CN1322898C

CN1322898C - Small interference RNA molecule SiRNA capable of attacking human hepatitis B virus and application thereof

Info

Publication number: CN1322898C
Application number: CNB031450407A
Authority: CN
Inventors: 丁佳逸; 郑树
Original assignee: HANGZHOU XINRUIJIA BIOLOGICAL MEDICINE TECHNOLOGY DEVELOPMENT Co Ltd
Current assignee: Lishui Reed Biological Medicine Co., Ltd.
Priority date: 2003-06-17
Filing date: 2003-06-17
Publication date: 2007-06-27
Anticipated expiration: 2023-06-17
Also published as: CN1566131A

Abstract

The present invention provides a small interference RNA molecule (SiRNA) for attacking human hepatitis B virus (HBV). The molecule is a double-chain RNA molecule, and the sequence of the molecule has at least 70% homological degree with a sequence (I) with the following characteristics: the lengths of a positive sense chain and an antisense chain are both 21 nucleotides; the 3' ends of the positive-sense chain and the antisense chain are respectively TT; basic groups on 19 nucleotides except for TT of the two chains are complemented into double chains. 19 nucleotide sequences of the positive-sense chain from the 5' end are completely consistent with 19 nucleotide sequences behind AA in No. 4 sequence of the sequence table; the proportion of the sum of the number of nucleotides whose basic groups are G and the number of nucleotides whose basic groups are C in each chain to the number of 19 nucleotides except for TT is larger than 25% and smaller than 75%. The antisense chain and the mutant of one nucleotide of the sequence (I) have no homology with known human genes and gene expression fragments. The SiRNA can be used for preparing medicines or preparations of preventing or treating hepatitis b and any disease relevant to hepatitis b virus infection.

Description

Attack the siRNA molecule (SiRNA) and the application thereof of human hepatitis B virus

Technical field

The present invention relates to the nucleic acid technical field, relate in particular to the siRNA molecule (SiRNA) and the application thereof of attacking human hepatitis B virus.

Background technology

Hepatitis B is a kind of disease of serious harm whole mankind health, the medicine that is used for the treatment of at present hepatitis B clinically has two classes, one class is the A Erfa interferon, it is to not directly effect of hepatitis B virus, and its antivirus action forms a kind of antiviral state by inducing hepatocyte and carries out; The second class lamivudine is a kind of inhibitor of nonspecific reverse transcriptase, thereby mainly to act on be that the reverse transcription process that suppresses RNA viruses reaches and suppresses duplicating of virus for it.Their common drawback is the poor specificity at hepatitis B virus, and the effective percentage that both treat hepatitis B lower (30%-40%), negative conversion rate to the surface antigen of hepatitis B virus is lower, and the A Erfa interferon is 6%, and the effect of lamivudine is also uncertain.

Summary of the invention

Technical problem to be solved by this invention provides the siRNA molecule (SiRNA) of attacking human hepatitis B virus.The present invention by the following technical solutions for this reason: it is double stranded rna molecule and its nucleotide sequence and the homology degree with nucleotide sequence (I) at least 70% of following feature: the length of positive-sense strand and antisense strand is 21 nucleotide, positive-sense strand and antisense strand 3 ' end separately is two successive deoxythymidylic acids (TT), article two, chain remove 3 ' end TT beyond 19 nucleotide on base complementrity form two strands, 19 nucleotide sequences after the positive-sense strand nucleotide that continuous two bases are adenine (A) in 19 nucleotide sequences of 5 ' end beginning and No. 4 nucleotide sequence of sequence table are in full accord, base in every chain is the nucleotide quantity of guanine (G) and nucleotide quantity sum that base is cytosine (C) ratio that accounts for 19 nucleotide quantity beyond the TT that removes 3 ' end for greater than 25% less than 75% (being the G/C ratio), and the mutant of the antisense strand of nucleotide sequence (I) and an one nucleotide and known person genoid and gene expression fragment do not have homology.

" no homology " this coding mutation body that is meant any one position of nucleotide sequence (I) antisense strand and its and known person genoid and gene expression fragment do not have 100% identical.

Above-mentioned siRNA molecule (SiRNA) is a synthetic, and positive-sense strand and antisense strand 3 ' end separately has two deoxythymidylic acids (TT), and the base of other 19 nucleotide can be adenine (A), guanine (G), cytosine (C) or uracil (U); Article two, chain is removed base complementrity (A and U, C and G) on 19 nucleotide beyond the TT of 3 ' end and is formed double-strandedly, but two TT of their 3 ' ends exist with the form of strand.

When No. 4 nucleotide sequence in the sequence table occurs that successive base is the nucleotide of adenine (A) more than two, above-mentioned " 19 nucleotide sequences after the nucleotide that continuous two bases of positive-sense strand No. 4 nucleotide sequence in 19 nucleotide sequences of 5 ' end Ji beginning and sequence table are adenine (A) are in full accord, " can be understood that " 19 nucleotide sequences after the nucleotide that any two successive bases of positive-sense strand No. 4 nucleotide sequence in 19 nucleotide sequences of 5 ' end beginning and sequence table are adenine (A) are in full accord.”

Human hepatitis B virus is to belong to a kind of little liver property DNA viruses of biting, the ring-shaped DNA molecule of the incomplete two strands that its genome is made up of about 3200 nucleotide, in sophisticated viral genome, it is made up of encode minus strand and incomplete normal chain of mRNA molecule of complete being used to.Though it belongs to DNA viruses, it is genomic duplicates with general DNA viruses differently, and quite similar with the retroviral (retrovirus) in the RNA viruses, needs to rely on a RNA intermediate to finish.Concrete process is behind virus infected cell, its dna molecular can be transported to nucleus, the ring-shaped DNA molecule of incomplete two strands can become maturation covalency shape there, closed ring-shaped DNA molecule (covalently closed circular DNA, ccc DNA), be that template utilizes endonuclear RNA transcriptase II to transcribe preceding gene RNA molecule longer slightly than minus strand of generation with the minus strand in this ccc dna molecular again, the effect of this molecule first is the template as the synthetic viral DNA of reverse transcription, the secondth, the same cAg (c antigen) and the multifunctional polymeric enzyme of being responsible for coding HBV with other mRNA molecule, therefore preceding gene RNA molecule is also referred to as cAg/polymerase (c/pol) mRNA molecule.

The breeding of hepatitis B virus except it is genomic duplicate, also need the synthetic of other function and structural protein, the mRNA molecule one of these structural protein of encoding has four kinds.The firstth, length is the e antigen mRNA of 3.5kb, and it also will be grown than c/ polymerase mRNA (preceding gene RNA) molecule, contains the promoter of e antigen protein, the e antigen protein of coding virus; The secondth, long is 2.4kb coding long surface protein (LHBs) mRNA, and it mainly synthesizes LHBs (being also referred to as pre-s1 protein); The 3rd is long mRNA for isometric in the 2.1kb coding and short surface protein, isometric in the coding (pre-s2 protein) and short surface protein (surface antigen protein that is often referred to); The 4th is the xmRNA that length has only the coding X protein of 0.7kb.The function of these mRNA encoded protein has nothing in common with each other, and X protein is that the replication relation of synthetic unique modulin of HBV and virus is close.Three kinds of surface antigens are the important structure albumen of virus, and wherein long surface antigen is virus and the bonded main molecules of hepatocyte still, plays crucial effect in the infectivity of virus.E antigen then with hepatitis B virus infection after being related closely of chronic hepatitis.

In the sequence table No. 4 nucleotide sequence be the inventor from the common sequences of ADR, the ADW of hepatitis B virus and three hypotypes of AYW obtain after through ordering common sequences.ADR, ADW are distributed in the hepatitis B virus hypotype that the Western Pacific prolongs the Asian-Pacific area hepatitis B country occurred frequently of bank, and AYW is the hepatitis B virus hypotype that mainly is distributed in many countries in Africa and Europe.Its process is the common sequences of at first finding out from the known viruse genome sequence of each hypotype separately (the the 1st, the 2nd, No. 3 sequence is respectively the common sequences of ADR, ADW and AYW hypotype in the sequence table), again these three common sequences are sorted, obtain the common sequences of ADR, ADW and three hypotypes of AYW, the used means that sort are the Clustal W that generally acknowledge, No. 4 sequence is exactly the positive-sense strand of this common sequences in the sequence table.The viral genome that wherein is used for ADR hypotype common sequences comprises in the number of landing of gene library: AB026811, AB026812, AB026813, AB026814, AB026815, AB033550, AB042282, AB042283, AB042284, AF411408, AF411409, AF411411, AF411412, AF458664, AF458665, AF461357, AF461361, AF461363; The ADW hypotype comprises: AB033551, AB033552, AB033553, AB033554, AB033555, AB033556, AB033557, AF100308, AF100309, AF282917, AF282918, HBVCGWITY, HBVP4CSX, HBVP6CSX, HPBADW1, HPBADW2, HPBADW3, HPBADWZ; AYW comprises: AB033558, AB033559, AB048701, AB048702, AB048703, HBVAYWGEN, HBVORFS, HBVP2CSX, HBVP3CSX, HBVP4PCXX, HBVP5PCXX, HBVP6PCXX, U95551, XXHEPA, XXHEPAV.

SiRNA molecule corresponding to this common sequences provided by the invention (SiRNA) can be specifically at human hepatitis B virus, it is by the preceding gene RNA molecule and the albumen messenger rna molecule of intracellular RISC (the RNA-induced silencing complex) hepatitis B virus of degrading effectively, the directly breeding of the duplicating of blocking virus genetic stew, virus, can also suppress it and cause a disease closely related proteinic synthesizing, find new effective means for treating acute and chronic hepatitis and liver cirrhosis closely-related and hepatocarcinoma with it.

When nucleotide sequence (I) also has following feature simultaneously, siRNA molecule provided by the present invention, the better effects if that it is realized:

1), the positive-sense strand of nucleotide sequence (I) is that base is a kind of in the nucleotide of guanine (G), cytosine (C) or uracil (U) from first nucleotide of 5 ' end Ji beginning; That is to say when occurring in No. 4 sequence of sequence table that successive base is the nucleotide of adenine (A) more than two, positive-sense strand from 19 nucleotide sequences of 5 ' end beginning and this more than two successive base be that 19 nucleotide sequences after latter two nucleotide in the nucleotide of adenine (A) are in full accord, ".

2), described continuous two bases are adenine (A) nucleotide is that the nucleotide at place is counted in following ranking in No. 4 sequence of sequence table, that is: in No. 1, No. 2 nucleotide sequence of sequence table, exist together mutually and be continuous two nucleotide that base is adenine (A) with this ranking number, and 19 nucleotide sequences after the nucleotide that these two bases are adenine (A) in No. the 1st, sequence table and No. 2 nucleotide sequence are in full accord or wherein have nucleotide different or two non-conterminous nucleotide differences are wherein arranged.

Following table has been listed the nucleotide sequence (I) that meets these conditions

Serial number positive-sense strand sequence antisense strand sequence MW* firing area #

5 UCUUCUCGAGGACUGGGGATT UCCCCAGUCCUCGAGAAGATT 13489.6 123-143

6 CCUCUUGUCCUCCAAUUUGTT CAAAUUGGAGGACAAGAGGTT 13459.4 342-362

7 GGUAUGUUGCCCGUUUGUCTT GACAAACGGGCAACAUACCTT 13474.5 458-478

8 GGAACCUCUAUGUUUCCCUTT AGGGAAACAUAGAGGUUCCTT 13459.4 542-562

9 CCUCUAUGUUUCCCUCNUGTT CANGAGGGAAACAUAGAGGTT 13466.5 546-566

10 CCUUCGGACGGAAACUGCATT UGCAGUUUCCGUCCGAAGGTT 13489.6 578-598

11 CUGCACCUGUAUUCCCAUCTT GAUGGGAAUACAGGUGCAGTT 13474.5 592-612

12 GUCUGUACAACAUCUUGAGTT CUCAAGAUGUUGUACAGACTT 13444.3 765-785

13 CAUCUUGAGUCCCUUUUUATT UAAAAAGGGACUCAAGAUGTT 13429.2 775-795

14 UUUUCUUUUGUCUUUGGGUTT ACCCAAAGACAAAAGAAAATT 13414.1 807-827

15 CUUCAUGGGAUAUGUAAUUTT AAUUACAUAUCCCAUGAAGTT 13414.1 873-893

16 CUUCCUGUAAACAGGCCUATT UAGGCCUGUUUACAGGAAGTT 13459.4 955-975

17 CAGGCCUAUUGAUUGGAAATT UUUCCAAUCAAUAGGCCUGTT 13444.3 966-986

18 UUGUGGGUCUUUUGGGNUUTT AANCCCAAAAGACCCACAATT 13451.4 998-1018

19 UGUGGNUAUCCUGCUUUAATT UUAAAGCAGGAUANCCACATT 13436.3 1036-1056

20 UGCCUUUAUAUGCAUGUAUTT AUACAUGCAUAUAAAGGCATT 13414.1 1055-1075

21 GCAGGCUUUCACUUUCUCGTT CGAGAAAGUGAAAGCCUGCTT 13474.5 1083-1103

22 CCUUUACCCCGUUGCCCGGTT CCGGGCAACGGGGUAAAGGTT 13519.8 1140-1160

23 CGGCCAGGUCUGUGCCAAGTT CUUGGCACAGACCUGGCCGTT 13519.8 1162-1182

24 GUGUUUGCUGACGCAACCCTT GGGUUGCGUCAGCAAACACTT 13489.6 1180-1200

25 CCCCCACUGGNUGGGGCUUTT AAGCCCCANCCAGUGGGGGTT 13526.9 1196-1216

26 CCUUUGUGGCUCCUCUGCCTT GGCAGAGGAGCCACAAAGGTT 13504.7 1244-1264

27 CUCCUAGCCGCUUGUUUUGTT CAAAACAAGCGGCUAGGAGTT 13474.5 1279-1299

28 UCCCGCGGACGACCCNUCUTT AGANGGGUCGUCCGCGGGATT 13526.9 1446-1466

29 GGUCUUACAUAAGAGGACUTT AGUCCUCUUAUGUAAGACCTT 13444.3 1646-1666

30 GAGGACUCUUGGACUCUCATT UGAGAGUCCAAGAGUCCUCTT 13474.5 1658-1678

31 UGUCAACGACCGACCUUGATT UCAAGGUCGGUCGUUGACATT 13474.5 1681-1701

32 CGACCGACCUUGAGGCAUATT UAUGCCUCAAGGUCGGUCGTT 13489.6 1687-1707

33 NGACUGGGAGGAGUUGGGGTT CCCCAACUCCUCCCAGUCNTT 13511.8 1727-1747

34 UUGGUCUGUUCACCAGCACTT GUGCUGGUGAACAGACCAATT 13474.5 1794-1814

35 CUUUUUCACCUCUGCCUAATT UUAGGCAGAGGUGAAAAAGTT 13444.3 1820-1840

36 UCAUCUCAUGUUCAUGUCCTT GGACAUGAACAUGAGAUGATT 13444.3 1839-1859

37 GCCUCCAAGCUGUGCCUUGTT CAAGGCACAGCUUGGAGGCTT 13504.7 1868-1888

38 GCUGUGCCUUGGGUGGCUUTT AAGCCACCCAAGGCACAGCTT 13504.7 1876-1896

39 GAAUUUGGAGCUUCUGUGGTT CCACAGAAGCUCCAAAUUCTT 13459.4 1922-1942

40 UUUGGAGCUUCUGUGGAGUTT ACUCCACAGAAGCUCCAAATT 13459.4 1925-1945

41 CAUUGUUCACCUCACCAUATT UAUGGUGAGGUGAACAAUGTT 13444.3 2039-2059

42 GCUAUUCUGUGUUGGGGUGTT CACCCCAACACAGAAUAGCTT 13474.5 2072-2092

43 UCUAGCCACCUGGGUGGGATT UCCCACCCAGGUGGCUAGATT 13504.7 2101-2121

44 UUAGUAGUCAGCUAUGUCATT UGACAUAGCUGACUACUAATT 13429.2 2150-2170

45 UAUGGGCCUAAAAAUCAGATT UCUGAUUUUUAGGCCCAUATT 13429.2 2176-2196

46 UCAGACAACUAUUGUGGUUTT AACCACAAUAGUUGUCUGATT 13429.2 2190-2210

47 CUAUUGUGGUUUCACAUUUTT AAAUGUGAAACCACAAUAGTT 13414.1 2198-2218

48 GAGAAACUGUUCUUGAGUATT UACUCAAGAACAGUUUCUCTT 13429.2 2235-2255

49 CUGUUCUUGAGUAUUUGGUTT ACCAAAUACUCAAGAACAGTT 13429.2 2241-2261

50 UGCCCCUAUCUUAUCAACATT UGUUGAUAAGAUAGGGGCATT 13444.3 2308-2328

51 CACUUCCGGAAACUACUGUTT ACAGUAGUUUCCGGAAGUGTT 13459.4 2325-2345

52 CUACUGUUGUUAGACGACGTT CGUCGUCUAACAACAGUAGTT 13459.4 2337-2357

53 GAAGAACUCCCUCGCCUCGTT CGAGGCGAGGGAGUUCUUCTT 13504.7 2373-2393

54 GAACUCCCUCGCCUCGCAGTT CUGCGAGGCGAGGGAGUUCTT 13519.8 2376-2396

55 GGUCUCAAUCGCCGCGUCGTT CGACGCGGCGAUUGAGACCTT 13519.8 2400-2420

56 UCGCCGCGUCGCAGAAGAUTT AUCUUCUGCGACGCGGCGATT 13504.7 2408-2428

57 GAUCUCAAUCUCGGGAAUCTT GAUUCCCGAGAUUGAGAUCTT 13459.4 2424-2444

58 UCUCGGGAAUCUCAAUGUUTT AACAUUGAGAUUCCCGAGATT 13444.3 2432-2452

59 UCUCAAUGUUAGUAUUCCUTT AGGAAUACUAACAUUGAGATT 13414.1 2441-2461

60 UGUUAGUAUUCCUUGGACUTT AGUCCAAGGAAUACUAACATT 13429.2 2447-2467

61 GGUGGGAAACUUUACGGGGTT CCCCGUAAAGUUUCCCACCTT 13489.6 2471-2491

62 CUUUACGGGGCUUUAUUCUTT AGAAUAAAGCCCCGUAAAGTT 13444.3 2480-2500

63 UUAAUUAUGCCUGCUAGGUTT ACCUAGCAGGCAUAAUUAATT 13429.2 2631-2651

64 UUAUGCCUGCUAGGUUUUATT UAAAACCUAGCAGGCAUAATT 13429.2 2635-2655

65 UAUUUGCCCUUGGAUAAAGTT CUUUAUCCAAGGGCAAAUATT 13429.2 2670-2690

66 CAAGAGCUACAGCAUGGGATT UCCCAUGCUGUAGCUCUUGTT 13474.5 2835-2855

67 GAGCUACAGCAUGGGAGGUTT ACCUCCCAUGCUGUAGCUCTT 13489.6 2838-2858

68 CCUCGANAAGGCAUGGGGATT UCCCCAUGCCUUNUCGAGGTT 13496.7 2869-2889

69 GGCAUGGGGACGAAUCUUUTT AAAGAUUCGUCCCCAUGCCTT 13474.5 2878-2898

70 UCUUUCUGUCCCCAAUCCUTT AGGAUUGGGGACAGAAAGATT 13459.4 2892-2912

71 CUCAGACAAUCCAGAUUGGTT CCAAUCUGGAUUGUCUGAGTT 13459.4 2958-2978

72 UCCAGAUUGGGACUUCAACTT GUUGAAGUCCCAAUCUGGATT 13459.4 2967-2987

73 UCGGCAGUCAGGAAGGCAGTT CUGCCUUCCUGACUGCCGATT 13504.7 3141-3161

74 GAGACACUCAUCCUCAGGCTT GCCUGAGGAUGAGUGUCUCTT 13489.6 3185-3205

75 CUGGAUCCUGCGCGGGACGTT CGUCCCGCGCAGGAUCCAGTT 13534.9 1398-1418

76 CUCCCUCGCCUCGCAGACGTT CGUCUGCGAGGCGAGGGAGTT 13534.9 2379-2399

77 UGAAAAAAGGAGACUAAAATT UUUUAGUCUCCUUUUUUCATT 13399 2612-2632

*: as N is arranged in the infructescence, the mean molecule quantity 339.2 of then getting A, U, C, four kinds of nucleotide of G calculates

#: firing area is meant the opposite position of this nucleotide sequence (I) in No. 4 sequence of sequence table.

For the numerical value of above said homology degree, with more than 80% for better.The said homology degree of the present invention is meant any SiRNA at human hepatitis B virus, the positive-sense strand or the antisense strand sequence of its nucleotide sequence and the said nucleotide sequence of the present invention (I), the identical rate of the nucleotide on the relevant position.

Effective inhibition human hepatitis B virus provided by the present invention duplicates siRNA molecule with protein expression and also comprises at any part of the sequence of above mentioned siRNA molecule (SiRNA) or whole formed siRNA molecules after chemical modification.

Described chemical modification comprises and mainly comprises following three classes: the first, the part of the phosphodiester bond that connects adjacent two nucleotide is modified or with other any chemical bond replacement.The part modification of typical phosphodiester bond is that the oxygen on the two keys of phosphoric acid is replaced as sulfur (sulfuration) or other element, or the oxygen on the phosphoric acid singly-bound is become nitrogen (nitrogenize) or other element and chemical group; Whole replacements to phosphodiester bond itself, also comprise partly and the part of two ribose that link to each other with it or whole change itself or its, typical illustration is that phosphodiester bond and two adjacent ribose are become peptide bond, make nucleic acid become peptide nucleic acid(PNA) (peptide nucleicacid, PNA); The second, the five-membered ring of ribose in the nucleoside is changed or its side chain on chemical group modify.The case history that changes ribose ring is as becoming five yuan ribose ring on the Morpholino ring of six rings; Modification on the ribose side chain is meant that mainly 2 ' in ribose is gone up OH becomes other element (as halogens), or with the H (for example alkyl) among the alternative OH of other chemical group.Three, the base ring on the nucleotide is done the whole change or the modification of side chain.

The siRNA molecule of attack human hepatitis B virus provided by the present invention (SiRNA) comprise to its modify formed SiRNA can be applied to prepare prevention or treatment human hepatitis B and with the medicine of hepatitis b virus infected relevant any disease or the application in the preparation.

Description of drawings

Fig. 1,12 kinds of SiRNA are to the effect of baicailin compound on hepatitis B.12 kinds of SiRNA of diagram 150nM act on the influence to HBsAg in its cell conditioned medium and HBeAg level after 72 hours of HepG2.2.15 cell.Matched group is that phalangeal cell deals with transfection reagent under condition of equivalent.Vertical coordinate represents that the antigen levels of SiRNA processed group accounts for the relative percentage of matched group.The meansigma methods of three repeated experiments of each data representation among the figure.

The effect of Fig. 2,12 kinds of interior hepatitis B virus DNA levels of SiRNA pair cell.12 kinds of SiRNA of diagram 150nM act on the influence of hepatitis B virus (HBV) dna level in the pair cell after 72 hours of HepG2.2.15 cell.Matched group is that phalangeal cell deals with transfection reagent under condition of equivalent.Vertical coordinate represents that the antigen levels of SiRNA processed group accounts for the relative percentage of matched group.The meansigma methods of three repeated experiments of each data representation among the figure.

The effect that three kinds of SiRNA of Fig. 3, variable concentrations express HBsAg.Diagram No. 2, No. 4 of variable concentrations and No. 6 molecular actions in the HepG2.2.15 cell after 72 hours to its cell conditioned medium in the influence of HBsAg level.Matched group is that phalangeal cell deals with transfection reagent under condition of equivalent.Vertical coordinate represents that the antigen levels of SiRNA processed group accounts for the relative percentage of matched group.The meansigma methods of three repeated experiments of each data representation among the figure.

The effect that three kinds of SiRNA of Fig. 4, variable concentrations express HBeAg.Diagram No. 2, No. 4 of variable concentrations and No. 6 molecular actions in the HepG2.2.15 cell after 72 hours to its cell conditioned medium in the influence of HBeAg level.Matched group is that phalangeal cell deals with transfection reagent under condition of equivalent.Vertical coordinate represents that the antigen levels of SiRNA processed group accounts for the relative percentage of matched group.The meansigma methods of three repeated experiments of each data representation among the figure.

The effect of hepatitis B virus DNA level in three kinds of SiRNA pair cells of Fig. 5, variable concentrations.Diagram No. 2, No. 4 of variable concentrations and No. 6 molecular actions in the HepG2.2.15 cell after 72 hours to its cell in the influence of dna level.Matched group is that phalangeal cell deals with transfection reagent under condition of equivalent.Vertical coordinate represents that the antigen levels of SiRNA processed group accounts for the relative percentage of matched group.The meansigma methods of three repeated experiments of each data representation among the figure.

The effect that Fig. 6, different SiRNA combinations are expressed HBsAg.Two kinds of SiRNA combination of diagram variable concentrations (No. 2 add No. 6, No. 2 add No. 4 add again No. 6) acts on the influence to HBsAg level in its cell conditioned medium after 72 hours of HepG2.2.15 cell.Matched group is that phalangeal cell deals with transfection reagent under condition of equivalent.Vertical coordinate represents that the antigen levels of SiRNA processed group accounts for the relative percentage of matched group.The meansigma methods of three repeated experiments of each data representation among the figure.

The effect that Fig. 7, different SiRNA combinations are expressed HBeAg.Two kinds of SiRNA combination of diagram variable concentrations (No. 2 add No. 6, No. 2 add No. 4 add again No. 6) acts on the influence to HBeAg level in its cell conditioned medium after 72 hours of HepG2.2.15 cell.Matched group is that phalangeal cell deals with transfection reagent under condition of equivalent.Vertical coordinate represents that the antigen levels of SiRNA processed group accounts for the relative percentage of matched group.The meansigma methods of three repeated experiments of each data representation among the figure.

The effect that Fig. 8, different SiRNA make up hepatitis B virus DNA level in the pair cell.Two kinds of SiRNA combination of diagram variable concentrations (No. 2 add No. 6, No. 2 add No. 4 add again No. 6) acts on the influence to its intracellular virus dna level after 72 hours of HepG2.2.15 cell.Matched group is that phalangeal cell deals with transfection reagent under condition of equivalent.Vertical coordinate represents that the antigen levels of SiRNA processed group accounts for the relative percentage of matched group.The meansigma methods of three repeated experiments of each data representation among the figure.

The specific embodiment

Embodiment 1 SiRNA's is synthetic

Synthetic can the trust of SiRNA openly externally carried out synthetic professional commercial company, such as U.S. Dharmacon company, specifically can pass through Www.dharmacon.comKnow that all SiRNA molecules all through 2 upward deprotection, desalination, purification and double-stranded processing of annealing formation, are dissolved in the distilled water of DEPC processing then.The synthetic method that it adopted is method in common, such as following document disclosed method: Scaringe SA, Wincott FE, and Caruthers MH.Novel RNA Synthesis MethodUsing 5 '-Silyl-2 '-Orthoester Protecting Groups.J Am Chem Soc 1998; 120:11820-11821.

The preparation method of siRNA molecule provided by the invention (SiRNA) also can adopt existing solid state chemistry synthetic method.This method can be referring to Wincott F, DiRenzo A, Shaffer C, Grimm S, Tracz D, Workman C, Sweedler D, Gonzalez C, Scaringe S and Usman N.Synthesis, deprotection, analysis and purification of RNA and ribozymes.Nucleic AcidsRes.1995,232677-84.

The siRNA molecule (SiRNA) of table sequence number 7 be an example in the past: whole chemosynthesis can roughly be divided into the synthesizing of process (1) oligomerization ribonucleic acid of four; (2) deprotection; (3) purifies and separates; (4) the desalination aseptic sterilization of annealing.

Concrete preparation manipulation is as follows:

(1), oligomerization ribonucleic acid is synthetic: SiRNA synthetic is that (for example: carry out Applied Biosystems EXPEDITE 8909), (order of positive-sense strand 5 '-GGUAUGUUGCCCGUUUGUCTT antisense strand 5 '-GACAAACGGGCAACAUACCTT) couples together the nucleotide of correspondence one by one according to the nucleotide sequence of the siRNA molecule SiRNA of sequence number 7 at automated DNA/RNA synthesizer.Because SiRNA is made up of with one 2 poly-deoxythymidylic acid one section 19 poly-oligomerization ribonucleic acid. so starting material is that 5 '-O-of being connected of solid phase (CPG) is to dimethoxytrityl-thymidine (1-2 umol). specifically each circulation is synthesized and can be divided into four and go on foot and finish (Fig. 1).The first step be with thymidine that solid phase is connected on protecting group eluting under the effect of 3% trichloroacetic acid of 5 '; Second step is under the effect of active catalyst S-ethyl tetrazolium, 5 '-O-being coupled on the last thymidine of sloughing protection dimethoxytrityl-thymidine phosphoramidite, forming two thymidine tris phosphites. coupling time and coupling circulation time number average provides program to finish by instrument producer; The 3rd step was that two thymidine tris phosphites with coupling are oxidized to two thymidine phosphotriesters under the effect of 0.05M iodine water; The 4th step was acetylation, with a small amount of unreacted active group on the solid phase (for example: hydroxyl and amido) thus under the effect of acetic anhydride, form ester or amide. reach sealing process, in order to reduce whole production of by-products.Repeat this circulation until finishing the synthetic of whole nucleotide sequences.

(1) deprotection: will synthesize good solid phase SiRNA and put into a bottle that can seal, and with the methylamine water solution (10M, 50% ethanol) that adds 1 milliliter, rest on room temperature. after two hours, take out solution, and solid phase CPG used ethanol once more; The mixed liquor drip washing of water and acetonitrile; and leacheate and the solution that takes out previously merged a place; its solvent is drained. in bottle, continue to add the tetrahydrofuran solution (1M) of 1 milliliter of tetrabutyl ammonium fluoride. solution was left standstill 12 hours in room temperature. slough the protecting group (comprising base, the silanization protecting group that nucleoside phosphorylase and nucleoside are 2 ') on all oligomerization ribonucleic acid.Pass through ethanol precipitation again, produce the crude product of SiRNA.

(2) purifies and separates: the crude product of SiRNA is dissolved in 2 milliliters the aqueous solution of ammonium acetate, passes through the separation of anti-phase C18 high pressure liquid chromatography then. the method for utilization gradient elution, collect the principal product (ammonium acetate of leacheate A:0.1M of SiRNA; The ammonium acetate of the 0.1M of leacheate b:20% and 80% acetonitrile). the solvent of the principal product of SiRNA is removed, and add 5 milliliter of 80% acetic acid aqueous solution, left standstill 15 minutes in room temperature. then this solution is carried out the separation (DEAE-5PW of anion exchange, anion-exchange column), can obtain purity in the SiRNA (gradient elution more than 90%, the Tris-HCl of leacheate A:0.025M, 0.025M NaClpH=8,5% acetonitrile; The Tris-HCl of leacheate b:0.025 M, 2.0M NaCl, pH=8,5% acetonitrile).

(3) the desalination aseptic sterilization of annealing: the SiRNA of purification is through dialysis, remove salt, the solution of SiRNA carries out filter-sterilized and drying crystalline. then the oligomerization ribonucleic acid of positive-sense strand and antisense strand is carried out its method of SiRNA. that annealing in process forms stable bifilar interlinkage and is oligomerization ribonucleic acid mixed dissolution (10mM Tris in the buffer solution of 1-2 milliliter with positive-sense strand and antisense strand, pH=7.5-8.0,50mM NaCl). this solution is heated to 95 ℃, slowly this solution cooling is caused room temperature (this process should be no less than hour) then. this solution is left in 4 ℃ of refrigerators preserve at last, so that can use at any time.

Purity and evaluation through the SiRNA behind the purification have two kinds of ways relatively more commonly used.One is identified the purity of SiRNA with the capillary gel electrophoresis method, this method can be referring to Paulus A, Ohms JI.Analysis ofoligonucleotides by capillary gelelectrophoresis. J Chromatogr1990,507:113-123.

It two is accurately to measure its molecular weight with the MALDI-TOF mass spectrum, thus the chemical constitution of definite SiRNA composition.

Said all SiRNA of the present invention can adopt method for preparing according to its sequence, and authentication method is the same.

The transfection of embodiment 2 SiRNA

1, the cell model of hepatitis B virus: what the cell model of hepatitis B was used is the HepG2.2.15 cell strain.This cell strain is to change the total length of AYW hypotype hepatitis B virus over to HepG2 by doctor's Acs laboratory in 1987 and form, and it is the external standard cell lines that is used for screening anti-hepatitis virus medicament always.RPMI 1640 culture medium that this cell contains 10% hyclone at external use are cultivated and are regularly screened the high expressed of keeping hepatitis virus with G418.(Sells MA，Chen ML，Acs G.Production of hepatitis B particles in HepG2 cells transfected with cloned hepatitis B virus DNA.PNAS1987；84：1005-1009.)

2, the transfection of SiRNA: SiRNA changes the HepG2.2.15 cell over to and carries out by means of the Oligofectamine of Invitrogen (www.invitrogen.com) company, and concrete steps are carried out to specifications.Be summarized as follows 5x10 ⁴The HepG2.2.15 cell inoculation on 24 well culture plates, spend the night, changed over to SiRNA in second day, continue then to cultivate 72 hours.

3, the antigenic mensuration of cell conditioned medium: after 72 hours, measure with the ELISA method by HBsAg in its culture supernatant and the level of HBeAg in the SiRNA effect for the HepG2.2.15 cell.Measure used HBsAg and HbeAgELISA medicine and close the manufacturing by Huamei Bio-Engrg Co., (www.sabc.com.cn), concrete steps are carried out according to the description that medicine closes.

4, the detection of hepatitis B virus DNA amount in the cell: intracellular hepatitis B virus DNA amount is measured with the technology of PCR in real time.Concrete grammar is summarized as follows: the QIAamp DNA Blood Mini Kit that produces with Qiagen company (www.qiagen.com) extracts total DNA of HepG2.2.15 cell earlier, then its concentration is adjusted to 2ng/ul.The amount of the DNA of hepatitis B virus detects with the ABI7700 quantitative PCR analyzer that Applied Biosystem makes.The sequence of two primers that PCR is used is respectively AGTGTGGATTCGCACTCC and GAGTTCTTCTTCTAGGGGACC, and the sequence of probe is CCAAATGCCCCTATCCTATCAACACTTC.The concrete operations step of PCR is all carried out (www.appliedbiosystems.com) according to the description of Applied Biosystem.

Embodiment 3 SiRNA duplicate and the synthetic inhibitory action of pathogenic associated protein virus in the hepatitis B virus cell model

1, the selection of hepatitis B virus cell model: it is the HepG.2.2.15 cell strain of standard that present embodiment adopts the hepatitis B virus cell model, and this cell strain is screened after by the full genome transfection of hepatitis B virus AYW hypotype by hepatoma carcinoma cell and forms.Hepatitis virus can be stablized in this cell strain and duplicates and breed, and can produce the various antigen proteins of virus.

2, the selection of SiRNA: as mentioned above, five kinds of mRNA molecules of hepatitis B virus gene group coexpression, each mRNA molecule all are responsible for the different albumen of coding, and the position of transcription initiation and termination also has nothing in common with each other on the genomic DNA molecule.So the position that the different proteic coding regions of encoding correspond to genomic DNA has nothing in common with each other, wherein the coding region of e antigen protein is from 1816 to 1902, C antigen is from 1903 to 2454, polymerase is from 2309 to 1625, pre-s1 protein is from 2850 to 3176, pre-s2 protein is from 3177 to 156, and S albumen is from 157 to 837, and X protein is from 1376 to 1840.In order to attack these different proteic mRNA of coding effectively, the present invention has selected 12 SiRNA molecules (seeing the following form) at each different coding district, except their possible combineds effect to preceding gene RNA molecule and e antigen mRNA, the coding region of different molecular actions is not quite similar, wherein be primarily aimed at the mRNA of surface antigen No. 1 and No. 2, No. 4 and No. 5 are at X protein, and No. 6 and No. 7 are at e antigen, No. 89 to No. 11 at cAg and polymerase at cAg.

Serial number	The SiRNA numbering	Positive-sense strand	Antisense strand	Attack the position	The mRNA molecule that may attack	The protein of main effect
Serial number	The SiRNA numbering	Positive-sense strand	Antisense strand	Attack the position	The mRNA molecule that may attack	The protein of main effect	7 12 24 75 31 35 38 40 50	N1 N2 N3 N4 N5 N6 N7 N8 N9	GGUAUGUUGCC CGUUUGUCTT GUCUGUACAAC AUCUUGAGTT GUGUUUGCUGA CGCAACCCTT CUGGAUCCUGC GCGGGACGTT UGUCAACGACC GACCUUGATT CUUUUUCACCU CUGCCUAATT GCUGUGCCUUG GGUGGCUUTT UUUGGAGCUUC UGUGGAGUTT UGCCCCUAUCU UAUCAACATT	GACAAACGGGCAA CAUACCTT CUCAAGAUGUUGU ACAGACTT GGGUUGCGUCAGC AAACACTT CGUCCCGCGCAGG AUCCAGTT UCAAGGUCGGUCG UUGACATT UUAGGCAGAGGUG AAAAAGTT AAGCCACCCAAGG CACAGCTT ACUCCACAGAAGC UCCAAATT UGUUGAUAAGAUA GGGGCATT	458-478 765-785 1180-120 0 1398-141 8 1681-170 1 1820-184 0 1876-189 6 1925-194 5 2308-232 8	2.1kb，2.4kb，3.5k b，C/pol 2.1kb，2.4kb，3.5k b，C/pol 2.1kb，2.4kb，3.5k b，C/pol 0.7kb，2.1kb，2.4k b，3.5kb，C/pol 0.7kb，2.1kb，2.4k b，3.5kb，C/pol 0.7kb，2.1kb，2.4k b，3.5kb，C/pol 0.7kb，2.1kb，2.4k b，3.5kb，C/pol 0.7kb，2.1kb，2.4k b，3.5kb，C/pol 3.5kb，C/pol	S S P X X E E C C，P

55 58 63

N10 N11 N12

GGUCUCAAUCG CCGCGUCGTT UCUCGGGAAUC UCAAUGUUTT UUAAUUAUGCC UGCUAGGUTT

CGACGCGGCGAUU GAGACCTT AACAUUGAGAUUC CCGAGATT ACCUAGCAGGCAU AAUUAATT

2400-242 0 2432-245 2 2631-265 1

3.5kb，C/pol 3.5kb，C/pol 3.5kb，C/pol

C，P C，P P

*: S=HBsAg, P=polymerase, X=X protein, E=HBeAg, C=cAg

3,12 SiRNA molecules are to the inhibitory action of virus replication and protein expression:

A, concentration are the effects of 12 kind SiRNA of 150nM to virus antigen expression and DNA amount:

12 kinds of SiRNA with 150nM acted on HepG2.2.15 after three days, observe them the surface antigen and the antigenic influence of e of virus are found that the antigenic expression of surface antigen and e is mainly by the SiRNA molecules in inhibiting at itself.In No. 1, No. 2 SiRNA molecules at surface antigen, the most effective with No. 2 molecules, its suppression ratio is about 60%, finds also that simultaneously at X protein No. 4 also have obvious suppression effect (Fig. 1) to HBsAg.E antigen mainly by No. 6 and No. 7 molecules in inhibiting at itself, is about 50% (Fig. 2).Other molecule all has partly inhibitory action to two class antigens.

Observing the time spent of doing of the interior hepatitis B virus DNA amount of these SiRNA pair cells finds, each SiRNA molecule also has nothing in common with each other to the inhibitory action of viral DNA quantity, wherein the inhibitory action with 2,6,7 and No. 10 molecules is the strongest, is respectively 87%, 88.5%, 88% and 88.4%.1,4,8, No. 11 levels to DNA viruses DNA also have obvious suppression effect (Fig. 3).

To being used for of virus antigen and DNA, every have obvious inhibiting molecule that viral DNA is also had the obvious suppression effect to virus antigen from these SiRNA molecules.

B, 2,4, No. 6 SiRNA express virus antigen and the concentration effect of the effect of DNA amount:

According to the result of above-mentioned SiRNA molecule to baicailin compound on hepatitis B and the effect of DNA amount, No. 2 molecules the strongest have been selected to the HBsAg inhibitory action, No. 6 molecules the strongest to HBeAg are added No. 4 molecules to X protein, make the depression effect of their variable concentrations to virus antigen and DNA amount.

Use 10nM, 20nM, 40nM, 80nM, 160nM, 2,4, No. 6 molecules of 320nM, find when acting on the amount of HepG2.2.15 cell HBsAg in the observation of cell supernatant after three days, No. 2 molecule is the strongest to this antigenic inhibitory action, and the fairly obvious and maximum depression effect of the inhibitory action of 10nM differs very little.When concentration inhibitory action 40 to 80nM the time reaches maximum, be about 62%, further increasing its inhibitory action of concentration does not only increase the trend that weakens is arranged on the contrary.No. 4 molecules of variable concentrations are quite similar to the binding mode of HBsAg and No. 2, but effect is than a little less than No. 2 10% to 20%.No. 6 molecule is expressed no obvious inhibitory action (Fig. 3) to the HBsAg taxi.

Find when observing the variation of HBeAg, have only No. 6 molecules that this antigen is had the obvious suppression effect, with No. 2, No. 4 molecules to the suppression mode of HBsAg similar be, the maximum depression effect of No. 6 molecules just occurs when low concentration, and its depression effect does not increase and descend on the contrary (Fig. 4) when the above concentration of 80nM.

Three kinds of molecules of variable concentrations are not quite similar to the binding mode of viral DNA amount, and the maximum depression effect of No. 2 molecules just occurs at 10nM, and No. 6 molecule then occurs at 20nM, and their both maximums suppress and can keep when high concentration.No. 4 molecules then are directly proportional with its concentration to the inhibitory action of viral DNA, reach during to 320nM the strongest (Fig. 5).

C, 2,4, No. 6 SiRNA express virus antigen and the joint effect of the effect of DNA amount:

From The above results as can be known, No. 2 and No. 6 SiRNA molecules all have the good restraining effect to the generation of viral DNA, but their itself only have inhibitory action to expression of its corresponding virus antigen.For this reason, the present invention has further observed to unite simultaneously and has used No. 2 and No. 6, and adds the effect to virus antigen expression and DNA generation for No. 2 and No. 6 No. 4.Found that, use simultaneously No. 2 and No. 6 molecules can suppress two kinds of antigenic expression of cell, this acting on reached maximum when their concentration separately is 20nM, both uses of uniting have superimposed effect to the expression of HBsAg, also to increase by 10% than single ceiling effect with No. 4, but this superimposed effect is also not obvious in the expression of HBeAg, and further adding No. 4 molecules can and then increase antigenic depression effect, but amplitude very big frequently (Fig. 6, Fig. 7).Because individual molecule has reached 80% to 90% to the inhibitory action of DNA amount, therefore two molecules are not obvious to the superimposed effect that DNA produces, it is also of no avail to add the third molecule, our result shows that No. 2 molecules of 40nM add that No. 6 molecules of 40nM surpass 90% (Fig. 8) to the suppression ratio that DNA produces.

The modification of embodiment 4 SiRNA

As mentioned above, the kind of modification can divide three major types and each apoplexy due to endogenous wind that many variations are arranged, so their synthetic methods separately are the same not to the utmost.Here enumerate two kinds of the most common SiRNA method of modifying.And following method does not limit the relevant protection domain that SiRNA is modified of the present invention.

1, sulfuration

Sulfuration is meant that an oxygen atom on the nucleoside phosphorylase diester linkage is transformed into sulphur atom forms nucleoside D2EHDTPA diester. do not change because other of whole SiRNA formed structure, so its is synthetic the same substantially with the SiRNA building-up process of embodiment 1.Only need the oxidation reaction in the building-up process is become vulcanization reaction, in this reaction, add suitable sulfuration reagent, 3H-1 for example, 2-benzodithiol-3-one 1,1-dioxide (claiming Beaucage reagent again).

2, the modification of 2 ' hydroxyl of nucleoside

The modification of 2 ' hydroxyl of nucleoside is meant 2 ' the hydroxyl that comes five Yuans sugar of substituted nucleosides to encircle with various saturated alkoxyls or unsaturated alcoxyl. wherein modal is the hydroxyl that comes 2 ' of substituted nucleosides with methoxyl group.The synthesis step basically identical of the SiRNA of the synthetic and embodiment 1 of 2 ' methoxylation of nucleoside of SiRNA only need replace 2 '-tertiary butyl dimethyl Si yl nucleosides phosphoramidite with 2 '-methoxyl group nucleoside phosphoramidites and get final product in coupling reaction.

Sequence table

＜110〉Hangzhou Xinruijia Biological Medicine Technology Development Co., Ltd.

＜120〉siRNA molecule (SiRNA) of attack human hepatitis B virus and application thereof

<160>77

<210>1

<211>3215

<212>DNA

<213>Hepadnavirus

<220>

<221>misc_feature

<222>1...3215

＜223〉double-stranded DNA is from the hepatitis B virus ADR subtype gene group common sequences that the complete genome DNA sequence of known hepatitis B virus ADR hypotype obtains, the sequence of the normal chain of classifying as

<400>1

aactccacaa cattccacca agctctgcta gaccccagag tgaggggcct atactttcct 60

gctggtggct ccagttccgg aacagtaaac cctgttccga ctactgcctc acccatatcg 120

tcaatcttct cgaggactgg ggaccctgca ccgaacatgg agaacacaac atcaggattc 180

ctaggacccc tgctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata 240

ccacagagtc tagactcgtg gtggacttct ctcaattttc tagggggagc acccacgtgt 300

cctggccaaa attcgcagtc cccaacctcc aatcactcac caacctcttg tcctccaatt 360

tgtcctggct atcgctggat gtgtctgcgg cgttttatca tattcctctt catcctgctg 420

ctatgcctca tcttcttgtt ggttcttctg gactaccaag gtatgttgcc cgtttgtcct 480

ctacttccag gaacatcaac taccagcacg ggaccatgca agacctgcac gattcctgct 540

caaggaacct ctatgtttcc ctcttgttgc tgtacaaaac cttcggacgg aaactgcact 600

tgtattccca tcccatcatc ctgggctttc gcaagattcc tatgggagtg ggcctcagtc 660

cgtttctcct ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc 720

actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacaacatc 780

ttgagtccct ttttacctct attaccaatt ttcttttgtc tttgggtata catttgaacc 840

ctaataaaac caaacgttgg ggctactccc ttaacttcat gggatatgta attggaagtt 900

ggggtacttt accgcaagaa catattgtac taaaantcaa gcaatgtttt cgaaaactgc 960

ctgtaaatag acctattgat tggaaagtat gtcagagaat tgtgggtctt ttgggctttg 1020

ctgccccttt tacacaatgt ggctatcctg ccttaatgcc tttatatgca tgtatacaat 1080

ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatatctga 1140

acctttaccc cgttgcccgg caacggtcag gtctctgcca agtgtttgct gacgcaaccc 1200

ccactggatg gggcttggct attggccatc gccgcatgcg tggaaccttt gtggctcctc 1260

tgccgatcca tactgcggaa ctcctagcag cttgttttgc tcgcagccgg tctggagcga 1320

aacttatcgg aacngacaac tctgttgtcc tctctcggaa atacacctcc tttccatggc 1380

tgctagggtg tgctgccaac tggatcctgc gcgggacgtc ctttgtctac gtcccgtcgg 1440

cgctgaatcc cgcggacgac ccgtctcggg gccgtttggg actctaccgt ccccttcttc 1500

atctgccgtt ccggccgacc acggggcgca cctctcttta cgcggtctcc ccgtctgtgc 1560

cttctcatct gccggtccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac 1620

cgtgaacgcc caccaggtct tgcccaaggt cttacataag aggactcttg gactctcagc 1680

aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaagg actgggagga 1740

gttgggggag gagattaggt taangntctt tgtactagga ggctgtaggc ataaattggt 1800

ctgttcacca gcaccatgca actttttcac ctctgcctaa tcatctcatg ttcatgtcct 1860

actgttcaag cctccaagct gtgccttggg tggctttggg gcatggacat tgacccgtat 1920

aaagaatttg gagcttctgt ggagttactc tcttttttgc cttctgactt ctttccttct 1980

attcgagatc tcctcgacac cgcctctgct ctgtatcggg aggccttaga gtctccggaa 2040

cattgttcac ctcaccatac agcactcagg caagctattc tgtgttgggg tgagttgatg 2100

aatctggcca cctgggtggg aagtaatttg gaagacccag catccaggga attagtagtc 2160

agctatgtca atgttaatat gggcctaaaa atcagacaac tactgtggtt tcacatttcc 2220

tgtcttactt ttggaagaga aactgttctt gagtatttgg tgtcttttgg agtgtggatt 2280

cgcactcctc ctgcttacag accaccaaat gcccctatct tatcaacact tccggaaact 2340

actgttgtta gacgacgagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga 2400

aggtctcaat cgccgcgtcg cagaagatct caatctcggg aatctcaatg ttagtatccc 2460

ttggactcat aaggtgggaa actttactgg gctttattct tctactgtac ctgtctttaa 2520

tcctgagtgg caaactccct cctttcctca cattcattta caggaggaca ttattaatag 2580

atgtcaacaa tatgtgggcc ctcttacagt taatgaaaaa aggagattaa aattaattat 2640

gcctgctagg ttctatccta accttaccaa atatttgccc ttggacaaag gcattaaacc 2700

atattatcct gaacatgcag ttaatcatta cttcaaaact aggcattatt tacatactct 2760

gtggaaggct ggcattctat ataagagaga aactacacgc agcgcctcat tttgtgggtc 2820

accatattct tgggaacaag agctacagca tgggaggttg gtcttccaaa cctcgacaag 2880

gcatggggac gaatctttct gttcccaatc ctctgggatt ctttcccgat caccagttgg 2940

accctgcgtt cggagccaac tcaaacaatc cagattggga cttcaacccc aacaaggatc 3000

actggccaga ggcaaatcag gtaggagcgg gagcattcgg gccagggttc accccaccac 3060

acggcggtct tttggggtgg agccctcagg ctcagggcat attgacaaca gtgccagtag 3120

cacctcctcc tgcctccacc aatcggcagt caggaagaca gcctactccc atctctccac 3180

ctctaagaga cagtcatcct caggccatgc agtgg

<160>77

<210>2

<211>3215

<212>DNA

<213>Hepadnavirus

<220>

<221>misc_feature

<222>1...3215

＜223〉double-stranded DNA is from the hepatitis B virus ADW subtype gene group common sequences that the complete genome DNA sequence of known hepatitis B virus ADW hypotype obtains, the sequence of the normal chain of classifying as

<400>2

ctccaccact ttccaccaaa ctcttcaaga tcccagagtc agggccctgt actttcctgc 60

tggtggctcc agttcaggaa cagtaanccc tgntcagaat actgtctctg ccatatcgtc 120

aatcttatcg aagactgggg accctgtacc gaacatggag aacatcgcat caggactcct 180

aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaaaaatcc tcacaatacc 240

acagagtcta gactcgtggt ggacttctct caattttcta gggggaacac ccgtgtgtct 300

tggccaaaat tcgcagtccc aaatctccag tcactcacca acctgttgtc ctccaatttg 360

tcctggttat cgctggatgt gtctgcggcg ttttatcatc ttcctctgca tcctgctgct 420

atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480

aattccagga tcatcaacna ccagcacngg accatgcaaa acctgcacna ctcctgctca 540

aggaacctct atgtttccct catgttgctg tacaaaacct acggacggaa actgcacctg 600

tattcccatc ccatcatctt gggctttcgc aaaataccta tgggagtggg cctcagtccg 660

tttctcttgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720

tgtctggctt tcagttatat ggatgatgtg gttttggggg ccaagtctgt acaacatctt 780

gagtcccttt atgccgctgt taccaatttt cttttgtctt tgggtataca tttaaaccct 840

cacaaaacaa aaagatgggg atantccctt aacttcatgg gatatgtaat tggnagttgg 900

ggcacattnc cacaggaaca tattgtacta aaaatcaaac aatgttttag gaaacttcct 960

gtaaacaggc ctattgattg gaaagtatgt caacgaattg tgggtctttt ggggtttgct 1020

gcccctttca cacaatgtgg atatcctgct ttaatgcctt tatatgcatg tatacaagcn 1080

aaacaggctt ttactttctc gccaacttac aaggcctttc taagtaaaca ntatctgaac 1140

ctttaccccg ttgctcggca acggccaggt ctgtgccaag tgtttgctga cgcaaccccc 1200

actggttggg gcttggccat aggccatcag cgcatgcgtg gaacctttgt gtctcctctg 1260

ccgatccata ctgcggaact cctagccgct tgttttgctc gcagcaggtc tggagcaaaa 1320

cttatcggga ctgacaattc tgtcgtcctc tcccgcaaat atacatcntt tccatggctg 1380

ctaggctgtg ctgccaactg gatcctgcgc gggacgtcct ttgtttacgt cccgtcggcg 1440

ctgaatcccg cggacgaccc ctcccggggc cgcttggggc tctaccgccc gcttctccgc 1500

ctgccgtacc gaccgaccac ggggcgcacc tctctttacg cggactcccc gtctgtgcct 1560

tctcatctgc cggaccgtgt gcacttcgct tcacctctgc acgtcgcatg gagaccaccg 1620

tgaacgccca ccggaacctg cccaaggtct tgcataagag gactcttgga ctttcagcaa 1680

tgtcaacgac cgaccttgag gcatacttca aagactgtgt gtttaatgag tgggaggagt 1740

tgggggagga gattaggtta aaggtctttg tactaggagg ctgtaggcat aaattggtct 1800

gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt catgtcctac 1860

tgttcaagcc tccaagctgt gccttgggtg gctttggggc atggacattg acccgtataa 1920

agaatttgga gcttctgtgg agttactctc ttttttgcct tctgacttct ttccttctat 1980

tcgagatctc ctcgacaccg cctctgctct gtatcgggag gccttagagt ctccggaaca 2040

ttgttcacct caccatacgg cactcaggca agctattctg tgttggggtg agttgatgaa 2100

tctagccacc tgggtgggaa gtaatttgga aganccagca tccagggaat tagtagtcag 2160

ctatgtcaan gttaatatgg gcctaaaaat cagacaacta ttgtggtttc acatttcctg 2220

tcttactttt ggaagagaaa ctgttcttga atatttggtg tcttttggag tgtggattcg 2280

cactcctcct gcatatagac caccaaatgc ccctatctta tcaacacttc cggaaactac 2340

tgttgttaga cgacgaggca ggtcccctag aagaagaact ccctcgcctc gcagacgaag 2400

gtctcaatcg ccgcgtcgca gaagatctca atctcgggaa tctcaatgtt agtattcctt 2460

ggactcataa ggtgggaaac tttacggggc tttattcttc tacggtacct ngctttaatc 2520

ctnaatggca aactccttct tttcctgaca ttcatttgca ggaggacatt nttgatagat 2580

gtaagcaatt tgtgggnccc cttacagtaa atgaaaacag gagactaaaa ttaattatgc 2640

ctgctaggtt ttatcccaat gttactaaat atttgccctt agataaaggn atcaaacctt 2700

attatccaga gcatgtagtt aatcattact tccagacgag acattattta catactcttt 2760

ggaaggcggg natnttatat aaaagagagn cnacacgtag cgcctcattt tgcgggtcac 2820

catattcttg ggaacaagat ctacagcatg ggaggttggt cttccaaacc tcgaaaaggc 2880

atggggacaa atctttctgt ccccaatcct ctgggattct tncccgatca ncagttggac 2940

cctgcattca aagccaactc agacaatcca gattgggacc tcaacccnca caaggacaac 3000

tggccggacg ccaacaaggt gggagtggga gcattcgggc cagggttcac cccnccccat 3060

gggggactgt tggggtggag ccctcaggct cagggcatac tcacaactgt gccagcagct 3120

cctcctcctg cctccaccaa tcggcagtca ggaaggcagc ctactcccnt ntctccacct 3180

ctaagagaca ctcatcctca ggccatgcag tggaa

<160>77

<210>3

<211>3182

<212>DNA

<213>Hepadnavirus

<220>

<221>misc_feature

<222>1...3182

<223>

Double-stranded DNA is from the hepatitis B virus AYW subtype gene group common sequences that the complete genome DNA sequence of known hepatitis B virus AYW hypotype obtains, the sequence of the normal chain of classifying as

<400>3

aactccacaa ccttccacca aactctgcaa gatcccagag tgagaggcct gtatttccct 60

gctggtggct ccagttcagg aacagtaaac cctgttccga ctactgcctc tcccatatcg 120

tcaatcttct cgaggattgg ggaccctgcg ctgaacatgg agaacatcac atcaggattc 180

ctaggacccc tgctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata 240

ccgcagagtc tagactcgtg gtggacttct ctcaattttc tagggggaac taccgtgtgt 300

cttggccaaa attcgcagtc cccaacctcc aatcactcac caacctcctg tcctccaact 360

tgtcctggtt atcgctggat gtgtctgcgg cgttttatca tcttcctctt catcctgctg 420

ctatgcctca tcttcttgtt ggttcttctg gactatcaag gtatgttgcc cgtttgtcct 480

ctaattccag gatcntcaac caccagcacg ggaccatgca gaacctgcac gactcctgct 540

caaggaacct ctatgtatcc ctcctgttgc tgtaccaaac cttcggacgg aaattgcacc 600

tgtattccca tcccatcatc ctgggctttc ggaaaattcc tatgggagtg ggcctcagcc 660

cgtttctcct ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc 720

actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 780

ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc 840

ctaacaaaac aaaaagatgg ggttattctt taaatttcat gggctatgtc attggatgtt 900

atgggtcatt gccacaagat cacatcatac anaaaatcaa agaatgtttt agaaaacttc 960

ctgttaacag gcctattgat tggaaagtct gtcaacgtat tgtgggtctt ttgggttttg 1020

ctgccccttt tacacaatgt ggttatcctg ctttaatgcc cttgtatgca tgtattcaat 1080

ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatacctga 1140

acctttaccc cgttgccagg caacggccag gtctgtgcca agtgtttgct gacgcaaccc 1200

ccactggctg gggcttggtc atgggccatc agcgcatgcg tggaaccttt ctggctcctc 1260

tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa 1320

acattctcgg gactgataac tctgttgttc tctcccgcaa atatacatcg tttccatggc 1380

tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg 1440

cgctgaatcc cgcggacgac ccttctcggg gccgcttggg gctctntcgt ccccttctcc 1500

gtctgccgtt ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc 1560

cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac 1620

cgtgaacgcc caccaattct tgcccaaggt cttacataag aggactcttg gactctctgc 1680

aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaaag actgggagga 1740

gttgggggag gagattagat taaaggtctt tgtactagga ggctgtaggc ataaattggt 1800

ctgcgcacca gcaccatgca actttttcac ctctgcctaa tcatctcttg ttcatgtcct 1860

actgttcaag cctccaagct gtgccttggg tggctttggg gcatggacat tgacccttat 1920

aaagaatttg gagctactgt ggagttactc tcgtttttgc cttctgactt ctttccttcn 1980

gtacgagatc ttctagatac cgcctcagct ctgtatcggg atgccttaga gtctcctgag 2040

cattgttcac ctcaccatac tgcactcagg caagcaattc tttgctgggg ggaactaatg 2100

actctagcta cctgggtggg tgntaatttg gaagatccag catctaggga cctagtagtc 2160

agttatgtca acactaatat gggcctaaag ttcaggcaac tattgtggtt tcacatttct 2220

tgtctcactt ttggaagaga aacggttata gagtatttgg tgtctttcgg agtgtggatt 2280

cgcactcctc cagcttatag accaccaaat gcccctatct tatcaacact tccggagact 2340

actgttgtta gacgacgagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga 2400

agatctcaat cgccgcgtcg cagaagatct caatctcggg aatctcaatg ttagtattcc 2460

ttggactcat aaggtgggaa actttacggg gctttattct tctactgtac ctgtctttaa 2520

ccctcattgg aaaacaccct cttttcctaa tatacattta caccaagaca ttatcaaaaa 2580

atgtgaacag tttgtaggcc cactcacagt caatgagaaa agaagactgc aattgattat 2640

gcctgctagg ttttatccaa atgttaccaa atatttgcca ttggataagg gtattaaacc 2700

ttattatcca gaacatctag ttaatcatta cttccaaacc agacattatt tacacactct 2760

atggaaggcg ggtatattat ataagagaga aacaacacat agcgcctcat tttgtgggtc 2820

accatattct tgggaacaag agctacagca tggggcagaa tctttccacc agcaatcctc 2880

tgggattctt tcccgaccac cagttggatc cagccttcag agcaaacacc gcaaatccag 2940

attgggactt caatcccaac aaggacacct ggccagacgc caacaaggta ggagctggag 3000

cattcgggct gggattcacc ccaccgcacg gaggcctttt ggggtggagc cctcaggctc 3060

agggcataat acaaaccttg ccagcaaatc cgcctcctgc ctctaccaat cgccagtcag 3120

gaaggcagcc taccccgctg tctccacctt tgagaaacac tcatcctcag gccatgcagt 3180

gg

<160>77

<210>4

<211>3215

<212>DNA

<213>Hepadnavirus

<220>

<221>misc_feature

<222>1...3215

＜223〉double-stranded DNA, the common sequences of the hepatitis B virus gene group that obtains from sequence 1, sequence 2, sequence 3, the sequence of the normal chain of classifying as

<400>4

aactccacaa cnttccacca aactctgcaa gatcccagag tgaggggcct gtactttcct 60

gctggtggct ccagttcagg aacagtaaac cctgttccga ctactgcctc tcccatatcg 120

tcaatcttct cgaggactgg ggaccctgca ccgaacatgg agaacatcac atcaggattc 180

ctaggacccc tgctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata 240

ccacagagtc tagactcgtg gtggacttct ctcaattttc tagggggaac acccgtgtgt 300

cttggccaaa attcgcagtc cccaacctcc aatcactcac caacctcttg tcctccaatt 360

tgtcctggtt atcgctggat gtgtctgcgg cgttttatca tcttcctctt catcctgctg 420

ctatgcctca tcttcttgtt ggttcttctg gactatcaag gtatgttgcc cgtttgtcct 480

ctaattccag gatcatcaac naccagcacg ggaccatgca aaacctgcac gactcctgct 540

caaggaacct ctatgtttcc ctcntgttgc tgtacaaaac cttcggacgg aaactgcacc 600

tgtattccca tcccatcatc ctgggctttc gcaaaattcc tatgggagtg ggcctcagtc 660

cgtttctcct ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc 720

actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacaacatc 780

ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc 840

ctaacaaaac aaaaagatgg ggntantccc ttaacttcat gggatatgta attggaagtt 900

ggggnacatt nccacaagaa catattgtac taaaaatcaa acaatgtttt agaaaacttc 960

ctgtaaacag gcctattgat tggaaagtat gtcaacgaat tgtgggtctt ttgggntttg 1020

ctgccccttt tacacaatgt ggntatcctg ctttaatgcc tttatatgca tgtatacaat 1080

ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatatctga 1140

acctttaccc cgttgcccgg caacggccag gtctgtgcca agtgtttgct gacgcaaccc 1200

ccactggntg gggcttggcc atnggccatc agcgcatgcg tggaaccttt gtggctcctc 1260

tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa 1320

aacttatcgg gactgacaac tctgttgtcc tctcccgcaa atatacatcn tttccatggc 1380

tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg 1440

cgctgaatcc cgcggacgac ccntctcggg gccgcttggg gctctaccgt ccccttctcc 1500

gtctgccgtt ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc 1560

cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac 1620

cgtgaacgcc caccagntct tgcccaaggt cttacataag aggactcttg gactctcagc 1680

aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaang actgggagga 1740

gttgggggag gagattaggt taaaggtctt tgtactagga ggctgtaggc ataaattggt 1800

ctgttcacca gcaccatgca actttttcac ctctgcctaa tcatctcatg ttcatgtcct 1860

actgttcaag cctccaagct gtgccttggg tggctttggg gcatggacat tgacccgtat 1920

aaagaatttg gagcttctgt ggagttactc tcttttttgc cttctgactt ctttccttct 1980

attcgagatc tcctcgacac cgcctctgct ctgtatcggg aggccttaga gtctccggaa 2040

cattgttcac ctcaccatac ngcactcagg caagctattc tgtgttgggg tgagttgatg 2100

aatctagcca cctgggtggg aagtaatttg gaaganccag catccaggga attagtagtc 2160

agctatgtca angttaatat gggcctaaaa atcagacaac tattgtggtt tcacatttcc 2220

tgtcttactt ttggaagaga aactgttctt gagtatttgg tgtcttttgg agtgtggatt 2280

cgcactcctc ctgcttatag accaccaaat gcccctatct tatcaacact tccggaaact 2340

actgttgtta gacgacgagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga 2400

aggtctcaat cgccgcgtcg cagaagatct caatctcggg aatctcaatg ttagtattcc 2460

ttggactcat aaggtgggaa actttacggg gctttattct tctactgtac ctgtctttaa 2520

tcctnantgg caaactccct cttttcctna cattcattta caggaggaca ttattaatag 2580

atgtnaacaa tttgtgggcc cncttacagt naatgaaaaa aggagactaa aattaattat 2640

gcctgctagg ttttatccna atgttaccaa atatttgccc ttggataaag gnattaaacc 2700

ttattatcca gaacatgtag ttaatcatta cttccaaacn agacattatt tacatactct 2760

ntggaaggcg ggnatnttat ataagagaga aacnacacgt agcgcctcat tttgtgggtc 2820

accatattct tgggaacaag agctacagca tgggaggttg gtcttccaaa cctcganaag 2880

gcatggggac gaatctttct gtccccaatc ctctgggatt ctttcccgat caccagttgg 2940

accctgcntt cagagccaac tcagacaatc cagattggga cttcaacccc aacaaggaca 3000

actggccaga cgccaacaag gtaggagcgg gagcattcgg gccagggttc accccaccnc 3060

acggnggnct tttggggtgg agccctcagg ctcagggcat antnacaacn gtgccagcag 3120

ctcctcctcc tgcctccacc aatcggcagt caggaaggca gcctactccc ntntctccac 3180

ctctaagaga cactcatcct caggccatgc agtgg

<160>77

<210>5

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

＜223〉two strands is classified the antisense strand sequence as in the table

<400>5

uccccagucc ucgagaagat t 21

<160>77

<210>6

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>6

caaauuggag gacaagaggt t 21

<160>77

<210>7

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>7

gacaaacggg caacauacct t 21

<160>77

<210>8

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>8

agggaaacau agagguucct t 21

<160>77

<210>9

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>9

cangagggaa acauagaggt t 21

<160>77

<210>10

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>10

ugcaguuucc guccgaaggt t 21

<160>77

<210>11

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>11

gaugggaaua caggugcagt t 21

<160>77

<210>12

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>12

cucaagaugu uguacagac t t 21

<160>77

<210>13

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>13

uaaaaaggga cucaagaug t t 21

<160>77

<210>14

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>14

acccaaagac aaaagaaaat t 21

<160>77

<210>15

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>15

aauuacauau cccaugaagt t 21

<160>77

<210>16

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>16

uaggccuguu uacaggaagt t 21

<160>77

<210>17

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>17

uuuccaauca auaggccugt t 21

<160>77

<210>18

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>18

aancccaaaa gacccacaat t 21

<160>77

<210>19

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>19

uuaaagcagg auanccacat t 21

<160>77

<210>20

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>20

auacaugcau auaaaggcat t 21

<160>77

<210>21

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>21

cgagaaagug aaagccugct t 21

<160>77

<210>22

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>22

ccgggcaacg ggguaaaggt t 21

<160>77

<210>23

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>23

cuuggcacag accuggccgt t 21

<160>77

<210>24

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>24

ggguugcguc agcaaacact t 21

<160>77

<210>25

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>25

aagccccanc cagugggggt t 21

<160>77

<210>26

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>26

ggcagaggag ccacaaaggt t 21

<160>77

<210>27

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>27

caagacaagc ggcuaggagt t 21

<160>77

<210>28

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>28

agangggucg uccgcgggat t 21

<160>77

<210>29

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>29

aguccucuua uguaagacct t 21

<160>77

<210>30

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>30

ugagagucca agaguccuct t 21

<160>77

<210>86

<211>31

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>31

ucaaggucgg ucguugacat t 21

<160>77

<210>32

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>32

uaugccucaa ggucggucgt t 21

<160>77

<210>33

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>33

ccccaacucc ucccagucnt t 21

<160>77

<210>34

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>34

gugcugguga acagaccaat t 21

<160>77

<210>35

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>35

uuaggcagag gugaaaaagt t 21

<160>77

<210>36

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>36

ggacaugaac augagaugat t 21

<160>77

<210>37

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>37

caaggcacag cuuggaggct t 21

<160>77

<210>38

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>38

aagccaccca aggcacagct t 21

<160>77

<210>39

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>39

ccacagaagc uccaaauuct t 21

<160>77

<210>40

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>40

acuccacaga agcuccaaat t 21

<160>77

<210>41

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>41

uauggugagg ugaacaaugt t 21

<160>77

<210>42

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>42

caccccaaca cagaauagc t t 21

<160>77

<210>43

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>43

ucccacccag guggcuagat t 21

<160>77

<210>44

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>44

ugacauagcu gacuacuaat t 21

<160>77

<210>45

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>45

ucugauuuuu aggcccauat t 21

<160>77

<210>46

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>46

aaccacaaua guugucugat t 21

<160>77

<210>47

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>47

aaaugugaaa ccacaauagt t 21

<160>77

<210>48

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>48

uacucaagaa caguuucuct t 21

<160>77

<210>49

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>49

accaaauacu caagaacagt t 21

<160>77

<210>50

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>50

uguugauaag auaggggcat t 21

<160>77

<210>51

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>51

acaguaguuu ccggaagugt t 21

<160>77

<210>52

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>52

cgucgucuaa caacaguagt t 21

<160>77

<210>53

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>53

cgaggcgagg gaguucuuct t 21

<160>77

<210>54

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>54

cugcgaggcg agggaguuct t 21

<160>77

<210>55

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>55

cgacgcggcg auugagacct t 21

<160>77

<210>56

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>56

aucuucugcg acgcggcgat t 21

<160>77

<210>57

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>57

gauucccgag auugagauct t 21

<160>77

<210>58

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>58

aacauugaga uucccgagat t 21

<160>77

<210>59

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>59

aggaauacua acauugagat t 21

<160>77

<210>60

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>60

aguccaagga auacuaacat t 21

<160>77

<210>61

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>61

ccccguaaag uuucccacct t 21

<160>77

<210>62

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>62

agaauaaagc cccguaaagt t 21

<160>77

<210>63

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>63

accuagcagg cauaauuaat t 21

<160>77

<210>64

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>64

uaaaaccuag caggcauaat t 21

<160>77

<210>65

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>65

cuuuauccaa gggcaaauat t 21

<160>77

<210>66

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>66

ucccaugcug uagcucuugt t 21

<160>77

<210>67

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>67

accucccaug cuguagcuct t 21

<160>77

<210>68

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>68

uccccaugcc uunucgaggt t 21

<160>77

<210>69

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>mise_feature

<222>1...21

<400>69

aaagauucgu ccccaugcct t 21

<160>77

<210>70

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>70

aggauugggg acagaaagat t 21

<160>77

<210>71

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>71

ccaaucugga uugucugagt t 21

＜120〉attack human hepatitis B virus less than disturbing RNA molecule (SiRNA) and using

<160>77

<210>72

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>72

guugaagucc caaucuggat t 21

<160>77

<210>73

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>73

cugccuuccu gacugccgat t 21

<160>77

<210>74

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>74

gccugaggau gagugucuct t 21

<160>77

<210>75

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>75

cgucccgcgc aggauccagt t 21

<160>77

<210>76

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>76

cgucugcgag gcgagggagt t 21

<160>77

<210>77

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>77

uuuuagucuc cuuuuuucat t 21

Claims

1, attacks the siRNA molecule (SiRNA) of human hepatitis B virus, it is characterized in that its nucleotides sequence classifies as

Positive-sense strand: GGUAUGUUGCCCGUUUGUCTT, antisense strand: GACAAACGGGCAACAUACCTT.

2, attack the siRNA molecule (SiRNA) of human hepatitis B virus, it is characterized in that its nucleotides sequence classifies as

Positive-sense strand: GUCUGUACAACAUCUUGAGTT, antisense strand: CUCAAGAUGUUGUACAGACTT.

3, attack the siRNA molecule (SiRNA) of human hepatitis B virus, it is characterized in that its nucleotides sequence classifies as

Positive-sense strand: GUGUUUGCUGACGCAACCCTT, antisense strand: GGGUUGCGUCAGCAAACACTT.

4, attack the siRNA molecule (SiRNA) of human hepatitis B virus, it is characterized in that its nucleotides sequence classifies as

Positive-sense strand: UGUCAACGACCGACCUUGATT, antisense strand: UCAAGGUCGGUCGUUGACATT.

5, attack the siRNA molecule (SiRNA) of human hepatitis B virus, it is characterized in that its nucleotides sequence classifies as

Positive-sense strand: CUUUUUCACCUCUGCCUAATT, antisense strand: UUAGGCAGAGGUGAAAAAGTT.

6, attack the siRNA molecule (SiRNA) of human hepatitis B virus, it is characterized in that its nucleotides sequence classifies as

Positive-sense strand: GCUGUGCCUUGGGUGGCUUTT, antisense strand: AAGCCACCCAAGGCACAGCTT.

7, attack the siRNA molecule (SiRNA) of human hepatitis B virus, it is characterized in that its nucleotides sequence classifies as

Positive-sense strand: UUUGGAGCUUCUGUGGAGUTT, antisense strand: ACUCCACAGAAGCUCCAAATT.

8, attack the siRNA molecule (SiRNA) of human hepatitis B virus, it is characterized in that its nucleotides sequence classifies as

Positive-sense strand: UGCCCCUAUCUUAUCAACATT, antisense strand: UGUUGAUAAGAUAGGGGCATT.

9, attack the siRNA molecule (SiRNA) of human hepatitis B virus, it is characterized in that its nucleotides sequence classifies as

Positive-sense strand: GGUCUCAAUCGCCGCGUCGTT, antisense strand: CGACGCGGCGAUUGAGACCTT.

10, attack the siRNA molecule (SiRNA) of human hepatitis B virus, it is characterized in that its nucleotides sequence classifies as

Positive-sense strand: UCUCGGGAAUCUCAAUGUUTT, antisense strand: AACAUUGAGAUUCCCGAGATT.

11, attack the siRNA molecule (SiRNA) of human hepatitis B virus, it is characterized in that its nucleotides sequence classifies as

Positive-sense strand: UUAAUUAUGCCUGCUAGGUTT, antisense strand: ACCUAGCAGGCAUAAUUAATT.

12, attack the siRNA molecule (SiRNA) of human hepatitis B virus, it is characterized in that its nucleotides sequence classifies as

Positive-sense strand: CUGGAUCCUGCGCGGGACGTT, antisense strand: CGUCCCGCGCAGGAUCCAGTT.