CN102083983B - Small RNS interference target site sequences of hepatitis B virus and small interference RNAs and the compositions and uses thereof - Google Patents
Small RNS interference target site sequences of hepatitis B virus and small interference RNAs and the compositions and uses thereof Download PDFInfo
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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
Technical field
The small nucleic acids that the invention relates to hepatitis B virogene disturbs target site sequence and small RNA and composition and application, or rather, the small nucleic acids that the present invention relates to hepatitis B virogene disturbs target site sequence, the small nucleic acids of expressing for suppressing hepatitis B virogene, and the pharmaceutical composition that contains this small RNA and this small RNA are in the application for the preparation of preventing and/or treating in the pharmaceutical composition of hepatitis B.
Background technology
Hepatitis B is called for short hepatitis B, is a kind of transmissible disease being caused by hepatitis B virus (hepatitis B virus, HBV).According to the World Health Organization (WHO), add up, whole world hepatitis B virus carriers has surpassed 2,000,000,000 people, wherein the state of an illness of 3.6 hundred million hepatitis B chronic patients worsens, and approximately has every year 1000000 people to die from liver failure, liver cirrhosis and the primary hepatocellular carcinoma of HBV due to infecting.China infects the maximum country of HBV number in world wide, hepatitis B virus (HBV) infection rate is up to 57.63%.The existing chronic viral hepatitis B patient 2,000 ten thousand in the whole nation, annual hepatitis B neopathy number approximately 500,000, has 23.7 ten thousand people to die from the disease that hepatitis B is relevant every year, wherein has 15.6 ten thousand people to die from liver cancer.According to Chinese hepatitis control foundation 2006, the direct medical treatment that China is used for the treatment of chronic hepatitis, liver cirrhosis, liver cancer every year expended approximately 30,000,000,000 yuans, and the direct economic loss that HBV causes is about 3,600 hundred million yuan/year.Everyone average annual medical treatment cost, up to 28864.89 yuans, is 2.12 times of China's GDP per capita in 2005.As can be seen here, hepatitis B is serious harm China people's health not only, but also be that country brings serious social economical burden.
The method for the treatment of hepatitis B mainly comprises the antiviral complex therapys such as body's immunity, protection liver cell, promotion liver cell regeneration and Chinese medicine, Primary Care and psychotherapeutics that copy, improve.The drug main of preventing and treating hepatitis B that generally acknowledge in the whole world is wanted to be divided into Interferon, rabbit, the large class of nucleoside analog 2.Interferon, rabbit has been used the long period as treating hepatitis B medicine, its treatment eventually last response rate only has 30%, even and if other antiviral are combined use or increase after dosage, its treatment eventually last response rate also only has 50%, and easily recurrence after drug withdrawal, decrement or minimizing frequency injection, untoward reaction obviously increases.In addition, life-time service can bring out interferon antibody, loses drug effect.Nucleoside analog is rapid-action, but eventually last response rate is not high yet, and nucleoside analog only suppresses the virus in cytoplasm of liver, to not effect of the CCC-DNA in liver cell nuclear, needs long term maintenance medication.The main problem that long-term prescription faces is virus variation and the resistance that causes, and after drug withdrawal, be also prone to virus bounce-back, recurrence rate is high.More seriously after a few patients drug withdrawal, occur that liver function sharply worsens, even dead.From gene level, suppress the generation of HBV and copy to reduce viral metabolism and hepatocellular infecting undoubtedly will be the ideal treatment means of hepatitis B.
Therefore, develop a kind of novel product that can prevent and/or treat hepatitis B and become problem in the urgent need to address.
Summary of the invention
The small nucleic acids that first object of the present invention is to provide hepatitis B virogene disturbs target site sequence.
Second object of the present invention is to provide a kind of small RNA of expressing for suppressing hepatitis B virogene.
The 3rd object of the present invention is to provide a kind of pharmaceutical composition that contains described small RNA.
The 4th object of the present invention is to provide described small RNA in the application for the preparation of preventing and/or treating in the pharmaceutical composition of hepatitis B.
Small nucleic acids disturbs and also can claim RNA to disturb (RNAinterference, RNAi), is to be closed the expression of corresponding gene or make the process of this gene silencing in mRNA level by double-stranded RNA (double-stranded RNA, dsRNA) molecule.RNA perturbation technique is called again Knockdown (knock-down) or gene silencing (gene silencing) visually, it is a kind of typical post-transcriptional gene regulation method, claim again PTGS (post-transcriptional gene silencing, PTGS).The report that relevant RNA disturbs the earliest appears at nineteen ninety, by two different research groups, report the RNA interference phenomenon in transgenic plant simultaneously, in nearly all eukaryotes such as nematode, fruit bat, zebra fish and mouse, observed RNA interference phenomenon again later.1999, it is the RNA fragment of 21-25 Nucleotide that Hamilton and Baulcombe have detected length in the plant that RNA interference occurs, it is necessary that these RNA fragments are proved to be RNA interference, is called small RNA (siRNA, Small interfering RNA).Small RNA (siRNA) and the relevant enzyme of cell source and the silencing complex (RNA-induced silencing complex, RISC) of Protein formation RNA induction.In RNA interfering process, positive-sense strand in small RNA (siRNA) is excluded from complex body, antisense strand instructs RISC to be attached to the corresponding site of said target mrna, the degraded of the rnase iii in mixture said target mrna then, thus close the expression of target gene.
In recent years, RNAi is very rapid in communicable disease and the development of malignant tumour field of gene as a kind of efficient sequence-specific gene knochout technique.Select without the sequence of homology, as suppressing sequence, can when suppressing virus replication, avoid the toxic side effect of normal tissue with human genome in viral genome.The method of the present clinical use of efficiency ratio of small RNA (siRNA) inhibition of gene expression (antibody or antisense widow close thuja acid) is high more than 1000 times, and has safe, special, efficient characteristic.Thereby utilize small RNA (siRNA) pharmacological agent hepatitis B, there is the incomparable advantage of conventional medicine.
Hepatitis B virus (HBV) belongs to Hepadnaviridae (hepadnaviridae), and 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 district).
S gene regions comprises S gene, pre-s1 gene and anterior s2 gene, their encode respectively S albumen, M albumen and L albumen; Wherein, S albumen and M albumen and hepatitis B virus to have a liking for liver property, genetic immunization and transcriptional activation closely related.
C gene regions comprises anterior c gene and C gene, their encode respectively nucleocapsid protein HBeAg and HBcAg, and this nucleocapsid HBeAg and HBcAg are the important component parts that forms hepatitis B virus core part, are the main bodys of virus replication.
P gene regions comprises P gene, coding P albumen, and P albumen, containing 816 amino acid, has 4 functional domains, comprises the archaeal dna polymerase, RNA enzyme H etc. with reverse transcriptase activity, participates in the whole process of hbv replication.
X gene district comprises X gene, it is open reading frame minimum in HBV viral genome, be positioned at the genomic 1374-1838bp of HBV place, the about 435-462bp of total length, code length is 154 amino acid whose albumen, this albumen can exert an influence to copying with propagation of self of virus directly or indirectly, and can the tune of infected cell in host cell be died and be exerted an influence with Carcinogenesis.
In order to realize first object of the present invention, the small nucleic acids that the invention provides hepatitis B virogene disturbs target site sequence, wherein, nucleotide sequence shown in this interference target site sequence one of comprises in SEQ ID Nos:2-11, and the length of described interference target site sequence is 15-27 Nucleotide.
In order to realize second object of the present invention, the present invention also provides a kind of small RNA, wherein, this small RNA is double stranded rna molecule, comprise positive-sense strand and antisense strand, and the length of described positive-sense strand and antisense strand is 15-27 Nucleotide, described positive-sense strand and antisense strand 3 ' end is separately two continuous deoxythymidylic acids, in described positive-sense strand and antisense strand, remove the complementary two strands that forms of 3 ' two continuous deoxythymidylic acids holding other Nucleotide in addition, wherein, the antisense strand of described small RNA can disturb target site sequence base complementrity with small nucleic acids provided by the invention, to suppress the expression of hepatitis B virogene.
In order to realize the 3rd object of the present invention, it is a kind of for preventing and/or treating the pharmaceutical composition of hepatitis B that the present invention also provides, and wherein, this pharmaceutical composition contains small RNA provided by the invention as activeconstituents.
In order to realize the 4th object of the present invention, the present invention also provides described small RNA in the application for the preparation of preventing and/or treating in the pharmaceutical composition of hepatitis B.
The present invention is by suppressing specifically the expression of one or several gene in S gene regions, C gene regions, P gene regions and the X gene district in hepatitis B virogene group, thereby reduction virus load, at gene level, to invading the hepatitis B virus of body, work, reach prevention and the object for the treatment of hepatitis B.Particularly, the inhibition activity that small RNA provided by the invention is expressed hepatitis B virogene is high, can effectively prevent and/or treat hepatitis B.
Embodiment
The small nucleic acids that the invention provides hepatitis B virogene disturbs target site sequence, wherein, nucleotide sequence shown in this interference target site sequence one of comprises in SEQ ID Nos:2-11, preferably include the nucleotide sequence shown in SEQ ID No:2, SEQ ID No:4, SEQ ID No:8 or SEQ ID No:10, and the length of described interference target site sequence is 15-27 Nucleotide, be preferably 19-21 Nucleotide.
Described small nucleic acids disturbs target site to refer to small RNA, coming in the process of inhibition of gene expression, in the mRNA sequence of this gene can with the nucleotide sequence of the antisense strand complementation of described small RNA.
One preferred embodiment in, described interference target site sequence is as shown in one of in SEQ ID Nos:12-21, more preferably, described interference target site sequence is as shown in SEQ ID No:12, SEQ ID No:14, SEQ ID No:18 or SEQ ID No:20.
The present invention also provides a kind of small RNA (siRNA), wherein, this small RNA is double stranded rna molecule, comprise positive-sense strand and antisense strand, and the length of described positive-sense strand and antisense strand is 15-27 Nucleotide, described positive-sense strand and antisense strand 3 ' end is separately two continuous deoxythymidylic acids, in described positive-sense strand and antisense strand, remove the complementary two strands that forms of 3 ' two continuous deoxythymidylic acids holding other Nucleotide in addition, wherein, the antisense strand of described small RNA can disturb target site sequence base complementrity with small nucleic acids provided by the invention, to suppress the expression of hepatitis B virogene.
Small RNA provided by the invention is the duplex molecule that comprises positive-sense strand and antisense strand, and the length of described positive-sense strand and antisense strand is respectively 15-27 Nucleotide; Under preferable case, the length of described positive-sense strand and antisense strand is respectively 19-21 Nucleotide.
In the present invention's preferred implementation in this respect, described small RNA has the nucleotide sequence shown in HBV-X1, HBV-X2, HBV-X3, HBV-X4, HBV-P1, HBV-P2, HBV-PS1, HBV-PS2, HBV-C1 or HBV-C2, or have the nucleotide sequence shown in HBV-X1, HBV-X2, HBV-X3, HBV-X4, HBV-P1, HBV-P2, HBV-PS1, HBV-PS2, HBV-C1 or HBV-C2 is carried out to the resulting nucleotide sequence of chemically modified, wherein
HBV-X1 positive-sense strand: 5 '-CGACCGACCUUGAGGCAUAdTdT-3 '
Antisense strand: 5 '-UAUGCCUCAAGGUCGGUCGdTdT-3 ';
HBV-X2 positive-sense strand: 5 '-CCUUGAGGCAUACUUCAAAdTdT-3 '
Antisense strand: 5 '-UUUGAAGUAUGCCUCAAGGdTdT-3 ';
HBV-X3 positive-sense strand: 5 '-GCGGGACGUCCUUUGUUUAdTdT-3 '
Antisense strand: 5 '-UAAACAAAGGACGUCCCGCdTdT-3 ';
HBV-X4 positive-sense strand: 5 '-CUAGGAGGCUGUAGGCAUAdTdT-3 '
Antisense strand: 5 '-UAUGCCUACAGCCUCCUAGdTdT-3 ';
HBV-P1 positive-sense strand: 5 '-GGAACAAGAUCUACAGCAUdTdT-3 '
Antisense strand: 5 '-AUGCUGUAGAUCUUGUUCCdTdT-3 ';
HBV-P2 positive-sense strand: 5 '-GAAAGUAUGUCAACGAAUUdTdT-3 '
Antisense strand: 5 '-AAUUCGUUGACAUACUUUCdTdT-3 ';
HBV-PS1 positive-sense strand: 5 '-GAUCCAGCCUUCAGAGCAAdTdT-3 '
Antisense strand: 5 '-UUGCUCUGAAGGCUGGAUCdTdT-3 ';
HBV-PS2 positive-sense strand: 5 '-CGUCAAUCUUCUCGAGGAUdTdT-3 '
Antisense strand: 5 '-AUCCUCGAGAAGAUUGACGdTdT-3 ';
HBV-C1 positive-sense strand: 5 '-GGGUGUUAAUUUGGAAGAUdTdT-3 '
Antisense strand: 5 '-AUCUUCCAAAUUAACACCCdTdT-3 ';
HBV-C2 positive-sense strand: 5 '-GGAAACUACUGUUGUUAGAdTdT-3 '
Antisense strand: 5 '-UCUAACAACAGUAGUUUCCdTdT-3 '.
Under preferable case, described small RNA has the nucleotide sequence shown in HBV-X1, HBV-X3, HBV-PS 1 or HBV-C1.
According to the present invention, described chemically modified is at least one in following modification:
(1) to connecting the modification of the phosphodiester bond of Nucleotide in the nucleotide sequence of described siRNA;
(2) modification to 2 '-OH of ribose in the nucleotide sequence of described siRNA;
(3) modification to base in the nucleotide sequence of described siRNA.
Described chemically modified is conventionally known to one of skill in the art, and the modification of described phosphodiester bond refers to modifies the oxygen in phosphodiester bond, comprises thiophosphoric acid modification, as shown in Equation 1; With borine phosphate modified, as shown in Equation 2.Two kinds of modifications can be stablized small RNA structure, keep high specific and the high-affinity of base pairing.
Described ribose is modified and is referred to the modification to 2 '-OH in Nucleotide pentose,, in the hydroxy position of ribose, introduces some substituting group that is, and for example, 2 '-fluoro is modified, as shown in Equation 3; 2 '-oxygen methyl is modified, as shown in Equation 4; 2 '-oxygen ethylidene methoxyl group is modified, as shown in Equation 5; 2,4 '-dinitrophenol(DNP) is modified, as shown in Equation 6; Lock nucleic acid (LNA), as shown in Equation 7; 2 '-amido modified, as shown in Equation 8; 2 '-deoxidation is modified, as shown in Equation 9.
Described base modification refers to be modified the base of Nucleotide, and for example, 5 '-bromouracil is modified, as shown in Equation 10; 5 '-iodouracil is modified, as shown in Equation 11; N-methyl uracil is modified, as shown in Equation 12; 2,6-diaminopurine is modified, as shown in Equation 13.
Under preferable case, the ability that described modification makes the small RNA after modification resist nuclease hydrolysis in cell strengthens.
In addition, in order to promote small RNA to enter cell, can be on the above basis of modifying, end at small RNA positive-sense strand is introduced the lipophilic groups such as cholesterol, lipophilic group comprises with covalent linkage is combined with small RNA, as end, introduce cholesterol, lipoprotein, vitamin-E etc., the cytolemma and the intracellular mRNA that are beneficial to by consisting of lipid bilayer have an effect.Meanwhile, small RNA also can carry out non covalent bond modification, as increased stability and biologic activity by hydrophobic bond or ionic linkage in conjunction with phospholipid molecule, polypeptide, cationic polymers etc.
The preparation method of small RNA provided by the invention comprises the design of small RNA sequence and the preparation of small RNA.
The design of described small RNA refers to that the relatively conservative MS-2 of Selective sequence (Genbank registration number is U95551) is for template.For the conserved regions of X, the P of hepatitis B virus, S, C gene, choose the nucleotide sequence of 19bp respectively, design corresponding small RNA.
The conserved regions of described X, P, S, C gene is respectively 1376-1820bp, 2309bp-1625bp, 2848-837bp, 1816bp-2454bp at HBV genome (Genbank registration number is U95551) relative position.
The described small RNA sequences Design for X, P, S, C gene is undertaken by following principle:
At 1376-1820bp, the 2309bp-1625bp of hepatitis B virus U95551 pnca gene group, choose the nucleotide sequence of 19bp length in the scope of 2848-837bp, 1816bp-2454bp.19bp nucleotide sequence choose following several the principles of main reference: (1) GC content is between 35-55%, (2) avoid in tumor-necrosis factor glycoproteins or low-complexity sequence area, (3) avoid occurring 4 above continuous base sequences, (4) avoid containing mononucleotide polymorphism site, (5) within avoiding the 50-100bp region in reading frame initiation codon and termination codon, in addition, also want composition and the thermodynamic property of analysis of nucleotide sequences.By BLAST, analyze, candidate's small RNA target site is carried out to sequence analysis with human genome sequencing, eliminating has the sequence of very large sequence homology (16 above bases) with other gene, to guarantee that candidate's small RNA target site can be to other irrelevant gene generation restraining effect, and only hepatitis B virogene is had to special restraining effect.
Finally 3 ' end of the 19bp nucleotide sequence obtaining is like this added to two deoxythymidine acid are as the positive-sense strand of small RNA sequence, at 3 ' end of the complementary sequence of this 19bp nucleotide sequence, add that two deoxythymidine acid are as the antisense strand of small RNA sequence.
According to the present invention, the preparation method of described small RNA is conventionally known to one of skill in the art, and for example, described small RNA can obtain by chemosynthesis, or obtains by the expression of plasmid and/or virus vector.
The synthetic method that can adopt chemosynthesis of described small RNA sequence, or entrust that to specialize in the synthetic biotech company of nucleic acid synthetic, as entrusted Shanghai GenePharma company to synthesize.
In general, the method for described chemosynthesis comprises following Four processes: (1) oligomerization ribonucleotide synthetic; (2) deprotection; (3) purifies and separates; (4) desalination.
For example, there are the concrete steps of chemosynthesis of small RNA of the nucleotide sequence shown in HBV-X1 as follows:
(1) oligomerization ribonucleotide is synthetic: the synthetic of oligomerization ribonucleotide at automated DNA/RNA synthesizer (is for example, Applied Biosystems EXPEDITE8909) on, carry out, according to the order of the nucleotide sequence shown in HBV-X1, corresponding Nucleotide is coupled together one by one.Because small RNA is comprised of one section of 19 oligomerization ribonucleotide and 2 deoxythymidylic acids.Therefore, initiator be 5 '-O-of connecting of solid phase to dimethoxy-thymidine, each concrete circulation is synthetic can be divided into four steps and complete, the first step is by protecting group wash-out under the effect of trichoroacetic acid(TCA) of 5 ' on the thymidine with being fixedly connected with; Second step is under the effect of active catalyst S-ethyl tetrazolium, 5 '-O-is coupled on de-de-protected upper thymidine dimethoxytrityl-thymidine phosphoramidite, form two thymidine tris phosphites, the program that coupling time, coupling number of times Jun An instrument producer provide completes; The 3rd step be by two thymidine tris phosphites of coupling under the effect of 0.05M iodine water, be oxidized to two thymidine phosphotriesters; The 4th step is acetylize; a small amount of unreacted active group in solid phase (for example, hydroxyl and amido) is formed to ester or acid amides under the effect of diacetyl oxide, thereby reach the effect of sealing; to reduce the generation of whole by product, repeat this circulation until complete the synthetic of whole nucleotide sequences.
(2) deprotection
Synthetic solid phase small RNA is put into the bottle that can seal to, and add the ethanol/amine (volume ratio is 1: 3) of 1 milliliter, then sealing, be placed in 55-70 ℃ of incubator, hatch 2-30 hour, take out solution, and solid phase is used to distilled water wash-out again, collect elutriant, and the dry solvent of removing.Then, add the tetrahydrofuran solution (1M) of 1 milliliter of tetrabutyl ammonium fluoride, room temperature is placed 4-12 hour, then through ethanol precipitation, obtains the crude product of small RNA.
(3) purifies and separates
The crude product of small RNA is dissolved in the ammonium acetate solution of 2 milliliters, then through the separation of reaction C18 high pressure liquid chromatography, uses the method for gradient elution, collect small RNA principal product (elutriant A:0.1M ammonium acetate; The 0.1M ammonium acetate of elutriant B:20% and 80% acetonitrile), remove the solvent in principal product, and add the acetic acid aqueous solution of 5 milliliter 80%, and in room temperature standing 15 minutes, the separation (DEAE-5PW that then this solution is carried out to anionresin, anion-exchange column), can obtain purity at more than 90% small RNA (gradient elution, the Tris-HCl of elutriant A:0.025M, the NaCl of 0.025M, pH=8,5% acetonitrile; The Tris-HCl of elutriant B:0.025M, the NaCl of 2.0M, pH=8,5% acetonitrile).
(4) desalination
The small RNA of purifying, through dialysis, is removed salt, subsequently small RNA solution is carried out to filter-sterilized and drying crystalline.Then the annealed processing of oligomerization Yeast Nucleic Acid of positive-sense strand and antisense strand is formed to stable double-stranded small RNA, its method is by oligomerization ribonucleotide mixed dissolution (10mMTris in the damping fluid of 1-2 milliliter of positive-sense strand and antisense strand, pH=7.5-8.0,50mM NaCl), this solution is heated to 95 ℃, then slowly this solution is cooled to room temperature, finally this solution is left in 4 ℃ of refrigerators and preserved, in order to using at any time.
Except chemosynthesis, small RNA also can obtain by the expression of plasmid and/or virus vector, obtains having the shRNA of hairpin structure, and its length is 50-90 Nucleotide.The structure of shRNA is:
Do are two ends restriction enzyme site (BamH for example? and EcoR?), centre is one section of loop sequence (for example GAAGCTTG), by clone technology, be inserted in the carrier of crossing with corresponding endonuclease digestion, then be incorporated in karyomit(e), can stably express small RNA.
For example: 5 '-GATCCG-positive-sense strand GAAGCTTG-antisense strand TTTTTTGGAATT-3 '
It is a kind of for preventing and/or treating the pharmaceutical composition of hepatitis B that the present invention also provides, and wherein, this pharmaceutical composition contains small RNA provided by the invention as activeconstituents.
According to the present invention, described pharmaceutical composition can be injection liquid.
In the present invention, described injection liquid contains pharmaceutically acceptable carrier and small RNA provided by the invention, the content of described small RNA and pharmaceutically acceptable carrier can in very large range change, under preferable case, with respect to the small RNA of 100 weight parts, the content of described pharmaceutically acceptable carrier is 100-10000000 weight part.
In the present invention, described pharmaceutically acceptable carrier is had no particular limits, can be pH value be the phosphoric acid buffer of 4.0-9.0, tri methylol amino methane hydrochloride damping fluid, physiological saline or pH that pH value the is 7.5-8.5 phosphate buffered saline buffer that is 5.5-8.5, under preferable case, described pharmaceutically acceptable carrier is the phosphoric acid buffer that pH value is 4.0-9.0.
According to the present invention, described injection liquid can also contain protective material and/or osmotic pressure regulator; Take described injection liquid as benchmark, and described protectant content is 0.01-30 % by weight, and described protective material is selected from one or more in inositol, sorbyl alcohol and sucrose; The content of described osmotic pressure regulator make the osmotic pressure of described injection liquid be 200-700 m osmole/kilogram, described osmotic pressure regulator is sodium-chlor and/or Repone K.
When injection is during medicinal compositions of the present invention, injection consumption can be the conventional dosage in this area, and described dosage can be according to various parameters, especially according to the severity of patient's to be treated age, body weight and illness, determine.
The present invention also provides described small RNA in the application for the preparation of preventing and/or treating in the pharmaceutical composition of hepatitis B.
Below in conjunction with embodiment, further illustrate the present invention, unless stated otherwise, the present invention's reagent used, substratum are commercial goods.
Embodiment 1
Synthesizing of small RNA
The relatively conservative hepatitis B virogene group (Genbank registration number is U95551) (SEQ ID NO.1) of Selective sequence is template.For the conserved regions of hepatitis B virus U95551 gene, choose the nucleotide sequence of 19bp respectively, design small RNA.
Describedly for X, P, S, C gene small RNA sequences Design, by following principle, undertaken:
At 1376-1820bp, the 2309bp-1625bp of hepatitis B virus U95551 pnca gene group, choose the nucleotide sequence of 19bp length in the scope of 2848-837bp, 1816bp-2454bp.19bp nucleotide sequence choose following several the principles of main reference: (1) GC content is between 35-55%, (2) avoid in tumor-necrosis factor glycoproteins or low-complexity sequence area, (3) avoid occurring 4 above continuous base sequences, (4) avoid containing mononucleotide polymorphism site, (5) within avoiding the 50-100bp region in reading frame initiation codon and termination codon, in addition, also want composition and the thermodynamic property of analysis of nucleotide sequences.By BLAST, analyze, candidate's small RNA target site is carried out to sequence analysis with human genome sequencing, eliminating has the sequence of very large sequence homology (16 above bases) with other gene, to guarantee that candidate's small RNA target site can be to other irrelevant gene generation restraining effect, and only hepatitis B virogene is had to special restraining effect.
Finally 3 ' end of the 19bp nucleotide sequence obtaining is like this added to two deoxythymidine acid are as the positive-sense strand of small RNA sequence, at 3 ' end of the complementary sequence of this 19bp nucleotide sequence, add that two deoxythymidine acid are as the antisense strand of small RNA sequence.
Nucleotide sequence shown in the target site sequence that the small RNA designing in the present embodiment is attacked one of comprises in SEQ ID Nos:2-11, concrete small nucleic acids disturbs target site sequence as shown in one of in SEQ ID Nos:12-21 (length is 19 Nucleotide).
The small RNA designing carries out chemosynthesis through Shanghai Ji agate (GenePharma) company, obtain small RNA HBV-X1 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
Described firing area refers to the opposite position of this small RNA in SEQ ID:NO.1.
Embodiment 2
The active detection of inhibition that hepatitis B virogene is expressed
(1) cultivation of HepG2.2.15 cell
With the DMEM perfect medium that contains 10% foetal calf serum, 2mM L-glutamine, 380ug/ml G418, on 24 porocyte culture plates with 1 * 10
5the density inoculation HepG2.2.15 cell (being derived from The People's Hospital of Peking University) of individual cells/well, is to cultivate in 37 ℃ and the CO2 content incubator that is 5% in temperature, within every 72 hours, goes down to posterity, changes fresh culture.In transfection first 24 hours, the trypsin digestion cell with 0.25%, counting, then with 1 * 10
5the concentration of individual cell/ml is inoculated into 24 orifice plates, every hole 1000 μ l.
(2) transfection of small RNA
Use the Lipofectamine of Invitrogen company
tMsmall RNA HBV-X1 to HBV-X4, the HBV-P1 to HBV-P2 that 2000 liposomes obtain embodiment 1, HBV-PS 1 to HBV-PS2 and HBV-C1 to HBV-C2 carry out respectively transfection, using and do not add small RNA as blank.
Concrete operation step is as follows: small RNA is dissolved in the sterilized water without RNA enzyme, and being mixed with concentration is the small RNA solution of 20 μ mol/L.Supernatant in the every porocyte of sucking-off, add 0.5ml the low blood serum medium of OptiMEM I (Invitrogen company, 31985-062).Respectively 3 μ l small RNA solution (20 μ mol/L) are diluted in the low blood serum medium of 50 μ lOpti-MEM I (Invitrogen company, 31985-062) in, by 1.0 μ lLipofectamine
tM2000 liposomes are diluted in the low blood serum medium of 50 μ l Opti-MEM I (Invitrogen company, 31985-062), then above-mentioned two kinds of solution are at room temperature hatched after 5 minutes and mix, mixing solutions is in room temperature after standing 20 minutes, and 100 these mixing solutionss of μ l are added to inoculation to be had in described 24 orifice plates of cell.The ultimate density of small RNA is 100nM.37 ℃ of cultivations of cell 4 hours, then add 1ml to contain the MEM perfect medium of 10% foetal calf serum, 2mM L-glutamine, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, then at 37 ℃, cultivate again 48 hours.
(3) fluorescence quantitative PCR method detects the restraining effect of small RNA to hepatitis B virus mrna expression
By the Real Time-PCR expression amount of hepatitis B virogene mRNA in the HEPG2.2.15 cell of small RNA HBV-X1 to HBV-X4, small RNA HBV-P1 to HBV-P2, small RNA HBV-PS1 to HBV-PS2 and small RNA HBV-C1 to HBV-C2 that detected respectively in (2) transfection, with the HEPG2.2.15 cell of untransfected small RNA in contrast.
Concrete steps are: the HepG2.2.15 cell of using the continuous expression hepatitis B virus of 1ml Trizol (GIBCOL company) cracking transfection small RNA, and extract total RNA, the concrete steps of extracting total RNA are: by the cell after transfection, in temperature, be 37 ℃ and CO
2content is to cultivate 48 hours in 5% incubator, centrifugal collecting cell then, and wash one time with the 2ml PBS of precooling; Described PBS consists of: NaCl 137mmol/L, KCl 2.7mmol/L, Na
2hPO
44.3mmol/L, KH
2pO
41.4mmol/L; Every hole adds 1ml Trizol, and room temperature is placed 5 minutes, cell generation cracking; Lysate is transferred in 1.5ml EP pipe; Add 200 μ l chloroforms, use hand concuss 15 seconds, room temperature is placed 3 minutes; 4 ℃ of 14000rpm are centrifugal 15 minutes; Get honest and upright and thrifty 500 μ l in liquid phase and be put in a new EP pipe, add 500 μ l Virahols, room temperature is placed 10 minutes; 12000rpm, 4 ℃ centrifugal 10 minutes, remove supernatant, the ethanol that is 75% by 1ml concentration is washed throw out once; 7600rpm, 4 ℃ are centrifugal 5 minutes; Remove supernatant, drying at room temperature RNA precipitation 10 minutes; Add 20 μ l ddH
2o dissolves RNA.
The DNase I (RNase-free) (TakaRa company) of 2 units is added in the DEPC water of the above-mentioned RNA of being dissolved with, and under 37 ℃ of conditions standing 30 minutes, to remove DNA residual in total RNA.After DNase I processes, adopt the PureLink Micro-to-Midi Total RNA Purification Kit of Invitrogen company to purify to total RNA, the concrete steps of purifying are: the ethanol that the concentration that adds 20 μ l in total RNA is 70%, vibration mixes, mixture is transferred on purification column, centrifugal 15 seconds of room temperature 12000rpm, discard filtered liquid, add 700 μ l cleaning buffer solution I (TakaRa company), centrifugal 15 seconds of room temperature 12000rpm, discard filtered liquid, add 500 μ l cleaning buffer solution II (TakaRa company), centrifugal 15 seconds of room temperature 12000rpm, discard filtered liquid, add again 500 μ l cleaning buffer solution II (TakaRa company), centrifugal 15 seconds of room temperature 12000rpm, abandon filtered liquid, centrifugal 1 minute of room temperature 12000rpm, purification column is transferred on RNA collection tube, add 30 μ l DEPC water, room temperature is placed 1 minute, centrifugal 2 minutes of room temperature 13000rpm, RNA sample is placed in to-80 ℃ of preservations.
The total RNA obtaining after purifying is carried out to reverse transcription reaction, in reverse transcription reaction, reversed transcriptive enzyme used is the M-MLV reversed transcriptive enzyme of Promega company, the concrete steps of reverse transcription reaction are: the total RNA after 1ug is purified mixes in test tube with the Oligo dT of 1ul (0.5ug), with DEPC water, cumulative volume is complemented to 16.25 μ l, test tube is heated, and the condition of heating comprises that Heating temperature is 70 ℃, and be 5 minutes heat-up time; Then test tube is cooled to rapidly to 0 ℃, and adds damping fluid (5 * MLVbuffer, 5 μ l, 10mM Dntp 1.25 μ l, RNasin 0.5 μ l, M-MLV1 μ l), under 42 ℃ of conditions, hatch 1 hour, obtain cDNA.
Using the cDNA obtaining as PCR, the template of reaction, carries out Real-time PCR reaction.Real-time PCR reaction system is: ddH
2o 17.5 μ l, 10mM Dntp 0.5 μ l, 10 * Taq buffer, 2.5 μ l, Taq 0.5 μ l, F primer0.5 μ l, R primer 0.5 μ l, Syber Green I 1 μ l, cDNA 2 μ l; The condition of PCR reaction is: 94 ℃ 2 minutes, 94 ℃ 15 seconds, 60 ℃ 30 seconds, totally 40 circulations.Set up GAPDH as reference gene simultaneously, calculate small RNA suppress active according to following formula, result is as shown in table 2.
Totally three pairs of Realtime PCR primers, choose respectively the primer for C gene, X gene, S and P gene according to added small RNA sample.Sequence is as following table:
Small RNA suppresses activity=[1-(the GAPDH copy number after copy number/small RNA transfection of the hepatitis B virogene after small RNA transfection)/(copy number/control wells GAPDH copy number of control wells hepatitis B virogene)] * 100%.
GAPDH:3-glyceraldehyde phosphate dehydrogenase gene.Glyceraldehyde 3-phosphate dehydro-genase gene is the house-keeping gene in cell, and stably express in cell is not subject to the impact of other additional factors, the internal reference therefore reacting as quantitative fluorescent PCR.
Table 2
As can be seen from Table 2, small RNA HBV-X1 to HBV-X4 provided by the invention, HBV-P1 to HBV-P2, HBV-PS1 to HBV-PS2 and HBV-C1 to HBV-C2 all can suppress the inhibiting rate of the expression of hepatitis B virogene, particularly HBV-X1, HBV-X3, HBV-PS1 and HBV-C1 more than 80%.
Embodiment 3
The active detection of inhibition that hepatitis B virus s antigen (HBsAg), e antigen (HBeAg) are expressed
(1) cultivation of HepG2.2.15 cell
With the MEM perfect medium that contains 10% foetal calf serum, 2mM L-glutamine, 380ug/ml G418, on 24 porocyte culture plates with 1 * 10
5the density inoculation HepG2.2.15 cell (being derived from The People's Hospital of Peking University) of individual cells/well, is to cultivate in 37 ℃ and the CO2 content incubator that is 5% in temperature, within every 72 hours, goes down to posterity, changes fresh culture.In transfection first 24 hours, the trypsin digestion cell with 0.25%, counting, then with 1 * 10
5the concentration of individual cell/ml is inoculated into 24 orifice plates, every hole 1ml.
(2) transfection of small RNA
Use the Lipofectamine of Invitrogen company
tMsmall RNA HBV-X1 to HBV-X4, HBV-P1 to HBV-P2, HBV-PS1 to HBV-PS2 and the HBV-C1 to HBV-C2 that 2000 liposomes obtain embodiment 1 carries out respectively transfection, usings and do not add small RNA as blank.
Concrete operation step is as follows: small RNA is dissolved in the sterilized water without RNA enzyme, and being mixed with concentration is the small RNA solution of 20 μ mol/L.Supernatant in the every porocyte of sucking-off, add 0.5ml the low blood serum medium of OptiMEM I (Invitrogen company, 31985-062).Respectively 3 μ l small RNA solution (20 μ mol/L) are diluted in the low blood serum medium of 50 μ l Opti-MEM I (Invitrogen company, 31985-062) in, by 1.0 μ l Lipofectamine
tM2000 liposomes are diluted in the low blood serum medium of 50 μ l Opti-MEM I (Invitrogen company, 31985-062), then above-mentioned two kinds of solution are at room temperature hatched after 5 minutes and mix, mixing solutions is in room temperature after standing 20 minutes, 100 these mixing solutionss of μ l are added to inoculation to be had in described 24 orifice plates of cell, shakes up gently.The ultimate density of small RNA is 100nM.37 ℃ of cultivations of cell 4 hours, then add 1ml to contain the MEM perfect medium of 10% foetal calf serum, 2mM L-glutamine, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, and then cultivate 48 hours.
(3) detect the content of culture supernatant s antigen (HBsAg)
In order to determine the impact of small RNA on HBV protein expression, use ELISA test kit to detect the content of the HBsAg of the cell conditioned medium of collecting.This test kit is biological purchased from Shanghai section China, and operation by specification carries out, and result represents with P/N value (absorbance value of the absorbance value/contrast of P/N value=sample), and calculates the inhibiting rate of small RNA to both by following formula.
Absorbance value deducts sample in the difference of the light absorption value gained at 630nm place for detecting the light absorption value of sample at 450nm place.
Inhibiting rate (%)=(blank hole P/N value-experimental port P/N value)/(P/N value-2.1, blank hole) * 100%, result is as shown in table 3.
(4) detect the content of culture supernatant e antigen (HBeAg)
In order to determine the impact of small RNA on HBV protein expression, use ELISA test kit to detect the content of the cell conditioned medium HBeAg collecting.This test kit is biological purchased from Shanghai section China, and operation by specification carries out, and result represents with P/N value (absorbance value of the absorbance value/contrast of P/N value=sample), and calculates the inhibiting rate of small RNA to both by following formula.
Absorbance value deducts sample in the difference of the light absorption value gained at 630nm place for detecting the light absorption value of sample at 450nm place.
Inhibiting rate (%)=(blank hole P/N value-experimental port P/N value)/(P/N value-2.1, blank hole) * 100%, result is as shown in table 4.
Table 3
As can be seen from Table 3, small RNA HBV-X1 to HBV-X4 provided by the invention, HBV-P1 to HBV-P2, HBV-PS1 to HBV-PS2 and HBV-C1 to HBV-C2 all can suppress the expression of hepatitis B virus S antigen; Particularly the inhibiting rate of small RNA HBV-X1, HBV-X3, HBV-PS1 and small RNA HBV-C1 is all more than 85%.
Table 4
As can be seen from Table 4, small RNA HBV-X1 to HBV-X4 provided by the invention, HBV-P1 to HBV-P2, HBV-PS1 to HBV-PS2 and HBV-C1 to HBV-C2 all can suppress the expression of hepatitis B virus E antigen; Particularly the inhibiting rate of HBV-X1, HBV-X3, HBV-PS1 and HBV-C1 is all more than 65%.
Embodiment 4
HBV Transgenic Mice experiment
Laboratory animal: C57BL/6j-TgN (AlblHBV) 44Bri hepatitis B virogene mouse, male, in age in 7-8 week, 20-25g, purchased from Department Of Medicine, Peking University Laboratory Animal Science portion; Animal credit number: SCXK (capital) 2006-0008; Raising condition is carried out according to SFP level animal standard.
The blood sampling of eye socket endocanthion, centrifugation serum, filters out serum HBsAg strong positive, the transgenic mice of the HBV DNA positive.Select the close mouse random packet of indices, 6 every group.Repeat 3 times.
The small RNA HBV-X1 to HBV-X4, HBV-P1 to HBV-P2, HBV-PS1 to HBV-PS2 and the HBV-C1 to HBV-C2 that respectively embodiment 1 are obtained carry out chemically modified, wherein, chemically modified refers to that 2 '-OH of the U of the positive-sense strand of small RNA HBV-X1 to HBV-X4, HBV-P1 to HBV-P2, HBV-PS1 to HBV-PS2 and HBV-C1 to HBV-C2, C and G Nucleotide pentose has been carried out to 2 '-fluoro to be modified, and 2 '-OH of antisense strand U and C Nucleotide pentose has carried out 2 '-oxygen methyl and modified.Respectively the small RNA 1.2mg (0.09 μ mol) after modifying is dissolved in the stroke-physiological saline solution of 1.5ml without RNA enzyme afterwards, being mixed with concentration is the small RNA solution of 60 μ mol/L, and mixes with the volume ratio of 1: 1 with liposome.With the small RNA after liposome, join in the stroke-physiological saline solution (small RNA concentration is 0.25mg/ml) of 5ml without RNA enzyme, obtain injection liquid.
Use respectively injection liquid by conventional tail vein injection, mouse to be injected, injection liquid/kg body weight that the volume of injection is 20ml, the physiological saline of blank group injection same volume, all only injects 1 time.MAIN OUTCOME MEASURES: after injected in mice the 3rd, 5,7 days, from eye socket endocanthion, get blood, the conventional centrifugal serum that obtains ,-20 ℃ of standby inspections, and within 7 days after administration, pluck after eyeball is got blood and put to death mouse, on ice chest, cut open the belly and get hepatic tissue, the standby inspection of homogenate.
(1) serum protein detects
After injected in mice the 3rd, 5,7 days, from eye socket endocanthion, get blood, the conventional centrifugal serum that obtains, utilize alanine aminotransferase/glutamic-oxal(o)acetic transaminase test kit (ALT/AST, IFCC recommends method, Beijing Zhongsheng Beikong Biological Science & Technology Co., Ltd. product), the application 7170A of Hitachi automatic clinical chemistry analyzer detects biochemical indicator.Concrete operations are in strict accordance with test kit specification sheets.Result is as shown in table 5 and table 6.
Table 5
As can be seen from Table 5, small RNA HBV-X1 to HBV-X4 provided by the invention, HBV-P1 to HBV-P2, HBV-PS1 to HBV-PS2 and HBV-C1 to HBV-C2 all can reduce the content of serum alt (alanine aminotransferase), and wherein the effect of HBV-X1, HBV-X3, HBV-PS1 and HBV-C1 is the most obvious.
Table 6
As can be seen from Table 6, small RNA HBV-X1 to HBV-X4 provided by the invention, HBV-P1 to HBV-P2, HBV-PS1 to HBV-PS2 and HBV-C1 to HBV-C2 all can reduce the content of AST in serum (glutamic-oxal(o)acetic transaminase), and wherein the effect of HBV-X1, HBV-X3, HBV-PS1 and HBV-C1 is the most obvious.
(2) serum HBsAg detects
After injected in mice the 14th day, to pluck eyeball and get blood, conventional centrifugation obtains serum, uses ELISA test kit to detect the content of HBsAg in serum.This test kit is purchased from French Biomerieux BV (NL), and operation by specification carries out.Result represents with P/N value (absorbance value/contrast absorbance value of P/N value=sample), and calculates the inhibiting rate of small RNA to HBsAg content by following formula.
Absorbance value deducts sample in the difference of the light absorption value gained at 630nm place for detecting the light absorption value of sample at 450nm place.
Inhibiting rate (%)=(blank hole P/N value-experimental port P/N value)/(P/N value-2.1, blank hole) * 100%, result is as shown in table 7.
Table 7
As can be seen from Table 7, small RNA HBV-X1 to HBV-X4 provided by the invention, HBV-P1 to HBV-P2, HBV-PS1 to HBV-PS2 and HBV-C1 to HBV-C2 all can suppress the content of HBsAg in serum.Wherein HBV-X1, HBV-X3, HBV-PS1 and HBV-C1 to the inhibiting rate of serum HBsAg content especially up to more than 75%.
(3) detection of hepatic tissue HBV-mRNA
After injected in mice the 3rd, 5,7 days, pluck after eyeball sacrificed by exsanguination, take out liver, cut the tissue block into about 100mg, put into homogenizer and fully grind, then according to the explanation of Trizol (GIBCOL company), use the total RNA of Trizol extracting hepatic tissue, the total RNA extracting is after DNA is removed in DNase enzymic digestion, and reverse transcription is cDNA, then with fluorescence quantitative PCR method, detects the restraining effect of small RNA to hepatitis B virus mrna expression
Concrete steps are:
1. Trizol (GIBCOL company) extracts total RNA.Mouse is put to death in dislocation, cuts open the belly and get hepatic tissue on ice chest, cuts the liver organization piece of about 100mg, adds 1ml Trizol reagent, with glass-Teflon or electric homogenizer homogenate; After homogenate, room temperature is placed 30min, completely separated to guarantee nucleoprotein complex body.; Lysate is transferred in the new 1.5ml EP pipe without RNase, added 200 μ L chloroform/1mLTrizol reagent, with hand concuss 15s, room temperature is placed 3min, 4 ℃ of centrifugal 10min of 12000g; Get supernatant and be put in a new EP pipe, add the Virahol of 1/2 volume, room temperature is placed 10min; 2000g, 4 ℃ of centrifugal 10min; Suck supernatant, with the ethanol of equal-volume ice-cold 75%, wash once, 10000g, 4 ℃ of centrifugal 5min, suck supernatant; Without the dry RNA precipitation of RNase ambient room temperature 10min, add 20 μ l DEPC water dissolution RNA.
2. the purifying of RNA.The DNase I (RNase-free) (TakaRa company) of 2 units is added in total RNA, and under 37 ℃ of conditions standing 30min, to remove DNA residual in total RNA; In total RNA, adding isopyknic concentration is 70% ethanol, and vibration mixes; Mixture is transferred on purification column, and the centrifugal 15s of room temperature 12000g, abandons filtered liquid; Add 700 μ L cleaning buffer solution I, the centrifugal 15s of room temperature 12000g, abandons filtered liquid; Add 500 μ L cleaning buffer solution II, the centrifugal 15s of room temperature 12000g, abandons filtered liquid; Add 500 μ L cleaning buffer solution II, the centrifugal 15s of room temperature 12000g, abandons filtered liquid again; The centrifugal 1min of room temperature 12000g, is transferred to purification column on RNA collection tube; Add 30 μ lDEPC water, room temperature is placed 1min; The centrifugal 2min of room temperature 13000g, abandons purification column, by RNA sample as for-80 ℃ of preservations.
3. RNA reverse transcription.Total RNA after 1 μ g (2 μ l) is purified is 70 ℃ of heating 5min in EP pipe; Microcentrifuge is instantaneous to be put into EP pipe rapidly on ice after centrifugal; The MgCl that adds successively 25mM
24 μ L, 10 * reverse transcription buffer2 μ L, the dNTP Mixture 2 μ L of 10mM, RNase inhibitor 0.5 μ L, reversed transcriptive enzyme 15U (1 μ g), Oligo primer 0.5 μ g, uses without RNase water and is settled to 20 μ L; Under 42 ℃ of conditions, hatch 15min, 95 ℃ of heating 5min, hatch 5min, obtain cDNA for 5 ℃.
Using the cDNA obtaining as PCR, the template of reaction, carries out Real-time PCR reaction.PCR test kit is purchased from Beijing Mei Laibo medical science and technology company limited.Real-time PCR reaction system is: ddH
2o 17.5 μ l, 10mM Dntp0.5 μ l, 10 * Taq buffer, 2.5 μ l, Taq 0.5 μ l, F primer 0.5 μ l, R primer 0.5 μ l, Syber Green I 1 μ l, cDNA2 μ l; The condition of PCR reaction is: 94 ℃ 2 minutes, 94 ℃ 15 seconds, 60 ℃ 30 seconds, totally 40 circulations.Set up GAPDH as internal reference simultaneously, calculate small RNA suppress active according to following formula, result is as shown in table 8.
Totally four pairs of Realtime PCR primers, choose respectively the primer for C gene, X gene, S and P gene according to added small RNA sample, and sequence is as following table:
Inhibition activity=[1-(copy number of copy number/administration group GAPDH of administration group hepatitis B virus gene)/(copy number of copy number/blank group GAPDH of blank group hepatitis B virus gene)] * 100% of small RNA.
Table 8
As can be seen from Table 8, small RNA HBV-X1 to HBV-X4 provided by the invention, HBV-P1 to HBV-P2, HBV-PS1 to HBV-PS2 and HBV-C1 to HBV-C2 all can suppress the expression of HBV gene in transgenic mice liver.Wherein HBV-X1, HBV-X3, HBV-PS1 and HBV-C1 to the inhibiting rate of HBV genetic expression in liver more than 70%.
Claims (6)
1. a small RNA, is characterized in that, the nucleotide sequence of this small RNA as shown in HBV-X2, wherein,
HBV-X2 positive-sense strand: 5 '-CCUUGAGGCAUACUUCAAAdTdT-3 '
Antisense strand: 5 '-UUUGAAGUAUGCCUCAAGGdTdT-3 '.
2. for preventing and/or treating a pharmaceutical composition for hepatitis B, it is characterized in that, this pharmaceutical composition contains small RNA claimed in claim 1 as activeconstituents.
3. pharmaceutical composition according to claim 2, wherein, described pharmaceutical composition is injection liquid, and described injection liquid also contains pharmaceutically acceptable carrier, with respect to the small RNA of 100 weight parts, the content of described pharmaceutically acceptable carrier is 100-10000000 weight part.
4. pharmaceutical composition according to claim 3, wherein, described pharmaceutically acceptable carrier is the pH value phosphoric acid buffer that is 4.0-9.0, tri methylol amino methane hydrochloride damping fluid or the physiological saline that pH value is 7.5-8.5.
5. pharmaceutical composition according to claim 3, wherein, described injection liquid also contains protective material and/or osmotic pressure regulator; The gross weight of described injection liquid of take is benchmark, and described protectant content is 0.01-30 % by weight, and described protective material is selected from one or more in inositol, sorbyl alcohol and sucrose; It is 200-700 milliliter mol/kg that the content of described osmotic pressure regulator makes the osmotic pressure of described injection liquid, and described osmotic pressure regulator is sodium-chlor and/or Repone K.
6. small RNA claimed in claim 1 is in the application for the preparation of preventing and/or treating in the pharmaceutical composition of hepatitis B.
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Families Citing this family (14)
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| HUE044815T2 (en) * | 2010-08-17 | 2019-11-28 | Sirna Therapeutics Inc | RNA INTERFERENCE MEDIATED INHIBITION OF HEPATITIS B VIRUS (HBV) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA) |
| CN111172162A (en) | 2011-04-21 | 2020-05-19 | 葛兰素史克公司 | Modulation of Hepatitis B Virus (HBV) expression |
| CN103333890B (en) * | 2012-12-21 | 2015-04-15 | 厦门成坤生物技术有限公司 | RNA interference preparation used for treating viral hepatitis B |
| CN104031917B (en) * | 2013-03-06 | 2018-08-24 | 石药集团中奇制药技术（石家庄）有限公司 | A kind of siRNA and its Liposomal formulation for HBx |
| CN105368860A (en) * | 2014-08-29 | 2016-03-02 | 石药集团中奇制药技术（石家庄）有限公司 | Recombinant plasmid carrier and construction method and application thereof |
| CN106636086A (en) * | 2015-10-30 | 2017-05-10 | 江苏命码生物科技有限公司 | siRNA capable of interfering hepatitis B virus replication, construction method and applications thereof |
| EP3719126A4 (en) | 2017-12-01 | 2021-10-20 | Suzhou Ribo Life Science Co., Ltd. | Nucleic acid, composition and conjugate containing nucleic acid, preparation method therefor and use thereof |
| AU2018374219C1 (en) | 2017-12-01 | 2023-05-11 | Suzhou Ribo Life Science Co., Ltd. | Double-stranded oligonucleotide, composition and conjugate comprising double-stranded oligonucleotide, preparation method therefor and use thereof |
| US11414665B2 (en) | 2017-12-01 | 2022-08-16 | Suzhou Ribo Life Science Co., Ltd. | Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof |
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| CN112390835A (en) * | 2019-08-14 | 2021-02-23 | 苏州瑞博生物技术股份有限公司 | Liver targeting compounds and conjugates |
| CN113891892B (en) * | 2019-08-29 | 2024-01-30 | 苏州瑞博生物技术股份有限公司 | Compounds and drug conjugates, methods of preparation and uses thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1566131A (en) * | 2003-06-17 | 2005-01-19 | 杭州新瑞佳生物医药技术开发有限公司 | Small interference RNA molecule SiRNA capable of attacking human hepatitis B virus and application thereof |
| CN1648250A (en) * | 2004-12-07 | 2005-08-03 | 浙江大学 | Smal linterference ribonucleic acid sequence of hepatitis B virus gene and preparing method |
| CN1667120A (en) * | 2004-12-30 | 2005-09-14 | 中山大学 | RNA interfered target sequence of HBV and use thereof |
| CN1276086C (en) * | 2003-08-13 | 2006-09-20 | 复旦大学 | A double-stranded DNA molecule and its use in preparing medicine for inhibiting hepatitis B virus replication |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030206887A1 (en) * | 1992-05-14 | 2003-11-06 | David Morrissey | RNA interference mediated inhibition of hepatitis B virus (HBV) using short interfering nucleic acid (siNA) |
| CN1289681C (en) * | 2003-02-20 | 2006-12-13 | 重庆医科大学 | Construction of hepatitis B virus siRNA expression vector and its application in antivirus treatment |
| CN1257284C (en) * | 2003-03-05 | 2006-05-24 | 北京博奥生物芯片有限责任公司 | Method of blocking expression of hepatitis B virus |
-
2009
- 2009-08-03 CN CN200980126405.7A patent/CN102083983B/en active Active
- 2009-08-03 WO PCT/CN2009/073049 patent/WO2010012244A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1566131A (en) * | 2003-06-17 | 2005-01-19 | 杭州新瑞佳生物医药技术开发有限公司 | Small interference RNA molecule SiRNA capable of attacking human hepatitis B virus and application thereof |
| CN1276086C (en) * | 2003-08-13 | 2006-09-20 | 复旦大学 | A double-stranded DNA molecule and its use in preparing medicine for inhibiting hepatitis B virus replication |
| CN1648250A (en) * | 2004-12-07 | 2005-08-03 | 浙江大学 | Smal linterference ribonucleic acid sequence of hepatitis B virus gene and preparing method |
| CN1667120A (en) * | 2004-12-30 | 2005-09-14 | 中山大学 | RNA interfered target sequence of HBV and use thereof |
Cited By (1)
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| US11896674B2 (en) | 2018-09-30 | 2024-02-13 | Suzhou Ribo Life Science Co., Ltd. | SiRNA conjugate, preparation method therefor and use thereof |
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