CN1903870B - Single chain DNA having therapeutic action against virus infection disease - Google Patents

Abstract

The present invention provides a batch of single-stranded deoxynucleotides, which can stimulate human cells to produce substances that inhibit the replication of viruses, including but not limited to hepatitis B virus, so that they can treat viral infectious diseases including but not limited to hepatitis B Hepatitis B caused by the hepatitis virus has a therapeutic effect.

Description

Single-stranded deoxynucleotides with therapeutic effect on viral infectious diseases

field of invention

The present invention provides a batch of single-stranded deoxynucleotides (oligonucleotides, hereinafter referred to as oligos) that have a therapeutic effect on viral infectious diseases, including but not limited to hepatitis B caused by hepatitis B virus infection. Can stimulate human cells to produce anti-viral substances, these anti-viral substances can inhibit the replication of viruses including but not limited to hepatitis B virus, and thus have effects on viral infectious diseases including but not limited to hepatitis B caused by hepatitis B virus Therapeutic effect.

Background of the invention

Viruses are a class of non-cellular microorganisms that are small in size and relatively simple in structure, and can only replicate in living cells. The structure of a virus includes nucleic acid, capsomer and envelope. The nucleic acid of the virus, that is, the genome of the virus is the core of the virus, which determines the genetics, mutation and replication of the virus. Capsomers and cell membranes protect the viral core and are antigenic. According to the type of viral nucleic acid, viruses are divided into DNA viruses and RNA viruses. DNA viruses include double-stranded DNA viruses and single-stranded DNA viruses; RNA viruses include single positive-strand RNA viruses, single-negative-strand RNA viruses, double-stranded RNA viruses, and retroviruses. DNA viruses include, but are not limited to: hepatitis B virus, TTV virus, adenovirus, papilloma virus, herpes zoster virus, smallpox virus, and vaccinia virus. RNA viruses include but are not limited to: influenza virus, swine fever virus, hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, rabies virus, Ebola virus, enterovirus and human immunodeficiency virus.

Viral hepatitis is an infectious liver inflammatory disease caused by hepatitis virus. There are seven types of viruses that cause viral hepatitis, namely hepatitis A virus (Hepatitis A virus, HAV), hepatitis B virus (Hepatitis B virus, HBV), hepatitis C virus (Hepatitis C virus, HCV), D Hepatitis D virus (HDV), hepatitis E virus (HEV), hepatitis G virus (HGV) and TTV (transfusion-transmitted viral hepatitis virus), which can cause A Type B, C, D, E, G and TTV hepatitis.

Hepatitis A virus (HAV) is a spherical, non-enveloped, single-stranded RNA virus. Hepatitis A virus hepatitis A is a kind of intestinal infectious disease caused by HAV (Atmar, R.L., et al. Detection of enteric viruses inoyster by using the polymerase chain reaction. Appl. Environ. Microbiol. 1993, 59 :631-635.). Symptoms of acute hepatitis A include fever, loss of appetite, weakness, hepatomegaly, and abnormal liver function. Only a small number of patients with acute hepatitis A develop jaundice. Viral hepatitis A generally does not turn into a chronic and pathogen-carrying state. Hepatitis A patients are most contagious within 10 days from the late incubation period to onset. Hepatitis A generally occurs in children and adolescents, and is less common in adults. The general incubation period is 2 to 6 weeks. Winter and spring are the peak periods of hepatitis A incidence (JENNIFER A. CUTHBERT et al. Hepatitis A: Old and New. CLINICAL MICROBIOLOGY REVIEWS, Jan. 2001, p. 38-58).

Hepatitis C virus (HCV) is a flavivirus with a diameter of about 50-60 nm and a lipid envelope, a single-stranded positive-sense RNA virus. Hepatitis C is mainly spread through blood and blood products. Data show that 90% of hepatitis infected due to blood transfusion belongs to hepatitis C (JANICE M.MATTHEWS-GREER, et al.Comparison of Hepatitis C Viral Loads in Patients with or without Human Immunodeficiency Virus.CLINICAL AND DIAGNOSTIC LABORATORYIMMUNOLOGY, pJuly2001, .690-694; Luo Kangxian et al., Subtypes of hepatitis C virus and their possible implications. Journal of Clinical Hepatobiliary Diseases, 1994, 10(2):61-62.) Hepatitis C and the occurrence of liver cirrhosis and hepatocellular carcinoma Closely related (NIZARN.ZEIN.Clinical Significance of Hepatitis C Virus Genotypes.CLINICAL MICROBIOLOGYREVIEWS, 2000, p.223-235; Yang Yongping, Cloning the C region gene of hepatitis C virus genome from Chinese patients and its expression in Escherichia coli Acta Virology, 1994, 10(2): 109-117).

Hepatitis D virus (HDV) is a defective virus whose nucleic acid is a single negative-sense circular RNA (Theo Heller and Jay H.Hoofnagle. Denying the wolf access to sheep's clothing. J. Clin. Invest. 2003, 112: 319 -321). As a defective virus, HDV needs the help of hepadnaviruses such as HBV to complete its life cycle. The transmission mode of hepatitis D virus is similar to that of hepatitis B virus, with vertical transmission, sexual contact and bodily fluid transmission as the main modes of transmission (Rizzetto, M., et al.1980.Transmission of the hepatitis B virus-associated deltaantigen to chimpanzees. J. Infect. Dis. 141:590-602). Infection with HDV can aggravate the clinical symptoms of HBV-infected patients (Hadler, S.C. et al. Delta virus infection and severe hepatitis. An epidemic in the Yucpa Indians of Venezuela. Ann. Intern. Med. 1984.100: 339-344.).

Hepatitis E virus (HEV) is a spherical non-enveloped virus with a diameter of 32-34nm. Its nucleic acid is a single positive-strand RNA containing 3 reading frames (Reyes, G.R., et al. Isolation of a cDNA from the virus responsible forenterically transmitted non-A, non-B hepatitis. Science. 1990, 247:1335-1339; Reyes, G.R., et al. Hepatitis E virus (HEV): the novel agent responsible for enterically transmitted non-A, non-B hepatitis. Gastroenterol .Jpn.1991, 26(Suppl.3):142-147.). Hepatitis E, like hepatitis A, is transmitted through the fecal-oral route, and large-scale outbreaks and epidemics are caused by polluted water sources. The pathological damage of hepatitis E is more obvious than that of hepatitis A, and the recovery is slow.

Hepatitis G virus (HGV/GBV-C) is an enveloped, single-stranded positive-sense RNA virus of the Flaviviridae family. It mainly replicates in the liver and is mainly transmitted through blood products, intravenous drug use, sexual contact, and vertical transmission from mother to child. Hepatitis G virus can cause acute and chronic hepatitis, and is even closely related to the occurrence of liver cancer and cirrhosis (Schmidt, B., K. Korn, and B. Fleckenstein. Molecular evidence for transmission of hepatitis G virus by blood transfusion.Lancet.1996 , 347:909; Iban~ez, A., M. Gime'nez-Barcons, et al. Prevalence and genotypes of GB virus C/hepatitis G virus (GBV-C/HGV) and hepatitis C virus (HCV) among human immunodeficiency virus (HIV) infected patients: evidence of GBV-C/HGV sexual transmission. J.Med.Virol.1998, 55:293-299; Wang Yongmei, research status of hepatitis G. Journal of Preventive Medicine Information, 2001, 17(4): 257-258; Sun Ke'er et al., Research Status of Hepatitis G Virus. Foreign Medical Virology Volume, 1997, 4(2): 33-36.).

Viral hepatitis transfusion transmitted virus (transfusion transmitted virus, TTV) is a non-enveloped, single-stranded DNA virus (C.-L.lin, et al. Fecal Excretion of a Novel Human Circovirus, TT Virus, inHealthy Children. Clinical and diagnostic laboratory immunology, 2000, p.960-963). The genome of TTV virus is circular DNA (Isa k.Mushahwar, et al.Molecular and biophysicalcharacterization of TT virus: Evidence for a new virus family infecting humans.Proc.Natl.Acad.Sci.USA Vol.96, pp.3177 -3182, March 1999. Microbiology). TTV virus can be transmitted through sexual contact, vertical transmission, etc., and the clinical symptoms of TTV infected patients are mild (Claudia Fornai, et al. High Prevalence of TT Virus (TTV) and TTV-Like Minivirus in Cervical Swabs. JOURNAL OF CLINICAL MICROBIOLOGY , 2001, p.2022-2024).

Hepatitis B virus (HBV) is a DNA virus belonging to the family Hepadnaviridae. Complete HBV consists of two parts, the envelope and the core. Hepatitis B surface antigen (HBsAg) is a major component of the envelope. HBsAg is synthesized in liver cells and released into the blood circulation in large quantities. It is not infectious in itself. The core part contains incomplete circular double-stranded DNA, DNA polymerase, core antigen (HBcAg) and e antigen (HBeAg), which is infectious. Hepatitis B virus is caused by hepatitis B virus, which seriously threatens human health and is an inflammatory damage disease of the liver. As of 2004, 400,000,000 people worldwide have been infected with hepatitis B virus (Lin KW, Kirchner JT.Hepatitis B.Am FamPhysician, 2004Jan1, 69(1):75-82.), among them, Asians account for most.

In order to develop therapeutic drugs for hepatitis B virus infection at the cellular level, Mary ann sells et al constructed HepG2.2.15 cells. HepG2.2.15 cells are liver cancer cells (HepG2 cells) stably transfected with HBV genome, which can stably secrete HBsAg, HBeAg and Dane particles. During the long-term subculture process, HepG2 cells can still maintain many functions of normal hepatocytes, such as ensuring the normal replication of HBV in them, expressing interferon, Toll-like receptors and other receptors (Volker Mersch -Sundermann. Use of a human-derived liver cell line for the detection of cytoprotective, antigetoxic and cogenotoxic agents. Toxicology, 2004, 198:329-340; Mary ann sells, Mei-long chen, and George_acs. Production of vi les part hepatitis Hep G2cells transfected with cloned hepatitis B virus DNA. Proc. Natl. Acad. Sci. USA, 1987, 84:1005-1009). Studies have shown that antiviral substances including but not limited to interferon can bind to the corresponding receptors of HepG2.2.15 cells, thereby exerting an antiviral effect. After acting on the corresponding receptors on the surface of HepG2.2.15 cells, interferon can induce cells to produce interferon regulatory factor 1 (IFNregulatory factor 1, IFN-1), RNase L (RNase L) and bis Stranded RNA activated protein kinase (double-strandedRNA activated protein kinase, PKR) expression to exert anti-virus activity including but not limited to HBV (Bachmaier, K. et al.IiNOS expression and nitrotyrosine formation in the myocardiumin response to inflammation is controlled by the interferon regulatory transcription factor1. Circulation, 1997, 96:585-591; Pitha, P.M., et al.Role of the interferon regulatory factors in virus-mediated signaling and regulation of cell. growth. Biochimie, 1991, 80: 6 658; Zhou, A. et al. Expression cloning of 2-5A-dependent RNAase: uniquely regulated mediator of interferon action. Cell, 1993, 72:753-765). Using HepG2.2.15 cells for in vitro experiments, the activity of antiviral substances can be expressed as the decrease of HBsAg, HBeAg and HBV nucleic acid content in the cell culture supernatant. Therefore, HepG2.2.15 cell is a better cell model for studying anti-HBV drugs and preparations. In the present invention, we use human peripheral blood mononuclear cell (PBMC) culture supernatant and HepG2.2.15 cell effect induced by single-stranded deoxynucleotide, according to the content of HBsAg, HbeAg and HBV nucleic acid detected in the supernatant To determine the biological effect of single-stranded deoxynucleotides against hepatitis virus.

The single-stranded deoxynucleotides described in the present invention can be chemically modified in various ways. These chemical modifications include, but are not limited to, phosphate backbone modifications, sugar ring modifications, and base modifications. The modification of the phosphate backbone may be a thio modification. Thiomodification refers to the replacement of non-bridging oxygen atoms in phosphodiester bonds in nucleotide fragments by sulfur atoms (Ekambar R.Kandimalla et al.Mixed backboneantisense oligonucleotides: design, biochemical and biological properties ofoligonucleotides containing2-5-ribo-and3 -5-deoxyribonucleotide segments. Nuclei.c Acids Research, 1997, 25 (2): 370-378), this substitution can occur in part or all of the phosphodiester bond of the deoxyribonucleotide. Modification of the phosphate backbone can be achieved by formation of other types of phospholipid linkages including, but not limited to, methylphosphonate, phosphoroselenoate, borylphosphonate, and phosphorodithioate. The modification of the sugar ring can be the modification of the 2' position of the pentose sugar. The 2' modification of pentose includes but not limited to methoxy, methoxyethoxy, propyleneoxy and fluoro modification (Ernst Urban, Christian R.Noe.Structural modifications of antisense oligonucleotides.Il Farmaco.2003,58:243-258 ). Base modification mainly refers to the inclusion of rare bases in single-stranded deoxynucleotides (Xiaolan Chen, Nancy Dudgeon, Long Shen and Jui H.Wang. Drug Discovery Target, 2005, 10(8):587-593), rare bases are Refers to some bases other than A, G, C, U, including dihydrouracil (DHU), pseudouracil (ψ, pseudouridine) and methylated purine (mG, mA), etc. In general pyrimidine nucleosides, the N-1 on the heterocycle is connected to the C-1' of the sugar ring to form a glycosidic bond, and the pseudouridine is connected to the C-1' of the sugar ring by C-5 on the heterocycle.

The addition of single-stranded deoxynucleotide bases refers to the single-stranded deoxynucleotide formed by adding 1 to 10 bases to one or both ends of the single-stranded deoxynucleotide provided by the present invention. The deletion at both ends refers to the single-stranded deoxynucleotide formed by deleting one or several bases at one or both ends of the single-stranded deoxynucleotide provided by the present invention.

The base change of the single-stranded deoxynucleotide refers to the single-stranded deoxynucleotide formed by changing the base of the single-stranded deoxynucleotide provided by the present invention.

Single-stranded deoxynucleotides include, but are not limited to, the sequences shown in the appended list, the sequences shown in Example 2, and the sequences deduced according to the formula shown in Example 2.

This study proves that the stimulation of human PBMC culture supernatant by single-stranded nucleotides can inhibit the production of HBsAg, HBeAg and HBV nucleic acids by HepG2.2.15 cells. Therefore, the results provided by the present invention indicate that single-stranded nucleotides can inhibit the replication of viruses and have a therapeutic effect on viral infections, and these viruses include but are not limited to HBV.

The single-stranded deoxynucleotides of the present invention can be used for the treatment of viral infectious diseases. The viruses that cause these viral infectious diseases include but are not limited to hepatitis B virus, TTV virus, adenovirus, papillomavirus, herpes zoster virus, Smallpox and vaccinia viruses, influenza virus, swine fever virus, hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, rabies virus, Ebola virus, enterovirus and Human Immunodeficiency Virus.

Contents of the invention

One of the objectives of the present invention is to provide artificially synthesized single-stranded deoxynucleotides, which can be used to treat viral infectious diseases. The viruses that cause such viral infections include but are not limited to hepatitis B virus.

The second object of the present invention is to provide technical methods and technical ideas for treating viral infectious diseases with any single-stranded deoxynucleotides. The viruses that cause such viral infections include but are not limited to hepatitis B virus.

The third object of the present invention is to provide single-stranded deoxynucleotide sequences that have a therapeutic effect on viral infectious diseases, including but not limited to viral infectious diseases caused by hepatitis B virus. Chemically modified core sequence, these chemical modifications include but not limited to phosphate backbone modification, sugar ring modification and base modification. These single-stranded deoxynucleotides are the core structure of single-stranded deoxynucleotides produced by adding/introducing or changing bases at both ends and in the middle.

The present invention will be further described in detail below in conjunction with specific preparation examples and biological effect examples, and with reference to the accompanying drawings. It should be understood that these examples are only for illustration of the present invention, but not to limit the scope of the present invention in any way.

Example

In the following examples, various processes and methods not described in detail are conventional methods well known in the art, for example, the solid-phase phosphoramidite triester method is used for synthesis.

In the following examples, the sources and trade names of the reagents used and/or those whose constituents must be listed are only indicated once. The same reagents used thereafter will not repeat the above content unless otherwise specified.

Embodiment 1, the synthesis of single-stranded deoxynucleotide

The solid-phase phosphoramidite triester method is used to synthesize DNA fragments. This method has the advantages of high efficiency and rapidity, and has been widely used in DNA chemical synthesis.

DNA chemical synthesis is different from enzymatic DNA synthesis. The enzymatic DNA synthesis process extends from the 5'→3' direction, while DNA chemical synthesis starts from the 3' end. Concrete reaction steps are as follows:

1. Deprotection group

Use trichloroacetic acid to remove the protective group DMT (dimethoxytrityl) of the nucleotides linked to CPG (Controlled Pore Glass) to obtain a free 5'-hydroxyl terminal for the next condensation reaction use.

2. Activation

The phosphoramidite-protected nucleotide monomer is mixed with tetrazolium activator and enters the synthesis column to form phosphoramidite tetrazolium active intermediate (its 3'-end has been activated, but the 5'-end is still protected by DMT protection), this intermediate will undergo a condensation reaction with the deprotected nucleotide on GPG.

3. Connection

When the phosphoramidite tetrazole active intermediate encounters the nucleotide that has been deprotected on CPG, it will undergo an affinity reaction with its 5'-hydroxyl group, condense and remove the tetrazole, and the synthesized oligonucleotide chain will extended by one base.

Four, closed

After the condensation reaction, in order to prevent the unreacted 5'-hydroxyl attached to the CPG from being extended in the subsequent cycle reaction, the terminal hydroxyl is often blocked by acetylation. Generally, the acetylation reagent is acetic anhydride and N-methyl Formed by mixing imidazole and so on.

5. Oxidation

During the condensation reaction, the nucleotide monomer is connected to the oligonucleotide connected to the CPG through a phosphite bond, and the phosphite bond is unstable and easily hydrolyzed by acid and alkali. Phosphoryls are converted to phosphotriesters, resulting in stable oligonucleotides.

After the above five steps, a deoxynucleotide is connected to the nucleotide of CPG, and then trichloroacetic acid is used to remove the protective group DMT on the 5'-hydroxyl group of the newly connected deoxynucleotide. , Repeat the above activation, ligation, closure, oxidation process to get a crude DNA fragment. Finally, it is cut, deprotected (generally A and C bases are protected with benzoyl group; G base is protected with isobutyryl group; T base is not required to be protected; phosphorous acid is protected with nitrile ethyl group), purification (commonly used There are HAP, PAGE, HPLC, C18, OPC and other methods), quantification and other synthetic post-processing to obtain oligonucleotide fragments that meet the experimental requirements.

Embodiment 2, programming

The design sequence is as follows:

1. Artificially synthesized single-stranded deoxynucleotides (X1=A, T, G;

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

1.5'-ggggTCgTTCgTCgTTgggggg-3'

2.5'-ggggATAACgTTgCgggggg-3'

3.5'-ggggTgCAACgTTCAgggggg-3'

4.5'-ggggTCCTACgTAggAgggggg-3'

5.5'-ggggTCCATgACgTTCCTgAAgggggg-3'

6.5'-gggggACgTCgCCgggggggg-3'

7.5'-ggATCCgTACgCATgggggg-3'

8.5′-gggggAATCgATTCgggggg-3′

9.5′-gggATgCATCgATgCATCgggggg-3′

10.5'-ggTgCgACgTCgCAgggggg-3'

11.5'-gggACgTACgTCgggggg-3'

12.5'-gggggATCgACgTCgATCgggggg-3'

13.5′-ggCgATCgATCgATCgggggggg-3′

14.5'-ggggTCgATCgATCgAgggggg-3'

15.5'-ggTCgCgATCgCgAgggggg-3'

16.5'-ggGGTCAACGTTGAggggggG-3'

17.5'-gTCgTTTTCgTCgACgAATTgggggggg-3'

18.5'-gTCgTTATCgTTTTTTCgTAgggggg-3'

19.5'-ggCgTTAACgACgggggg-3'

20.5'-gTCggCACgCgACgggggg-3'

21.5'-ggTgCgACgTCgCAgggggg-3'

22.5'-gTCTATTTTgTACgTACgTgggg-3'

23.5'-gACgTCgACgTCgACgTCAggggg-3'

24.5'-ggggTCgATCgTTgCTAgCgggggg-3'

25.5'-gggggACgTTATCgTATTggggggg-3'

26.5'-ggggTCgTCgTTTgTCgTgTgTCgTTgggggg-3'

27.5'-ACgATCgATCgATCgggggg-3'

28.5'-AgACgTCTAACgTCggggg-3'

29.5'-ggggTgCTggCCgTCgTTgggggg-3'

30.5'-ggggTCgTTgCCgTCgggggg-3'

31.5'-ACCggTATCgATgCCggTgggggg-3'

32.5'-TTCgTTgCATCgATgCATCgTTgggggg-3'

2. The artificially synthesized single-stranded deoxynucleotide (X=A, T; Y=A, T; L, M =A, T, C, G; n is 0-6)

33.5'-ggggACgATACgTCggggggg-3'

34.5'-ggggACgATATCgATgggggg-3'

35.5'-ggACgATCgATCgTgggggg-3'

36.5'-TCggggACgATCgTCgggggg-3'

37.5′-gggggATCgATATCgATCgggggg-3′

38.5'-ggATCgATCgATCgATgggggg-3'

39.5′-ggTgCATCgATCgATgCAgggggg-3′

40.5′-ggTgCATCgTACgATgCAgggggg-3′

41.5'-ggTgCgATCgATCgCAgggggg-3'

42.5'-ggggggggTCgATCgATgggggg-3'

43.5'-ggggTCgTCgAACgTTgggggg-3'

44.5'-TgTCgTTCCTTgTCgTT-3'

45.5'-TTCgCTTCgCTTTTCgCTTCgCTT-3'-3'

46.5'-ACCgCCAAggAgAAgCCgCAggAggg-3'

47.5'-TACAACggCgAggAATACC-3'

48.5'-gTACAACggCgAggAATACCT-3'

49.5'-ACCgTCgTTgCCgTCggCCC-3'

50.5'-TgCTggCCgTCgTT-3'

51.5'-gTCggCACgCgACg-3'

52.5'-gTCggCACgCgACgCCCCCC-3'

53.5'-TCCCgCTggACgTT-3'

54.5'-TTACCggTTAACgTTggCCggCC-3'

55.5'-ACCggTTAACgTTgTCCCCgggg-3'

56.5'-CgTTgACgATCgTCCCATggCggg-3'

57.5'-TCTgCggCCTTCgTCg-3'

58.5'-TAgTAACCggTCCggCgCCCCC-3'

59.5'-TTgCAgCgCTgCCggTggg-3'

60.5'-CggCCCATCgAgggCgACggC-3'

61.5'-TCATCgACTCTCgAgCgTTC-3'

62.5'-ATCgTCgACTCTCgTgTTCTC-3'

63.5'-TgCAgCTTgCTgCTTgCTgCTTC-3'

64.5'-ggTgCgACgTCgCAgATgAT-3'

65.5'-ggTCgAACgTTCgAgATgAT-3'

66.5'-gggggCgTCgTTTTCgTCgACgAATT-3'

67.5'-actcgagacgcccgttgatagctt-3'

68.5'-AACgTTggCgTCgACgTCAgCgCC-3'

69.5'-gACgTCgACgTTgACgCT-3'

70.5'-ggCgTTAACgTTAgCgCT-3'

71.5'-AgCgCTAgCgCTgACgTT-3'

72.5'-CTAgACgTTCAAgCgTT-3'

73.5'-gACgATCgTCgACgATCgTC-3'

74.5'-gTCgTTCgTAgTCgACTACgAgTT-3'

75.5'-AAAAgACgTCgACgTCgACgTCTTTT-3'

76.5'-TgCgACgATCgTCgCACgATCggAT-3'

77.5'-TgCgACgTCgCACAgCgT-3'

3. Artificially synthesized single-stranded deoxynucleotides (L, M=A, T, C, G; n is 0-6) conforming to the formula of (TCG)n(L)nCG(M)n(G)n

78.5'-TCgTTgCCgTCgg-3'

79.5'-TCgTTgCCgTCggg-3'

80.5'-TCgTTgCCgTCgggg-3'

81.5'-TCgTTgCCgTCggggg-3'

82.5'-TCgTTgCCgTCgggggg-3'

83.5'-TCgTTgCCgTCgggggggg-3'

84.5'-TCgTTgCCgTCgggggggg-3'

85.5'-TCgTTgCCgTCgggggggggg-3'

86.5'-TCgTCgggTgCATCgATgCAgggggg-3'

87.5'-TCgTCgggTgCAACgTTgCAgggggg-3'

88.5'-TCgTCgggTgCgTCgACgCAgggggg-3'

89.5'-TCgTCgggTgCgATCgCAgggggg-3'

90.5'-TCgTCgggTgCgACgATCgTCgCAgggggg-3'

91.5'-TCgTCgTgCgACgTCgCAgggggg-3'

92.5'-TCgTCgCAgAACgTTCTgggggg-3'

93.5'-TCgTgCgACgTCgCAgggggg-3'

94.5'-TCgTgCgACgATCgTCgCAgggggg-3'

95.5'-TCgTATgCATCgATgCATAggAgg-3'

96.5'-TCgTgCATCgATgCAgggggg-3'

97.5'-TCgAAACgTTTCgggggg-3'

98.5'-TCggACgATCgTCgggggg-3'

99.5'-TCgAgCgATCgCTCgAgggggg-3'

100.5'-TCgTCgCTTTgTCgTTgggg-3'

101.5'-TCgTCgTTTTgTCgTTgggg-3'

102.5'-TCgTCgggTgCgACgTCgCAg-3'

103.5'-TCgTCgggTgCgACgTCgCAgg-3'

104.5'-TCgTCgggTgCgACgTCgCAggg-3'

105.5'-TCgTCgggTgCgACgTCgCAgggg-3'

106.5'-TCgTCgggTgCgACgTCgCAggggg-3'

107.5'-TCgTCgggTgCgACgTCgCAgggggg-3'

108.5'-TCgTCgggTgCgACgATCgTCgggggg-3'

109.5'-TCgTCgTTTgCATCgATgCAgggggggg-3'

110.5'-TCgTCgTTTTgACgATCgTCgggggg-3'

111.5'-TCgTTCggggTgCCg-3'

112.5'-TCgTTCggggTACCgATgggg-3'

113.5'-TCgTTgCgCTCCCATgCCgggggg-3'

114.5'-TCgTCgTTTCgTCgTTgggg-3'

115.5'-TCgTTgTCgTTTCgCTgCCggCggggg-3'

116.5'-TgCTTgggTggCAgCTgCCAgggggg-3'

117.5'-TgCTgCTTTgCTgCTTgggg-3'

118.5'-AACgTTCgACgTCgAACgggggggg-3'

119.5'-AACgACgACgTTggggg-3'

4. Synthetic single-strand deoxynucleotides (X1=A, T, G; X2=A, T; L, M=A, T, C) that meet the formula of (TCG)n(L)nX1X2CG(M)n , G; n is 0-6)

120.5'-TCgTAACgTTgTTTTTAACgTT-3'

121.5'-TCgTCgTATACgACgATCgTT-3'

122.5'-TCgTCgTTTgCgTTgTCgTT-3'

123.5'-TCCTgTCgTTTTgTCgTT-3'

124.5'-TCgTCgTTgTCgTTCgCT-3'

125.5'-TCgTCgTTACCgATgACgTCgCCgT-3'

126.5'-TCgTCgTTTgCATCgATgCAgTCgTCgTT-3'

127.5'-TCgCCTCgTCgCCTTCgAgC-3'g-3'

128.5'-TCgTgTgCgTgCCgTTggg-3'T-3'

129.5'-TCgTCgAgggCgCCggTgAC-3'

130.5'-TCgTCgCCggTgggggTgTg-3'

131.5'-TCgTCgTACgCAATTgTCTT-3'

132.5'-TCgCCCACCggTgggggggg-3'

133.5'-TCgTCgCAgACCggTCTgggg-3'

134.5'-TCgTCgCggCCggCgCCCCC-3'

135.5'-TCgTCgCggCCgCgAgggggg-3'

136.5'-TCgAggACAAgATTCTCgTgC-3'

137.5'-TCgAggACAAgATTCTCgTgCAggCC-3'

138.5'-TCgTgCAggCCAACgAggCCg-3'

139.5'-TCgTTgCCgTCggCCC-3'

140.5'-TCggCACgCgACgTgCTggCCgTCgTTTCC-3'

141.5'-TCgTTgCCgTCggCCCCCCCCC-3'

142.5'-TCgTTgCCgTCggCCCCCC-3'

143.5'-TCgTTgCCgTCggCCCCC-3'

144.5'-TCgTTgCCgTCggCCCC-3'

145.5'-TCgTTgCCgTCggCCCCCCC-3'

146.5'-TCgAggACAAgATTCTCgT-3'

147.5'-TCggCACgCgACgTgCTggCCgTCgTT-3'

148.5'-TCgTCgCgCCgTCACgggggg-3'

149.5'-TCgTgTgCgTgCCgTTggg-3'

150.5'-TCgTCgCCgTTgggCggg-3'

151.5'-TCgTCgACgTCgTTgggCggg-3'

152.5'-TCgCAgTTgTCgTAACgTTgggCggg-3'

153.5'-TCgTCgTTggTATgTT-3'

154.5'-TCgTCgTCgTCgTTgTCgTT-3'

155.5'-TCgTCgTCgTCgTTgTCgTTgggg-3'

156.5'-TCgTTCggggTgCCg-3'

157.5'-TCgTTCggggTAACgATT-3'

158.5'-TCgTTCggggTAACgTT-3'

159.5'-TCgTTCggggTACCgAT-3'

160.5'-TCgTACggCCgCCgTACggCggg-3'

161.5'-TCgCgTCgACTCCCCTCgAgggg-3'

162.5'-TCgTCgTCgACTCgTggTCggggg-3'

163.5'-TCgggCgCCCgATCgggggg-3'

164.5'-TCgTCggTCTTTCgAAATT-3'

165.5'-TCgTgACgTCCTCgAgTT-3'

166.5'-TCgTCTTTCgACTCgTTCTC-3'

167.5'-TCgTCgTTTTgCgTTCTC-3'

168.5'-TCgACTTTCgTCgTTCTgTT-3'

169.5'-TCgTCgTTTCgTCgTTCTC-3'

170.5'-tcgtcgggtgcgacgtcgca-3'

171.5'-TCgTTCTCgACTCgTTCTC-3'

172.5'-TCgACgTTCgTCgTTCgTCgTTC-3'

173.5'-TCgTCgACgTCgTTCgTTCTC-3'

174.5'-TCgTgCgACgTCgCAgATgAT-3'

175.5'-TCgTCgAgCgCTCgATCggAT-3'

176.5'-TCgTCgTTTCgTAgTCgTTgACgTCggg-3'

177.5'-TCgTCggACgTTTTCCgACgTTCT-3'

178.5'-TCgTCgTTTTCgTCgTTTTCgTCgTT-3'

179.5'-TCgTCgTTTgTCgTgTgTCgTT-3;

180.5'-TCgTCgTTggTCggggTCgTTggggTCgTT-3'

181.5'-TCgTCgTTTCgTCTCTCgTT-3'

182.5'-TCgTCgTTTTgCTgCgTCgTT-3'

183.5'-TCgAgCgTTTTCgCTCgAATT-3'

4. The artificially synthesized single-stranded deoxynucleotide containing the sequence formula of TTCGTCG

184.5'-TTCgTCgTTTgATCgATgTTCgTTgggggg-3'

185.5'-TTCgTCgTTgTgATCgATgggggg-3'-3'

186.5'-TATCgATgTTTTCgTCgTCgTTgggggg-3'

187.5'-TCgACTTTCgTCgTTCTgTT-3'

188.5'-TCgTCgTTTCgTCgTTCTC-3'

189.5'-TCgACgTTCgTCgTTCgTCgTTC-3'

190.5'-TCgTCgTTTTCgTCgTTTTCgTCgTT-3'

The sequence of the single-stranded deoxynucleotide (Oligo) used in embodiment 3,4,5 is as follows:

Oligo1 Seq No 89: 5'-TCgTCgggTgCgATCgCAgggggg-3'

Oligo2 seq No 92: 5'-TCgTCgCAgAACgTTCTgggggg-3'

Oligo4 seq No 95: 5'-TCgTATgCATCgATgCATAggAgg-3'

Oligo3 seq No 174: 5'-TCgTgCgACgTCgCAgATgAT-3'

Oligo5 seq No 164: 5'-TCgTCggTCTTTCgAAATT-3'

Oligo6 seq No 36:5′-TCggggACgATCgTCgggggg-3′

Oligo7 seq No 172: 5'-TCgACgTTCgTCgTTCgTCgTTC-3'

Oligo8 seq No 173: 5'-TCgTCgACgTCgTTCgTTCTC-3'

Oligo9 seq No 98: 5'-TCggACgATCgTCgggggg-3'

Oligo10 seq No 99: 5'-TCgAgCgATCgCTCgAgggggg-3'

Oligo11 seq No 107: 5'-TCgTCgggTgCgACgTCgCAgggggg-3'

Oligo12 seq No 170: 5'-TCgTCgggTgCgACgTCgCA-3'

Oligo13 seq No 102: 5'-TCgTCgggTgCgACgTCgCAg-3'

Oligo14 seq No 103: 5'-TCgTCgggTgCgACgTCgCAgg-3'

Oligo15 seq No 104: 5'-TCgTCgggTgCgACgTCgCAggg-3'

Oligo16 seq No 105: 5'-TCgTCgggTgCgACgTCgCAgggg-3'

Oligo17 seq No 106: 5'-TCgTCgggTgCgACgTCgCAggggg-3'

Oligo18 seq No 3: 5'-ggggTgCAACgTTCAgggggg-3'

Oligo19 seq No 1:5'-ggggTCgTTCgTCgTTgggggg-3'

Oligo20 seq No 66: 5'-gggggCgTCgTTTTCgTCgACgAATT-3'

Example 3, Single-stranded deoxynucleotides stimulate the inhibitory effect of human PBMC culture supernatant on the secretion of HBsAg from HepG2.2.15 cells

1. Isolation of Human Peripheral Blood Mononuclear Cells

1) Instruments and equipment: cryogenic refrigerators, carbon dioxide incubators, ultra-clean benches, inverted microscopes, liquid nitrogen tanks, distilled water devices, vacuum pumps, cell culture bottles, bacteria filters, filter bottles, straws of various specifications, Sampler, dropper, hemocytometer, horizontal centrifuge, etc.

2) Reagents and materials: Heparin-anticoagulated human whole blood was purchased from Changchun Central Blood Bank. Polysucrose-diatrizoate: specific gravity 1.077±0.001, purchased from Beijing Dingguo Biotechnology Co., Ltd. RPMI1640 culture solution: 10.4 grams of RPMI1640 (GIBCOBRL) of L-glutamine, 2.0 grams of sodium bicarbonate, 100,000 units of gentamicin, add triple distilled water to 1000 ml, filter the bacteria with a filter membrane of 0.22 microns, and separate Pack.

3) Method: Mononuclear cells from human peripheral blood were separated with Ficoll-diatrizoate lymphocyte stratification solution. The volume ratio of layered liquid to heparin anticoagulated peripheral blood is about 1:2. Centrifuge horizontally (1,000×g, 20 min). Aspirate the cell layer containing mononuclear cells with a pipette and place into another centrifuge tube. Add an equal volume of serum-free medium. Centrifuge at 1,000×g for 15 minutes and discard the supernatant. Repeat the wash twice. Discard the supernatant, resuspend the cells with 2ml culture medium, and count the cells.

2. Collection of human PBMC culture supernatant stimulated by single-stranded deoxynucleotides

Dilute the isolated PBMC (lymphocytes) with 10% FBS IMDM to a final concentration of 3× ¹⁰⁶ /ml, add 2ml to each well of the 12-well plate, that is, 6× ¹⁰⁶ /2ml/well; add each single-chain deoxygenated Nucleotides were added to a final concentration of 6 μg/ml, and three replicate wells were set up. Place the 12-well plate in a 5% CO ₂ incubator at 37°C for 48 hours, collect the culture supernatant, mix the culture supernatant collected from 3 duplicate wells, aliquot, and store at -20°C.

3. Inhibitory effect of human PBMC culture supernatant stimulated by single-stranded deoxynucleotides on the secretion of HBsAg from HepG2.2.15 cells

1) Inoculate 1×10 ⁵ cells/ml of HepG2.2.15 cells in a 24-well cell culture plate, 1 ml per well, and culture at 37°C with 5% CO ₂ . At 24 hours after culture, add single-stranded deoxynucleotide-stimulated Human PBMC culture supernatant was diluted to 1:64.

2) At 48h, 72h, and 96h after culture, the culture supernatant was collected respectively, and the content of HBsAg was detected with a kit (Suzhou Xinbo Biotechnology Co., Ltd.). The determination of the HBsAg content was performed according to the instructions provided by the kit.

3) Calculate the inhibition rate of HBsAg secretion: inhibition rate=(control well concentration-experimental well concentration)/control well concentration×100%

4. Results

The human PBMC culture supernatant stimulated by single-stranded deoxynucleotides can effectively inhibit the secretion of HBsAg from HepG2.2.15 cells (Table 1).

5 Conclusion

The human PBMC culture supernatant stimulated by single-stranded deoxynucleotides can inhibit the replication of HBV, so the single-stranded deoxynucleotides provided by the present invention have antiviral biological activity.

Example 4, Single-stranded deoxynucleotides stimulate the inhibitory effect of human PBMC culture supernatant on the secretion of HBeAg from HepG2.2.15 cells

1. Isolation of Human Peripheral Blood Mononuclear Cells

1) Instruments and equipment: cryogenic refrigerators, carbon dioxide incubators, ultra-clean benches, inverted microscopes, liquid nitrogen tanks, distilled water devices, vacuum pumps, cell culture bottles, bacteria filters, filter bottles, straws of various specifications, Sampler, dropper, hemocytometer, horizontal centrifuge, etc.

2) Reagents and materials: Heparin-anticoagulated human whole blood was purchased from Changchun Central Blood Bank. Polysucrose-diatrizoate: specific gravity 1.077±0.001, purchased from Beijing Dingguo Biotechnology Co., Ltd. RPMI1640 culture solution: 10.4 grams of RPMI1640 (GIBCOBRL) of L-glutamine, 2.0 grams of sodium bicarbonate, 100,000 units of gentamicin, add triple distilled water to 1000 ml, filter the bacteria with a filter membrane of 0.22 microns, and separate Pack.

3) Method: Mononuclear cells from human peripheral blood were separated with Ficoll-diatrizoate lymphocyte stratification solution. The volume ratio of layered liquid to heparin anticoagulated peripheral blood is about 1:2. Centrifuge horizontally (1,000×g, 20 min). Aspirate the cell layer containing mononuclear cells with a pipette and place into another centrifuge tube. Add an equal volume of serum-free medium. Centrifuge at 1,000×g for 15 minutes, discard the supernatant. Repeat the wash twice. Discard the supernatant, resuspend the cells with 2ml culture medium, and count the cells.

2. Collection of human PBMC culture supernatant stimulated by single-stranded deoxynucleotides

Dilute the isolated PBMC (lymphocytes) with 10% FBS IMDM to a final concentration of 3× ¹⁰⁶ /ml, add 2ml to each well of the 12-well plate, that is, 6× ¹⁰⁶ /2ml/well; add each single-chain deoxygenated Nucleotides were added to a final concentration of 6 μg/ml, and three replicate wells were set up. Place the 12-well plate in a 5% CO ₂ incubator at 37°C for 48 hours, collect the culture supernatant, mix the culture supernatant collected from 3 duplicate wells, aliquot, and store at -20°C.

3. Inhibition of the human PBMC culture supernatant stimulated by single-stranded deoxynucleotides on the secretion of HBsAg from HepG2.2.15 cells 1) Inoculate 1×10 ⁵ /ml HepG2.2.15 cells in a 24-well cell culture plate, 1ml per well , 37 ℃ 5% CO ₂ culture, in the first 24h after culture, add single-stranded deoxynucleotides to stimulate human PBMC culture supernatant to make the dilution factor 1:64.

2) At 48h, 72h, and 96h after culturing, collect the culture supernatants of each group in different time periods, and use a kit (Suzhou Xinbo Biotechnology Co., Ltd.) to detect the content of HBeAg, and the determination of HBeAg content is provided by the kit operation manual.

3) Calculate the inhibition rate of HBeAg secretion: inhibition rate=(control well concentration-experimental well concentration)/control well concentration×100%

4. Results

Various single-stranded deoxynucleotides can effectively inhibit the secretion of HBeAg from HepG2.2.15 cells by stimulating human PBMC culture supernatant at a dilution of 1:64 (Table 2).

5. Conclusion: The human PBMC culture supernatant stimulated by single-stranded deoxynucleotides can inhibit the replication of HBV, so the single-stranded deoxynucleotides provided by the present invention have antiviral biological activity.

Embodiment 5, single-stranded deoxynucleotide stimulates the inhibition of human PBMC culture supernatant to HepG2.2.15 cells secreting HBV DNA

1. Isolation of Human Peripheral Blood Mononuclear Cells

1) Instruments and equipment: cryogenic refrigerators, carbon dioxide incubators, ultra-clean benches, inverted microscopes, liquid nitrogen tanks, distilled water devices, vacuum pumps, cell culture bottles, bacteria filters, filter bottles, straws of various specifications, Sampler, dropper, hemocytometer, horizontal centrifuge, etc.

2) Reagents and materials: Heparin-anticoagulated human whole blood was purchased from Changchun Central Blood Bank. Polysucrose-diatrizoate: specific gravity 1.077±0.001, purchased from Beijing Dingguo Biotechnology Co., Ltd. RPMI1640 culture solution: 10.4 grams of RPMI1640 (GIBCOBRL) of L-glutamine, 2.0 grams of sodium bicarbonate, 100,000 units of gentamicin, add triple distilled water to 1000 ml, filter the bacteria with a filter membrane of 0.22 microns, and separate Pack.

3) Method: Mononuclear cells from human peripheral blood were separated with Ficoll-diatrizoate lymphocyte stratification solution. The volume ratio of layered liquid to heparin anticoagulated peripheral blood is about 1:2. Centrifuge horizontally (1,000×g, 20 min). Aspirate the cell layer containing mononuclear cells with a pipette and place into another centrifuge tube. Add an equal volume of serum-free medium. Centrifuge at 1,000×g for 15 minutes and discard the supernatant. Repeat the wash twice. Discard the supernatant, resuspend the cells with 2ml culture medium, and count the cells.

2. Collection of human PBMC culture supernatant stimulated by single-stranded deoxynucleotides

Dilute the isolated PBMC (lymphocytes) with 10% FBS IMDM to a final concentration of 3× ¹⁰⁶ /ml, add 2ml to each well of the 12-well plate, that is, 6× ¹⁰⁶ /2ml/well; add each single-chain deoxygenated Nucleotides were added to a final concentration of 6 μg/ml, and three replicate wells were set up. Place the 12-well plate in a 5% CO ₂ incubator at 37°C for 48 hours, collect the culture supernatant, mix the culture supernatant collected from 3 duplicate wells, aliquot, and store at -20°C.

3. Inhibitory effect of human PBMC culture supernatant stimulated by single-stranded deoxynucleotides on HepG2.2.15 cells secreting HBV DNA

1) Inoculate 1×10 ⁵ cells/ml HepG2.2.15 cells in a 24-well cell culture plate, 1ml per well, and add single-stranded deoxynucleotides to stimulate the human PBMC culture supernatant to make the dilution factor 24 hours after culture for 1:64.

2) 48h, 72h, and 96h after culturing, collect the culture supernatant of each group in different time periods respectively, and detect the content of HBV DNA with HBV DNA fluorescent quantitative PCR kit (Shanghai Shenyou Biotechnology Co., Ltd.), HBV DNA The determination of the content was performed according to the instructions provided by the kit.

3) Calculate HBV DNA secretion inhibition rate: inhibition rate=(control well log (copy number)-experimental well log (copy number))/control well log (copy number)×100%

4. Results

Each single-stranded deoxynucleotide stimulates human PBMC culture supernatant to effectively inhibit the secretion of HBV DNA from HepG2.2.15 cells at a dilution of 1:64 (Table 3).

5 Conclusion

The human PBMC culture supernatant can be stimulated by the single-stranded deoxynucleotides to inhibit the replication of HBV, so the single-stranded deoxynucleotides provided by the present invention have antiviral biological activity.

Table 1 Inhibitory effect of human PBMC supernatants stimulated by single-stranded deoxynucleotides on the secretion of HBsAg from HepG2.2.15 cells

Table 2 Inhibitory effect of human PBMC supernatants stimulated by single-stranded deoxynucleotides on the secretion of HBeAg from HepG2.2.15 cells

Table 3 Inhibitory effect of human PBMC supernatant stimulated by single-stranded deoxynucleotides on HepG2.2.15 cells secreting HBV DNA

sequence listing

<110>OrganizationName: Changchun Huapu Biotechnology Co., Ltd.

<120>Title: Single-stranded deoxynucleotides that have therapeutic effects on viral infectious diseases

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

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<211>23

<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

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<211>20

<212>DNA

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<400>168

<210>169

<211>19

<212>DNA

<213>Artificial

<400>169

<210>170

<211>20

<212>DNA

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<400>170

<210>171

<211>19

<212>DNA

<213>Artificial

<400>171

<210>172

<211>23

<212>DNA

<213>Artificial

<400>172

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<211>21

<212>DNA

<213>Artificial

<400>173

<210>174

<211>21

<212>DNA

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<211>21

<212>DNA

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<212>DNA

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<400>176

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<211>24

<212>DNA

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<400>177

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<212>DNA

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<212>DNA

<213>Artificial

<400>179

<210>180

<211>30

<212>DNA

<213>Artificial

<400>180

<210>181

<211>20

<212>DNA

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<400>181

<210>182

<211>21

<212>DNA

<213>Artificial

<400>182

<210>183

<211>21

<212>DNA

<213>Artificial

<400>183

<210>184

<211>30

<212>DNA

<213>Artificial

<400>184

<210>185

<211>24

<212>DNA

<213>Artificial

<400>185

<210>186

<211>28

<212>DNA

<213>Artificial

<400>186

<210>187

<211>20

<212>DNA

<213>Artificial

<400>187

<210>188

<211>19

<212>DNA

<213>Artificial

<400>188

<210>189

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<212>DNA

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<400>190

Claims (14)

1. single chain deoxynucleotide, it is the dna molecular of strand, its length is 6-50 base, and meets (TCG) n (L) nCG (M) n (G) n formula, L wherein, M=A, T, C, G; N is 0-6, and the sequence of wherein said single chain deoxynucleotide is SEQ ID No:89.

2. according to the described single chain deoxynucleotide of claim 1, its phosphodiester bond can be unvulcanised, partial vulcanization or complete cure, also can be the phosphodiester bond of other form.

3. according to claim 1 or 2 described single chain deoxynucleotides, they can stimulate people's cell to produce antiviral substance.

4. according to claim 1 or 2 described single chain deoxynucleotides, they can stimulate the material of people's cell generation resisting DNA virus.

5. according to claim 1 or 2 described single chain deoxynucleotides, they can stimulate people's cell to produce the material of hepatitis virus resisting.

6. according to claim 1 or 2 described single chain deoxynucleotides, they can stimulate people's cell to produce the material of anti-hepatitis B virus.

7. according to claim 1 or 2 described single chain deoxynucleotides, the material that they stimulate people's cell to produce can suppress duplicating of virus.

8. according to claim 1 or 2 described single chain deoxynucleotides, the material that they stimulate people's cell to produce can suppress duplicating of dna virus.

9. according to claim 1 or 2 described single chain deoxynucleotides, the material that they stimulate people's cell to produce can suppress duplicating of hepatitis virus.

10. according to claim 1 or 2 described single chain deoxynucleotides, the material that they stimulate people's cell to produce can suppress duplicating of hepatitis B virus.

11. according to claim 1 or 2 described single chain deoxynucleotides, they have antiviral effect, can treat disease of viral infection.

12. according to claim 1 or 2 described single chain deoxynucleotides, they have the effect of resisting DNA virus, can treat dna virus infection property disease.

13. according to claim 1 or 2 described single chain deoxynucleotides, they have the effect of hepatitis virus resisting, can treat hepatites virus infections property disease.

14. according to claim 1 or 2 described single chain deoxynucleotides, they have the effect of anti-hepatitis B virus, can treat the hepatitis B that is caused by hepatitis b virus infected.