CN102083983A - Small RNS interference target site sequences of hepatitis B virus and small interference RNAs and the compositions and uses thereof - Google Patents

Abstract

The small RNA interference target site sequences of hepatitis B virus gene, the small interference RNAs(siRNA) used to inhibit the expression of hepatitis B virus gene, the drug compositions containing the small interference RNAs, and the uses of the small interference RNAs in preparing the drug composition used to prevent and treat hepatitis B virus. The said small interference RNAs have high inhibition activities to the expression of hepatitis B virus gene, and can effectively prevent and treat hepatitis B.

Description

Small RNS interference target site sequences of hepatitis B virus and small interference RNAs and the compositions and uses thereof

Hepatitis B virogene small nucleic acids interference target site sequence and

Small RNA and composition and applied technical field

The present invention is on the small nucleic acids interference target site sequence of hepatitis B virogene and small RNA and composition and application, more specifically, target site sequence is disturbed the present invention relates to the small nucleic acids of hepatitis B virogene, small nucleic acids for suppressing hepatitis B virogene expression, and the pharmaceutical composition containing the small RNA and the small RNA are preparing the application in being used to prevent and/or treat the pharmaceutical composition of hepatitis B.Background technology

Virus B hepatitis abbreviation hepatitis B is one kind by hepatitis type B virus（Hepatitis B virus, HBV) caused by infectious disease.According to the World Health Organization（WHO) count, global hepatitis carrier has exceeded 2,000,000,000 people, wherein the state of an illness of 3.6 hundred million hepatitis B chronic patients deteriorates, hepatic failure, hepatic sclerosis and primary hepatoma caused by 1,000,000 people die from HBV infection are there are about every year.China is the most country of infection HBV numbers in world wide, and hepatitis type B virus (HBV) infection rate is up to 57.63%.The existing chronic hepatitis B patient 20,000,000 in the whole nation, annual hepatitis B neopathy number about 500,000 has 23.7 ten thousand people to die from the related disease of hepatitis B, wherein there is 15.6 ten thousand people to die from liver cancer every year.According to Chinese hepatitis preventing and treating foundation 2006, China was used for direct about 30,000,000,000 yuans of the medical treatment consuming for treating chronic hepatitis, hepatic sclerosis, liver cancer every year, and the direct economic loss that HBV is caused is about 360,000,000,000 yuan/year.Everyone is up to 28864.89 yuans by average annual health care costs, is 2.12 times of China's GDP per capita in 2005.As can be seen here, hepatitis B not only seriously endangers the health of our people, but also brings serious social economical burden for country.

Treating the method for hepatitis B mainly includes antiviral duplication, improves body's immunity, protection liver cell, promotes the complex treatment such as liver cell regeneration and Chinese medicine, Primary Care and psychotherapy.The generally acknowledged medicine for preventing and treating hepatitis B in the whole world can be divided mainly into interferon, the major class of nucleoside analog 2.Interferon has used the long period as treating hepatitis B medicine, it is treated last response rate eventually and there was only 30%, even if with other antiviral drugs be used in combination or incremental dose after, it is treated last response rate eventually and also there was only 50%, and easily recurred after drug withdrawal, decrement or reduction frequency injection, adverse reaction substantially increases.In addition, interferon antibody can be induced by being used for a long time, drug effect is lost.Nucleoside analog is rapid-action, but last response rate is not also high eventually, and nucleoside analog only suppresses the virus in cytoplasm of liver, the CCC-DNA in liver cell nuclear is not acted on, it is necessary to maintain medication for a long time.The main problem that long-term prescription faces is virus variation and the drug resistance that triggers, and viral rebound, high recurrence rate also easily occurs after being discontinued.More seriously there is liver function after a few patients drug withdrawal drastically to deteriorate, in addition it is dead.Suppress HBV generation and duplication to reduce viral metabolism and will be undoubtedly the ideal treatment means of hepatitis B to infecting for liver cell from gene level.

Therefore, the problem of a kind of new product that can prevent and/or treat hepatitis B turns into the urgent need to address is developed.The content of the invention First purpose of the present invention is to provide the small nucleic acids interference target site sequence of hepatitis B virogene.

Second object of the present invention is to provide a kind of small RNA for being used to suppress hepatitis B virogene expression.Third object of the present invention is to provide a kind of pharmaceutical composition containing the small RNA.

Fourth object of the present invention is to provide the small RNA and is preparing the application in being used to prevent and/or treat the pharmaceutical composition of hepatitis B.

Small nucleic acids interference can also claim RNA to disturb (RN A interference, RNAi), be by double-stranded RNA

(double-stranded RNA, dsRNA) molecule closes the expression of corresponding gene in mRNA level in-site or makes the process of the gene silencing.RNA perturbation techniques are visually referred to as Knockdown again（) or gene silencing knock-down（Gene silencing), it is a kind of typical post-transcriptional gene regulation method, also known as PTGS（post-transcriptional gene silencing, PTGS).The report of relevant RNA interference appears in nineteen ninety earliest, report the RNA interference phenomenons in genetically modified plants simultaneously by two different research groups, observed RNA interference phenomenons in nearly all eucaryote such as nematode, drosophila, zebra fish and mouse again later.1999, Hamilton and Baulcombe detected RNA fragment of the length for 21-25 nucleotides in the plant for occurring RNA interference, and these RNA fragments are proved to be necessary to RNA interference, referred to as small RNA（siRNA, Small interfering RNA ).Small RNA

(siRNA) with the silencing complex of relevant enzyme and protein the formation RNA inductions of cell source（RNA-induced silencing complex, RISC ).In RNA interfering processes, small RNA（SiRNA the positive-sense strand in) is excluded from complex, and antisense strand instructs RISC to be attached to the corresponding site of said target mrna, is then degraded said target mrna by the rnase iii in compound, so as to close the expression of target gene.

In recent years, RNAi is extremely rapid in communicable disease and the development of malignant tumour field of gene as a kind of efficient sequence specific gene occluding technique.The toxic side effect of normal tissue can be avoided while suppressing virus replication as sequence is suppressed with sequence of the human genome without homology in selection viral genome.Small RNA（SiRNA) method of the efficiency of inhibition of gene expression than present Clinical practice（Antibody or antisense widow close thuja acid）It is high more than 1000 times, and with safe, special, efficient characteristic.Thus utilize small RNA（SiRNA) drug therapy hepatitis B, with the incomparable advantage of conventional medicine.

Hepatitis type B virus（HBV thermophilic liver DNA) is belonged to Genome is about 3.2kb, is partially double stranded cyclic DNA;Hepatitis B virogene group contains 4 opening code-reading frames (ORF, S gene regions, C gene regions, P gene regions and X gene area）.

S gene regions include S genes, pre-s1 gene and anterior s2 gene, and they are separately encoded S protein, M albumen and L albumen；Wherein, S protein and the addicted to liver property of M albumen and hepatitis type B virus, genetic immunization and transcriptional activation are closely related.

C gene regions include anterior c gene and C genes, and they are separately encoded nucleocapsid protein HBeAg and HBcAg, and nucleocapsid HBeAg and HBcAg are the important components for constituting hepatitis B virus core part, are the main bodys of virus replication.

P gene regions include P genes, encode P albumen, and P albumen is containing 816 amino acid, with 4 functional domains, including the archaeal dna polymerase with reverse transcriptase activity, RNase H etc., participate in the overall process of hbv replication.

X gene area includes X gene, is minimum open reading frame in HBV viral genomes, positioned at the 1374-1838 bp of HBV gene group at, total length about 435-462 bp, code length for 154 amino acid albumen, the albumen can directly or Influence is produced on the virus duplication of itself and propagation indirectly, and the tune of infected cell in host cell can be died and Carcinogenesis generation influence.

In order to realize first purpose of the present invention, target site sequence is disturbed the invention provides the small nucleic acids of hepatitis B virogene, wherein, the interference target site sequence includes SEQ ID Nos:Nucleotide sequence shown in one of 2-11, and the length of the interference target site sequence is 15-27 nucleotides.

In order to realize second object of the present invention, present invention also offers a kind of small RNA, wherein, the small RNA is double stranded rna molecule, including positive-sense strand and antisense strand, and the length of the positive-sense strand and antisense strand is 15-27 nucleotides, the respective 3' ends of positive-sense strand and antisense strand are two continuous deoxythymidylic acids, other nucleotide complementaries formation double-strand beyond two continuous deoxythymidylic acids at 3' ends is removed in the positive-sense strand and antisense strand, wherein, the small nucleic acids that the antisense strand of the small RNA can be provided with the present invention disturb target site sequence base complementrity, to suppress the expression of hepatitis B virogene.

In order to realize third object of the present invention, present invention also offers a kind of pharmaceutical composition for being used to preventing and/or treating hepatitis B, wherein, the pharmaceutical composition contains the small RNA of the invention provided as active component.

In order to realize fourth object of the present invention, the application in being used to prevent and/or treat the pharmaceutical composition of hepatitis B is being prepared present invention also offers described small RNA.

Expression of the invention by specifically suppressing one or several genes in the S gene regions in hepatitis B virogene group, C gene regions, P gene regions and X gene area, so as to reduce virus load, the hepatitis type B virus for invading body is worked in gene level, the purpose of prevention and treatment hepatitis B is reached.Particularly, the inhibitory activity that the small RNA that the present invention is provided is expressed hepatitis B virogene is high, can effectively prevent and/or treat hepatitis B.Embodiment

Target site sequence is disturbed the invention provides the small nucleic acids of hepatitis B virogene, wherein, the interference target site sequence includes SEQ ID Nos:Nucleotide sequence shown in one of 2-11, preferably includes SEQ ID No: 2、 SEQ ID No: 4、 SEQ ID No:8 or SEQ ID No:Nucleotide sequence shown in 10, and the length of the interference target site sequence is 15-27 nucleotides, preferably 19-21 nucleotides.

Small nucleic acids interference target site refers to during inhibition of gene expression is carried out using small RNA, nucleotide sequence that can be complementary with the antisense strand of the small RNA in the mRNA sequence of the gene.

In one preferred embodiment, the interference target site sequence such as SEQ ID Nos:Shown in one of 12-21, more preferably, the interference target site sequence such as SEQ ID No: 12、 SEQ ID No: 14、 SEQ ID No:18 or SEQ ID No:Shown in 20.

Present invention also offers a kind of small RNA（SiRNA), wherein, the small RNA is double stranded rna molecule, including positive-sense strand and antisense strand, and the length of the positive-sense strand and antisense strand is 15-27 nucleotides, the respective 3' ends of positive-sense strand and antisense strand are two continuous deoxythymidylic acids, other nucleotide complementaries formation double-strand beyond two continuous deoxythymidylic acids at 3' ends is removed in the positive-sense strand and antisense strand, wherein, the small nucleic acids that the antisense strand of the small RNA can be provided with the present invention disturb target site sequence base complementrity, to suppress the expression of hepatitis B virogene. The small RNA that the present invention is provided is to include the duplex molecule of positive-sense strand and antisense strand, and the length of the positive-sense strand and antisense strand is respectively 15-27 nucleotides；Under preferable case, the length of the positive-sense strand and antisense strand is respectively 19-21 nucleotides.

In the preferred embodiment of the present invention in this respect, the small RNA has HBV-X1, HBV-X2, nucleotide sequence shown in HBV- X3, HBV- X4, HBV- PI, HBV- P2, HBV-PSK HBV- PS2, HBV- CI or HBV- C2, or with the nucleotide sequence obtained by being chemically modified to the nucleotide sequence shown in HBV-X1, HBV-X2, HBV-X3, HBV-X4, HBV-P1 HBV-P2, HBV-PS1, HBV-PS2, HBV-Cl or HBV-C2, wherein

HBV-X1 positive-sense strands:5 ,-CGACCGACCUUGAGGCAUAdTdT-3'

Antisense strand：5 ,-UAUGCCUCAAGGUCGGUCGdTdT-3'；

HBV-X2 positive-sense strands:5 ,-CCUUGAGGCAUACUUCAAAdTdT-3 '

Antisense strand：5 ,-UUUGAAGUAUGCCUCAAGGdTdT-3 '；

HBV-X3 positive-sense strands:5 ,-GCGGGACGUCCUUUGUUUAdTdT-3 '

Antisense strand： 5'- UAAACAAAGGACGUCCCGCdTdT-3'；

HBV-X4 positive-sense strands: 5'- CUAGGAGGCUGUAGGCAUAdTdT-3 '

Antisense strand： 5'- UAUGCCUACAGCCUCCUAGdTdT-3'；

HBV-P1 positive-sense strands： 5'- GGAACAAGAUCUACAGCAUdTdT-3'

Antisense strand：5 ,-AUGCUGUAGAUCUUGUUCCdTdT-3'；

HBV-P2 positive-sense strands：5,-G A AAGU AUGUC A ACG A AUUdTdT -3,

Antisense strand：5,-A AUUCGUUG AC AU ACUUUCdTdT -3 '；

HBV-PS1 positive-sense strands： 5' - GAUCCAGCCUUCAGAGCAAdTdT-3'

Antisense strand：5 ,-UUGCUCUGAAGGCUGGAUCdTdT-3'；

HBV-PS2 positive-sense strands： 5' - CGUCAAUCUUCUCGAGGAUdTdT-3'

Antisense strand：5 ,-AUCCUCGAGAAGAUUGACGdTdT-3'；

HBV-C1 positive-sense strands：5 ,-GGGUGUUAAUUUGGAAGAUdTdT-3'

Antisense strand：5 ,-AUCUUCCAAAUUAACACCCdTdT-3'；

HBV-C2 positive-sense strands：5 ,-GGAAACUACUGUUGUUAGAdTdT-3 '

Antisense strand：5 ,-UCUAACAACAGUAGUUUCCdTdT-3'.

Under preferable case, the small RNA has HBV-X1, HBV-X3, HBV-PS1 or the nucleotide sequence shown in HBV-Cl.

According to the present invention, the chemical modification is at least one of following modification：

(1) to the modification for the phosphodiester bond that nucleotides is connected in the nucleotide sequence of the siRNA；

(2) to the modification of the 2'-OH of ribose in the nucleotide sequence of the siRNA；

(3) to the modification of base in the nucleotide sequence of the siRNA.

The chemical modification is known to those skilled in the art, and the modification of the phosphodiester bond refers to phosphodiester bond In oxygen modified, including D2EHDTPA modification, as shown in Equation 1；With boron protective embankment phosphate modified, as shown in Equation 2.Two kinds of modifications can stablize small RNA structure, keep the high specific and high-affinity of base pairing.

(1) (2)

The ribose modification refers to the modification to 2'-OH in nucleotides pentose, and gp introduces some substituents in the hydroxy position of ribose, for example, 2'- fluoro is modified, as shown in Equation 3；2'- oxygen methyl is modified, as shown in Equation 4；2'- oxygen ethylenemethoxy is modified, as shown in Equation 5；2,4'- dinitrophenol are modified, as shown in Equation 6；Lock nucleic acid (LNA), as shown in Equation 7;2'- is amido modified, as shown in Equation 8；2'- deoxidations are modified, as shown in Equation 9.

H,

(3) (4)

Ζ〇 DHP

(c) 2-Ο-ΜΟΕ

(5) (6)

(7) (8) (9) described base modification refers to modify the base of nucleotides, for example, 5'- bromouracils are modified, as shown in Equation 10;5'- iodouracils are modified, as shown in Equation 11；N- methyl uracils are modified, as shown in Equation 12；2,6-diaminopurine is modified, as shown in Equation 13.

(12) (13)

Under preferable case, the ability that the modification makes the small RNA after modification resist nuclease hydrolysis in the cell strengthens.In addition, in order to promote small RNA to enter cell, on the basis of being modified more than, the lipophilic groups such as cholesterol are introduced in the end of small RNA positive-sense strand, lipophilic group includes being combined with small RNA with covalent bond, such as end introduce cholesterol, lipoprotein, vitamin E, in favor of by the cell membrane being made up of lipid bilayer with Intracellular mRNA has an effect.Meanwhile, small RNA can also carry out non-covalent bond modification, such as increase stability and biological activity by hydrophobic bond or ionic bond combination phospholipid molecule, polypeptide, cationic polymer.

The preparation method for the small RNA that the present invention is provided includes the design of small RNA sequence and the preparation of small RNA.

The design of the small RNA refers to select sequence conservative hepatitis B virus strain relatively（Genbank registration numbers are U95551) it is template.The conserved region of X, P, S, C gene of hepatitis type B virus is directed to respectively, 19bp nucleotide sequence is chosen, and designs corresponding small RNA.

The conserved region of X, P, S, C gene is in HBV gene group（Genbank registration numbers are U95551) relative position is respectively 1376-1820bp, 2309bp-1625bp, 2848-837bp, 1816bp-2454bp.

The small RNA sequences Design for X, P, S, C gene is carried out by following principle：

The nucleotide sequence of 19bp length is chosen in the range of 1376-1820bp, 2309bp-1625bp of hepatitis type B virus U95551 pnca gene groups, 2848-837bp, 1816bp-2454bp.The selection Primary Reference following items principle of 19bp nucleotide sequences：（1) G/C content is between 35-55 %,（2) avoid in repetitive sequence or low-complexity sequence area,（3) the continuous base sequence of more than 4 is avoided the occurrence of,（4) avoid containing mononucleotide polymorphism site,（5) avoid within the 50-100bp regions in reading frame initiation codon and termination codon, in addition, also want the composition and macroscopic property of analysis of nucleotide sequences.Analyzed by BLAST, candidate's small RNA target site is subjected to sequence analysis with human genome sequencing, exclusion has very big sequence homology with other genes（More than 16 bases）Sequence, to ensure that to other irrelevant genes inhibitory action will not occur for candidate's small RNA target site, and only there is special inhibitory action to hepatitis B virogene.

Finally using the 3' ends for the 19bp nucleotide sequences being achieved in that plus the sour positive-sense strand as small RNA sequence of two deoxythymidines, two deoxythymidines acid are added in the 3' ends of the complementary series of this 19bp nucleotide sequence as the antisense strand of small RNA sequence.

According to the present invention, the preparation method of the small RNA is known to those skilled in the art, for example, the small RNA can be obtained by chemical synthesis, or is obtained by the expression of plasmid and/or viral vector.

Biotech company's synthesis of nucleic acid synthesis is specialized in the method that the synthesis of the small RNA sequence can use chemical synthesis, or commission, and such as commission Shanghai GenePharma companies are synthesized.

In general, the method for the chemical synthesis includes following four process：（1) synthesis of Oligoribonucleotides；(2) it is deprotected；（3) purifies and separates；（4) desalination.

For example, the chemical synthesis of the small RNA with the nucleotide sequence shown in HBV-X1 is comprised the following steps that：(1) synthesis of Oligoribonucleotides：The synthesis of Oligoribonucleotides is in automated DNA/RNA synthesizers（For example, Applied Biosystems EXPEDITE8909) on carry out, the order of the nucleotide sequence according to HBV-X1 connects corresponding nucleotides one by one.Because small RNA is made up of one section of 19 Oligoribonucleotides and 2 deoxythymidylic acids.Therefore, starting material is the 5'-0- of solid diffusivity to dimethoxy-thymidine, and each specific circulation synthesis can be divided into four steps to complete, and the first step is will to be eluted with the protection group of 5' on the thymidine being fixedly connected in the presence of trichloroacetic acid；Second step is in the presence of active catalyst S- ethyl tetrazoliums, by 5, -0- to dimethoxytrityl - Thymidine phosphoramidite is coupled to be taken off on a de-protected upper thymidine, forms two thymidine tris phosphites, and Coupling time, coupling number of times are completed by the program of instrument producer offer;3rd step be by two thymidine tris phosphites of coupling 0.05M iodine water effect under, be oxidized to the ester of two thymidine phosphates three；4th step is acetylation, by a small amount of unreacted active group in solid phase（For example, hydroxyl and amido）Ester or acid amides are formed in the presence of acetic anhydride, so as to reach the effect of closing, to reduce the generation of overall accessory substance, this circulation is repeated until completing the synthesis of whole nucleotide sequences.

(2) it is deprotected

Synthetic solid phase small RNA is put into can be sealedly in bottle to one, and adds 1 milliliter of ethanol/amine (volume ratio is 1:3), then seal, be placed in 55-7CTC incubators, be incubated 2-30 hours, take out solution, and solid phase is eluted with distilled water again, collect eluent, and dry removal solvent.Then, the tetrahydrofuran solution of 1 milliliter of tetrabutyl ammonium fluoride is added（1M), room temperature is placed 4-12 hours, then by ethanol precipitation, obtains the crude product of small RNA.

(3) purifies and separates

In the ammonium acetate solution that the crude product of small RNA is dissolved in 2 milliliters, then by the separation of reaction C18 high pressure liquid chromatographies, with the method for gradient elution, small RNA principal product is collected（Eluent A:0.1M ammonium acetates；Eluent B:20% 0.1M ammonium acetates and 80 % acetonitrile）, the solvent in principal product is removed, and 5 milliliter of 80 % acetic acid aqueous solution is added, it is being stored at room temperature 15 minutes, then this solution is carried out to separation (DEAE-5PW, anion-exchange column of anion exchange）, you can obtain small RNA of the purity in 90 more than %（Gradient elution, eluent A:The % of 0.025M Tris-HCl'0.025M NaCl, pH=8,5 acetonitrile;Eluent B:0.025M Tris-HCl, 2.0M NaCl, pH=8,5 % acetonitrile）.

(4) desalination

The small RNA of purifying removes salt by dialysis, and small RNA solution then is carried out into filtering sterilization and drying crystalline.Then the oligonucleotides of positive-sense strand and antisense strand are formed to stable double-strand small RNA by annealing, its method is the Oligoribonucleotides mixed dissolution by positive-sense strand and antisense strand in 1-2 milliliters of buffer solution（10mM Tris, pH=7.5-8.0,50mM NaCl), this solution is heated to 95 °C, this solution is slowly then cooled to room temperature, finally this solution is stored in 4 °C of refrigerators and preserved, in case using at any time.

In addition to chemical synthesis, small RNA can also be obtained by the expression of plasmid and/or viral vector, obtain the shRNA with hairpin structure, and its length is 50-90 nucleotides.ShRNA structure is：

Two ends are restriction enzyme site（Such as BamHAnd EcoR), centre is one section of loop sequence（Such as GAAGCTTG), inserted it into by clone technology in the carrier crossed with corresponding endonuclease digestion, then be incorporated into chromosome, you can stable expression small RNA.

For example：5 ,-GATCCG- positive-sense strand GAAGCTTG- antisense strands TTTTTTGGAATT-3'

Present invention also offers a kind of pharmaceutical composition for being used to preventing and/or treating hepatitis B, wherein, the pharmaceutical composition contains the small RNA of the invention provided as active component.

According to the present invention, described pharmaceutical composition can be parenteral solution.

In the present invention, the parenteral solution contains the small RNA that pharmaceutically acceptable carrier and the present invention are provided, described The content of small RNA and pharmaceutically acceptable carrier can change in very large range, under preferable case, relative to the small RNA of 100 parts by weight, and the content of the pharmaceutically acceptable carrier is 100-10000000 parts by weight.

In the present invention, the pharmaceutically acceptable carrier is had no particular limits, can be pH value be 4.0-9.0 phosphate buffer, pH value be 7.5-8.5 trihydroxy methyl amido first protective embankment hydrochloric acid salt buffer, physiological saline or pH be 5.5-8.5 phosphate buffer, under preferable case, the pharmaceutically acceptable carrier is the phosphate buffer that pH value is 4.0-9.0.

According to the present invention, the parenteral solution can also contain protective agent and/or osmotic pressure regulator；On the basis of the parenteral solution, protectant content is 0.01-30 weight %, one or more of the protective agent in inositol, sorbierite and sucrose；The content of the osmotic pressure regulator make the osmotic pressure of the parenteral solution for 200-700 m osmoles/kilogram, the osmotic pressure regulator is sodium chloride and/or potassium chloride.

When injecting Pharmaceutical composition of the present invention, injection consumption can be dosage commonly used in the art, the dosage can according to various parameters, the age in particular according to patient to be treated, the order of severity of body weight and illness determine.

The application in being used to prevent and/or treat the pharmaceutical composition of hepatitis B is being prepared present invention also offers described small RNA.

The present invention is further illustrated with reference to embodiment, unless stated otherwise, reagent, culture medium used in the present invention are commercial goods.Embodiment 1

The synthesis of small RNA

Select sequence conservative hepatitis B virogene group relatively（Genbank registration numbers are U95551) (SEQ ID NO. 1) be template.The conserved region of hepatitis type B virus U95551 genes is directed to respectively, 19bp nucleotide sequence is chosen, and designs small RNA.

It is described to be carried out for X, P, s, C gene small RNA sequences Design by following principle：

The nucleotide sequence of 19bp length is chosen in the range of 1376-1820bp, 2309bp-1625bp, 2848-837bp 1816bp-2454bp of hepatitis type B virus U95551 pnca gene groups.The selection Primary Reference following items principle of 19bp nucleotide sequences：（1) G/C content is between 35-55 %,（2) avoid in repetitive sequence or low-complexity sequence area,（3) the continuous base sequence of more than 4 is avoided the occurrence of,（4) avoid containing mononucleotide polymorphism site,（5) avoid within the 50-100bp regions in reading frame initiation codon and termination codon, in addition, also want the composition and macroscopic property of analysis of nucleotide sequences.Analyzed by BLAST, candidate's small RNA target site is subjected to sequence analysis with human genome sequencing, exclusion has very big sequence homology with other genes（More than 16 bases）Sequence, to ensure that to other irrelevant genes inhibitory action will not occur for candidate's small RNA target site, and only there is special inhibitory action to hepatitis B virogene.

Finally using the 3' ends for the 19bp nucleotide sequences being achieved in that plus the sour positive-sense strand as small RNA sequence of two deoxythymidines, two deoxythymidines acid are added in the 3' ends of the complementary series of this 19bp nucleotide sequence as the antisense strand of small RNA sequence. The target site sequence that the small RNA designed in the present embodiment is attacked includes SEQ ID Nos:Nucleotide sequence shown in one of 2-11, specific small nucleic acids interference target site sequence such as SEQ ID Nos:Shown in one of 12-21（Length is 19 nucleotides）.

Designed small RNA is through the lucky agate in Shanghai（GenePharma) company carries out chemical synthesis, obtains small RNA HBV- XI to HBV- X4, HBV-P1 to HBV- P2, HBV-PS1 to HBV- PS2 and HBV- C1 to HBV-C2, their nucleotide sequence is as shown in table 1.Table 1

The firing area refers to the small RNA in SEQ ID:Opposite position in NO.l.Embodiment 2

The inhibitory activity detection expressed hepatitis B virogene

(1) culture of HepG2.2.15 cells

With containing 10 % hyclones, 2mM L- glutamines, 380ug/ml G418 DMEM complete mediums, with lxlO in 24 porocyte culture plates⁵The density inoculation HepG2.2.15 cells of individual cells/well（From The People's Hospital of Peking University）, cultivated in the incubator that temperature is 37 °C and C02 contents are 5 %, passage in every 72 hours, replacing Fresh culture.24 hours before transfection, with 0.25% trypsin digestion cell, count, then with lxlO⁵Individual cell/ml concentration is inoculated into 24 orifice plates, per the μ 1 of hole 1000.

(2) transfection of small RNA

Small RNA HBV-X1 to HBV- X4, HBV- P1 to HBV- P2, HBV- PS1 to HBV- PS2 and the HBV- C1 to HBV- C2 obtained using the liposomes of Lipofectamine 2000 of Invitrogen companies to embodiment 1 is transfected respectively, to be used as blank control without small RNA.

Concrete operation step is as follows：In the sterilized water that small RNA is dissolved in no RNase, concentration is configured to for 2 (mol/L small RNA solution.Suction out the low blood serum mediums of OptiMEM I that 0.5ml is added per the supernatant in the cell of hole（Invitrogen companies, 31985-062).Respectively by the small RNA solution of 3 μ 1（The ω of 20 μ η ι ο 1/ are diluted in the low blood serum mediums of Opti-MEM I of 50 μ 1（Invitrogen companies, 31985-062) in, the liposomes of 1, Ο μ Lipofectamine 2000 are diluted in the low blood serum mediums of Opti-MEM I of 50 μ 1（Invitrogen companies, 31,985 62) in, then by above two solution be incubated at room temperature after 5 minutes mix, mixed solution is in being stored at room temperature after 20 minutes, and Ι Ο Ο μ Ι, the mixed solution is added in 24 orifice plate for being inoculated with cell.The ultimate density of small RNA is 100 η Μ.37 °C of cell is cultivated 4 hours, add lml containing 10% hyclone, 2mM L- glutamines, 100U/ml penicillin, 10 (the MEM complete mediums of ^g/ml streptomysins, are then further cultured for 48 hours under 37 °C.

(3) fluorescence quantitative PCR method detection small RNA is detected respectively to the inhibitory action that hepatitis type B virus mRNA is expressed by Real Time-PCR（2) expression quantity of hepatitis B virogene mRNA in small RNA HBV-X1 to HBV-X4, small RNA HBV-P1 to HBV-P2, small RNA HB V-PS 1 to HB V-PS2 and small RNA HBV-C1 to HBV-C2 HEPG2.2.15 cells has been transfected in, control is used as using the HEPG2.2.15 cells of untransfected small RNA.

Ju Ti Walk are suddenly：Use lml Tri_Z0The HepG2.2.15 cells of the continuous expression hepatitis type B virus of l (GIBCOL companies) cracking transfection small RNAs, and total serum IgE is extracted, extract concretely comprising the following steps for total serum IgE：By the cell after transfection temperature be 37 °C and C0₂Content is then centrifuged for collecting cell, and washed one time with the 2ml PBS of precooling to cultivate 48 hours in 5 % incubator；The composition of the PBS is： NaCl 137mmol/L, KC1 2.7mmol/L, Na₂HP0₄ 4.3mmol/L, KH₂P0₄1.4mmol/L;Lml Trizol are added per hole, room temperature is placed 5 minutes, and cell is cracked；Lysate is transferred in 1.5ml EP pipes；The chloroforms of 200 μ 1 are added, are acutely shaken with hand 15 seconds, room temperature is placed 3 minutes；4 °C of 14000rpm is centrifuged 15 minutes；Take honest and upright and thrifty 500 μ 1 in liquid phase to be put in a new Ε Ρ pipes, add the isopropanols of 500 μ 1, room temperature is placed 10 minutes；12000 rpm, 4 °C centrifuge 10 minutes, remove supernatant, are washed sediment once for 75% ethanol with 1 ml concentration；7600 rpm, 4 °C centrifuge 5 minutes；Remove supernatant, drying at room temperature RNA precipitate 10 minutes；Add the ddH of 20 μ 1₂0 dissolving RNA.

By DNase I (RNase-free) (TakaRa companies of 2 units）Add into the above-mentioned DEPC water for being dissolved with RNA, and 30 minutes are stood under the conditions of 37 °C, to remove the DNA remained in total serum IgE.After DNase I processing, using the PureLink Micro-to-Midi Total RNA Purification Kit of Invitrogen companies total serum IgE is purified, purification is concretely comprised the following steps：The concentration that 20 μ 1 are added in total serum IgE is 70% ethanol, and vibration is well mixed, and mixture is transferred on purification column, and room temperature 12000rpm is centrifuged 15 seconds, discards filtered fluid, adds the cleaning buffer solution I (TakaRa of 700 μ 1 Company）, room temperature 12000rpm centrifuges 15 seconds, discards filtered fluid, add cleaning buffer solution II (the TakaRa companies of 500 μ 1）, room temperature 12000rpm centrifuge 15 seconds, discard filtered fluid, add cleaning buffer solution II (the TakaRa companies of 500 μ 1）, room temperature 12000rpm centrifuge 15 seconds, abandon filtered fluid, room temperature 12000rpm is centrifuged 1 minute, and purification column is transferred on RNA collecting pipes, adds 3CV1 DEPC water, room temperature is placed 1 minute, and room temperature 13000rpm is centrifuged 2 minutes, and RNA sample is placed in into -80 °C of preservations.

Reverse transcription reaction is carried out to the total serum IgE that is obtained after purification, in reverse transcription reaction, reverse transcriptase used is the M-MLV reverse transcriptases of Promega companies, reverse transcription reaction is concretely comprised the following steps：Total serum IgE after lug is purified is mixed with lul (0.5ug) Oligo dT in test tube, cumulative volume is complemented into 16.25 μ 1 with DEPC water, test tube is heated, it is 70 °C that the condition of heating, which includes heating-up temperature, and the heat time is 5 minutes；Then test tube is rapidly cooled to 0 °C, and adds buffer solution (5xMLV buffer 5 μ 1,10mM Dntp 1.25 μ 1, RNasin 0.5 μ 1, M-MLV 1 μ 1), be incubated 1 hour under the conditions of 42 °C, obtain cDNA.

The template that obtained cDNA is reacted as PCR, carries out Real-time PCR reactions.Real-time PCR reaction systems are：<1(1Η₂The μ 1 of 0 17.5 μ 1,10mM Dntp 0.5 μ 1, lOxTaq buffer 2.5 μ 1, Τ 0.5 μ 1, F primer 0.5 μ 1, R primer 0.5 μ 1, Syber Green I Ι μ, ο Ν Α 2;PCR reaction condition be：94 °C 2 minutes, 94 °C 15 seconds, 60 °C 30 seconds, totally 40 circulation.GAPDH is set up simultaneously as reference gene, small RNA inhibitory activity is calculated according to following formula, as a result as shown in table 2.

Totally three pairs of Realtime PCR primers, according to added small RNA sample choose respectively for C genes, X gene, S and P genes primer.Sequence such as following table：

Small RNA inhibitory activity=[1- (the GAPDH copy numbers after the copy number of the hepatitis B virogene after small RNA transfection/small RNA transfection）I (copy number of control wells hepatitis B virogene/control wells GAPDH copy numbers)] χ ο ο %.

GAPDH:Glyceraldehyde 3-phosphate dehydro-genase gene.3- Pity acid glycerol aldehyde dehydrogenase genes are the house-keeping genes in cell, and stable expression, is not influenceed by other additional factors in cell, therefore the internal reference reacted as quantitative fluorescent PCR.

Table 2

HBV-PS2 51 %

HBV-C1 91 %

HBV-C2 46%

As can be seen from Table 2, the small RNA HBV-X1 that the present invention is provided can suppress the expression of hepatitis B virogene to HBV-PS2 and HBV-C1 to HBV-C2 to HBV-X4, HBV-P1 to HBV-P2. HBV-PS1, and particularly HBV-X1, HBV-X3. HBV-PS1 HBV-C1 inhibiting rate are all more than 80%.Embodiment 3

To hepatitis type B virus s antigens (HBsAg), the inhibitory activity detection of e antigens (HBeAg) expression

(1) culture of HepG2.2.15 cells

With containing 10 % hyclones, 2mM L- glutamines, 380ug/ml G418 MEM complete mediums, with lxlO in 24 porocyte culture plates⁵The density inoculation HepG2.2.15 cells of individual cells/well (are derived from The People's Hospital of Peking University）, cultivated in the incubator that temperature is 37 °C and C02 contents are 5 %, passage in every 72 hours, replacing fresh culture.24 hours before transfection, with 0.25% trypsin digestion cell, count, then with lxlO⁵Individual cell/ml concentration is inoculated into 24 orifice plates, per hole lml.

(2) transfection of small RNA

The small RNA HBV-X1 obtained using the liposomes of Lipofectamine 2000 of Invitrogen companies to embodiment 1 is transfected to HBV-X4, HBV-P1 respectively to HBV-P2, HBV-PS1 to HBV-PS2 and HBV-C1 to HBV-C2, to be used as blank control without small RNA.

Concrete operation step is as follows：In the sterilized water that small RNA is dissolved in no RNase, concentration is configured to for 2 (mol/L small RNA solution.Suction out the low blood serum mediums of OptiMEM I that 0.5ml is added per the supernatant in the cell of hole（Invitrogen companies, 31985-062).Respectively by the small RNA solution of 3 μ 1（The ω of 20 μ η ι ο 1/ are diluted in the low blood serum mediums of Opti-MEM I of 50 μ 1（Invitrogen companies, 31985-062) in, the liposomes of 1, Ο μ Lipofectamine 2000 are diluted in the low blood serum mediums of Opti-MEM I of 50 μ 1（Invitrogen companies, 31985 62) in, then above two solution was incubated after 5 minutes at room temperature and mixed, mixed solution is in being stored at room temperature after 20 minutes, Ι Ο Ο μ Ι, the mixed solution is added in 24 orifice plate for being inoculated with cell, is gently shaken up.The ultimate density of small RNA is 100 η Μ.37 °C of cell is cultivated 4 hours, add lml containing 10 % hyclones, 2mM L- glutamines, 100U/ml penicillin, 10 (then the MEM complete mediums of ^g/ml streptomysins are further cultured for 48 hours.

(3) content of detection culture supernatant s antigens (HBsAg)

In order to determine influence of the small RNA to HBV protein expressions, the HBsAg of the cell conditioned medium of collection content is detected using ELISA kit.The kit is biological purchased from China of Shanghai section, and operation by specification is carried out, as a result with P/N values (The absorbance value of absorbance value/control of/1^ values=sample) represent, and small RNA is calculated to both inhibiting rates by following equation.

Absorbance value subtracts the difference obtained by light absorption value of the sample at the 630nm for light absorption value of the detection sample at 450nm.Inhibiting rate（%)=(blank control wells P/N values-experimental port P/N values)/(blank control wells P/N values -2.1) X 100%, knot Fruit is as shown in table 3.

(4) content of detection culture supernatant e antigens (HBeAg)

In order to determine influence of the small RNA to HBV protein expressions, the cell conditioned medium HBeAg of collection content is detected using ELISA kit.The kit is biological purchased from China of Shanghai section, and operation by specification is carried out, and is as a result represented with P/N values (absorbance value of absorbance value/control of P/I^t=sample), and calculates small RNA to both inhibiting rates by following equation.

Absorbance value subtracts the difference obtained by light absorption value of the sample at the 630nm for light absorption value of the detection sample at 450nm.Inhibiting rate（%)=(blank control wells P/N values-experimental port P/N values)/(blank control wells P/N values -2.1) xl00%, as a result as shown in table 4.Table 3

As can be seen from Table 3, the small RNA HBV-X1 that provides of the present invention can suppress the expression of hepatitis B virus S antigen to HBV-PS2 and HBV-C1 to HBV-C2 to HBV-X4, HBV-P1 to HBV-P2, HBV-PS1；Particularly small RNA HBV-X1, HBV-X3 HBV-PS1 and small RNA HBV ~ C1 inhibiting rate are all more than 85%.Table 4

HBV-PS2 45 %

HBV-Cl 66%

HBV-C2 53 %

As can be seen from Table 4, the small RNA HBV-X1 that provides of the present invention can suppress the expression of hepatitis B virus E antigen to HBV-PS2 and HBV-C1 to HBV-C2 to HBV-X4, HBV-P1 to HBV-P2. HBV-PS1；Particularly HBV-X1, HBV-X3, HBV-PSl standing grain mouthful HBV-Cl inhibiting rate is all more than 65%.Embodiment 4

HBV Transgenic Mice is tested

Experimental animal：C57BL/6j-TgN (AlblHBV) 44Bri hepatitis B virogene mouse, male, 7-8 week old, 20-25g, purchased from Laboratory Animal Science portion of Department Of Medicine, Peking University；Animal credit number：SCXK (capital） 2006-0008;Rearing conditions are performed according to SFP grades of minimal standards.

Eye socket endocanthion is taken a blood sample, and centrifuges serum, filters out serum HBsAg strong positive, transgenic mice positive HBV DNA.The close mouse of selection indices is grouped at random, every group 6.It is repeated 3 times.

The small RNA HBV-X1 obtained respectively to embodiment 1 is chemically modified to HBV-P2, HBV-PSl to HBV-PS2 and HBV-C1 to HBV-C2 to HBV-X4, HBV-P1, wherein, chemical modification refers to that 2'- fluoro has been carried out to U, C of small RNA HBV-X1 to HBV-X4, HBV-P1 to HBV-P2, HBV-PSl to HBV-PS2 and HBV-C1 to HBV-C2 positive-sense strand and the 2'-OH of G nucleotide pentose to be modified, and the 2'-OH of antisense strand U and C nucleotide pentose has carried out the modification of 2'- oxygen methyl.The small RNA 1.2mg (0.09 μ η ι ο 1) after modification is dissolved in sterile salines of the 1.5ml without RNase respectively afterwards, be configured to concentration for 6 (^mol/L small RNA solution, and with liposome with 1:1 volume ratio mixing.Sterile salines of the 5ml without RNase is added to the small RNA after liposome（Small RNA concentration is 0.25mg/ml) in, obtain parenteral solution.

Mouse is injected by conventional tail vein injection using parenteral solution respectively, the volume of injection is 20ml parenteral solution/kg body weight, blank control group is injected the physiological saline of same volume, only injected 1 time.MAIN OUTCOME MEASURES：The 3rd after mouse injection, 5,7 days, take blood from eye socket endocanthion, conventional centrifugal obtains serum, -20 °C of standby inspections, and in 7 days after administration plucked eyeball and take mouse is put to death after blood, cut open the belly on ice chest and take hepatic tissue, the standby inspection of homogenate.

(1) haemocyanin is detected

The 3rd after mouse injection, 5,7 days, blood is taken from eye socket endocanthion, conventional centrifugal obtains serum, utilize ALT/glutamic-oxalacetic transaminease kit (ALT/ AST, IFCC recommends method, Beijing Zhongsheng Beikong Biological Science ＆ Technology Co., Ltd. product）, biochemical indicator is detected using Hitachi's 7170A automatic clinical chemistry analyzers.Concrete operations are in strict accordance with kit specification.As a result as shown in table 5 and table 6.Table 5 HBV-X1 135 mmol/L

HBV-X2 247 mmol/L

HBV-X3 148 mmol/L

HBV-X4 240 mmol/L

The mmol/L of 4 HBV-P1 of embodiment 185

HBV-P2 192 mmol/L

HBV-PSl 174 mmol/L

HBV-PS2 223 mmol/L

HBV-Cl 168 mmol/L

As can be seen from Table 5, the small RNA HBV-X1 that the present invention is provided to HBV-X4, HBV-P1 to HBV-P2. HBV-PS1 can reduce serum alt (ALT to HBV-PS2 and HBV-C1 to HBV-C2 to the mmol/L of HBV-C2 246）Content, wherein HBV-X1, HBV-X3, HBV-PSl and HBV-Cl effect is the most obvious.

Table 6

As can be seen from Table 6, small RNA HBV- XI to HBV- X4, HBV- PI to HBV-P2, HBV-PSl to HBV-PS2 and the HBV-Cl to HBV-C2 that the present invention is provided can reduce AST (glutamic-oxalacetic transamineases in serum）Content, wherein HBV-X1, HBV-X3, HBV-PSl standing grain B HBV-Cl effect is the most obvious.

(2) serum HBsAg is detected

The 14th day after mouse injection, pluck eyeball and take blood, the isolated serum of conventional centrifugal is detected using ELISA kit to the content of HBsAg in serum.The kit is purchased from French Biomerieux BV (NL), and operation by specification is carried out.As a result represented with P/N values (absorbance value of P/f^S=sample/control absorbance value), and inhibiting rate of the small RNA to HBsAg contents is calculated by following equation.

Absorbance value subtracts the difference obtained by light absorption value of the sample at the 630nm for light absorption value of the detection sample at 450nm.Inhibiting rate（%)=(blank control wells P/N values-experimental port P/N values)/(blank control wells P/N values -2.1) X 100%, as a result as shown in table 7.Table 7 Embodiment numbering small RNA numbers the inhibiting rate to the HBsAg contents in serum of transgenic mice

( )

HBV-X1 81 %

HBV-X2 22%

HBV-X3 75 %

HBV-X4 15 %

Embodiment 4

HBV-P1 61 %

HBV-P2 20%

HBV-PS1 77 %

HBV-PS2 23 %

HBV-C1 76%

HBV-C2 19%

As can be seen from Table 7, small RNA HBV- XI to HBV- X4, the HBV- PI that the present invention is provided are extremely

HBV-P2, HBV-PS1 can suppress the content of HBsAg in serum to HBV-PS2 and HBV-C1 to HBV-C2.Wherein HBV-X1, HBV-X3, HBV-PS1 and HBV 1 is more up to more than 75% to the inhibiting rate of serum HBsAg content.

(3) hepatic tissue HBV-mRNA detection

The 3rd after mouse injection, 5,7 days, pluck after eyeball sacrificed by exsanguination, take out liver, about lOOmg tissue block is cut into, homogenizer is put into and is fully ground, then according to the explanation of Trizol (GIBCOL companies), hepatic tissue total serum IgE is extracted using Trizol, after the total serum IgE of extraction removes DNA through DNase enzymic digestions, reverse transcription is cDNA, then detects the inhibitory action that small RNA is expressed hepatitis type B virus mRNA with fluorescence quantitative PCR method

Concretely comprise the following steps：

1. Trizol (GIBCOL companies) extracts total serum IgE.Mouse is put to death in dislocation, is cut open the belly on ice chest and is taken hepatic tissue, cuts about lOOmg liver organization block, adds lml Trizol reagents, is homogenized with glass-Teflon or electric homogenizer；Room temperature places 30min after homogenate, to ensure that nucleoprotein complex is kept completely separate.；Lysate is transferred in the new 1.5ml EP pipes without RNase, adding 20, (L chloroforms/lmL Trizol reagents, 15s is acutely shaken with hand, and room temperature places 3min, 12000_g4 °C of centrifugation lOmin;Take supernatant to be put in a new EP pipes, add the isopropanol of 1/2 volume, room temperature places lOmin;2000 g, 4 °C of centrifugation lOmin;Supernatant is sucked, is washed once with isometric ice-cold 75% ethanol, 10000 g, 4 °C of centrifugation 5min suck supernatant；RNA precipitate 10min is dried without RNase ambient room temperatures, adding 2, (^1 DEPC water dissolves RNA.

2. RNA purifying.By DNase I (RNase-free) (TakaRa companies of 2 units）Addition stands 30min into total serum IgE, and under the conditions of 37 °C, to remove the DNA remained in total serum IgE;The ethanol that isometric concentration is 70% is added in total serum IgE, vibration is well mixed；Mixture is transferred on purification column, room temperature 12000g centrifugation 15s abandon filtered fluid；700 μ cleaning buffer solution I, room temperature 12000g centrifugation 15s are added, filtered fluid is abandoned；500 μ cleaning buffer solution II, room temperature 12000g centrifugation 15s are added, filtered fluid is abandoned；Adding 50, (L cleaning buffer solution II, room temperature 12000g centrifugation 15s, abandon filtered fluid；Room temperature 12000g centrifuges lrnin, and purification column is transferred on RNA collecting pipes；The DEPC water of 30 μ 1 is added, room temperature places lrnin;Room temperature 13000g centrifuges 2min, purification column is abandoned, by RNA sample as -80 °C Preserve.

3. RNA reverse transcriptions.By 1 μ_βTotal serum IgE after (2 L) purification heats 5min for 70 °C in EP pipes;EP pipes are put on ice rapidly after micro centrifuge brief centrifugation；Sequentially add 25mM MgCl₂4 L, 10x reverse transcription buffer the lOmM g of dNTP Mixture 2 L, RNase inhibitor 0.5 L, reverse transcriptase 15U (l g), Oligo bow I things 0.5,2 (L are settled to without RNase water;15min, 95 °C of heating 5min, 5 °C of incubation 5min are incubated under the conditions of 42 °C, cDNA is obtained.

The template that obtained cDNA is reacted as PCR, carries out Real-time PCR reactions.PCR kit is purchased from Beijing Mei Laibo medical science and technologies Co., Ltd.Real-time PCR reaction systems are： ddH₂The μ 1 of 0 17.5 μ 1,10mM Dntp 0.5 μ 1, lOxTaq buffer 2.5 μ 1, Taq 05 μ 1, F primer 05 μ 1, R primer 0.5 μ 1, Syber Green I Ι μ, ε Ν Α 2_;PCR reaction condition be：94 °C 2 minutes, 94 °C 15 seconds, 60 °C 30 seconds, totally 40 circulation.GAPDH is set up simultaneously as internal reference, small RNA inhibitory activity is calculated according to following formula, as a result as shown in table 8.

Totally four pairs of Realtime PCR primers, according to added small RNA sample choose respectively for C genes, X gene, S and P genes primer, sequence such as following table：

The inhibitory activity of the small RNA=[1- (copy of administration group hepatitis B virus gene/administration group GAPDH copy numbers）I (copy number of blank control group hepatitis B virus gene/blank control group GAPDH copy number)] 100 % of χ.

Table 8

HBV-PSl to HBV-PS2 and HBV-C1 to HBV-C2 can suppress the expression of HBV gene in transgenic mice liver.The inhibiting rate that wherein HBV-X1, HBV-X3, HBV-PSl and HBV-Cl are expressed HBV gene in liver is more than 70%.

Claims (9)

1. Claims

   1st, the small nucleic acids interference target site sequence of hepatitis B virogene, it is characterised in that small nucleic acids interference target site sequence includes SEQ ID Nos:Nucleotide sequence shown in one of 2-11, and the length of small nucleic acids interference target site sequence is 15-27 nucleotides.

   2nd, small nucleic acids interference target site sequence according to claim 1, wherein, the length of small nucleic acids interference target site sequence is 19-21 nucleotides.

   3rd, small nucleic acids interference target site sequence according to claim 1, wherein, small nucleic acids interference target site sequence includes SEQ ID No: 2、 SEQ ID No: 4、 SEQ ID No:8 or SEQ ID No:Nucleotide sequence shown in 10.

   4th, small nucleic acids interference target site sequence according to claim 1 or 2, wherein, small nucleic acids interference target site sequence such as SEQ ID Nos:Shown in one of 12-21.

   5th, small nucleic acids interference target site sequence according to claim 4, wherein, small nucleic acids interference target site sequence such as SEQ ID No: 12、 SEQ ID No: 14、 SEQ ID No:18 or SEQ ID No:Shown in 20.

   6, a kind of small RNA, it is characterized in that, the small RNA is double stranded rna molecule, including positive-sense strand and antisense strand, and the length of the positive-sense strand and antisense strand is 15-27 nucleotides, the respective 3' ends of positive-sense strand and antisense strand are two continuous deoxythymidylic acids, other nucleotide complementaries formation double-strand beyond two continuous deoxythymidylic acids at 3' ends is removed in the positive-sense strand and antisense strand, wherein, the antisense strand of the small RNA can disturb target site sequence base complementrity with the small nucleic acids described in claim 1 or 4, to suppress the expression of hepatitis B virogene.7th, small RNA according to claim 6, wherein, the length of the positive-sense strand and antisense strand is respectively

   19-21 nucleotides.

   8th, small RNA according to claim 7, wherein, the small RNA has the nucleotide sequence shown in HBV-X1, HBV-X2, HBV-X3, HBV-X4, HBV-Pl, HBV-P2, HBV-PSK HBV-PS2, HBV-Cl or HBV-C2, or with to HBV-X1, HBV-X2, HBV-X3, HBV-X4, HBV-Pl HBV-P2, HBV-PS1, HBV-PS2, nucleotide sequence shown in HBV-Cl or HBV-C2 is chemically modified resulting nucleotide sequence, wherein

   HBV-X1 positive-sense strands： 5'- CGACCGACCUUGAGGCAUAdTdT-3'

   Antisense strand： 5'- UAUGCCUCAAGGUCGGUCGdTdT-3'； HBV-X2 positive-sense strands:5, '-CCUUGAGGCAUACUUCAAAdTdT-3' antisense strands：5 ,-UUUGAAGUAUGCCUCAAGGdTdT-3'；

   HBV-X3 positive-sense strands:5, '-GCGGGACGUCCUUUGUUUAdTdT-3 '

   Antisense strand：5 ,-UAAACAAAGGACGUCCCGCdTdT-3'；

   HBV-X4 positive-sense strands:5, '-CUAGGAGGCUGUAGGC AUAdTdT-3 '

   Antisense strand：5 ,-UAUGCCUACAGCCUCCUAGdTdT-3'；

   HBV-P1 positive-sense strands：5 ,-GGAACAAGAUCUACAGCAUdTdT-3'

   Antisense strand：5 ,-AUGCUGUAGAUCUUGUUCCdTdT-3'；

   HBV-P2 positive-sense strands：5 ,-GAAAGUAUGUCAACGAAUUdTdT-3'

   Antisense strand：5 ,-AAUUCGUUGACAUACUUUCdTdT-3'；

   HBV-PS1 positive-sense strands：5 ,-GAUCCAGCCUUCAGAGCAAdTdT-3 '

   Antisense strand：5 ,-UUGCUCUGAAGGCUGGAUCdTdT-3'；

   HBV-PS2 positive-sense strands：5 ,-CGUCAAUCUUCUCGAGGAUdTdT-3'

   Antisense strand：5 ,-AUCCUCGAGAAGAUUGACGdTdT-3'；

   HBV-C1 positive-sense strands： 5' - GGGUGUUAAUUUGGAAGAUdTdT -3 '

   Antisense strand：5 ,-AUCUUCCAAAUUAACACCCdTdT-3'；

   HBV-C2 positive-sense strands：5 ,-GGAAACUACUGUUGUUAGAdTdT-3 ' antisense strand：5 ,-UCUAACAACAGUAGUUUCCdTdT-3'9, small RNA according to claim 8, wherein, the small RNA have HBV-X1, HBV-X3,

   Nucleotide sequence shown in HBV-PS1 or HBV-C1.

   10th, small RNA according to claim 8, wherein, the chemical modification is at least one of following modification：

   (1) to the modification for the phosphodiester bond that nucleotides is connected in the nucleotide sequence of the small RNA；

   (2) to the 2'-OH of the ribose in the nucleotide sequence of small RNA modification；

   (3) to the modification of the base in the nucleotide sequence of the small RNA.

   11st, it is a kind of to be used to preventing and/or treating the pharmaceutical composition of hepatitis B, it is characterised in that the pharmaceutical composition contains small RNA described in any one in claim 6-10 as active component.

   12nd, pharmaceutical composition according to claim 11, wherein, described pharmaceutical composition is parenteral solution, the parenteral solution also contains pharmaceutically acceptable carrier, relative to the small RNA of 100 parts by weight, the content of the pharmaceutically acceptable carrier is 100-10000000 parts by weight. 13rd, pharmaceutical composition according to claim 12, wherein, the pharmaceutically acceptable carrier be pH value be 4.0-9.0 phosphate buffer, pH value be 7.5-8.5 trihydroxy methyl amido first protective embankment hydrochloric acid salt buffer, physiological saline or pH be 5.5-8.5 phosphate buffer.

   14th, pharmaceutical composition according to claim 12, wherein, the parenteral solution also contains protective agent and/or osmotic pressure regulator；On the basis of the gross weight of the parenteral solution, protectant content is 0.01-30 weight %, one or more of the protective agent in inositol, sorbierite and sucrose；The content of the osmotic pressure regulator make the osmotic pressure of the parenteral solution for 200-700 m osmoles/kilogram, the osmotic pressure regulator is sodium chloride and/or potassium chloride.

   15th, the small RNA described in any one in claim 6-10 is preparing the application in being used to prevent and/or treat the pharmaceutical composition of hepatitis B.