CN1847256A

CN1847256A - Antiviral prepn containing both CpG single-strand deoxyoligonucleotide and ribavirin

Info

Publication number: CN1847256A
Application number: CNA2005100645375A
Authority: CN
Inventors: 王丽颖; 包木胜; 于永利
Original assignee: Changchun Huapu Biotechnology Co Ltd
Current assignee: Huapu Biotechnology Jiangsu Co ltd
Priority date: 2005-04-13
Filing date: 2005-04-13
Publication date: 2006-10-18
Anticipated expiration: 2025-04-13
Also published as: WO2006108358A1; CN1847256B

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

Contain the antivirus action that CpG strand deoxy-oligonucleotide and ribavirin combined utilization produce

Invention field

The present invention relates to a kind of single chain deoxynucleotide and ribavirin of the CpG of containing dinucleotides, particularly relate to the single chain deoxynucleotide and the ribavirin combined utilization that contain the CpG dinucleotides, can be used for the treatment and the prevention of virus infection and virus infection relative disease when single chain deoxynucleotide that contains the CpG dinucleotides involved in the present invention and ribavirin combined utilization.The virus of indication of the present invention includes but not limited to influenza virus, foot and mouth disease virus, dengue fever virus, japanese encephalitis virus, hepatitis C virus, hepatitis B virus, Human Immunodeficiency Virus and papilloma virus.The present invention also provides the sequence of the single chain deoxynucleotide that contains the CpG dinucleotides.

Background of invention

CpG ODN is the deoxy-oligonucleotide single stranded DNA of containing of synthetic of one or more CpG dinucleotides, CpG wherein is the dinucleotides that is connected into by phosphoric acid by cytosine(Cyt) and guanine, and C represents cytosine(Cyt), and G represents guanine, p represents phosphoric acid, and cytosine(Cyt) is positioned at 5 ' end.Because sequence is the difference of the sequence of CpG both sides especially, CpG ODN can have diversified form.Some CpG ODN has clear and definite immunoregulation effect, shows clinical value (Weiner GJ.The immunobiology and clinical potential of immunostimulatory CpGoligodeoxynucleotides.J Leukoc Biol 2000 Oct preferably; 68 (4): 455-63).The effect of some CpG ODN has species specificity, promptly a kind of animal is showed the CpG ODN of biological action, then may not show biologic activity (the Gunther Hartmann of same property or intensity another kind of animal, et al.Delineation of a CpGPhosphorothioate Oligodeoxynucleotide for Activating Primate Immune ResponsesIn Vitro and In Vivol The Journal of Immunology, 2000,164:1617-1624).

According to the characteristics of function, CpG ODN is divided into three types: A type CpG ODN (CpG-A ODN), Type B CpG ODN (CpG-B ODN) and C type CpG ODN (CpG-C ODN).Mainly show as the CpG ODN that activates dendritic cell, natural killer cell, stimulation dendritic cell secretion interferon activity and be classified as A type CpG ODN.A type CpG ODN is supposed to be used for tumour, disease of viral infection and other treatment of diseases with interferon therapy.Mainly show as the activation bone-marrow-derived lymphocyte, strengthen the active CpG ODN of humoral immunoresponse(HI) and be classified as Type B CpG ODN.Type B CpG ODN is the molecule adjuvant, can strengthen the immune effect of vaccine.Have both the main active CpGODN of A type CpG ODN and Type B CpG ODN and be classified as C type CpG ODN.

Experiment shows that CpG ODN has antiviral biologic activity.Transvaginal is used CpG ODN can make mouse or cavy improve ability (the Richard B.Pyles et al.Use ofImmunostimulatory sequence-Containing Oligonucleotides as Topical Therapy forGenital Herpes Simplex Virus Type 2 Infection.Journal of Virology of opposing II herpes simplex virus type (HSV-2), November 2002, Vol.76, No.22 are p.11387-11396).Use lotus amount (the Cho JY that CpG ODN can reduce its respiratory syncytial virus (RSV) to mouse, et al.Immunostimulatory DNA sequences inhibit respiratorysyncytiai viral load, airway inflammation, and mucus secretion.J Allergy ClinImmunol 2001 Nov; 108 (5): 697-702).The mouse of injection CpG ODN is after the HSV-2 through lethal quantity attacks, virus titer obviously reduces (Harandi AM in the vaginal secretions, Eriksson K, Holmgren J.A protectiverole of locally administered immunostimulatory CpG oligodeoxynucleotide in amouse model of genital herpes infection.J Virol 2003 Jan; 77 (2): 953-62).

Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, Ribavirin), also claim Virazole, it is a kind of nucleoside analog, antiviral activity (Gilbert with wide spectrum, 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 RW, 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 U S is Apr A.1973; 70 (4): 1174-8.), be used to the treatment (Gilbert of multiple disease of viral infection, B E and Knight V.Biochemistry andclinical applications of ribavirin.Antimicrob.Agents Chemother.1986,30 (2): 201-205).Ribavirin and interferon-alpha combined utilization are to treat effective ways (the Davis GL of the chronic hepatitis C that is caused by infection with hepatitis C virus, Esteban-Mur R, Rustgi V, Hoefs J, Gordon SC, TrepoC, Shiffman ML, Zeuzem S, Craxi A, Ling MH, Albrecht J.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 Nov19；339(21)：1493-9.)。Ribavirin and long-acting interferon-alpha (pegylated interferon, peginterferon) combined utilization has tangible curative effect (Torriani FJ, Rodriguez-Torres M to the treatment hepatitis C, RockstrohJK, Lissen E, 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).

Influenza virus is the pathogenic agent that causes human influenza.At 1918-1919, the whole world has 20,000,000 people to die from popular (Patterson, the K.D.﹠amp of influenza virus; Pyle, G.F. (1991) Bull.Hist.Med.65,4-213).Vaccination and application antiviral are two class main method (Bridges, C.B., Fukuda, K., Cox, the N.J.﹠amp of control influenza virus; Singleton, J.A. (2001) Morbid.Mortal.Wkly.Rep.50 1-44), but fact proved, these two class methods all can not be controlled popular (Webby, the R.J.﹠amp of influenza effectively; Webster, R.G. (2001) Philos.Trans.R.Soc.London 356,1817-1828).In Susceptible population, vaccinated short-term (half a year) protection ratio is about 39% (Fukuda, K., N.J. (1999) Morbid.Mortal.Wkly.Rep.48,1-28, Castle, S.C.Clinical relevance of age-related immune dysfunction.ClinInfect Dis.2000 Aug; 31 (2): 578-85.Epub 2000 Sep 14.Review).Because the continuous variation of influenza antigen [hemagglutinin (HA) and Sialidase (NA)] property, existing vaccine almost all will be reconstructed structure every year again.Because the bigger and new anti-medicine strains of influenza viruses of side effect constantly occurs, the effect of the anti-influenza virus medicament that some get the Green Light also undesirable (Luscher-Mattli, M.Influenza chemotherapy:a review of thepresent state of art and of new drugs in development.Arch Virol.2000; 145 (11): 2233-48.Review.).

Hepatitis C virus (HCV) is the pathogenic agent that causes hepatitis C, has infected about more than 100,000,000 seven thousand ten thousand people in the whole world.Liver cancer and liver cirrhosis are that HCV infects two severe complications that cause.Worldwide, adopting alpha-interferon and ribavirin combined utilization is the method for treatment infection with hepatitis C virus, its efficient 40%[Jon Cohen.Science, 1999,285:26-30 of being about].Hepatitis C virus is a kind of tunicary sub-thread positive chain RNA virus, belongs to flaviviridae.Other two kinds of viruses of flaviviridae, dengue fever 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.).Dengue fever virus, japanese encephalitis virus also are the RNA viruses of sub-thread normal chain.Owing to still do not have the cell and the animal model of research hepatitis C virus, the result who adopts dengue fever virus, japanese encephalitis virus to do the experiment acquisition can represent on suitable degree with hepatitis C virus and be the result that the cell experiment obtains.

(foot-and-mouthdisease FMD) is the deadly infectious disease that is caused by foot and mouth disease virus to foot and mouth disease.Pig, ox, sheep all can infect foot and mouth disease virus and foot and mouth disease takes place, and pathologies such as bubble, ulceration and speck appear in the lighter at positions such as mouth, tongue, lip, hoof, breast, and death takes place weight person.The generation of foot and mouth disease and popular meeting cause huge direct and indirect financial loss [Jiang Pengfei, Zhao Qizu, Xie Qingge, foot and mouth disease progress Scientia Agricultura Sinica 1999,32 (6): 93～100].International Office of Epizootics (OIE) classifies foot and mouth disease as the category-A livestock contagious disease.Countries in the world are all attached great importance to prevention and control foot and mouth disease.Except that massacring the domestic animal that infects foot and mouth disease, vaccination is the major measure [M.Woolhouse, 2001] of prevention and control foot and mouth disease.Traditional aftosa vaccine refuels the deactivation vaccine that adjuvant emulsion makes by the foot and mouth disease virus of the preliminary purification of deactivation, and the susceptible domestic animal is had suitable provide protection, but exists deactivation insufficient and cause the possibility of foot and mouth disease.In addition, the great-hearted foot and mouth disease virus of employing in producing this vaccine process is a kind of very important potential contagium.

Twentieth century is since the beginning of the eighties, and many countries have dropped into a large amount of human and material resources and financial resources are developed novel aftosa vaccine, as recombinant protein vaccine, and synthetic peptide vaccine, [Broekhuigsen M P, 1987 such as live vector vaccine and D N A vaccine; Morgan D O, 1990; Ward, G., Rieder, E.﹠amp; Mason, P.W.Plasmid DNA encodingreplicating foot-and-mouth disease virus genomes induces antiviral immuneresponses in swine.Journal of Virology 1997,71,7442-7447.; Berinstein A, TamiC, Taboga O, Smitsaart E, Carrillo E.Protective immunity against foot-and-mouthdisease virus induced by a recombinant vaccinia virus.Vaccine.2000 Apr28; 18 (21): 2231-8.].Some vaccines have shown effect preferably, but major part is still among breadboard exploration.

Foot and mouth disease virus (foot-and-mouth disease virus, FMDV) be the pathogenic agent of foot and mouth disease, be the member of Picornaviridae (Picornaviridae) Hostis (Aphthovirus), 7 serotypes have been had now found that, be O, A, C type (title Europe class), SAT1, SAT2, SAT3 type (South Africa 1,2,3 types also claim African type) and AsiaI type (the Asia I type claims Asian type).O type foot and mouth disease virus is a popular wider serotype in the world wide in recent years.Studies show that, at humoral immunization in the human body foot-and-mouth disease virus resistant infects, play a major role.Be in the carcass, the ability that the level of foot and mouth disease virus neutralizing antibody and its opposing foot and mouth disease virus are attacked is tangible positive correlation [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.].Based on this observation, a large amount of experiments have been done in many laboratories, on foot and mouth disease virus, seek the target spot that can stimulate body to produce virucidin, there is structure [the Crowther JR of five candidates in discovery in the coat protein (VP) of foot and mouth disease virus, Farias S, Carpenter WC, Samuel AR.Identification of a fifth neutralizable site on type O 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): 26-34.], wherein three are arranged in coat protein 1 (Vp1).Experimental results show that, isolated Vp1 albumen has good immunogenicity [Bachrach HL from foot and mouth disease virus, 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, NovellaIS, Andreu D, Sobrino F.Antigenic specificity of porcine T cell response againstfoot-and-mouth disease virus structural proteins:identification of T helperepitopes in VP1.Virology.1994Nov 15; 205 (1): 24-33.].The neutrality antibody that produces with the proteic peptide section of Vp1 (141-160 and 200-213) immune guinea pig and ox can watch for animals and avoid infecting foot and mouth disease virus [Bittle JL, Houghten RA, Alexander H, Shinnick TM, Sutcliffe JG, Lerner RA, Rowlands DJ, BrownF.Protection against foot-and-mouth disease by immunization with a chemicallysynthesized peptide predicted from the viral nucleotide sequence.Nature.1982 Jul1; 298 (5869): 30-3.; Pfaff E, Mussgay M, Bohm HO, Schulz GE, Schaller H.Antibodiesagainst a preselected peptide recognize and neutralize foot and mouth diseasevirus.EMBO is 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.]." ring " shape structure that VP1 140-160 amino acid constitutes is exposed to virus surface, R-G-D sequence wherein is at the foot and mouth disease virus high conservative, be the key structure of foot and mouth disease virus, may mediate foot and mouth disease virus and enter infected cells [Jackson T, 2002 in conjunction with cell surface receptor; Almeida MR, 1998].Can induce the high-caliber neutralizing antibody of generation according to VP1 ring texture synthetic peptide section.Also there are some researches show, use the Vp1 protein immune animal of separating from foot and mouth disease virus or gene engineering method obtains, can generating unit divide the protection effect.The albumen that Vp1 albumen and some is derived from other microorganisms by genetic engineering means merges and can improve the proteic immunogenicity of Vp1 [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.].

Japanese encephalitis is that the japanese encephalitis virus by Flavivirus causes.Most of people can not fall ill after infecting japanese encephalitis virus.Most to send out patient's clinical symptom lighter, common fever, headache, feels sick, burnout, suffers from abdominal pain or the disturbance of consciousness and mental symptom occur.Severe cases is many to begin with burst high fever, headache, vomiting, and meninges then occurring stimulates phenomenon, and spasm, abnormal behaviour, myotony appear in 3-5 day, consciousness considerable change even stupor, death.

Japanese encephalitis is many countries in the Asia, remain the prevailing disease of serious threat health.Because the enforcement of protective inoculation, the case in Japan, Korea S and Taiwan has reduced much, but many countries that it is contiguous comprise that states such as China's Mainland, Philippines, Indonesia, Malaysia, India, Nepal still have many Japanese encephalitis patients, and idol has the popular of this disease.

Hepatitis B virus is a kind of dna virus.Complete hepatitis B virus particles diameter is 42nm, can be divided into coating and core two portions.Protein on the coating is called hepatitis B surface antigen(HBsAg) (HBsAg), and it is synthetic in liver cell, disengages then in the blood circulation, and itself there is no infectivity.The bifilar DNA of ring-type, archaeal dna polymerase, cAg (HBcAg) and e antigen (HBeAg) are contained in the core, are the main bodys of virus replication, and are infectious.

Acquired immune deficiency syndrome (AIDS) is owing to infected a kind of lethality transmissible disease that causes behind the human immunodeficiency virus (being called for short HIV).HIV mainly destroys the immunity system of human body, and therefore the various pathogenic agent that make body lose protection capability gradually and can not resist the external world very easily infect infectious diseases and the tumour that the general health people is difficult for trouble, finally cause death.China's acquired immune deficiency syndrome (AIDS) epidemic situation was steady ascendant trend in 2000, compared with last one year, and report HIV the infected number increases by 11.2%, and the acquired immune deficiency syndrome (AIDS) case increases by 1.3%.By the end of the year 2000, the accumulation report HIV the infected of 31 provinces, autonomous regions and municipalities in the whole nation (not comprising Taiwan Province, Hong Kong and Macao Special Administrative Region) 22517 examples, wherein acquired immune deficiency syndrome (AIDS) case 880 examples.Report death 496 examples (dead 466 examples of AIDS).Year 2000 report acquired immune deficiency syndrome (AIDS) death 110 examples, dead 17 examples of HIV the infected.

Hiv virus (HIV) is the RNA retrovirus that has coating, belongs to Retroviridae in the classification.HIV is spherical in shape or avette, and diameter 100-130nm is made up of coating and two parts of core.Envelope protein comprises outer membrane glycoprotein (gp120) and transmembrane glycoprotein (gp41).The core is made up of the nucleic acid gene group RNA and the enzyme of nucleocapsid protein, two identical copies.

The present world has developed some and can effectively suppress the medicine of hiv virus, and these medicines can have been alleviated acquired immune deficiency syndrome (AIDS) patient's symptom to a certain extent, prolongs patient's life and improves its quality of life.But these medicines are very expensive, and very big side effect is arranged, and some medicine uses the back that hiv virus has not just been had effect for a long time.At present, our country does not also have the formal entry or produces these medicines.Up to now, also do not develop the thoroughly medicine and the viral vaccine that effectively prevents AIDS of treatment of AIDS in the world.

Papilloma virus belongs to the Papillomavirus of papovaviridae (Papovaviridae), it comprise the papilloma virus of multiple animal and human papillomavirus (Human papillomavirus, HPV).HPV is a kind of little dna virus, and diameter 45～55nm, capsid are the three-dimensional symmetry of icosahedron, contain 72 shell particulates, do not have cyst membrane.The HPV genome is a closed loop distrand DNA, molecular weight 5 * 106 dalton.Can be divided into early stage district (E district), late region (L district) and three zones of non-coding region (NCR) by function.E divides into E1～E7 open reading frame, main coding and virus replication, transcribe, regulation and control and cell transformation proteins associated.L distinguishes L1 and L2, encode respectively main capsid protein and less important capsid protein.NCR is the dna fragmentation of E district and L interval-6.4～1.0bp, can be responsible for the regulation and control of transcribing and duplicating.

Show according to the epidemiology survey data: the infection rate of global papilloma virus is about 9-13%, and promptly annual nearly 600,000,000 3 thousand ten thousand people infect papilloma virus.Papilloma virus and a lot of mucocutaneous disease are closely related.The infection of papilloma virus can cause the verruca plana of skin, phallic pointed condyloma, and the infection of even more serious is papilloma virus and the cancer of cervical cancer, penile cancer and anus are closely related.The U.S. has about 1% the active grownup of sexual life to suffer from pointed condyloma every year, also has at least 15% subclinical infection person in addition.(L Koutsky.Epidemiology of genital human papillomavirus infection.Am JMed，May 5，1997；102(5A)：3-8)。According to The World Health Organization (WHO), annual de novo cervical cancer patient 4.5 ten thousand people in the whole world, its patient of 80% is positioned at developing country, and cervical cancer is women second killer who is only second to mammary cancer.Wherein, the annual new cases 13.5 ten thousand of China account for 1/3rd of the world.Because human papillomavirus (HPV) infects and to increase, the cervical cancer sickness rate is obviously risen and be tending towards rejuvenation in recent years.The patient that cervical cancer is died from (the rich tumour of giving birth to is seeked advice from) whole world every year has 232000 people, wherein 192000 people of developing country

Other studies confirm that, herpes simplex II C-type virus C and human papillomavirus are the important biomolecule factors that causes cervical cancer, is the most common pathogenic agent of genital system infection.Sickness rate by the woman uterus cancer of the simple sore rash of uterine neck II C-type virus C infection is higher than 8 times of healthy womens.Human papillomavirus causes the danger of cervical cancer bigger than herpes simplex II C-type virus C.

The human papillomavirus that identifies at present (HPV) has surpassed 100 types, and different according to each C-type virus C and its pathogenic hazardness are divided into low risk, high-risk-type with HPV.Low risk, mainly cause benign lesions such as pointed condyloma, wherein the infection of HPV 6 types accounts for more than 70%, secondly is HPV11 type (BrownDR.Schroeder JM, Fife KH, etal.Detection of multiplehuman papillomavirus types in condylomata acuminata lesions from otherwise healthy andimmunosuppressed patients.J Clin Microhiol, 1999,37 (10): 3316-3322); High-risk-type, main relevant with the generation of malignant tumours such as cervical cancer, carcinoma vulvae, penile cancer, based on the HPV16 type.(human papillomavirus Progress on Molecular Biology Deng Yan flies in April, 2000 foreign medical science physiology, pathology science and clinical fascicle, the 20th volume, the 2nd phase.)

Take effective vaccine, prevention and treatment parillomarvirus infections are the desirable approach of prevention and treatment cervical cancer and venereal disease complication.Scientist is carrying out unremitting exploration always, attempts to search out a kind of effective vaccine, as dna vaccination, vector-viral vaccine and synthetic peptide vaccine etc.Up to now, do not develop effective papillomavirus vaccine in the world as yet.

Summary of the invention

What one of purpose of the present invention provided synthetic contains CpG single chain deoxynucleotide (CpG ODN), and they are made of the oligonucleotide single strand dna that contains one or more CpG.The phosphodiester bond of CpG ODN can be non-sulfurized, and partial vulcanization also can be complete cure.The CpG single chain deoxynucleotide that contains of synthetic can be through chemically modified.

Two of purpose of the present invention provides the antiviral effect of CpG ODN and the antiviral effect of CpG ODN and the generation of ribavirin combined utilization.

Three of purpose of the present invention provides with CpG ODN and the treatment of ribavirin combined utilization and prophylaxis of viral infections and viral method and the imagination of feeling the relative disease that causes.

Four of purpose of the present invention provides the method for the disease for the treatment of and preventing to include but not limited to be caused by influenza virus, foot and mouth disease virus, dengue fever virus, japanese encephalitis virus, hepatitis C virus, hepatitis B virus, Human Immunodeficiency Virus and parillomarvirus infections with CpG ODN and ribavirin combined utilization.

In addition, it is pointed out that aspect and creationary beneficial effect that other has substantive distinguishing features of the present invention can directly be known by inference to those skilled in the art on the basis of the application's contextual disclosure.

Embodiment

In the context of the present invention, employed term generally has the implication of those of ordinary skill in the art's common sense unless otherwise indicated.Especially, following term has following implication:

Preferably, CpG ODN of the present invention has sequence as follows:

CpG ODN 1：5’-ggggacgatatcgatgggggg-3’

CpG ODN 2：5’-ggggtcgttcgtcgttgggggg-3’

CpG ODN 3：5’-ggggtgcaacgttcagggggg-3’

CpG ODN 4：5’-tcgtgcgacgatcgtcgcagggggg-3’

CpG ODN 5：5’-tcgagcgatcgctcgagggggg-3’

CpG ODN 6：5’-ggggtgctggccgtcgttgggggg-3’

CpG ODN 7：5’-tcgaaacgtttcgggggg-3’

CpG ODN 8：5’-tcgtcgggtgcgacgtcgcagggggg-3’

CpG ODN 9：5’-tcggacgatcgtcgggggg-3’

CpG ODN 10：5’-tcgtcgggtgcgatcgcagggggg-3’

CpG ODN 11：5’-tcgtgcgacgtcgcagatgat-3’

CpG ODN 12：5’-tcgtatgcatcgatgcatagggagg-3’

CpG ODN 13：5’-tcgtcgtttcgtcgttgggg-3’

CpG ODN 14：5′-tcgtcgggtgcatcgatgcagggggg-3′

CpG ODN 15：5’-tcgtcgcagaacgttctgggggg-3’

CpG ODN 16：5’-tcgtcgggtgcgacgtcgca-3’

CpG ODN 17：5’-gtcgttttcgtcgacgaattgggggggg-3’

CpG ODN 18：5’-tcgtcgtcgactcgtggtcggggg-3’

CpG ODN 19：5′-tcggggacgatcgtcgggggg-3′

CpG ODN 20：5’-gggggcgtcgttttcgtcgacgaatt-3’

Phosphodiester bond wherein can be partial vulcanization, and all sulfurized also can be unvulcanized.

CpG ODN of the present invention can for example adopt the solid phase phosphoramidite triester method to produce by known method production.Following embodiment has at length exemplified a kind of method that contains CpG strand deoxy-oligonucleotide of producing synthetic of the present invention.

When the disease of prevention and treatment virus infection and virus infection initiation, a drug dosage of these CpG ODN is the 1-5000 microgram.The consumption of ribavirin is the 300-1000 milligram.

The application mode of CpG ODN comprises mucomembranous surface (comprising respiratory tract, digestive tube and urogenital mucous membrane) application, and subcutaneous, intramuscular injection is used, gastrointestinal applications, and use in the abdominal cavity, and modes such as intravenous injection are used.

The application mode of ribavirin comprises mucomembranous surface (comprising respiratory tract, digestive tube and urogenital mucous membrane) application, and subcutaneous, intramuscular injection is used, gastrointestinal applications, and use in the abdominal cavity, and modes such as intravenous injection are used.

Below in conjunction with concrete preparation embodiment and biology effect embodiment, and the present invention is described in further detail with reference to accompanying drawing.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.

Embodiment

In following embodiment, various processes of Xiang Ximiaoshuing and method are not ordinary methods as known in the art, for example synthetic solid phase phosphoramidite triester method that adopts.

In following embodiment, the source of agents useful for same, trade(brand)name and/or be necessary to list its moiety person are all only indicated once.Do not giving unnecessary details foregoing if no special instructions at used identical reagent thereafter.

The preparation of embodiment 1 CpG ODN

Adopt the synthetic CpG ODN of solid phase phosphoramidite triester method

1, reagent and material:

Trichoroacetic acid(TCA) (Trichloroacetic Acid, TCA), controlled pore glass (Controlled Pore Glass), DMT (dimethoxytrityl), tetrazole activator, diacetyl oxide, N-Methylimidazole, A, T, four kinds of nucleotide monomers of C, G, ABI dna synthesizer, high performance liquid chromatography chromatograph etc.

2, method:

1) deprotection base

With trichoroacetic acid(TCA) (Trichloroacetic Acid; TCA) slough the blocking group dimethoxytrityl (DMT) that is attached at the Nucleotide on the controlled pore glass (Controlled Pore Glass); obtain free 5 '-hydroxyl terminal, for next step condensation reaction.

2) activation

With the nucleotide monomer and the tetrazole activator mix of phosphoramidite protection and enter synthetic post; (its 3 '-end is activated to form phosphoramidite tetrazolium active intermediate; but 5 '-end is protected by DMT still), this intermediate will with the Nucleotide generation condensation reaction of the base of deprotection on the controlled pore glass.

3) connect

When phosphoramidite tetrazolium active intermediate runs on the controlled pore glass the Nucleotide of deprotection base, will with its 5 '-hydroxyl generation affinity reaction, condensation is also sloughed tetrazolium, this moment, the synthetic oligonucleotide chain prolonged a base forward.

4) sealing

Be extended in circulating reaction subsequently for 5 ' of the reaction-hydroxyl that has neither part nor lot in that prevents to be connected on the controlled pore glass after the condensation reaction; often seal this terminal hydroxy group by acetylize, general acetylation reagent mixes formation with diacetyl oxide and N-Methylimidazole etc.

5) oxidation

Nucleotide monomer is to be connected with oligonucleotide on being connected in controlled pore glass by inferior phosphide key during condensation reaction, and inferior phosphide key instability, easily by acid, basic hydrolysis, this moment, the tetrahydrofuran solution of iodine commonly used was converted into phosphotriester with inferior phosphinylidyne, obtained stable oligonucleotide.

Through after above five steps; a deoxynucleotide is just linked on the Nucleotide of controlled pore glass; after sloughing blocking group DMT on the deoxynucleotide 5 '-hydroxyl that newly connects with trichoroacetic acid(TCA) equally again, repeat above activation, connection, sealing, oxidising process and can obtain a dna fragmentation crude product.At last to its cut, the deprotection base (generally adopts the benzoyl protection to A, C base; The G base is protected with isobutyryl; The T base needn't be protected; Phosphorous acid is with nitrile ethyl protection), purifying (commonly used have HAP, PAGE, HPLC, C18, methods such as OPC), synthetic aftertreatment such as quantitative can obtain meeting the oligonucleotide fragment of requirement of experiment.

Unvulcanized CpG ODN is synthetic on ABI 3900 dna synthesizers; The synthetic employing substitution method of full sulfuration and partial vulcanization CpG strand deoxy-oligonucleotide, synthetic on ABI 394 dna synthesizers.

The CpG ODN sequence of using among embodiment 2, embodiment 3, embodiment 4, embodiment 5, the embodiment 6 is all as follows:

CpG ODN 1：5’-ggggacgatatcgatgggggg-3’

CpG ODN 2：5’-ggggtcgttcgtcgttgggggg-3’

CpG ODN 3：5’-ggggtgcaacgttcagggggg-3’

CpG ODN 4：5’-tcgtgcgacgatcgtcgcagggggg-3’

CpG ODN 5：5’-tcgagcgatcgctcgagggggg-3’

CpG ODN 6：5’-ggggtgctggccgtcgttgggggg-3’

CpG ODN 7：5’-tcgaaacgtttcgggggg-3’

CpG ODN 8：5’-tcgtcgggtgcgacgtcgcagggggg-3’

CpG ODN 9：5’-tcggacgatcgtcgggggg-3’

CpG ODN 10：5’-tcgtcgggtgcgatcgcagggggg-3’

CpG ODN 11：5’-tcgtgcgacgtcgcagatgat-3’

CpG ODN 12：5’-tcgtatgcatcgatgcatagggagg-3’

CpG ODN 13：5’-tcgtcgtttcgtcgttgggg-3’

CpG ODN 14：5′-tcgtcgggtgcatcgatgcagggggg-3′

CpG ODN 15：5’-tcgtcgcagaacgttctgggggg-3’

CpG ODN 16：5’-tcgtcgggtgcgacgtcgca-3’

CpG ODN 17：5’-gtcgttttcgtcgacgaattgggggggg-3’

CpG ODN 18：5’-tcgtcgtcgactcgtggtcggggg-3’

CpG ODN 19：5′-tcggggacgatcgtcgggggg-3′

CpG ODN 20：5’-gggggcgtcgttttcgtcgacgaatt-3’

The anti-folliculus Stomatovirus effect of embodiment 2 CpG ODN and CpG ODN and ribavirin combined utilization relatively

1, the amplification of folliculus Stomatovirus (VSV)

1) plant and instrument and material: the suction pipe of cryogenic refrigerator, carbonic acid gas incubator, Bechtop, inverted microscope, liquid nitrogen container, water distillation apparatus, vacuum pump, Tissue Culture Flask, all size, sample injector, dropper etc.

2) RPMI1640 substratum: contain RPMI1640 (GIBCOBRL) 10.4 grams of L-glutaminate, 2.0 gram sodium bicarbonates, 10 unit gentamicins add tri-distilled water to 1000 milliliters of volumes.0.22 the degerming of filter membrane vacuum pump suction filtration, the packing of micron.

3) method: with the RPMI1640 culture medium culturing Vero E6 cell that contains 10% calf serum (Invitrogen is through 56 ℃ of processing in 30 minutes).After in the 100ml culturing bottle, growing up to individual layer, change nutrient solution.Add the nutrient solution that contains folliculus Stomatovirus (VSV), its content is 10TCID50.If virus-free contrast.After about 24-48 hour, pathology all takes place in the Vero E6 cell that infects VSV, collects sick cell and supernatant, it is put-20 ℃ and spends the night.Inferior morning, it is melted, fiercely blow and beat the centrifugal supernatant of collecting with suction pipe.After measuring TCID50, supernatant is stored in-20 ℃ standby.

2, the mensuration of folliculus Stomatovirus (VSV) TCID50

2) RPMI1640 substratum: contain RPMI1640 (GIBCOBRL) 10.4 grams of L-glutaminate, 2.0 gram sodium bicarbonates, 10 unit gentamicins add tri-distilled water to 1000 milliliters of volumes.Filter membrane Entkeimung, packing with 0.22 micron.

3) contain the RPMI1640 substratum of 10% calf serum: 10 milliliters of calf serums (Invitrogen handled through 56 ℃ in 30 minutes), 90 milliliters of RPMI1640 substratum.

4) 0.5% Viola crystallina dye liquor: Viola crystallina 0.5 gram, NaCl 0.85 gram is dissolved in 50 milliliters of anhydrous diethyl ethers.Add 3 milliliters of formaldehyde, 47 ml distilled waters.

5) Viola crystallina destainer: 50 milliliters of ethylene glycol monomethyl ethers mix with 50 ml distilled waters.

6) 0.25% trypsin Trypsin)

7) method: with the well-grown Vero E6 of 0.25% tryptic digestion cell, 3-5 minute.Centrifugal (1000rpm, 5 minutes) washed cell.RPMI1640 cultivation keynote cell concn with 10% calf serum is 4 * 105/milliliter.Add the cell suspension in the flat culture plates in 96 holes (Costar), every hole 100 microlitres are established three multiple holes.37 ℃ of CO ₂Incubator was cultivated after 24 hours, and cell forms individual layer.Change nutrient solution, what wherein contain doubling dilution treats titration VSV (Changchun institute of Biological Products).If there is not the VSV contrast.After 24 hours, judge cytopathic degree with the method for violet staining.Nutrient solution is abandoned in suction, and every hole adds 200 μ l, 0.5% Viola crystallina dye liquor, 37 ℃, 15 minutes.Flowing water washes out the Viola crystallina dye liquor.Every hole adds 200 μ l Viola crystallina destainers, the vibrator vibration, dyestuff is deviate from cell fully, with spectrophotometer at colorimetric (the Cytokines.A practical approach of 540nm wavelength place, second edition, edited by F.R.BALKWILLThe practical approach series.).With X10 reciprocal that 50% cytopathic viral dilution degree the occurs TCID50/ milliliter numerical value of viral liquid for this reason.

3, the separation of human peripheral blood single nucleus cell

1) plant and instrument and equipment: the suction pipe of cryogenic refrigerator, carbonic acid gas incubator, Bechtop, inverted microscope, liquid nitrogen container, water distillation apparatus, vacuum pump, Tissue Culture Flask, bacterial filter, filtration bottle, all size, sample injector, dropper, blood counting chamber, horizontal whizzer etc.

2) reagent and material: people's whole blood of anticoagulant heparin is available from Central Blood Ban, Changchun City.Ficoll-Hypaque: proportion 1.077 ± 0.001, available from Beijing ancient cooking vessel state Bioisystech Co., Ltd.RPMI1640 nutrient solution: the RPMI1640 of L-glutaminate (GIBCOBRL) 10.4 gram, sodium bicarbonate 2.0 grams, gentamicin 100,000 units add tri-distilled water to 1000 milliliter.0.22 the degerming of filter membrane suction filtration, the packing of micron.

3) method: with the mononuclearcell of Ficoll-Hypaque lymphocyte layering liquid separation of human peripheral blood.The volume ratio of layering liquid and anticoagulant heparin peripheral blood is about 2: 1.Level centrifugal (1,000xg, 20min).Draw the liquid band that contains mononuclearcell with suction pipe, insert in other centrifuge tube.Add isopyknic serum-free medium.1, the centrifugal 15min of 000g abandons supernatant.Twice of repeated washing.Abandon supernatant,, and carry out cell counting with 2ml nutrient solution re-suspended cell.

4, the mensuration of the effect of CpG ODN and CpG ODN and the anti-folliculus Stomatovirus of ribavirin combined utilization

Final concentration with the RPMI1640 substratum mediator peripheral blood mononuclear cell that contains 10% calf serum is 3 * 10 ⁶Individual/milliliter.Add the cell suspension in 12 well culture plates, every hole 2ml.Add CpG ODN (1-20) to final concentration 6 μ g/ml.37 ℃, 5% carbonic acid gas incubator was cultivated 48 hours, collected supernatant.

Set up CpG group, ribavirin group, CpG+ ribavirin group, IMDM group.The Vero E6 cell inoculation that growth conditions is good is in 96 well culture plates, every hole 1.3 * 10 ⁴Individual cell, 37 °, 5%CO ₂Cultivate after 24 hours, the CpG group adds human PBMC's culture supernatant of the CpG ODN stimulation of dilution in 1: 100, the ribavirin group adds ribavirin (to final concentration 10 μ mol/l), CpG+ ribavirin group adds the human PBMC's culture supernatant and the ribavirin (Zhejiang Cheng Yi Pharmaceutical Co., Ltd) of the CpG ODN stimulation of dilution in 1: 100, and the IMDM group only adds IMDM.Continue to cultivate after 1.5 hours, add the VSV virus of 250TCID50/ml, establish normal VERO cell control group simultaneously, cultivated 44 hours.Judge cytopathic degree with the method for violet staining.Nutrient solution is abandoned in suction.Every hole adds 200 μ l, 0.5% Viola crystallina dye liquor, 37 ℃, hatches 15 minutes.Flowing water washes out the Viola crystallina dye liquor.Every hole adds 200 microlitre Viola crystallina destainers, and the vibrator vibration is deviate from dyestuff fully in cell, measure the spectrophotometric value with spectrophotometer at the 540nm wavelength.

5, result: CpG ODN group and CpG ODN compare with ribavirin combined utilization group, the OD value of CpG ODN and ribavirin combined utilization group is higher than CpG ODN group significantly, the result shows, CpG ODN has tangible antivirus action, and CpG ODN and ribavirin combined utilization have more significant antivirus action.OD value between two groups relatively sees Table 1.

The anti-folliculus Stomatovirus effect of table 1 CpG ODN and CpG ODN and ribavirin combined utilization relatively

The resisiting influenza virus effect of embodiment 3 CpG ODN and CpG ODNN and ribavirin combined utilization relatively

1, the separation of human peripheral blood single nucleus cell

2) reagent and material: people's whole blood of anticoagulant heparin is available from Central Blood Ban, Changchun City.Ficoll-Hypaque: proportion 1.077 ± 0.001, available from Beijing ancient cooking vessel state Bioisystech Co., Ltd.DMEM nutrient solution: 1000ml contains gentamicin 100,000 units.0.22 the degerming of filter membrane suction filtration, the packing of micron.

2, the resisiting influenza virus effect of CpG ODN and CpG ODN and ribavirin combined utilization is measured

Final concentration with DMEM nutrient solution mediator peripheral blood mononuclear cell is 3 * 10 ⁶Individual/milliliter.Add this cell suspension in 12 well culture plates, every hole 2ml.Add CpG ODN to final concentration 6 μ g/ml.37 ℃, 5% carbonic acid gas incubator was cultivated 48 hours, collected supernatant, detected the activity of its resisiting influenza virus.

Set up CpG group, ribavirin group, CpG+ ribavirin group, DMEM group.Regulate good Vero E6 cell concn to 3 * 10 of growth conditions with 0.3%BSA DMEM ⁵Individual/ml.With cell inoculation in the flat culture plate in 96 holes, every hole 1.3 * 10 ⁴Individual cell, 37 ° of and 5%CO ₂Cultivate after 24 hours, the CpG group adds human PBMC's culture supernatant of the CpG ODN stimulation of dilution in 1: 100, the ribavirin group adds ribavirin (to final concentration 10 μ mol/l), CpG+ ribavirin group adds the human PBMC's culture supernatant and the ribavirin (Zhejiang Cheng Yi Pharmaceutical Co., Ltd) of the CpG ODN stimulation of dilution in 1: 100, and the DMEM group only adds DMEM.Continue to cultivate after 1.5 hours, every hole adds influenza virus liquid (Changchun Biological Products Institute) the 200 μ l of 0.3%BSA DMEM (4 μ g/ml pancreatin) dilution, establishes normal VERO cell contrast simultaneously.37 ℃, 5%CO2 incubator were cultivated 44 hours, judged cytopathic degree with the method for violet staining.Nutrient solution is abandoned in suction.Every hole adds 200 μ l, 0.5% Viola crystallina dye liquor, 37 ℃, 15 minutes.Flowing water washes out the Viola crystallina dye liquor.Every hole adds 200 microlitre Viola crystallina destainers, and the vibrator vibration is deviate from dyestuff fully in cell, measure the spectrophotometric value with spectrophotometer at the 540nM wavelength.

3 results: CpG ODN group and CpG ODN compare with ribavirin combined utilization group, the OD value of CpG ODN and ribavirin combined utilization group is higher than CpG ODN group significantly, the result shows, CpG ODN has tangible resisiting influenza virus effect, and CpG ODN and ribavirin combined utilization have more significant resisiting influenza virus effect.OD value between two groups relatively sees Table 2.

The resisiting influenza virus effect of table 2 CpG ODN and CpG ODNN and ribavirin combined utilization relatively

Experiment in the mouse body of embodiment 4 CpG and ribavirin resisiting influenza virus

1 material: female Kunming mouse, the 14-16 gram, influenza virus H1N1 type lung adapted strain (FM1) is available from the Changchun institute of Biological Products.

2 methods: set up normal mouse contrast, model control group (negative control group), ribavirin group, CpG group, ribavirin to add the CpG group.CpG organizes mouse subcutaneous injection CpG ODN 80 μ g (CpG ODN 1-20), after 48 hours, influenza virus is diluted to 10LD with 0.3%BSA DMEM ₅₀, except that the normal mouse control group, other respectively organizes mouse behind etherization, and nasal cavity gives the 10LD of 20 μ l ₅₀Influenza virus.After 2 hours, adopt the method afford ribavirin group mouse of irritating stomach, 50mg ribavirin/kg body weight, every mouse 0.2-0.4ml, every day 3 times.Adopt hypodermic mode to give CpG group mouse 80 μ gCpG ODN, injection in per two days once.Ribavirin adds the CpG group, gives ribavirin and CpG, and dosage and administering mode are with ribavirin group and CpG group.After 4 days, dissect mouse, get lung, weigh, judge the pulmonary lesion index.Pulmonary lesion index (Biochemical and Biophysical Research Communications 279,158-161 (2000) .Inhibitory Effects of an Antisense Oligonucleotide in an Experimentally InfectedMouse Model of Influenza A Virus.) is judged index:

-obvious pathology do not arranged

+ 25% lung tissue generation pathology

++ 25-50% lung tissue generation pathology

+++50-75% lung tissue generation pathology

++ ++＞75% lung tissue generation pathology

3 results: the pulmonary lesion index of CpG+ ribavirin group mouse significantly is lower than CpG group and ribavirin group.Illustrate that CpG and ribavirin combined utilization have better effect when using separately than CpG and ribavirin.The tuberculosis varying index relatively sees Table 3 between each group.

4 conclusions: CpG ODN has tangible resisiting influenza virus effect, and CpG ODN and ribavirin combined utilization have more significant resisiting influenza virus effect.

The foot-and-mouth disease virus resistant effect of embodiment 5 CpGODN and CpGODNN and ribavirin combined utilization relatively

1, the separation of human peripheral blood single nucleus cell

1) plant and instrument and equipment:

The suction pipe of cryogenic refrigerator, carbonic acid gas incubator, Bechtop, inverted microscope, liquid nitrogen container, water distillation apparatus, vacuum pump, Tissue Culture Flask, bacterial filter, filtration bottle, all size, sample injector, dropper, blood counting chamber, horizontal whizzer etc.

2) reagent and material: O type foot and mouth disease virus is provided by Inner Mongol bio-pharmaceuticals factory, and antiviral experiment is carried out in Inner Mongol bio-pharmaceuticals factory.People's whole blood of anticoagulant heparin is available from Central Blood Ban, Changchun City.Ficoll-Hypaque: proportion 1.077 ± 0.001, available from Beijing ancient cooking vessel state Bioisystech Co., Ltd.IMDM nutrient solution: 1000ml contains gentamicin 100,000 units.0.22 the degerming of filter membrane suction filtration, the packing of micron.

3) method: with the mononuclearcell of Ficoll-Hypaque lymphocyte layering liquid separation of human peripheral blood.The volume ratio of layering liquid and anticoagulant heparin peripheral blood is about 2: 1.Level centrifugal (1,000xg, 20min).Draw the liquid band that contains mononuclearcell with suction pipe, insert in other centrifuge tube.Add isopyknic serum free medium.1, the centrifugal 15min of 000g abandons supernatant.Twice of repeated washing.Abandon supernatant, with 2ml substratum re-suspended cell and carry out cell counting.

2, the foot-and-mouth disease virus resistant effect of CpG ODN and CpG ODN and ribavirin combined utilization is measured

Final concentration with the IMDM nutrient solution mediator peripheral blood mononuclear cell that contains 10% foetal calf serum is 3 * 10 ⁶Individual/milliliter.Add this cell suspension in 12 well culture plates, every hole 2ml.Add CpG ODN to final concentration 6 μ g/ml.37 ℃, 5% carbonic acid gas incubator was cultivated 48 hours, collected supernatant, detected the activity of its foot-and-mouth disease virus resistant.

Set up CpG group, ribavirin group, CpG+ ribavirin group, IMDM group.Regulate good bhk cell concentration to 3 * 10 of growth conditions with 5% foetal calf serum IMDM ⁵Individual/ml.With cell inoculation in the flat culture plate in 96 holes, every hole 1.3 * 10 ⁴Individual cell, 37 °, 5%CO ₂Cultivate after 24 hours, the CpG group adds human PBMC's culture supernatant of the CpG ODN stimulation of dilution in 1: 100, the ribavirin group adds ribavirin (to final concentration 10 μ mol/l), CpG+ ribavirin group adds the human PBMC's culture supernatant and the ribavirin (Zhejiang Cheng Yi Pharmaceutical Co., Ltd) of the CpG ODN stimulation of dilution in 1: 100, and the IMDM group only adds IMDM.Continue to cultivate after 1.5 hours, every hole adds hoof-and-mouth disease venom (Inner Mongol bio-pharmaceuticals factory) the 200 μ l of 2%FBS IMDM dilution, establishes normal Vero cell contrast.37 ℃, 5%CO2 incubator were cultivated 44 hours, judged cytopathic degree with the method for violet staining.Nutrient solution is abandoned in suction.Every hole adds 200 μ l, 0.5% Viola crystallina dye liquor, 37 ℃, 15 minutes.Flowing water washes out the Viola crystallina dye liquor.Every hole adds 200 microlitre Viola crystallina destainers, and the vibrator vibration is deviate from dyestuff fully in cell, measure the spectrophotometric value with spectrophotometer at the 540nm wavelength.

3 results: CpG ODN group and CpG ODN compare with ribavirin combined utilization group, the OD value of CpG ODN and ribavirin combined utilization group is higher than CpG ODN group significantly, the result shows, CpG ODN has tangible foot-and-mouth disease virus resistant effect, and CpG ODN and ribavirin combined utilization have more significant foot-and-mouth disease virus resistant effect.OD value between two groups relatively sees Table 4.

The foot-and-mouth disease virus resistant effect of table 4 CpGODN and CpGODNN and ribavirin combined utilization relatively

Embodiment 6 CpG ODN and CpG ODNN and the effect of the anti-sub-thread positive chain RNA of ribavirin combined utilization japanese encephalitis virus are relatively

1, the separation of human peripheral blood single nucleus cell is with embodiment 2.

2, obtain CpG ODN (6 μ g/ml) stimulation human peripheral blood PBMC culture supernatant with embodiment 2.

3, the anti-sub-thread positive chain RNA japanese encephalitis virus effect of CpG ODN and CpG ODN and ribavirin combined utilization is measured

1) material: japanese encephalitis virus is available from the Changchun institute of Biological Products.10%FCSRPMI1640 complete culture solution: with embodiment 2.

2) method:

Final concentration with the RPMI1640 substratum mediator peripheral blood mononuclear cell that contains 10% calf serum is 3 * 10 ⁶Individual/milliliter.Add the cell suspension in 12 well culture plates, every hole 2ml.Add CpG ODN to final concentration 6 μ g/ml.37 ℃, 5% carbonic acid gas incubator was cultivated 48 hours, collected supernatant.

Set up CpG group, ribavirin group, CpG+ ribavirin group, IMDM group.The bhk cell that growth conditions is good is inoculated in 96 well culture plates, every hole 1.3 * 10 ⁴Individual cell, 37 °, 5%CO ₂Cultivate after 24 hours, the CpG group adds human PBMC's culture supernatant of the CpG ODN stimulation of dilution in 1: 100, the ribavirin group adds ribavirin (to final concentration 10 μ mol/l), CpG+ ribavirin group adds the human PBMC's culture supernatant and the ribavirin (Zhejiang Cheng Yi Pharmaceutical Co., Ltd) of the CpG ODN stimulation of dilution in 1: 100, and the IMDM group only adds IMDM.Continue to cultivate after 1.5 hours, add the japanese encephalitis virus of 100TCID50/ml, establish normal bhk cell contrast simultaneously, cultivated 44 hours.Examine under a microscope cytopathy (CPE), 25% cytopathy note "+" occurs, the cytopathy note " ++ " of 26-50% occurs, the cytopathy note " +++" of 51-75% occurs, the cytopathy note " ++ ++ " of 76-100% occurs.

3) result's (CPE method): CpG ODN group and CpG ODN compare with ribavirin combined utilization group, the cytopathy of CpG ODN and ribavirin combined utilization group is markedly inferior to CpG ODN group, the result shows, CpG ODN has tangible Japanese ence phalitis Viruses effect, and CpG ODN and ribavirin combined utilization have more significant Japanese ence phalitis Viruses effect.Cytopathic between two groups relatively sees Table 5.

Table 5 CpG ODN and CpG ODNN and the effect of the anti-sub-thread positive chain RNA of ribavirin combined utilization japanese encephalitis virus are relatively

Above-mentioned experimental result shows that CpG ODN of the present invention has the effect of tangible Japanese ence phalitis Viruses, and CpG ODN and ribavirin combined utilization have the effect of more significant Japanese ence phalitis Viruses.Hepatitis C virus and dengue fever virus, the same RNA viruses that is both sub-thread normal chain of japanese encephalitis virus, therefore, CpG ODN and ribavirin combined utilization can have function of resisting hepatitis C virus equally.

The subordinate list part

CpG ODN	The OD value
	The OD value		CpG ODN induces supernatant	CpGODN induces supernatant+Ribavirin
	1	0.512±0.03	CpG ODN induces supernatant	CpGODN induces supernatant+Ribavirin	0.908±0.05
2	1	0.512±0.03	0.498±0.04	1.120±0.13	0.908±0.05
2	3	0.625±0.09	0.498±0.04	1.120±0.13	1.340±0.08
4	3	0.625±0.09	0.523±0.07	1.180±0.10	1.340±0.08
4	5	0.465±0.09	0.523±0.07	1.180±0.10	0.988±0.03
6	5	0.465±0.09	0.533±0.01	0.877±0.11	0.988±0.03
6	7	0.476±0.06	0.533±0.01	0.877±0.11	1.005±0.12
8	7	0.476±0.06	0.516±0.07	0.998±0.08	1.005±0.12
8	9	0.487±0.10	0.516±0.07	0.998±0.08	1.560±0.14
10	9	0.487±0.10	0.678±0.11	1.345±0.09	1.560±0.14
10	11	0.576±0.07	0.678±0.11	1.345±0.09	1.123±0.13
12	11	0.576±0.07	0.765±0.14	1.407±0.09	1.123±0.13
12	13	0.816±0.12	0.765±0.14	1.407±0.09	1.654±0.14
14	13	0.816±0.12	0.569±0.09	1.213±0.11	1.654±0.14
14	15	0.659±0.17	0.569±0.09	1.213±0.11	1.457±0.19
16	15	0.659±0.17	0.789±0.13	1.345±0.08	1.457±0.19
16	17	0.499±0.05	0.789±0.13	1.345±0.08	1.180±0.15
18	17	0.499±0.05	0.522±0.07	1.398±0.11	1.180±0.15
18	19	0.609±0.14	0.522±0.07	1.398±0.11	1.478±0.18
20	19	0.609±0.14	0.712±0.18	1.589±0.16	1.478±0.18

IMDM group 0.213 ± 0.07

Ribavirin group 0.398 ± 0.09

Normal VERO cell: 1.625 ± 0.11

CpG ODN	The OD value
	The OD value		CpGODN induces supernatant	CpGODN induces supernatant+Ribavirin
	1	0.678±0.12	CpGODN induces supernatant	CpGODN induces supernatant+Ribavirin	1.412±0.16
2	1	0.678±0.12	0.569±0.09	1.345±0.11	1.412±0.16
2	3	0.729±0.07	0.569±0.09	1.345±0.11	1.456±0.19
4	3	0.729±0.07	0.801±0.06	1.678±0.18	1.456±0.19
4	5	0.599±0.04	0.801±0.06	1.678±0.18	1.342±0.09
6	5	0.599±0.04	0.499±0.06	1.234±0.06	1.342±0.09
6	7	0.612±0.04	0.499±0.06	1.234±0.06	1.367±0.06
8	7	0.612±0.04	0.579±0.05	1.258±0.03	1.367±0.06
8	9	0.578±0.03	0.579±0.05	1.258±0.03	1.298±0.06
10	9	0.578±0.03	0.621±0.06	1.312±0.07	1.298±0.06
10	11	0.587±0.02	0.621±0.06	1.312±0.07	1.199±0.05
12	11	0.587±0.02	0.703±0.05	1.389±0.08	1.199±0.05
12	13	0.698±0.06	0.703±0.05	1.389±0.08	1.378±0.07
14	13	0.698±0.06	0.709±0.08	1.409±0.09	1.378±0.07
14	15	0.567±0.06	0.709±0.08	1.409±0.09	1.298±0.08
16	15	0.567±0.06	0.721±0.09	1.476±0.09	1.298±0.08
16	17	0.691±0.05	0.721±0.09	1.476±0.09	1.309±0.07
18	17	0.691±0.05	0.580±0.04	1.199±0.05	1.309±0.07
18	19	0.615±0.07	0.580±0.04	1.199±0.05	1.099±0.08
20	19	0.615±0.07	0.745±0.08	1.305±0.07	1.099±0.08
20			0.745±0.08	1.305±0.07

DMEM group 0.245 ± 0.08

Ribavirin group 0.423 ± 0.09

Normal VERO cell contrast 1.328 ± 0.13

Experiment in the mouse body of table 3 CpG ODN and CpG ODN and ribavirin combined utilization resisiting influenza virus

CpG ODN	Cytopathy
	Cytopathy		CpG ODN group	CpG ODN+Ribavirin group
	1	++	CpG ODN group	CpG ODN+Ribavirin group	+
2	1	++	++	-	+
2	3	++	++	-	-
4	3	++	+++	+	-
4	5	++	+++	+	-
6	5	++	++	-	-
6	7	++	++	-	-
8	7	++	++	-	-
8	9	++	++	-	-
10	9	++	++	+	-
10	11	++	++	+	-
12	11	++	++	-	-
12	13	++	++	-	-
14	13	++	+++	-	-
14	15	++	+++	-	+
16	15	++	++	-	+
16	17	++	++	-	-
18	17	++	+++	+	-
18	19	++	+++	+	-
20	19	++	++	-	-

Model group (negative control) ++ ++

The normal cell group-

The ribavirin group +++

CpG ODN	The 0D value
	The 0D value		CpGODN induces supernatant	CpGODN induces supernatant+Ribavirin
	1	0.678±0.08	CpGODN induces supernatant	CpGODN induces supernatant+Ribavirin	1.325±0.07
2	1	0.678±0.08	0.895±0.09	1.654±0.12	1.325±0.07
2	3	0.567±0.05	0.895±0.09	1.654±0.12	1.189±0.10
4	3	0.567±0.05	0.901±0.10	1.679±0.11	1.189±0.10
4	5	0.697±0.09	0.901±0.10	1.679±0.11	1.345±0.09
6	5	0.697±0.09	0.713±0.06	1.513±0.10	1.345±0.09
6	7	0.890±0.09	0.713±0.06	1.513±0.10	1.690±0.11
8	7	0.890±0.09	0.541±0.06	1.167±0.10	1.690±0.11
8	9	0.900±0.14	0.541±0.06	1.167±0.10	1.791±0.18
10	9	0.900±0.14	0.734±0.06	1.398±0.09	1.791±0.18
10	11	0.499±0.04	0.734±0.06	1.398±0.09	1.011±0.02
12	11	0.499±0.04	0.590±0.05	0.999±0.01	1.011±0.02
12	13	0.609±0.08	0.590±0.05	0.999±0.01	1.187±0.06
14	13	0.609±0.08	0.714±0.06	1.310±0.08	1.187±0.06
14	15	0.824±0.12	0.714±0.06	1.310±0.08	1.456±0.19
16	15	0.824±0.12	0.766±0.11	1.388±0.16	1.456±0.19
16	17	0.578±0.09	0.766±0.11	1.388±0.16	1.213±0.12
18	17	0.578±0.09	0.643±0.07	1.312±0.09	1.213±0.12
18	19	0.719±0.06	0.643±0.07	1.312±0.09	1.398±0.09
20	19	0.719±0.06	0.890±0.09	1.432±0.12	1.398±0.09

IMDM group 0.267 ± 0.05

Ribavirin group 0.561 ± 0.09

Normal bhk cell 1.782 ± 0.14

CpG ODN	Cytopathy
	Cytopathy		CpGODN induces supernatant	CpGODN induces supernatant+Ribavirin
	1	+	CpGODN induces supernatant	CpGODN induces supernatant+Ribavirin	-
2	1	+	+	-	-
2	3	+	+	-	-
4	3	+	+	-	-
4	5	+	+	-	-
6	5	+	+	-	-
6	7	+	+	-	-
8	7	+	+	-	-
8	9	+	+	-	-
10	9	+	+	-	-
10	11	+	+	-	-
12	11	+	+	-	-
12	13	+	+	-	-
14	13	+	+	-	-
14	15	+	+	-	-
16	15	+	+	-	-
16	17	+	+	-	-
18	17	+	+	-	-
18	19	+	+	-	-
20	19	+	+	-	-

The IMDM group ++ ++

The ribavirin group ++

Normal bhk cell-

The CpG sequence

Implementation sequence is as follows:

(G)n(L)nX1X2CGY1Y2(M)n(G)n

X1=A, T, G; X2=A, T; Y1=A, T; Y2=A, T, C; L, M=A, T, C, G; N is 0-6

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

5’-ggggATAACgTTgCgggggg-3’(SEQ ID NO：2)[143]

5’-ggggTgCAACgTTCAgggggg-3’(SEQ ID NO：3)[402]

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

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

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

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

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

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

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

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

5′-gggggATCgACgTCgATCgggggg-3’(SEQ ID NO：12)[322]

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

5’-ggggTCgATCgATCgAgggggg-3′(SEQ ID NO：14)[113]

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(G)n(L)nCG(XY)nCG(M)n(G)n

X=A, T; Y=A, T; L, M=A, T, C, G; N is 0-6

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

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

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

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

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

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

5′-ggTgCATCgATCgATgCAgggggg-3′(SEQ ID NO：39)[101]

5′-ggTgCATCgTACgATgCAgggggg-3′(SEQ ID NO：40)[100]

5’-ggTgCgATCgATCgCAgggggg-3′(SEQ ID NO：41)[134]

5’-gggggggTCgATCgATgggggg-3’(SEQ ID NO：42)[519]

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

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

5’-TTCgCTTCgCTTTTCgCTTCgCTT-3’-3’(SEQ ID NO：45)[212]

5’-ACCgCCAAggAgAAgCCgCAggAggg-3’(SEQ ID NO：46)[166]

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

5’-gggggCgTCgTTTTCgTCgACgAATT-3’(SEQ ID NO：66)[278]

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

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

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

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

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

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

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

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

5’-AAAAgACgTCgACgTCgACgTCTTTT-3’(SEQ ID NO：75)[489]

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

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

(TCG)n(L)nCG(M)n(G)n

L, M=A, T, C, G; N is 0-6

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

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

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

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

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

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

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

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

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

5’-TCgTCgggTgCAACgTTgCAgggggg-3’(SEQ ID NO：87)[564]

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

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

5’-TCgTCgggTgCgACgATCgTCgCAgggggg-3’(SEQ ID NO：90)[465]

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

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

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

5’-TCgTgCgACgATCgTCgCAgggggg-3’(SEQ ID NO：94)[178]

5’-TCgTATgCATCgATgCATAgggAgg-3’(SEQ ID NO：95)[410]

5’-TCgTgCATCgATgCAgggggg-3’(SEQ ID NO：96)[444]

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

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

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

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

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

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

5’-TCgTCgggTgCgACgATCgTCgggggg-3’(SEQ ID NO：103)[290]

5’-TCgTCgTTTgCATCgATgCAggggggg-3’(SEQ ID NO：104)[627]

5’-TCgTCgTTTTgACgATCgTCgggggg-3’(SEQ ID NO：105)[500]

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

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

5’-TCgTTgCgCTCCCATgCCgggggg-3’(SEQ ID NO：108)[319]

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

5’-TCgTTgTCgTTTCgCTgCCggCggggg-3’(SEQ ID NO：110)[417]

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

5’-TgCTgCTTTgCTgCTTgggg-3’(SEQ ID NO：112)[421]

5’-AACgTTCgACgTCgAACggggggg-3’(SEQ ID NO：113)[453]

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

5’-tcgtcgggtgcgacgtcgca-3’(SEQ ID NO：165)[612]

(TCG)n(L)nX1X2CG(M)n

X1=A, T, G; X2=A, T; L, M=A, T, C, G; N is 0-6

Implementation sequence is as follows:

5’-TCgTAACgTTgTTTTTAACgTT-3’(SEQ ID NO：115)[470]

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

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

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

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

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

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

5’-TCgCCTCgTCgCCTTCgAgC-3’g-3’(SEQ ID NO：122)[102]

5’-TCgTgTgCgTgCCgTTggg-3’T-3’(SEQ ID NO：123)[406]

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

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

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

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

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

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

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

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

5’-TCgAggACAAgATTCTCgTgCAggCC-3’(SEQ ID NO：132)[570]

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

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

5’-TCggCACgCgACgTgCTggCCgTCgTTTCC-3’(SEQ ID NO：135)[370]

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

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

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

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

5’-TCgTTgCCgT℃ggCCCCCCC-3’(SEQ ID NO：140)[501]

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

5’-TCggCACgCgACgTgCTggCCgTCgTT-3’(SEQ ID NO：142)[509]

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

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

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

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

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

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

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

5’-TCgTCgTCgTCgTTgTCgTTgggg-3’(SEQ ID NO：150)[309]

5’-TCgTTCggggTgCCg-3’(SEQ ID NO：151)[409]

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

5’-TCgTCgAgCgCTCgATCggAT-3’(SEQ ID NO：170)[211]

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

5’-TCgTCggACgTTTTCCgACgTTCT-3’(SEQ ID NO：172)[308]

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

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

5’-TCgTCgTTggTCggggTCgTTggggTCgTT-3’(SEQ ID NO：175)[405]

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

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

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

The sequence that comprises TTCGTCG

5’-TTCgTCgTTTgATCgATgTTCgTTgggggg-3’(SEQ ID NO：179)[507]

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

5’-TATCgATgTTTTTCgTCgTCgTTgggggg-3’(SEQ ID NO：181)[202]

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

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

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

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

Claims

1, synthetic contain the CpG single chain deoxynucleotide, they are made of the oligonucleotide single strand dna that contains one or more CpG.

2, synthetic contain the CpG single chain deoxynucleotide, their phosphodiester bond can be non-sulfurized, partial vulcanization also can be complete cure.The CpG single chain deoxynucleotide that contains of synthetic can be through chemically modified.

3, the CpG single chain deoxynucleotide that contains of synthetic includes but not limited to the sequence shown in the SEQ ID NO:1-179.

4, the antiviral effect that is produced according to each the described CpG of containing single chain deoxynucleotide irritation cell among the claim 1-3.

5, according to each described antiviral effect that contains CpG single chain deoxynucleotide and the generation of ribavirin combined utilization among the claim 1-3.

6, contain the treatment of CpG single chain deoxynucleotide and the treatment of ribavirin combined utilization and prophylaxis of viral infections and susceptible malicious method and technology method and the technology imagination that infects the relative disease that causes of virus with artificial synthetic.

7, according to the described ribavirin of claim 5-6, it can be a this compound of ribavirin, also can be through the ribavirin of chemically modified and the derivative of ribavirin.

8, according to the described CpG strand deoxy-oligonucleotide that contains of claim 1-3, uniting the antiviral effect that the back produces with ribavirin, these viruses can include but not limited to influenza virus, foot and mouth disease virus, dengue fever virus, japanese encephalitis virus, hepatitis C virus, hepatitis B virus, Human Immunodeficiency Virus and papilloma virus.

9, according to the described ribavirin of claim 5-7, in the antiviral effect that produces when containing CpG strand deoxy-oligonucleotide combined utilization, these viruses can include but not limited to influenza virus, foot and mouth disease virus, dengue fever virus, japanese encephalitis virus, hepatitis C virus, hepatitis B virus, Human Immunodeficiency Virus and papilloma virus.

10, according to the described CpG strand deoxy-oligonucleotide that contains of claim 1-3, when uniting with ribavirin to the treatment of diseases that includes but not limited to by virus cause and the effect of prevention by influenza virus, foot and mouth disease virus, dengue fever virus, japanese encephalitis virus, hepatitis C virus, hepatitis B virus, Human Immunodeficiency Virus and parillomarvirus infections.

11, ribavirin in accordance with claim, when containing CpG strand deoxy-oligonucleotide combined utilization to include but not limited to the treatment of diseases that influenza virus, foot and mouth disease virus, dengue fever virus, japanese encephalitis virus, hepatitis C virus, hepatitis B virus, Human Immunodeficiency Virus and parillomarvirus infections cause and the effect of prevention by virus.

12, contain treatment of CpG single chain deoxynucleotide and ribavirin combined utilization and prevention are included but not limited to the disease that influenza virus, foot and mouth disease virus, dengue fever virus, japanese encephalitis virus, hepatitis C virus, hepatitis B virus, Human Immunodeficiency Virus and parillomarvirus infections cause by virus technological method and technology imagination.