID NO: 65) [133]

[0254] 5' -gggggCgTCgTTTTCgTCgACgAATT-3,(SEQ ID NO :66) [278] [0254] 5 '-gggggCgTCgTTTTCgTCgACgAATT-3, (SEQ ID NO: 66) [278]

[0255] 5,-actcgagacgcccgttgatagctt-3,(SEQ ID NO :67)355 [244] [0255] 5, -actcgagacgcccgttgatagctt-3, (SEQ ID NO: 67) 355 [244]

[0256] 5,-AACgTTggCgTCgACgTCAgCgCC-3,(SEQ ID NO :68) [623] [0256] 5, -AACgTTggCgTCgACgTCAgCgCC-3, (SEQ ID NO: 68) [623]

[0257] 5,-gACgTCgACgTTgACgCT-3,(SEQ ID NO :69) [485] [0257] 5, -gACgTCgACgTTgACgCT-3, (SEQ ID NO: 69) [485]

[0258] 5,-ggCgTTAACgTTAgCgCT-3,(SEQ ID NO :70) [579] [0258] 5, -ggCgTTAACgTTAgCgCT-3, (SEQ ID NO: 70) [579]

[0259] 5,-AgCgCTAgCgCTgACgTT-3,(SEQ ID NO :71) [232] [0259] 5, -AgCgCTAgCgCTgACgTT-3, (SEQ ID NO: 71) [232]

[0260] 5,-CTAgACgTTCAAgCgTT-3,(SEQ ID NO :72) [233] [0260] 5, -CTAgACgTTCAAgCgTT-3, (SEQ ID NO: 72) [233]

[0261] 5,-gACgATCgTCgACgATCgTC-3,(SEQ ID NO :73) [344] [0261] 5, -gACgATCgTCgACgATCgTC-3, (SEQ ID NO: 73) [344]

[0262] 5,-gTCgTTCgTAgTCgACTACgAgTT-3,(SEQ ID NO :74) [379] [0262] 5, -gTCgTTCgTAgTCgACTACgAgTT-3, (SEQ ID NO: 74) [379]

[0263] 5' -AAAAgACgTCgACgTCgACgTCTTTT-3,(SEQ ID NO :75) [489] [0263] 5 '-AAAAgACgTCgACgTCgACgTCTTTT-3, (SEQ ID NO: 75) [489]

[0264] 5,-TgCgACgATCgTCgCACgATCggAT-3,(SEQ ID NO :76) [479] [0264] 5, -TgCgACgATCgTCgCACgATCggAT-3, (SEQ ID NO: 76) [479]

[0265] 5,-TgCgACgTCgCACAgCgT-3,(SEQ ID NO :77) [492] [0265] 5, -TgCgACgTCgCACAgCgT-3, (SEQ ID NO: 77) [492]

[0266] (TCG) η (L) nCG (M) η (G) η[0267] L, M = A, T, C,G ;n 为0—6 [0266] (TCG) η (L) nCG (M) η (G) η [0267] L, M = A, T, C, G; n is 0-6

[0268] 5,-TCgTTgCCgTCgg-3,(SEQ ID NO :78) [619] [0268] 5, -TCgTTgCCgTCgg-3, (SEQ ID NO: 78) [619]

[0269] 5,-TCgTTgCCgTCggg-3,(SEQ ID NO :79) [577] [0269] 5, -TCgTTgCCgTCggg-3, (SEQ ID NO: 79) [577]

[0270] 5,-TCgTTgCCgTCgggg-3,(SEQ ID NO :80) [533] [0270] 5, -TCgTTgCCgTCgggg-3, (SEQ ID NO: 80) [533]

[0271] 5,-TCgTTgCCgTCggggg-3,(SEQ ID NO :81) [537] [0271] 5, -TCgTTgCCgTCggggg-3, (SEQ ID NO: 81) [537]

[0272] 5,-TCgTTgCCgTCgggggg-3,(SEQ ID NO :82)[481] [0272] 5, -TCgTTgCCgTCgggggg-3, (SEQ ID NO: 82) [481]

[0273] 5,-TCgTTgCCgTCggggggg-3,(SEQ ID NO :83) [177] [0273] 5, -TCgTTgCCgTCggggggg-3, (SEQ ID NO: 83) [177]

[0274] 5,-TCgTTgCCgTCgggggggg-3,(SEQ ID NO :84)[111] [0274] 5, -TCgTTgCCgTCgggggggg-3, (SEQ ID NO: 84) [111]

[0275] 5,-TCgTTgCCgTCggggggggg-3,(SEQ ID NO :85) [105] [0275] 5, -TCgTTgCCgTCggggggggg-3, (SEQ ID NO: 85) [105]

[0276] 5' -TCgTCgggTgCATCgATgCAgggggg-3' (SEQ ID NO :86) [664] [0276] 5 '-TCgTCgggTgCATCgATgCAgggggg-3' (SEQ ID NO: 86) [664]

[0277] 5' -TCgTCgggTgCAACgTTgCAgggggg-3,(SEQ ID NO :87) [564] [0277] 5 '-TCgTCgggTgCAACgTTgCAgggggg-3, (SEQ ID NO: 87) [564]

[0278] 5,-TCgTCgggTgCgTCgACgCAgggggg-3,(SEQ ID NO :88) [542] [0278] 5, -TCgTCgggTgCgTCgACgCAgggggg-3, (SEQ ID NO: 88) [542]

[0279] 5,-TCgTCgggTgCgATCgCAgggggg-3,(SEQ ID NO :89)[450] [0279] 5, -TCgTCgggTgCgATCgCAgggggg-3, (SEQ ID NO: 89) [450]

[0280] 5' -TCgTCgggTgCgACgATCgTCgCAgggggg-3,(SEQ ID NO :90) [465] [0280] 5 '-TCgTCgggTgCgACgATCgTCgCAgggggg-3, (SEQ ID NO: 90) [465]

[0281] 5,-TCgTCgTgCgACgTCgCAgggggg-3,(SEQ ID NO :91) [498] [0281] 5, -TCgTCgTgCgACgTCgCAgggggg-3, (SEQ ID NO: 91) [498]

[0282] 5,-TCgTCgCAgAACgTTCTgggggg-3,(SEQ ID NO :92) [527] [0282] 5, -TCgTCgCAgAACgTTCTgggggg-3, (SEQ ID NO: 92) [527]

[0283] 5,-TCgTgCgACgTCgCAgggggg-3,(SEQ ID NO :93) [112] [0283] 5, -TCgTgCgACgTCgCAgggggg-3, (SEQ ID NO: 93) [112]

[0284] 5' -TCgTgCgACgATCgTCgCAgggggg-3,(SEQ ID NO :94) [178] [0284] 5 '-TCgTgCgACgATCgTCgCAgggggg-3, (SEQ ID NO: 94) [178]

[0285] 5' -TCgTATgCATCgATgCATAgggAgg-3,(SEQ ID NO :95) [410] [0285] 5 '-TCgTATgCATCgATgCATAgggAgg-3, (SEQ ID NO: 95) [410]

[0286] 5' -TCgTgCATCgATgCAgggggg-3,(SEQ ID NO :96)[444] [0286] 5 '-TCgTgCATCgATgCAgggggg-3, (SEQ ID NO: 96) [444]

[0287] 5,-TCgAAACgTTTCgggggg-3,(SEQ ID NO :97) [532] [0287] 5, -TCgAAACgTTTCgggggg-3, (SEQ ID NO: 97) [532]

[0288] 5,-TCggACgATCgTCgggggg-3,(SEQ ID NO :98) [598] [0288] 5, -TCggACgATCgTCgggggg-3, (SEQ ID NO: 98) [598]

[0289] 5,-TCgAgCgATCgCTCgAgggggg-3,(SEQ ID NO :99) [555] [0289] 5, -TCgAgCgATCgCTCgAgggggg-3, (SEQ ID NO: 99) [555]

[0290] 5,-TCgTCgCTTTgTCgTTgggg-3,(SEQ ID NO :100)[418] [0290] 5, -TCgTCgCTTTgTCgTTgggg-3, (SEQ ID NO: 100) [418]

[0291] 5,-TCgTCgTTTTgTCgTTgggg-3,(SEQ ID NO :101) [208] [0291] 5, -TCgTCgTTTTgTCgTTgggg-3, (SEQ ID NO: 101) [208]

[0292] 5,-TCgTCgggTgCgACgTCgCAgggggg-3,(SEQ ID NO :102) [302] [0292] 5, -TCgTCgggTgCgACgTCgCAgggggg-3, (SEQ ID NO: 102) [302]

[0293] 5' -TCgTCgggTgCgACgATCgTCgggggg-3,(SEQ ID NO :103) [290] [0293] 5 '-TCgTCgggTgCgACgATCgTCgggggg-3, (SEQ ID NO: 103) [290]

[0294] 5' -TCgTCgTTTgCATCgATgCAggggggg-3,(SEQ ID NO :104) [627] [0294] 5 '-TCgTCgTTTgCATCgATgCAggggggg-3, (SEQ ID NO: 104) [627]

[0295] 5' -TCgTCgTTTTgACgATCgTCgggggg-3,(SEQ ID NO :105) [500] [0295] 5 '-TCgTCgTTTTgACgATCgTCgggggg-3, (SEQ ID NO: 105) [500]

[0296] 5,-TCgTTCggggTgCCg-3,(SEQ ID NO :106) [103] [0296] 5, -TCgTTCggggTgCCg-3, (SEQ ID NO: 106) [103]

[0297] 5,-TCgTTCggggTACCgATgggg-3,(SEQ ID NO :107) [578] [0297] 5, -TCgTTCggggTACCgATgggg-3, (SEQ ID NO: 107) [578]

[0298] 5' -TCgTTgCgCTCCCATgCCgggggg-3,(SEQ ID NO :108) [319] [0298] 5 '-TCgTTgCgCTCCCATgCCgggggg-3, (SEQ ID NO: 108) [319]

[0299] 5,-TCgTCgTTTCgTCgTTgggg-3,(SEQ ID NO :109) [205] [0299] 5, -TCgTCgTTTCgTCgTTgggg-3, (SEQ ID NO: 109) [205]

[0300] 5' -TCgTTgTCgTTTCgCTgCCggCggggg-3,(SEQ ID NO :110) [417] [0300] 5 '-TCgTTgTCgTTTCgCTgCCggCggggg-3, (SEQ ID NO: 110) [417]

[0301] 5,-TgCTTgggTggCAgCTgCCAgggggg-3,(SEQ ID NO :111)[427] [0301] 5, -TgCTTgggTggCAgCTgCCAgggggg-3, (SEQ ID NO: 111) [427]

[0302] 5' -TgCTgCTTTgCTgCTTgggg-3,(SEQ ID NO :112) [421] [0302] 5 '-TgCTgCTTTgCTgCTTgggg-3, (SEQ ID NO: 112) [421]

[0303] 5' -AACgTTCgACgTCgAACggggggg-3,(SEQ ID NO :113) [453] [0303] 5 '-AACgTTCgACgTCgAACggggggg-3, (SEQ ID NO: 113) [453]

[0304] 5,-AACgACgACgTTggggg-3,(SEQ ID NO :114) [580] [0304] 5, -AACgACgACgTTggggg-3, (SEQ ID NO: 114) [580]

[0305] 5,-tcgtcgggtgcgacgtcgca-3,(SEQ ID NO :165) [612][0306] (TCG) η (L) nXlX2CG (M) η [0305] 5, -tcgtcgggtgcgacgtcgca-3, (SEQ ID NO: 165) [612] [0306] (TCG) η (L) nXlX2CG (M) η

[0307] Xl = A, Τ, G ;X2 = A,T ;L,M = A, T, C,G ;n 为0—6 [0307] Xl = A, Τ, G; X2 = A, T; L, M = A, T, C, G; n is 0-6

[0308] 设计序列如下: [0308] Design of the following sequence:

[0309] 5' -TCgTAACgTTgTTTTTAACgTT-3,(SEQ ID NO :115) [470] [0309] 5 '-TCgTAACgTTgTTTTTAACgTT-3, (SEQ ID NO: 115) [470]

[0310] 5,-TCgTCgTATACgACgATCgTT-3,(SEQ ID NO :116) [502] [0310] 5, -TCgTCgTATACgACgATCgTT-3, (SEQ ID NO: 116) [502]

[0311] 5,-TCgTCgTTTgCgTTgTCgTT-3,(SEQ ID NO :117) [601] [0311] 5, -TCgTCgTTTgCgTTgTCgTT-3, (SEQ ID NO: 117) [601]

[0312] 5,-TCCTgTCgTTTTgTCgTT-3,(SEQ ID NO :118) [625] [0312] 5, -TCCTgTCgTTTTgTCgTT-3, (SEQ ID NO: 118) [625]

[0313] 5,-TCgTCgTTgTCgTTCgCT-3,(SEQ ID NO :119) [430] [0313] 5, -TCgTCgTTgTCgTTCgCT-3, (SEQ ID NO: 119) [430]

[0314] 5,-TCgTCgTTACCgATgACgTCgCCgT-3,(SEQ ID NO :120) [480] [0314] 5, -TCgTCgTTACCgATgACgTCgCCgT-3, (SEQ ID NO: 120) [480]

[0315] 5,-TCgTCgTTTgCATCgATgCAgTCgTCgTT-3,(SEQ ID NO :121) [108] [0315] 5, -TCgTCgTTTgCATCgATgCAgTCgTCgTT-3, (SEQ ID NO: 121) [108]

[0316] 5,-TCgCCTCgTCgCCTTCgAgC-3,g_3,(SEQ ID NO :122) [102] [0316] 5, -TCgCCTCgTCgCCTTCgAgC-3, g_3, (SEQ ID NO: 122) [102]

[0317] 5,-TCgTgTgCgTgCCgTTggg-3,T_3,(SEQ ID NO :123) [406] [0317] 5, -TCgTgTgCgTgCCgTTggg-3, T_3, (SEQ ID NO: 123) [406]

[0318] 5,-TCgTCgAgggCgCCggTgAC-3,-3,(SEQ ID NO :124) [560] [0318] 5, -TCgTCgAgggCgCCggTgAC-3, -3, (SEQ ID NO: 124) [560]

[0319] 5,-TCgTCgCCggTgggggTgTg-3,3,(SEQ ID NO :125) [629] [0319] 5, -TCgTCgCCggTgggggTgTg-3,3, (SEQ ID NO: 125) [629]

[0320] 5,-TCgTCgTACgCAATTgTCTT-3,3,(SEQ ID NO :126) [440] [0320] 5, -TCgTCgTACgCAATTgTCTT-3,3, (SEQ ID NO: 126) [440]

[0321] 5,-TCgCCCACCggTgggggggg-3,3,(SEQ ID NO :127) [207] [0321] 5, -TCgCCCACCggTgggggggg-3,3, (SEQ ID NO: 127) [207]

[0322] 5,-TCgTCgCAgACCggTCTgggg-3,(SEQ ID NO :128) [615] [0322] 5, -TCgTCgCAgACCggTCTgggg-3, (SEQ ID NO: 128) [615]

[0323] 5,-TCgTCgCggCCggCgCCCCC-3,(SEQ ID NO :129) [610] [0323] 5, -TCgTCgCggCCggCgCCCCC-3, (SEQ ID NO: 129) [610]

[0324] 5,-TCgTCgCggCCgCgAggggg-3,(SEQ ID NO :130) [206] [0324] 5, -TCgTCgCggCCgCgAggggg-3, (SEQ ID NO: 130) [206]

[0325] 5,-TCgAggACAAgATTCTCgTgC-3,(SEQ ID NO :131) [119] [0325] 5, -TCgAggACAAgATTCTCgTgC-3, (SEQ ID NO: 131) [119]

[0326] 5' -TCgAggACAAgATTCTCgTgCAggCC-3,(SEQ ID NO :132) [570] [0326] 5 '-TCgAggACAAgATTCTCgTgCAggCC-3, (SEQ ID NO: 132) [570]

[0327] 5,-TCgTgCAggCCAACgAggCCg-3,(SEQ ID NO :133) [631] [0327] 5, -TCgTgCAggCCAACgAggCCg-3, (SEQ ID NO: 133) [631]

[0328] 5,-TCgTTgCCgTCggCCC-3,(SEQ ID NO :134) [115] [0328] 5, -TCgTTgCCgTCggCCC-3, (SEQ ID NO: 134) [115]

[0329] 5' -TCggCACgCgACgTgCTggCCgTCgTTTCC-3,(SEQ ID NO :135) [370] [0329] 5 '-TCggCACgCgACgTgCTggCCgTCgTTTCC-3, (SEQ ID NO: 135) [370]

[0330] 5,-TCgTTgCCgTCggCCCCCCCCC-3,(SEQ ID NO :136) [309] [0330] 5, -TCgTTgCCgTCggCCCCCCCCC-3, (SEQ ID NO: 136) [309]

[0331] 5,-TCgTTgCCgTCggCCCCCC-3,(SEQ ID NO :137) [506] [0331] 5, -TCgTTgCCgTCggCCCCCC-3, (SEQ ID NO: 137) [506]

[0332] 5,-TCgTTgCCgTCggCCCCC-3,(SEQ ID NO :138) [404] [0332] 5, -TCgTTgCCgTCggCCCCC-3, (SEQ ID NO: 138) [404]

[0333] 5,-TCgTTgCCgTCggCCCC-3,(SEQ ID NO :139) [203] [0333] 5, -TCgTTgCCgTCggCCCC-3, (SEQ ID NO: 139) [203]

[0334] 5,-TCgTTgCCgT0C ggCCCCCCC-3,(SEQ ID NO :140) [501] [0334] 5, -TCgTTgCCgT0C ggCCCCCCC-3, (SEQ ID NO: 140) [501]

[0335] 5,-TCgAggACAAgATTCTCgT-3,(SEQ ID NO :141) [305] [0335] 5, -TCgAggACAAgATTCTCgT-3, (SEQ ID NO: 141) [305]

[0336] 5' -TCggCACgCgACgTgCTggCCgTCgTT-3,(SEQ ID NO :142) [509] [0336] 5 '-TCggCACgCgACgTgCTggCCgTCgTT-3, (SEQ ID NO: 142) [509]

[0337] 5,-TCgTCgCgCCgTCACgggggg-3,(SEQ ID NO :143)[630] [0337] 5, -TCgTCgCgCCgTCACgggggg-3, (SEQ ID NO: 143) [630]

[0338] 5,-TCgTgTgCgTgCCgTTggg-3,(SEQ ID NO :144)[106] [0338] 5, -TCgTgTgCgTgCCgTTggg-3, (SEQ ID NO: 144) [106]

[0339] 5,-TCgTCgCCgTTgggCggg-3,(SEQ ID NO :145) [117] [0339] 5, -TCgTCgCCgTTgggCggg-3, (SEQ ID NO: 145) [117]

[0340] 5,-TCgTCgACgTCgTTgggCggg-3,(SEQ ID NO :146) [280] [0340] 5, -TCgTCgACgTCgTTgggCggg-3, (SEQ ID NO: 146) [280]

[0341] 5,-TCgCAgTTgTCgTAACgTTgggCggg-3,(SEQ ID NO :147)[299] [0341] 5, -TCgCAgTTgTCgTAACgTTgggCggg-3, (SEQ ID NO: 147) [299]

[0342] 5,-TCgTCgTTggTATgTT-3,(SEQ ID NO :148) [613] [0342] 5, -TCgTCgTTggTATgTT-3, (SEQ ID NO: 148) [613]

[0343] 5,-TCgTCgTCgTCgTTgTCgTT-3,(SEQ ID NO :149) [306] [0343] 5, -TCgTCgTCgTCgTTgTCgTT-3, (SEQ ID NO: 149) [306]

[0344] 5,-TCgTCgTCgTCgTTgTCgTTgggg-3,(SEQ ID NO :150) [309][0345] 5,-TCgTTCggggTgCCg-3,(SEQ ID NO :151) [409] [0344] 5, -TCgTCgTCgTCgTTgTCgTTgggg-3, (SEQ ID NO: 150) [309] [0345] 5, -TCgTTCggggTgCCg-3, (SEQ ID NO: 151) [409]

[0346] 5,-TCgTTCggggTAACgATT-3,(SEQ ID NO :152) [508] [0346] 5, -TCgTTCggggTAACgATT-3, (SEQ ID NO: 152) [508]

[0347] 5,-TCgTTCggggTAACgTT-3,(SEQ ID NO :153) [540] [0347] 5, -TCgTTCggggTAACgTT-3, (SEQ ID NO: 153) [540]

[0348] 5,-TCgTTCggggTACCgAT-3,(SEQ ID NO :154) [401] [0348] 5, -TCgTTCggggTACCgAT-3, (SEQ ID NO: 154) [401]

[0349] 5,-TCgTACggCCgCCgTACggCggg-3,(SEQ ID NO :155) [607] [0349] 5, -TCgTACggCCgCCgTACggCggg-3, (SEQ ID NO: 155) [607]

[0350] 5,-TCgCgTCgACTCCCCTCgAgggg-3,(SEQ ID NO :156) [380] [0350] 5, -TCgCgTCgACTCCCCTCgAgggg-3, (SEQ ID NO: 156) [380]

[0351] 5,-TCgTCgTCgACTCgTggTCggggg-3,(SEQ ID NO :157) [656] [0351] 5, -TCgTCgTCgACTCgTggTCggggg-3, (SEQ ID NO: 157) [656]

[0352] 5,-TCgggCgCCCgATCgggggg-3,(SEQ ID NO :158) [310] [0352] 5, -TCgggCgCCCgATCgggggg-3, (SEQ ID NO: 158) [310]

[0353] 5,-TCgTCggTCTTTCgAAATT-3,(SEQ ID NO :159) [109] [0353] 5, -TCgTCggTCTTTCgAAATT-3, (SEQ ID NO: 159) [109]

[0354] 5,-TCgTgACgTCCTCgAgTT-3,(SEQ ID NO :160) [330] [0354] 5, -TCgTgACgTCCTCgAgTT-3, (SEQ ID NO: 160) [330]

[0355] 5,-TCgTCTTTCgACTCgTTCTC-3,(SEQ ID NO :161) [605] [0355] 5, -TCgTCTTTCgACTCgTTCTC-3, (SEQ ID NO: 161) [605]

[0356] 5,-TCgTCgTTTTgCgTTCTC-3,(SEQ ID NO :162) [504] [0356] 5, -TCgTCgTTTTgCgTTCTC-3, (SEQ ID NO: 162) [504]

[0357] 5,-TCgACTTTCgTCgTTCTgTT-3,(SEQ ID NO :163) [407] [0357] 5, -TCgACTTTCgTCgTTCTgTT-3, (SEQ ID NO: 163) [407]

[0358] 5,-TCgTCgTTTCgTCgTTCTC-3,(SEQ ID NO :164) [550] [0358] 5, -TCgTCgTTTCgTCgTTCTC-3, (SEQ ID NO: 164) [550]

[0359] 5,-TCgTTCTCgACTCgTTCTC-3,(SEQ ID NO :166) [277] [0359] 5, -TCgTTCTCgACTCgTTCTC-3, (SEQ ID NO: 166) [277]

[0360] 5,-TCgACgTTCgTCgTTCgTCgTTC-3,(SEQ ID NO :167) [667] [0360] 5, -TCgACgTTCgTCgTTCgTCgTTC-3, (SEQ ID NO: 167) [667]

[0361] 5,-TCgTCgACgTCgTTCgTTCTC-3,(SEQ ID NO :168) [705] [0361] 5, -TCgTCgACgTCgTTCgTTCTC-3, (SEQ ID NO: 168) [705]

[0362] 5,-TCgTgCgACgTCgCAgATgAT-3,(SEQ ID NO :169) [114] [0362] 5, -TCgTgCgACgTCgCAgATgAT-3, (SEQ ID NO: 169) [114]

[0363] 5' -TCgTCgAgCgCTCgATCggAT-3,(SEQ ID NO :170) [211] [0363] 5 '-TCgTCgAgCgCTCgATCggAT-3, (SEQ ID NO: 170) [211]

[0364] 5,-TCgTCgTTTCgTAgTCgTTgACgTCggg-3,(SEQ ID NO :171) [204] [0364] 5, -TCgTCgTTTCgTAgTCgTTgACgTCggg-3, (SEQ ID NO: 171) [204]

[0365] 5' -TCgTCggACgTTTTCCgACgTTCT-3,(SEQ ID NO :172) [308] [0365] 5 '-TCgTCggACgTTTTCCgACgTTCT-3, (SEQ ID NO: 172) [308]

[0366] 5,-TCgTCgTTTTCgTCgTTTTCgTCgTT-3,(SEQ ID NO :173) [340] [0366] 5, -TCgTCgTTTTCgTCgTTTTCgTCgTT-3, (SEQ ID NO: 173) [340]

[0367] 5,-TCgTCgTTTgTCgTgTgTCgTT-3 ; (SEQ ID NO :174) [503] [0367] 5, -TCgTCgTTTgTCgTgTgTCgTT-3; (SEQ ID NO: 174) [503]

[0368] 5' -TCgTCgTTggTCggggTCgTTggggTCgTT-3,(SEQ ID NO :175) [405] [0368] 5 '-TCgTCgTTggTCggggTCgTTggggTCgTT-3, (SEQ ID NO: 175) [405]

[0369] 5,-TCgTCgTTTCgTCTCTCgTT-3,(SEQ ID NO :176)[614] [0369] 5, -TCgTCgTTTCgTCTCTCgTT-3, (SEQ ID NO: 176) [614]

[0370] 5,-TCgTCgTTTTgCTgCgTCgTT-3,(SEQ ID NO :177) [505] [0370] 5, -TCgTCgTTTTgCTgCgTCgTT-3, (SEQ ID NO: 177) [505]

[0371] 5,-TCgAgCgTTTTCgCTCgAATT-3,(SEQ ID NO :178) [530] [0371] 5, -TCgAgCgTTTTCgCTCgAATT-3, (SEQ ID NO: 178) [530]

[0372] 包含TTCGTCG的序列 [0372] comprising the sequence of TTCGTCG

[0373] 5' -TTCgTCgTTTgATCgATgTTCgTTgggggg-3,(SEQ ID NO :179) [507] [0373] 5 '-TTCgTCgTTTgATCgATgTTCgTTgggggg-3, (SEQ ID NO: 179) [507]

[0374] 5,-TTCgTCgTTgTgATCgATgggggg-3,-3,(SEQ ID NO :180) [210] [0374] 5, -TTCgTCgTTgTgATCgATgggggg-3, -3, (SEQ ID NO: 180) [210]

[0375] 5' -TATCgATgTTTTTCgTCgTCgTTgggggg-3,(SEQ ID NO :181) [202] [0375] 5 '-TATCgATgTTTTTCgTCgTCgTTgggggg-3, (SEQ ID NO: 181) [202]

[0376] 5,-TCgACTTTCgTCgTTCTgTT-3,(SEQ ID NO :182) [303] [0376] 5, -TCgACTTTCgTCgTTCTgTT-3, (SEQ ID NO: 182) [303]

[0377] 5,-TCgTCgTTTCgTCgTTCTC-3,(SEQ ID NO :183) [491] [0377] 5, -TCgTCgTTTCgTCgTTCTC-3, (SEQ ID NO: 183) [491]

[0378] 5,-TCgACgTTCgTCgTTCgTCgTTC-3,(SEQ ID NO :184) [590] [0378] 5, -TCgACgTTCgTCgTTCgTCgTTC-3, (SEQ ID NO: 184) [590]

[0379] 5,-TCgTCgTTTTCgTCgTTTTCgTCgTT-3,(SEQ ID NO :185) [633] [0379] 5, -TCgTCgTTTTCgTCgTTTTCgTCgTT-3, (SEQ ID NO: 185) [633]

Claims (5)

1.人工合成的含CpG单链脱氧核苷酸与利巴韦林混合或联合应用,用于制备治疗和预防病毒感染及病毒感染引起的相关疾病的制剂的用途,所述的单链脱氧核苷酸的序列为SEQID No 26 :5, _tcgtcgggtgcgacgtcgcagggggg_3,。 1. The single-stranded deoxyribonucleic synthetic CpG-containing single-stranded deoxynucleotide or mixed in combination with ribavirin, for the preparation of the treatment and prevention of viral infection and the preparation of disease associated with viral infection, the nucleotide sequence of SEQID No 26: 5, _tcgtcgggtgcgacgtcgcagggggg_3 ,.
2.按照权利要求1所述的用途,其中所述的病毒为流感病毒、口蹄疫病毒、登革热病毒、日本脑炎病毒、丙型肝炎病毒、乙型肝炎病毒、人类免疫缺陷性病毒或乳头瘤病毒。 2. The use according to claim 1, wherein said virus is influenza virus, foot and mouth disease virus, dengue virus, Japanese encephalitis virus, hepatitis C virus, hepatitis B virus, human immunodeficiency virus, or papilloma virus .
3. 一种制剂组合物,包含权利要求1所述的单链脱氧核苷酸和利巴韦林。 3. A preparation composition, comprising a single-stranded deoxynucleotide claims and claim ribavirin 1.
4.权利要求3所述的制剂组合物用于制备治疗和预防病毒感染及病毒感染引起的相关疾病的制剂的用途。 4. The preparation composition as claimed in claim 3 for the preparation of the treatment and prevention of viral infections and the preparation of related diseases caused by viral infections.
5.根据权利要求4所述的用途,其中所述的病毒为流感病毒、口蹄疫病毒、登革热病毒、日本脑炎病毒、丙型肝炎病毒、乙型肝炎病毒、人类免疫缺陷性病毒或乳头瘤病毒。 5. The use as claimed in claim 4, wherein said virus is influenza virus, foot and mouth disease virus, dengue virus, Japanese encephalitis virus, hepatitis C virus, hepatitis B virus, human immunodeficiency virus, or papilloma virus .
CN 200510064537 2005-04-13 2005-04-13 Antiviral prepn containing both CpG single-strand deoxyoligonucleotide and ribavirin CN1847256B (en)

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