CN1566131A

CN1566131A - Small interference RNA molecule SiRNA capable of attacking human hepatitis B virus and application thereof

Info

Publication number: CN1566131A
Application number: CN 03145040
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: 2005-01-19
Anticipated expiration: 2023-06-17
Also published as: CN1322898C

Abstract

The invention provides a small interference RNA molecule SiRNA capable of attacking human hepatitis B virus, which is double chain RNA molecule whose sequence has at least 70% of consanguinity degree with the sequence (I), wherein the antisense chain of the sequence (I) and one nucleic acid mutant has no consanguinity with the known human gene and gene expression segment. The SiRNA can be used for preparing medicament or preparation for prevention or treating hepatitis B and any diseases relating to hepatitis B viral 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 universe 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, rabbit, 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 reversed transcriptive enzyme, 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 both treat efficient lower (30%-40%) of hepatitis B, negative conversion rate to the surface antigen of hepatitis B virus is lower, and the A Erfa Interferon, rabbit 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 VITAMIN B4 (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(Cyt) (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 genetic 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 genetic 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 VITAMIN B4 (A), guanine (G), cytosine(Cyt) (C) or uridylic (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 two above successive bases of No. 4 nucleotide sequence appearance are the Nucleotide of VITAMIN B4 (A) in the sequence table, 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 beginning and sequence table are VITAMIN B4 (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 VITAMIN B4 (A) are in full accord.”

Human hepatitis B virus is to belong to a kind of little liver property dna virus 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 virus, it is genomic duplicates with general dna virus 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 promotor 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 x mRNA 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 unique modulin of HBV synthetic and virus is close.Three kinds of surface antigens are the important structure albumen of virus, and wherein long surface antigen is virus and liver cell bonded main molecules 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 contriver 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 West Pacific Ocean 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 liver cancer 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(Cyt) (C) or uridylic (U) from first Nucleotide of 5 ' end beginning; That is to say when two above successive bases of appearance are the Nucleotide of VITAMIN B4 (A) in No. 4 sequence of sequence table, 19 nucleotide sequences after positive-sense strand latter two Nucleotide in the Nucleotide that 19 nucleotide sequences of 5 ' end beginning and these two above successive bases are VITAMIN B4 (A) are in full accord, ".

2), described continuous two bases are VITAMIN B4 (A) Nucleotide is that the Nucleotide at place is counted in following ranking in No. 4 sequence of sequence table, that is: in No. the 1st, sequence table, No. 2 nucleotide sequence, exist together mutually and be continuous two Nucleotide that base is VITAMIN B4 (A) with this ranking number, and 19 nucleotide sequences after the Nucleotide that these two bases are VITAMIN B4 (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

Sequence 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 molecular-weight average 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 corresponding 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 chemically modified.

Described chemically modified 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 sulphur (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-ring of ribose in the nucleosides 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 0H 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.Control group is that phalangeal cell deals with transfection reagent under condition of equivalent.Ordinate zou represents that the antigen levels of SiRNA treatment group accounts for the relative percentage of control group.The mean value 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.Control group is that phalangeal cell deals with transfection reagent under condition of equivalent.Ordinate zou represents that the antigen levels of SiRNA treatment group accounts for the relative percentage of control group.The mean value of three repeated experiments of each data representation among the figure.

The effect that three kinds of SiRNA of Fig. 3, different concns express HBsAg.Diagram No. 2, No. 4 of different concns and No. 6 molecularitys in the HepG2.2.15 cell after 72 hours to its cell conditioned medium in the influence of HBsAg level.Control group is that phalangeal cell deals with transfection reagent under condition of equivalent.Ordinate zou represents that the antigen levels of SiRNA treatment group accounts for the relative percentage of control group.The mean value of three repeated experiments of each data representation among the figure.

The effect that three kinds of SiRNA of Fig. 4, different concns express HBeAg.Diagram No. 2, No. 4 of different concns and No. 6 molecularitys in the HepG2.2.15 cell after 72 hours to its cell conditioned medium in the influence of HBeAg level.Control group is that phalangeal cell deals with transfection reagent under condition of equivalent.Ordinate zou represents that the antigen levels of SiRNA treatment group accounts for the relative percentage of control group.The mean value 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, different concns.Diagram No. 2, No. 4 of different concns and No. 6 molecularitys in the HepG2.2.15 cell after 72 hours to its cell in the influence of dna level.Control group is that phalangeal cell deals with transfection reagent under condition of equivalent.Ordinate zou represents that the antigen levels of SiRNA treatment group accounts for the relative percentage of control group.The mean value 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 different concns (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.Control group is that phalangeal cell deals with transfection reagent under condition of equivalent.Ordinate zou represents that the antigen levels of SiRNA treatment group accounts for the relative percentage of control group.The mean value 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 different concns (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.Control group is that phalangeal cell deals with transfection reagent under condition of equivalent.Ordinate zou represents that the antigen levels of SiRNA treatment group accounts for the relative percentage of control group.The mean value 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 different concns (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.Control group is that phalangeal cell deals with transfection reagent under condition of equivalent.Ordinate zou represents that the antigen levels of SiRNA treatment group accounts for the relative percentage of control group.The mean value of three repeated experiments of each data representation among the figure.

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, purifying 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 synthesis 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.1995,23:2677-84.

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 Yeast Nucleic Acid of four; (2) deprotection; (3) purifies and separates; (4) the desalination aseptic sterilization of annealing.

Concrete preparation manipulation is as follows:

(1), oligomerization Yeast Nucleic Acid is synthetic: SiRNA synthetic is that (for example: carry out Appl ied 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 one section 19 poly-oligomerization Yeast Nucleic Acid and one 2 poly-deoxythymidylic acid.Therefore initiator is that 5 '-O-of connecting of solid phase (CPG) is to dimethoxytrityl-thymidine (1-2umol).Concrete each circulation is synthetic can be divided into for four steps and to finish (Fig. 1).The first step be with thymidine that solid phase is connected on protecting group wash-out under the effect of 3% trichoroacetic acid(TCA) of 5 '; Second step was coupled to 5 '-O-on the last thymidine of sloughing protection to dimethoxytrityl-thymidine phosphoramidite under the effect of active catalyst S-ethyl tetrazolium, formed two thymidine tris phosphites.Coupling time and coupling round-robin 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 acetylize, and a small amount of unreacted active group on the solid phase (for example: hydroxyl and amido) is formed ester or acid amides under the effect of diacetyl oxide.Thereby 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 aqueous methylamine solution (10M, 50% ethanol) that adds 1 milliliter, rest on room temperature.After two hours, take out solution, and solid phase CPG is used ethanol once more; The mixed solution 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 nucleosides are 2 ') on all oligomerization Yeast Nucleic Acid.Pass through ethanol sedimentation 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 of utilization gradient elution, the principal product (ammonium acetate of leacheate A:0.1M of collection 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 added 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, anion-exchange column) of anionresin, can obtain purity at the SiRNA more than 90% (gradient elution, the Tris-HCl of leacheate A:0.025M, 0.025M NaClpH=8,5% acetonitrile; The Tris-HCl of leacheate b:0.025M, 2.0M NaCl, pH=8,5% acetonitrile).

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

Purity and evaluation through the SiRNA behind the purifying 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 structure 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.The RPMI1640 substratum that this cell contains 10% foetal calf serum at external use is cultivated and is regularly screened the high expression level 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 5 * 10 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 specification sheets 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 determinator 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 specification sheets of Applied Biosystem.

Embodiment 3 SiRNA duplicate and pathogenic associated protein synthetic restraining effect 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 liver cancer 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 transcription initiation and terminated position also have 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 actings in conjunction to preceding gene RNA molecule and e antigen mRNA, the coding region of different molecularitys 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 polysaccharase at cAg.

Sequence SiRNA compiles the mRNA that may attack and mainly acts on

Number number positive-sense strand antisense strand is attacked the protein of position molecule

GGUAUGUUGCC?GACAAACGGGCAA 2.1kb，2.4kb，3.5k

7 N1 CGUUUGUCTT CAUACCTT 458-478 b，C/pol S

GUCUGUACAAC?CUCAAGAUGUUGU 2.1kb，2.4kb，3.5k

12 N2 AUCUUGAGTT ACAGACTT 765-785 b，C/pol S

GUGUUUGCUGA?GGGUUGCGUCAGC?1180-120?2.1kb，2.4kb，3.5k

24 N3 CGCAACCCTT AAACACTT 0 b，C/pol P

CUGGAUCCUGC?CGUCCCGCGCAGG?1398-141?0.7kb，2.1kb，2.4k

75 N4 GCGGGACGTT AUCCAGTT 8 b，3.5kb，C/pol X

UGUCAACGACC?UCAAGGUCGGUCG?1681-170?0.7kb，2.1kb，2.4k

31 N5 GACCUUGATT UUGACATT 1 b，3.5kb，C/pol X

CUUUUUCACCU?UUAGGCAGAGGUG?1820-184?0.7kb，2.1kb，2.4k

35 N6 CUGCCUAATT AAAAAGTT 0 b，3.5kb，C/pol E

GCUGUGCCUUG?AAGCCACCCAAGG?1876-189?0.7kb，2.1kb，2.4k

38 N7 GGUGGCUUTT CACAGCTT 6 b，3.5kb，C/pol E

UUUGGAGCUUC?ACUCCACAGAAGC?1925-194?0.7kb，2.1kb，2.4k

40 N8 UGUGGAGUTT UCCAAATT 5 b，3.5kb，C/pol C

UGCCCCUAUCU?UGUUGAUAAGAUA?2308-232

50 N9 UAUCAACATT GGGGCATT 8 3.5kb，C/pol C，P

GGUCUCAAUCG?CGACGCGGCGAUU?2400-242

55 N10 CCGCGUCGTT GAGACCTT 0 3.5kb，C/pol C，P

UCUCGGGAAUC?AACAUUGAGAUUC?2432-245

58 N11 UCAAUGUUTT CCGAGATT 2 3.5kb，C/pol C，P

UUAAUUAUGCC?ACCUAGCAGGCAU?2631-265

63 N12 UGCUAGGUTT AAUUAATT 1 3.5kb，C/pol P

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

3,12 SiRNA molecules are to the restraining effect 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 inhibiting rate 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 restraining effect 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 restraining effect of viral DNA quantity, wherein the restraining effect 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 virus 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 restraining effect, No. 6 molecules the strongest to HBeAg are added No. 4 molecules to X protein, make the retarding effect of their different concns 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 liquor after three days, No. 2 molecule is the strongest to this antigenic restraining effect, and the fairly obvious and maximum retarding effect of the restraining effect of 10nM differs very little.When concentration restraining effect 40 to 80nM the time reaches maximum, be about 62%, further increasing its restraining effect of concentration does not only increase the trend that weakens is arranged on the contrary.No. 4 molecules of different concns 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 restraining effect (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 retarding effect of No. 6 molecules just occurs when lower concentration, and its retarding effect does not increase and descend on the contrary (Fig. 4) when the above concentration of 80nM.

Three kinds of molecules of different concns are not quite similar to the binding mode of viral DNA amount, and the maximum retarding 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 density.No. 4 molecules then are directly proportional with its concentration to the restraining effect 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 restraining effect 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 maximum 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 retarding effect, but amplitude very big frequently (Fig. 6, Fig. 7).Because individual molecule has reached 80% to 90% to the restraining effect 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 inhibiting rate that DNA produces.

The modification of embodiment 4 SiRNA

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

1, sulfuration

Sulfuration is meant that a Sauerstoffatom on the nucleoside phosphorylase diester linkage is transformed into sulphur atom forms nucleosides thiophosphoric acid 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 oxidizing 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 nucleosides

The modification of 2 ' hydroxyl of nucleosides is meant 2 ' the hydroxyl that comes five Yuans sugar of substituted nucleosides to encircle with various saturated alkoxyl groups 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 nucleosides of SiRNA only need replace 2 '-tertiary butyl dimethyl Si yl nucleosides phosphoramidite with 2 '-methoxyl group nucleoside phosphoramidites and get final product in coupled 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, the hepatitis B virus that obtains from the complete genome DNA sequence of known hepatitis B virus ADR hypotype

ADR subtype gene group common sequences, 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, the hepatitis B virus that obtains from the complete genome DNA sequence of known hepatitis B virus ADW hypotype

ADW subtype gene group common sequences, 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?uguacagact?t?21

<160>77

<210>13

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>1...21

<400>13

uaaaaaggga?cucaagaugt?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

caaaacaagc?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

＜1 10〉Hangzhou Xinruijia Biological Medicine Technology Development Co., Ltd.

<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?cagaauagct?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>misc_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

<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, attack the siRNA molecule (SiRNA) of human hepatitis B virus, 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 VITAMIN B4 (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(Cyt) (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 genetic expression fragment do not have homology.

2, the siRNA molecule of attack human hepatitis B virus as claimed in claim 1 (SiRNA), it is characterized in that it with nucleotide sequence (I) also have simultaneously following be characterized as good:

1), the positive-sense strand of nucleotide sequence (I) is that base is a kind of in the Nucleotide of guanine (G), cytosine(Cyt) (C) or uridylic (U) from first Nucleotide of 5 ' end beginning;

2), described continuous two bases are VITAMIN B4 (A) Nucleotide is that the Nucleotide at place is counted in following ranking in No. 4 sequence of sequence table, that is: in No. the 1st, sequence table, No. 2 nucleotide sequence, exist together mutually and be continuous two Nucleotide that base is VITAMIN B4 (A) with this ranking number, and 19 nucleotide sequences in No. the 1st, sequence table and No. 2 nucleotide sequence after the Nucleotide that these two bases are VITAMIN B4 (A) are in full accord or wherein have Nucleotide different or two non-conterminous Nucleotide differences are wherein arranged.

3, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UCUUCUCGAGGACUGGGGATT, antisense strand: UCCCCAGUCCUCGAGAAGATT.

4, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CCUCUUGUCCUCCAAUUUGTT, antisense strand: CAAAUUGGAGGACAAGAGGTT.

5, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

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

6, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GGAACCUCUAUGUUUCCCUTT, antisense strand: AGGGAAACAUAGAGGUUCCTT.

7, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CCUCUAUGUUUCCCUCNUGTT, antisense strand: CANGAGGGAAACAUAGAGGTT.

8, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CCUUCGGACGGAAACUGCATT, antisense strand: UGCAGUUUCCGUCCGAAGGTT.

9, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CUGCACCUGUAUUCCCAUCTT, antisense strand: GAUGGGAAUACAGGUGCAGTT.

10, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

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

11, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CAUCUUGAGUCCCUUUUUATT, antisense strand: UAAAAAGGGACUCAAGAUGTT.

12, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UUUUCUUUUGUCUUUGGGUTT, antisense strand: ACCCAAAGACAAAAGAAAATT.

13, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CUUCAUGGGAUAUGUAAUUTT, antisense strand: AAUUACAUAUCCCAUGAAGTT.

14, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CUUCCUGUAAACAGGCCUATT, antisense strand: UAGGCCUGUUUACAGGAAGTT.

15, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CAGGCCUAUUGAUUGGAAATT, antisense strand: UUUCCAAUCAAUAGGCCUGTT.

16, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UUGUGGGUCUUUUGGGNUUTT, antisense strand: AANCCCAAAAGACCCACAATT.

17, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UGUGGNUAUCCUGCUUUAATT, antisense strand: UUAAAGCAGGAUANCCACATT.

18, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UGCCUUUAUAUGCAUGUAUTT, antisense strand: AUACAUGCAUAUAAAGGCATT.

19, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GCAGGCUUUCACUUUCUCGTT, antisense strand: CGAGAAAGUGAAAGCCUGCTT.

20, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CCUUUACCCCGUUGCCCGGTT, antisense strand: CCGGGCAACGGGGUAAAGGTT.

21, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CGGCCAGGUCUGUGCCAAGTT, antisense strand: CUUGGCACAGACCUGGCCG.

22, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

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

23, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CCCCCACUGGNUGGGGCUUTT, antisense strand: AAGCCCCANCCAGUGGGGGTT.

24, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CCUUUGUGGCUCCUCUGCCTT, antisense strand: GGCAGAGGAGCCACAAAGGTT.

25, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CUCCUAGCCGCUUGUUUUGTT, antisense strand: CAAAACAAGCGGCUAGGAGTT.

26, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UCCCGCGGACGACCCNUCUTT, antisense strand: AGANGGGUCGUCCGCGGGATT.

27, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GGUCUUACAUAAGAGGACUTT, antisense strand: AGUCCUCUUAUGUAAGACCTT.

28, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GAGGACUCUUGGACUCUCATT, antisense strand: UGAGAGUCCAAGAGUCCUCTT.

29, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

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

30, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CGACCGACCUUGAGGCAUATT, antisense strand: UAUGCCUCAAGGUCGGUCGTT.

31, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: NGACUGGGAGGAGUUGGGGTT, antisense strand: CCCCAACUCCUCCCAGUCNTT.

32, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UUGGUCUGUUCACCAGCACTT, antisense strand: GUGCUGGUGAACAGACCAATT.

33, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

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

34, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UCAUCUCAUGUUCAUGUCCTT, antisense strand: GGACAUGAACAUGAGAUGATT.

35, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GCCUCCAAGCUGUGCCUUGTT, antisense strand: CAAGGCACAGCUUGGAGGCTT.

36, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

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

37, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GAAUUUGGAGCUUCUGUGGTT, antisense strand: CCACAGAAGCUCCAAAUUCTT.

38, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

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

39, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CAUUGUUCACCUCACCAUATT, antisense strand: UAUGGUGAGGUGAACAAUGTT.

40, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GCUAUUCUGUGUUGGGGUGTT, antisense strand: CACCCCAACACAGAAUAGCTT.

41, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UCUAGCCACCUGGGUGGGATT, antisense strand: UCCCACCCAGGUGGCUAGATT.

42, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UUAGUAGUCAGCUAUGUCATT, antisense strand: UGACAUAGCUGACUACUAATT.

43, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UAUGGGCCUAAAAAUCAGATT, antisense strand: UCUGAUUUUUAGGCCCAUATT.

44, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UCAGACAACUAUUGUGGUUTT, antisense strand: AACCACAAUAGUUGUCUGATT.

45, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CUAUUGUGGUUUCACAUUUTT, antisense strand: AAAUGUGAAACCACAAUAGTT.

46, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GAGAAACUGUUCUUGAGUATT, antisense strand: UACUCAAGAACAGUUUCUCTT.

47, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CUGUUCUUGAGUAUUUGGUTT, antisense strand: ACCAAAUACUCAAGAACAGTT.

48, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

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

49, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CACUUCCGGAAACUACUGUTT, antisense strand: ACAGUAGUUUCCGGAAGUGTT.

50, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CUACUGUUGUUAGACGACGTT, antisense strand: CGUCGUCUAACAACAGUAGTT.

51, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GAAGAACUCCCUCGCCUCGTT, antisense strand: CGAGGCGAGGGAGUUCUUCTT.

52, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GAACUCCCUCGCCUCGCAGTT, antisense strand: CUGCGAGGCGAGGGAGUUCTT.

53, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

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

54, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UCGCCGCGUCGCAGAAGAUTT, antisense strand: AUCUUCUGCGACGCGGCGATT.

55, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GAUCUCAAUCUCGGGAAUCTT, antisense strand: GAUUCCCGAGAUUGAGAUCTT.

56, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

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

57, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UCUCAAUGUUAGUAUUCCUTT, antisense strand: AGGAAUACUAACAUUGAGATT.

58, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UGUUAGUAUUCCUUGGACUTT, antisense strand: AGUCCAAGGAAUACUAACATT.

59, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GGUGGGAAACUUUACGGGGTT, antisense strand: CCCCGUAAAGUUUCCCACCTT.

60, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CUUUACGGGGCUUUAUUCUTT, antisense strand: AGAAUAAAGCCCCGUAAAGTT.

61, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

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

62, effective inhibition human hepatitis B virus as claimed in claim 2 duplicates the siRNA molecule (SiRNA) with protein expression, it is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UUAUGCCUGCUAGGUUUUATT, antisense strand: UAAAACCUAGCAGGCAUAATT.

63, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UAUUUGCCCUUGGAUAAAGTT, antisense strand: CUUUAUCCAAGGGCAAAUATT.

64, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CAAGAGCUACAGCAUGGGATT, antisense strand: UCCCAUGCUGUAGCUCUUGTT.

65, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GAGCUACAGCAUGGGAGGUTT, antisense strand: ACCUCCCAUGCUGUAGCUCTT.

66, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CCUCGANAAGGCAUGGGGATT, antisense strand: UCCCCAUGCCUUNUCGAGGTT.

67, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GGCAUGGGGACGAAUCUUUTT, antisense strand: AAAGAUUCGUCCCCAUGCCTT.

68, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UCUUUCUGUCCCCAAUCCUTT, antisense strand: AGGAUUGGGGACAGAAAGATT.

69, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CUCAGACAAUCCAGAUUGGTT, antisense strand: CCAAUCUGGAUUGUCUGAGTT.

70, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UCCAGAUUGGGACUUCAACTT, antisense strand: GUUGAAGUCCCAAUCUGGATT.

71, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UCGGCAGUCAGGAAGGCAGTT, antisense strand: CUGCCUUCCUGACUGCCGATT.

72, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: GAGACACUCAUCCUCAGGCTT, antisense strand: GCCUGAGGAUGAGUGUCUCTT.

73, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

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

74, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: CUCCCUCGCCUCGCAGACGTT, antisense strand: CGUCUGCGAGGCGAGGGAGTT.

75, the siRNA molecule of attack human hepatitis B virus as claimed in claim 2 (SiRNA) is characterized in that its nucleotide sequence (I) is

Positive-sense strand: UGAAAAAAGGAGACUAAAATT, antisense strand: UUUUAGUCUCCUUUUUUCATT.

76, as the siRNA molecule (SiRNA) of arbitrary described attack human hepatitis B virus in the claim 1 to 75, it is characterized in that described homology degree with more than 80% for good.

77, as any part of the siRNA molecule (SiRNA) of arbitrary described attack human hepatitis B virus in the claim 1 to 75 or whole formed small interference ribonucleic acid molecules after chemically modified.

78, as the siRNA molecule (SiRNA) of the described attack human hepatitis B virus of claim 77, described chemically modified comprises to the part modification of the phosphodiester bond that connects Nucleotide or with other any chemical bond and replacing.

79, as the siRNA molecule (SiRNA) of the described attack human hepatitis B virus of claim 77, described chemically modified comprises that the OH on to these SiRNA molecule ribose 2 does the modification and the change of any chemistry.

80, as the siRNA molecule (SiRNA) of the described attack human hepatitis B virus of claim 77, described chemically modified comprises does chemically modified or change to any position on the base.

81, as the siRNA molecule (SiRNA) of arbitrary described attack human hepatitis B virus in the claim 1 to 75 preparation prevention or treatment human hepatitis B and with the medicine of hepatitis b virus infected relevant any disease or the application in the preparation.

82, as the siRNA molecule (SiRNA) of arbitrary described attack human hepatitis B virus in the claim 76 preparation prevention or treatment human hepatitis B and with the medicine of hepatitis b virus infected relevant any disease or the application in the preparation.

83, as the siRNA molecule (SiRNA) of arbitrary described attack human hepatitis B virus in the claim 77 preparation prevention or treatment human hepatitis B and with the medicine of hepatitis b virus infected relevant any disease or the application in the preparation.

84, as the siRNA molecule (SiRNA) of arbitrary described attack human hepatitis B virus in the claim 78 preparation prevention or treatment human hepatitis B and with the medicine of hepatitis b virus infected relevant any disease or the application in the preparation.

85, as the siRNA molecule (SiRNA) of arbitrary described attack human hepatitis B virus in the claim 79 preparation prevention or treatment human hepatitis B and with the medicine of hepatitis b virus infected relevant any disease or the application in the preparation.

86, as the siRNA molecule (SiRNA) of arbitrary described attack human hepatitis B virus in the claim 80 preparation prevention or treatment human hepatitis B and with the medicine of hepatitis b virus infected relevant any disease or the application in the preparation.