CN1631443A - Targeted small interference RNA formulation for preventing and treating Hepatitis C and its preparation method - Google Patents

Targeted small interference RNA formulation for preventing and treating Hepatitis C and its preparation method Download PDF

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CN1631443A
CN1631443A CN 200410051562 CN200410051562A CN1631443A CN 1631443 A CN1631443 A CN 1631443A CN 200410051562 CN200410051562 CN 200410051562 CN 200410051562 A CN200410051562 A CN 200410051562A CN 1631443 A CN1631443 A CN 1631443A
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hcv
sirna
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CN100344331C (en
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程度
李宝健
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Tuopu Gene Tech Co Ltd Guangzhou
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Abstract

The invention discloses a targeted small interference RNA formulation for preventing and treating Hepatitis C and its preparation method, wherein the preparation comprises small interfering RNAs, hepatocyte targeted nucleinic acid leading-in carrier, the hepatocyte targeted nucleinic acid leading-in carrier comprises hepatocyte targeted molecules and/or nucleinic acid bonded molecules, by establishing nucleinic acid highly effective transfer hepatocyte architecture to the targeted hepatocyte carrier, siRNA capable of effectively inhibiting type C hepatitis viruse reproduction and infection can be screened at in vitro cell level, the siRNA is led into hepatocyte through in vivo system or local administration. The invention also provides an administration technology for targeting the liver tissues.

Description

Be used to prevent or treat target small-interfering RNA preparation of hepatitis C and preparation method thereof
Technical field
The present invention relates to a kind of target small-interfering RNA preparation that is used to prevent or treat hepatitis C, the invention still further relates to the preparation method of siRNA preparation.
Background technology
About 75% develops into chronic hepatitis after the HCV actute infection, and wherein about 20% final final result is liver cirrhosis, liver cell tumor or liver failure.Nearly 1.7 hundred million patients in the world wide, China's number of the infected is approximately 5,000 ten thousand, is inferior clinical symptom because of HCV infects, and the effective strength may be more.HCV infects has become one of social public health problem of serious threat human health.Still there is not special effectively preventing method so far.
HCV is the sub-thread positive chain RNA virus, belongs to the flaviviridae hepatitis virus and belongs to.The HCV genome mutation is bigger, is divided into genotype, hypotype, separated strain, four levels of quasispecies.HCV will have six genotype in the world wide, more than 80 sub-gene type, and popularity distributes relevant with geographical and crowd.China is mainly II/1b, the III/2a type.The about 9.6Kb of HCV genome total length contains single opening code-reading frame (ORF), and both sides are noncoding region (UTR).5 ' UTR contains internal ribosome entry site (IRES), and is very conservative; 3 ' UTR is relevant with replication initiation.Opening code-reading frame about 3010-3033 amino acid whose polyprotein precursor of encoding.This amyloid protein precursor generates 10 maturation proteins or enzyme under the combined effect of host signal peptidase and virus protease, be followed successively by Core-E1-E2-P7-NS2-NS3-NS4A-NS4B-NS5A-NS5B in order.
Structural protein comprise core protein Core and two envelope protein E1, E2, and they are virus envelope and ribosomal main component.What be positioned at the structural protein downstream is the embrane-associated protein of about 7KD, called after p7, and function is not quite clear.NS2-NS5B albumen is that viral genome is duplicated necessary non-structural area albumen.NS2 albumen and part NS3 albumen have unique proteinase activity, selective hydrolysis NS2/3 bonding pad.NS3 albumen is multi-functional, and 180 aminoacid of N end have the serine proteinase enzymatic activity, cutting NS3/4A, NS4A/4B, NS4B/NS5A, NS5A/NS5B bonding pad; 451 aminoacid of C-terminal have the helicase activity that ATP relies on, and are to duplicate necessary zone.NS4A albumen is as the proteic molecule that acts on altogether of NS3, and NS4B albumen is memebrane protein.NS5A albumen exists with phosphorylation form in cell, can and regulate its function in conjunction with NS5B albumen.NS5B albumen has the rna polymerase activity that RNA relies on, and is responsible for duplicating of HCV geneome RNA.
At present, the treatment that HCV is infected only limits to use separately IFN-a, perhaps unites use with the broad-spectrum antiviral medicament ribavirin.Yet the effect of treatment is unsatisfactory, and certain side effect is arranged.
RNAi technology (RNA interference technique), in brief, be exactly when RNA fragment (dsRNA) transfered cell long or after in organizing 21-23 nucleotide, the latter can make purpose mRNA be cut into the long fragment of 21-23 nucleotide, thereby degraded mRNA hinders or has stoped the process of being translated into the respective egg white matter by mRNA.Illustrate, the mRNA sequence relevant according to virus replication, design the long RNA fragment (dsRNA) of 21-23 nucleotide, after virus is invaded organism, dsRNA is imported organism, cause the degraded of the mRNA that virus replication is relevant, thereby blocked duplicating of virus, reach the effect of removing virus.
RNAi finds the only short 5 years time, because its high efficiency and high degree of specificity, RNAi becomes rather ideal cellular level gene knockout instrument, and becomes important means indispensable in the genome times afterwards comprehensively functional genomics research rapidly.By taking siRNA in mammalian cell, to induce strong specific RNA interference effect; this breakthrough progress has been started the new world of human disease treatment; applied research in the treatment of diseases of viral infection such as SARS, AIDS enlivens and gos deep into very much, and has obtained challenging achievement.In the treatment of AIDS, the advantageous manner that RNA disturbs may be become antagonism HIV viral infection, to duplicate.People have designed multiple siRNA at the different phase of HIV biocycle, at HIV virus gag, tat, the isogenic siRNA of rev, nef can be in order to the control virus replication, siRNA at the surperficial HIV acceptor gene of host cell can enter in the host cell in order to control virus, the course of infection that suppresses virus, the RNAi technology also is used for the exploration of anti-other multiple viruses except that being used for anti-HIV research.They comprise hepatitis B virus (HBV), hepatitis C virus (HCV), respiratory syncytial virus (RSV), influenza virus (influenzavirus), poliovirus (poliovirus) etc.
On December 20th, 2002, Small RNA; RNAi is chosen as first of the year ten big technological achievements by the Science magazine, and the Nature magazine also is chosen as one of annual major scientific and technological achievement with Small RNA.The breakthrough of RNA research is a biomedical sector over nearly 20 years, can with the Human Genome Project (human genomicsprogram, one of the most great achievement of HGP) mentioning in the same breath.The topmost function of RNAi is to regulate and to close expression of gene, and then the various senior vital movement of regulating cell.People will advance the research of gene function greatly to the further investigation of small RNA molecular, and new treatment approach is opened up in the radical cure of diseases such as more various viral diseases and tumor.Have extremely important theory and practical significance, it will produce far-reaching influence to the development of biology and medical science.
Viral vector is mainly derived from Mus and human DNA, RNA viruses, comprise retrovirus, adenovirus (Ad), adeno-associated virus (AAV), slow virus, herpes simplex virus (HSV), vaccinia virus, baculovirus, Epstein-Barr virus (EBV), vesicular stomatitis virus (VSV) and human cytomegalic inclusion disease virus (CMV) etc., and some of them have entered clinical experimental stage.Other carriers then also are in conceptual phase as bacteria carrier and artificial carrier.
Through the viral vector of transforming, transfection efficiency is higher, but is that preparation is complicated, and immunogenicity is arranged, and can not use repeatedly in the body, and safety exists hidden danger and non-guidance quality, has some difficulties in clinical practice.Non-virus carrier is a nucleic acid drug, especially the important channel of antisensenucleic acids, plasmid DNA and siRNA nucleic acid drug.Usually utilize hydrophilic or hydrophobic polyvalent cation polymer to condense recombiant plasmid or antisense oligonucleotide, form microgranule, by cell endocytic.Although the non-virus carrier transfection efficiency still is lower than viral carrier, non-virus carrier is owing to have low toxicity, low immunoreation, targeting and be easy to advantage such as assembling.
Liposome is the single or multiple lift spherical particle that drug encapsulation is formed in the class lipid bilayer, and similar cellularity has biomembranous characteristic and function.It can coated water-soluble and fat-soluble two types medicine.Be applied to transgenic liposome DNA complex and divide positive, neutral fat plastid, negative liposome and pH-sensitive lipid body.Also constantly there is novel liposome to be pushed out, as two four ammonium compoundss etc.Liposome is after Polyethylene Glycol (PEG) is modified, can reduce the mutual gathering between liposome-DNA composite particles, reduced the interaction with blood constituent, made liposome DNA composite particles become granule, do not influenced the affinity of liposome and cell simultaneously by bulky grain.RNA/DNA oligonucleoside chain chimeric molecule once was used to the single base mutation in the DNA plerosis gene.
Cationic polymer can concentrate and be rich in anionic DNA, adheres to then on the mucoitin sulfuric acid of cell surface, is entered in the cell by cell endocytic, thereby makes plasmid expression.The cationic polymer kind is a lot, as polymine (polyethylenimine, PEI), polypropylene imines tree ridge portion (polypropyleniminedendrimers), polyamide resin ridge portion (polyamidoamine dendrimer), the carbowax modifier of poly-D-lysine, poly histidine, poly arginine, protamine, poly-glucosamine (chitosan) etc. and above-mentioned polymer.Wherein research early be poly-D-lysine, find that afterwards the polymine performance is better.PEI can suppress lysosome, and protonated in the phagocytic vacuole sour environment, positive charge increases, and provides bigger protective effect to DNA, helps nucleic acid and flees from phagocytic vacuole.Because the transfer complex that DNA and cationic polymer form is to enter cell by endocytosis, easily degraded by lysosome, therefore adding the phagocytic vacuole releasing agent helps improving transfection efficiency.The most frequently used phagocytic vacuole releasing agent is chloroquine and amphiphatic molecule peptide.
What use the earliest in the polypeptide gene delivery system is two oleoyl melittins (dioleoylmelittin), forms annular particles with plasmid DNA, and transfecting eukaryotic cells has effectively been set up the method for polypeptide transgenic carrier.Basic amino acid polypeptide length 16～18 aminoacid are enough to transfer DNA to cell.Cation poly-D-lysine or arginine peptide chain compression dna molecular, 6 to 8 cationic amino acid residues of minimum need; Can increase transfection efficiency after adding cysteine and histidine, as Cys-His-(Lys) (6)-His-Cys oligopeptide etc.Peptide carrier is part district, the cation district in conjunction with DNA, nuclear localization signal, amphiphatic molecule functional areas, is the synthetic small peptide of quaternity.But as THALWHT oligopeptide specific bond airway epithelia, the THALWHT oligopeptide that Laser Scanning Confocal Microscope is found labelling is with after cell combines, and quilt is swallowed in the cell; The synthetic polypeptide that comprises CTHALWHTC sequence and DNA bound fraction can arrive gene transfer in the airway epithelia cell effectively.The surface protein granule that adds phagocytic vacuole releasing agent, cationic polymer and virus helps to improve transfection efficiency.The advantage of peptide carrier is that available Peptide synthesizer is synthetic, is convenient to use and gene engineering research production in the body, has overcome the big defective of complexity, inhomogeneity and systematic error of part oligopeptide and poly-D-lysine or PEI coupling preparation.Peptide carrier is the trend of non-virus carrier miniaturization.Weak point is that the cation load is on the low side relatively.
Summary of the invention
The object of the present invention is to provide a kind of target small-interfering RNA preparation that is used to prevent or treat hepatitis C, another object of the present invention is to provide the method for preparing the siRNA preparation.
The present invention is used to prevent or the siRNA preparation for the treatment of hepatitis C imports carrier by the nucleic acid of siRNA, liver cell targeting and forms; The nucleic acid of described liver cell targeting imports carrier and is made up of liver cell targeting molecule and/or nucleic acid binding molecule.
SiRNA of the present invention can be made up of the single siRNA that acts on a target sequence, also can be made up of a plurality of siRNAs of the target sequence on a plurality of target sequences that act on a gene or a plurality of gene; Described target sequence is the rna gene group sequence of hepatitis C virus, or the receptor CD81 or the Fas gene of the hepatitis C virus of the sequence of morbidity host's self endogenous gene such as human liver cell, can be by being made up of a sequence in the sequence table at least.
SiRNA of the present invention can be the double-stranded RNA of the 21-23 base pair of chemosynthesis; Also can be carrier or the double-stranded RNA of expressing the 21-23 base pair of framework expression, described carrier or expression framework are rna plymerase iii promoter and the expression of rna plymerase iii terminator regulation and control siRNA in mammalian cell, and the rna plymerase iii promoter comprises the U6 promoter and the people H1 promoter in people source or Mus source.
Large protein or middle albumen or small protein or part S gene order expressed proteins that the S gene regions that liver cell targeting molecule of the present invention is a hepatitis B virus is expressed; Described nucleic acid binding molecule is to be rich in basic amino acid and amphoteric amino acids molecule, sequence signature is KKALLALALHHLALLAHHLALALKKA or CCKKHHHKKHKKHGGLLALALHHLALLAHHLALAL, the nucleic acid binding molecule can be that linear molecule also can be the molecule (sequence signature is seen Fig. 1) of ramiform, and liver cell targeting molecule and nucleic acid binding molecule can prepare respectively and also can connect into fusion rotein by 2-10 GG or other aminoacid and form; It can be the polymine (PEI) that Polyethylene Glycol (PEG) is modified.
Liver cell targeting molecule of the present invention or nucleic acid binding molecule can be synthetic by chemical method, also can express in yeast cells or mammalian cell or insect baculovirus.
The production system of expressing in yeast cells of the present invention comprises yeast cells and Yeast expression carrier, and wherein yeast cells can be Pichia sp. cell, brewing yeast cell, and Yeast expression carrier is pICZ α or pYES2/CT or other Yeast expression carrier; The mammalian cell expression production system comprises people's Chinese hamster ovary celI and mammalian cell exogenous gene expression carrier; The insect cell expression production system comprises insect cell and rhabdovirus expression vector.
The present invention is used to prevent or treat the preparation method of the siRNA preparation of hepatitis C, the targeting proteins of recombinant production is behind ultracentrifugation or column purification, dialysis back lyophilizing, be dissolved in the pH5-7PBS solution and 30-100: the siRNA of 1 ratio mixes, use the electricity of 2-4mm to swash cup, under the condition of 220V and 500-1500uF, handle, make siRNA and targeting proteins form complex, can be used for the interior and in-vitro transfection of body of the hepatocellular siRNA of targeting.
The present invention is used to prevent or treat the preparation method of the siRNA preparation of hepatitis C, the fusion molecule of targeting proteins and nucleic acid binding molecule behind ultracentrifugation or column purification, the lyophilizing of dialysis back; Be dissolved in the pH5-7PBS solution and 30-150: the siRNA of 1 ratio mixes, and makes siRNA and fusion molecule form complex, can be used in the body of the hepatocellular siRNA of targeting and in-vitro transfection.
The present invention is used to prevent or treat the preparation method of the siRNA preparation of hepatitis C, the ramiform nucleic acid binding molecule of chemosynthesis and siRNA press 10: 1 mixed, after leaving standstill certain hour, form complex, mix with targeting proteins again, finally form targeting proteins/ramiform nucleic acid binding molecule/siRNA complex, can be used for the interior and in-vitro transfection of body of the hepatocellular siRNA of targeting.
The present invention is used to prevent or treat the preparation method of the siRNA preparation of hepatitis C, get an amount of lactosylation or glycosyl galactose albumen and be dissolved in dimethyl sulfoxide (DMSO) adding TEA, under the protection of nitrogen, stirred 1 minute, PEG (PEG3400-N-hydroxy succinimide) is with 2-5: 1 part by weight adds, at room temperature stirred 4 hours, after organic solvent is evaporated completely, obtain the PEG of lactosylation or glycosyl galactose albumen chelating; The PEG of lactosylation for preparing or glycosyl galactose albumen chelating is dissolved in DMSO, adds the TEA of 4-8 times of volume, stirs, and add PEI under stirring condition, and adding PEI quality is 20-40 a times of PEG, reaction overnight under the stirring condition; The lactosylation that obtains or the PEG-PEI of glycosyl galactose albumen chelating and PEI are dissolved in 5mM HEPES buffer by 1: 1 mixed, and it is 8 5mM HEPES buffer that synthetic siRNA also is dissolved in pH value; Both mix by 2: 1 mass ratio, and vibration mixing 30 seconds obtains the hepatocellular siRNA preparation of targeting.
The present invention is used to prevent or the siRNA preparation for the treatment of hepatitis C can mix with liposome and uses.
The present invention is used for preventing or the siRNA preparation for the treatment of hepatitis C can be injected directly into hepatic tissue by hepatic portal vein, or by the respiratory tract spraying or instil and make the siRNA preparation absorb the back through pulmonary to arrive the liver position; Also can or instil and be administered systemically by the injection of intravenously administrable approach.
The siRNA preparation imports before viral infection and behind the viral infection in cultured cell in vitro or the body, reduces filtering out the siRNA preparation that can effectively prevent and treat hepatitis C virus by the viral genome copy number; Hepar damnification by animal model alleviates and filters out the siRNA preparation that can effectively prevent and treat hepatitis C virus; By heavy dose of and repeat to import the siRNA preparation and in animal model, filter out the siRNA preparation that does not influence heart, liver, spleen, kidney, lymph node and blood system normal function.
The target small-interfering RNA preparation that the present invention treats the viral hepatitis C can improve the drug level of liver, improves medicine hepatitis C treatment of diseases effect, not obvious toxic and side effects.Treatment viral hepatitis C's target small-interfering RNA preparation can suppress the HCV virus replication and infect the protective effect that reaches hepatic tissue.
Description of drawings
The architectural feature of Fig. 1 ramiform nucleic acid binding molecule.
Fig. 2 siRNA, targeted molecular and the formed complex of nucleic acid binding molecule can effectively mediate the efficient transfection hepatocyte of siRNA.A: the preparation transfection hepatocyte that targeted molecular, nucleic acid binding molecule and fluorescently-labeled siRNA form; B: fluorescently-labeled siRNA transfection hepatocyte.
Fig. 3 pCI-HCV-Luc recombiant plasmid figure.
The special siRNA of Fig. 4 HCV strikes the expression of low HCV-Luc fusion gene, effectively suppresses the expression of LUC Photinus pyralis LUC Photinus pyralis FL in the hepatic tissue.
The specific embodiment
L albumen is made up of 110 aminoacid, is Pre-S1 albumen in the proteic N-terminal 108-119 of L amino acids, is positioned at the surface in L albumen, play and the bonded effect of surface of hepatocytes receptor-specific, and be that HBV viral infection hepatocyte is necessary.From the genome of human hepatitis B virus, clone coding Lprotein and the DNA sequence of the Lprotein that partly encodes, clone's sequence is connected in the T carrier, correct through sequence verification, coded sequence is cloned into Yeast expression carrier or baculovirus expression carrier or mammalian cell expression vector, expression vector is transfected in corresponding yeast or insecticide or the zooblast again, produce the recombiant protein that L protein gene or L protein part dna sequence encoding are arranged of reorganization, recombiant protein is behind ultracentrifugation or chromatographic column purification, because targeted molecular (Lprotein) has hepatocellular targeting, after liposome or nucleic acid are connected in conjunction with polypeptide, phenotype goes out hepatic targeting characteristics efficiently, thereby prepares the high efficiency siRNA preparation of targeting hepatocyte or hepatic tissue; Or mix with liposome that is combined with siRNA or ramiform polypeptide, prepare the high efficiency siRNA preparation of targeting hepatocyte or hepatic tissue.
Above-mentioned liver targeting L albumen or partial L protein sequence also can be synthetic by chemical method, and adopt the liposome of similar way and siRNA or ramiform polypeptide to form complex, prepare the high efficiency siRNA preparation of targeting hepatocyte or hepatic tissue.The L albumen that the present invention uses can be the L albumen of the HBV of wild type, also can be the reorganization L albumen that can escape immune system recognition of sudden change.
Designed rna gene group at hepatitis C virus, 175 siRNA have been designed, comprising the siRNA in targeting 5 ' noncoding region, 3 ' noncoding region, core protein C district, E1 district, E2/NS1 district, NS2a district, NS2b district, NS3 district, NS4a district, NS4b district, NS5a district, NS5b district.And adopt the above-mentioned siRNA that synthesized of chemical method and in vitro transcription.Adopt the prepared polypeptide of the present invention at room temperature or under the condition that electricity swashs to combine, contain HCV at in-vitro transfection and duplicate the hepatocyte of unit, also inject or import in the hepatic tissue of live body through the mode of being administered systemically of lung by intravenous injection or local organization with designed siRNA.Hepatocyte transfection HCV duplicates before the unit and distinguishes the preparation that the pair cell transfection is made up of targeted molecular, nucleic acid binding protein and siRNA afterwards.
The targeting molecule is the big antigen in surface of hepatitis B virus in the preparation of being made up of targeted molecular, nucleic acid binding protein and siRNA, has good hepatic targeting.The nucleic acid binding molecule that is adopted has two kinds: linear nucleic acid in conjunction with the nucleic acid of polypeptide and ramiform in conjunction with polypeptide.Linear nucleic acid is KKALLALALHHKKALLALALHHLALLAHHLALALKKAGG-NH in conjunction with the sequence signature of polypeptide 2, can merging with the liver targeted molecular effectively in conjunction with the siRNA molecule, this fusion rotein can pass through yeast or insect baculovirus or mammalian cell expression production, just is prepared into the efficient transfecting formulations of siRNA of hepatic targeting behind the purification.The polypeptide of ramiform can be efficiently in conjunction with the siRNA molecule, and can be connected with targeting proteins by non-covalent bond, therefore, the present invention has synthesized the polypeptide of ramiform by chemical method, the sequence signature structure is seen Fig. 1, and with after siRNA combines, the reorganization liver targeting proteins with preparation mixes ramiform nucleic acid again in conjunction with polypeptide, form complex, thereby be prepared into the efficient transfecting formulations of siRNA of hepatic targeting.
Estimate the efficient transfecting formulations of siRNA of liver targeting by the efficient of fluorescently-labeled siRNA transfectional cell.Targeted molecular, nucleic acid binding molecule and fluorescently-labeled siRNA form complex, complex and hepatocyte are cultivated altogether, after 24 hours or 37 hours, after using PBS solution washing cell, examine under a microscope the fluorescence situation of cultured cell, with fluorescently-labeled siRNA is contrast, do not failing to observe fluorescence in the siRNA cells transfected in conjunction with targeted molecular, nucleic acid binding protein as can be seen from Fig. 2, and behind siRNA, targeted molecular and the nucleic acid binding protein complex transfectional cell, can be observed cell has tangible fluorescence.
After successfully siRNA efficiently being imported hepatocyte, we have designed the fusion gene of hepatitis c virus gene sequence and firefly luciferase gene (Luc gene), and with fusion gene cloning to mammalian expression vector pCI, enough build up the pCI-HCV-Luc recombiant plasmid, the mPNA that this plasmid is transcribed out contains HCV mRNA and can translate activated LUC Photinus pyralis LUC Photinus pyralis FL, the mRNA of this fusion gene if the special siRNA of HCV can degrade just detects less than LUC Photinus pyralis LUC Photinus pyralis FL.The special siRNA of this plasmid and HCV is by forming complex with targeting proteins/nucleic acid binding protein, and imports in the Mus body by intravenous injection, and as can be known, the special siRNA of HCV can effectively suppress the expression of LUC Photinus pyralis LUC Photinus pyralis FL in the hepatic tissue from Fig. 3 and Fig. 4.
After the medication of having set up siRNA drug targeting liver organ, we will further estimate the preventive and therapeutic effect of siRNA to hepatitis C in vitro and in vivo.Estimate the siRNA molecule to reducing the inhibitory action of intracellular virus genome copy number by real-time quantitative RT-PCR.Find that therefrom the siRNA duplex molecule can effectively suppress the infection of hepatitis C virus and duplicates.
Designed the special siRNA of a series of test evaluation HCV drug effect and safety in animal body.At least one siRNA in the claim and targeted molecular, nucleic acid binding protein be mixed with in the siRNA body import preparation.The siRNA for preparing different time before and after virus attack passes through vein or respiratory tract or local hepatic tissue administration to the siRNA preparation of various dose.The siRNA processing time is that HCV duplicates first transfection or HCV virus attack animal (was administered once every 24-48 hour) before 4-72 hour, HCV duplicates first transfection or HCV virus attack animal (being administered once every 24 hours-48 hours).In animal model, import the complex of the special siRNA/ targeted molecular/nucleic acid binding protein of HCV, estimated the siRNA molecule to reducing the inhibitory action of viral genome copy number in the hepatic tissue sample by real-time quantitative RT-PCR respectively at 24 hours, 48 hours, 72 hours and 96 hours; Pathological index by hepatic tissue is estimated the protective effect of siRNA molecule to liver, and the result shows that the siRNA preparation of siRNA of the present invention and preparation can effectively suppress duplicating of hepatitis C virus in vivo and infects, and has effectively protected hepatic tissue.
This class siRNA material form and formulation preparation method not only suitable hepatitis C control, and be applicable to other liver class treatment of diseases, as hepatocarcinoma etc.
Embodiment 1: the preparation of targeting proteins.Design primer forward primer 5 ' atgggaggtt ggtcttccaaacct ' 3 and downstream primer 5 ' aatgtatacccaaagacaaaagaaaatt ' 3, with HBV DNA is template, reaction system is: 5 μ l, 10 * RT Buffer, 10 μ l MgCl2,10 μ l dNTPmixture, 1 μ l forward primer, 1 μ l downstream primer, HBV DNA 2ng, TaqDNA polymerase 1 μ l adds water to 50 μ l.The PCR reaction condition is: 94 ℃ of pre-degeneration 5min, and 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 2min carry out 30cycle, and 72 ℃ are extended 10min.The PCR product is with PCR purify Kit purification, and is connected with pMD18-T Vector, transforms, and the picking monoclonal is identified positive colony, checks order.The dna clone of the targeting proteins that sequence verification is correct, imports expression vector in the Pichia sp. cell after the enzyme action checking is correct to Yeast expression carrier pICZ α.At first, cut Yeast expression carrier pICZ α DNA more than 1 hour, make it linearisation with Sacl.Moisturizing adds 300ul phenol: chloroform to 300ul: isoamyl alcohol (25: 24: 1) is put upside down mixing.Centrifugal 12000rpm, 5 minutes.Get supernatant, add 750ml ethanol, 30ul NaAc ,-20 ℃, 30 minutes.Centrifugal, 12000rpm, 10 minutes.80% ethanol cleans, and dries up, and is dissolved in 10ul water.
Prepare the exponential phase yeast again: (1 chooses single colony inoculation in 30 ℃ of overnight incubation of 5ml YPD fluid medium.(2) the bacterium liquid 0.1-0.5ml that gets incubated overnight is inoculated in the fresh YPD fluid medium of 50ml, and 30 ℃ are cultured to OD600=1.3-1.5 again.(3) at 4 ℃, centrifugal 5 minutes of 1500g is with 0 ℃ of resuspended thalline of sterilized water of 50ml.(4) centrifugal the same, be resuspended in 0 ℃ of sterilized water of 25ml again.(5) centrifugal the same, be resuspended in 0 ℃ of 1M sorbitol solution of 20ml.(6) centrifugal the same, be resuspended in 0 ℃ of 1M sorbitol solution of 1ml again, place on ice (use on the same day).
By electric-shocking method expression vector is imported yeast cells at last: (1) injects 0 ℃ of electric shock pond with the cell 80 μ l and the 5-90 μ g linearisation DNA of preparation, and mixing was put 5 minutes on ice.(2) handle cell according to the yeast pulse parameter.(voltage: 1500V; Electric capacity: 25 μ F; Resistance: 400 Ω; Number of times: 1 time; Time: 8.9s).After promptly add 0 ℃ of 1M sorbitol solution of 1ml to the electric shock pond in, mixing.(3) again with liquid transposition 1.5ml EP pipe in the pond, cultivated 1-2 hour for 30 ℃.(4) get 100 μ l bacterium liquid and coat the YPDS flat board that contains ZeocinTM 100 μ g/ml, residue bacterium liquid full coat is distributed in the YPDS flat board that another contains ZeocinTM 100 μ g/ml, cultivates 3-4 days for 30 ℃.
Picking yeast transformant list colony inoculation places the 50ml conical flask in 2.5mlBMGY, at 28-30 ℃, the 250-300rpm shake-flask culture is to OD600=2-6 (approximately 16-18 hour), and yeast cells reaches exponential phase.3000rpm, under the room temperature, centrifugal thalline 5 minutes removes supernatant, and thalline is resuspended among the 10-20mlBMMY, to OD600=1.Place the two-layer gauze of 100ml conical flask middle cover, continue shake-flask culture.Added 100% methanol to final concentration 0.5% every 24 hours, keep inducing.Add at every turn and collect a subculture (the i.e. different time sections 24,48,72 of cultivation before the methanol, 96 hours, sampling respectively), get culture 2ml at every turn, centrifugal 3 minutes of 12000rpm gets supernatant and stores in-20 ℃, and SDS-PAGE analyzes Recombinant Protein Expression amount and molecular weight.Adopt the purification kit of Invitrogen company to carry out purification, its process is as follows:
1 resin treatment:
(1) get 2 milliliters of affine resins, 1000 left the heart 1 minute, removed supernatant;
(2) add 6 ml water mixings, 1000 left the heart 1 minute, removed supernatant;
(3) add 6 milliliters of Binding buffer mixings, 1000 left the heart 1 minute, removed supernatant;
2 protein purifications:
Fermentation liquid and mixed with resin about (1) 8 milliliter, room temperature was placed 1 hour;
(2) 1000 left the heart 1 minute, removed supernatant;
(3) add 8 milliliters of Wash buffer mixings, 1000 left the heart 1 minute, removed supernatant;
(4) add 2 milliliters of Elution buffer mixings, 1000 left the heart 1 minute, collected supernatant.
Collect to such an extent that obtain the albumen of purification after the supernatant lyophilization.
Embodiment 2: the preparation of the fusion rotein that targeted molecular and nucleic acid binding molecule are formed.Design forward primer 5 ' atgggaggtt ggtcttccaa acct ' 3 and downstream primer 5 ' aatgtatacccaaagacaaaagaaaatt ' 3 are template with HBVDNA, and reaction system is: 5 μ l, 10 * RT Buffer, 10 μ l MgCl 2, 10 μ l dNTPmixture, 1 μ l forward primer, 1 μ l downstream primer, HBV DNA 2ng, TaqDNA polymerase 1 μ l adds water to 50 μ l.The PCR reaction condition is: 94 ℃ of pre-degeneration 5min, and 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 2min carry out 30cycle, and 72 ℃ are extended 10min.The PCR product is with PCR purify Kit purification, and is connected with pMD18-T Vector, transforms, and the picking monoclonal is identified positive colony, checks order.Encoded K KALLALALHHKKALLALALHHLALLAHHLALALKKAGG-NH 25 ' ggtggcgccaagaagttcgccctccaccacgccctcctcgccctccaccacctcgc cctcgccctcctcgccaagaagcaccacctcgccctcgccctcctcgccaagaag3 ' of polypeptide, sequence inserts targeted molecular and forms fusion gene behind synthetic.The coding targeting proteins that sequence verification is correct and the fusion gene cloning of nucleic acid binding protein, import expression vector in the Pichia sp. cell after the enzyme action checking is correct to Yeast expression carrier pICZ α.The method of the Yeast expression carrier transformed yeast cell of reorganization and the purification process of recombiant protein are with embodiment 1.
Embodiment 3: ramiform nucleic acid is in conjunction with the preparation of polypeptide.
Synthetic can the carrying out on ABI431A solid phase automatic peptide synthesizer of ramiform polypeptide also can be by manual synthetic, and sequential structure is seen Fig. 1.The Fmoc method of employing standard; select the 0.125mmolFmoc-MAP-Branch-PSC resin for use; according to sequence peptide chain is extended one by one to the N end from the C end; each amino acid whose consumption is 0.5mmol; aminoacid: resin=4: 1; first aminoacid is connected to and uses DMAP on the resin, and amino acid whose activation is removed the Fmoc blocking group with 20% piperidine solution after using HOBt and DCC coupling.The step that recommend according to PE company the synthetic back of polypeptide; synthetic adding behind the ice bath is in condition of ice bath; under 10ml cutting liquid in to adopt its composition of cutting liquid B be crystallization phenol 0.75mg EDT0.25ml; after thioanisole 0.5ml deionized water 0.5ml TFA10ml mixeding liquid temperature to be cut rises to room temperature; lasting stirring reaction is 1.5 hours under the room temperature condition; peptide chain cracking from the resin is got off; remove the kinds of protect group simultaneously. reaction mixture G4 glass hourglass bucket is filtered to filter resin, successively with 1mlTFA 5 10mlDCM flushing reaction bulb, resin and funnel.Filtrate low pressure at normal temperatures is evaporated to ether sedimentation polypeptide after 1-2ml adds 50ml pre-cooling at least, and 4 degree are placed and spent the night, and filter through the G6 glass funnel, and vacuum was drained 3 hours at least, and gained is that polypeptide thick product-20 ℃ stores for future use.With MAP dissolving crude product concentration is 15mg/ml purification on explore100 type medium pressure liguid chromatograph after the filtration of 0.45m fibrous filter membrane, according to its dissolution characteristics is fat-soluble employing POROS-50-R1, the purification column of inserts, mobile phase is: A (10% ethanol+0.1%TFA), (90% ethanol+0.1%TFA).Gradient is 1.5 column volumes of 100% mobile phase A balance pillar; 100%A-100%B10 column volume; 0.5 column volume of 100%B, applied sample amount is 2ml, collects polypeptide solution at the main peak place.Use the resin column desalination, stand-by after the lyophilization.
Embodiment 4: the preparation of targeted molecular, nucleic acid binding molecule and preparation that siRNA forms.
Use two kinds of methods to prepare the compound formulation of targeting proteins/nucleic acid binding protein/siRNA.
1. at first ramiform nucleic acid binding molecule combines by non-covalent bond with siRNA.An amount of siRNA is dissolved in the sterilized water that does not contain the RNA enzyme, and mixing gently because finite volume, can adopt the siRNA of high concentration, and general siRNA is 8-15 μ g/ μ L.Get an amount of siRNA and ramiform nucleic acid binding molecule by 1: the mixed of 4-8, at room temperature incubation is 30 minutes, to form the siRNA/ polypeptide complex.
Then, targeted molecular is dissolved in the PBS solution of 500ul, adds an amount of siRNA again, after dissolving mixing fully, use Gene PulserII electroprotion system (Bio-Rad company provides) instrument, electricity swashs under the condition of 220V and 950uF, makes targeted molecular parcel siRNA.
At last, siRNA/ nucleic acid binding molecule and two kinds of complex mixings of targeted molecular/siRNA with above-mentioned preparation, at room temperature placed 30 minutes, be used in the body by interacting between two kinds of complex, being prepared into and the preparation of targeted molecular/nucleic acid binding molecule/siRNA of external targeting transfection siRNA.
2. use the fusion rotein of the targeted molecular/nucleic acid binding molecule of embodiment 2 preparations to prepare siRNA preparation (preparation is by targeted molecular, nucleic acid binding molecule and siRNA).An amount of siRNA is dissolved in the sterilized water that does not contain the RNA enzyme, and mixing gently because finite volume, can adopt the siRNA of high concentration, and general siRNA is 8-15 μ g/ μ L.The fusion rotein of getting an amount of siRNA and targeted molecular/nucleic acid binding molecule is by 1: the mixed of 10-15, at room temperature incubation is 30 minutes, with the complex of formation targeted molecular/nucleic acid binding molecule/siRNA, thereby be prepared into the siRNA preparation of forming by targeted molecular, nucleic acid binding molecule and siRNA.
Embodiment 5. targeted moleculars/nucleic acid binding molecule efficiently mediates siRNA transfection hepatocyte.
Fluorescently-labeled siRNA and targeted molecular/nucleic acid binding molecule is prepared into complex according to the method described in the embodiment 4.In the previous day of transfection, 4-5 * 10 4Cell inoculation is on 24 orifice plates, and 0.5mL contains FBS and antibiotic cell culture medium, should be able to make cell density reach the 40-70% of final densities in 24 hours.Dilution is fully mixed by targeted molecular, nucleic acid binding molecule, the formed preparation of siRNA in the culture medium of the serum-free of 100 μ l, joins in the cell culture medium, gently mixing.Cell behind 37 ℃ of incubation 24h-120h, the fluorescence condition diagram 2 of observation of cell under fluorescence microscope.
Embodiment 6 targeting proteins/nucleic acid binding protein/siRNA strikes expression of exogenous gene in the low hepatic tissue.
Designed the fusion gene of HCV core gene order and firefly luciferase gene (Luc gene), and with fusion gene cloning to mammalian expression vector pCI, enough build up the pCI-HCV-Luc recombiant plasmid, the mRNA that this plasmid is transcribed out contains the mRNA of HCV core gene and can translate activated LUC Photinus pyralis LUC Photinus pyralis FL, the mRNA of this fusion gene if the special siRNA of HCV core can degrade just detects less than LUC Photinus pyralis LUC Photinus pyralis FL.Prepare the preparation of forming by siRNA, targeted molecular and the nucleic acid binding molecule of HCV core gene specific according to the method described in the embodiment 4, import in the body by intravenous injection, after 3 days with animal euthanasia, separate hepatic tissue, extract total RNA, by the expression of RT-PCR quantitative analysis firefly luciferase mRNA.As can be known, the special siRNA of HCV can effectively suppress the expression of LUC Photinus pyralis LUC Photinus pyralis FL from Fig. 3 and Fig. 4.
Embodiment 7: the applying transgene Mus estimate siRNA suppress infection with hepatitis C virus with duplicate drug effect and safety.
(1) sets up the animal model of hepatitis C.
Operational model is: Alb-uPA transgenic (4 placed in-line Mus urokinase genes of albumin gene promoter regulation, make the urokinase overexpression, causing the fibrin unit very few and quicken hepatocellular death) SCID mice (Reconstruction in Sever Combined Immunodeciency Mice) implants isolating human liver cell, and liver source human albumin is used for transplanting.Liver HCV toxaemia like the mankind is arranged, and sustainable 35 days is the animal model that anti-HCV medicament is screened in comparatively suitable being used to.
Operational model also can be: select the healthy adult tree shrew for use, middle remote tree shrew kind, true West Asia kind, male and female half and half, body weight 95-148g.Tree shrew is through tail intravenous inoculation HCV positive serum 0.5ml, and the next day is inoculated for the second time.
Symptom: it is poor spirit to occur once, appetite descends and loose stool (continuing a week approximately), HCV-RNA appears in inoculation 2-3 week serum, as seen hepatic tissue portal area lymphocytic infiltration, Histological changes such as the change of hepatocyte fat, acidophilia's change, as seen positive material is granular and is distributed in the cytoplasm, and mitochondrion, smooth endoplasmic reticulum toxic change in the visible hepatocyte.
(2) arrangement of laboratory animal
Select the pure-blood of sex, age unanimity as far as possible, laboratory animal is carried out random packet.The preparation formed of siRNA by HCV core gene specific, targeted molecular and nucleic acid binding molecule of preparation among the embodiment 4 is injected directly in the hepatocyte by intravenous injection or lumbar injection or liver.
(3) preparation of the siRNA of HCV core gene specific, targeted molecular and nucleic acid binding molecule composition suppresses the HCV virus replication and infects the protective effect that reaches hepatic tissue.Get hepatic tissue cell and blood sample extraction RNA at different times (the 1st, 4,7,10,14,21 day) respectively, carry out the RT-PCR check.Because NS5b, NS5a, NS4b or the NS4a district of 5 of HCV ' end are the most conservative, how to design the detection primer according to them, detect the copy number of HCV RNA, targeting proteins/nucleic acid binding protein/siRNA preparation can import in the hepatocyte by effectively that HCV is special siRNA, and has effectively suppressed the HCV virus replication.
HCV-siRNA.WorkFile
Organization?Applicant
----------------------
Street: Room 805, No. 11 8 buildings, gem road, Guangzhou Economic and Technological Development Zone
City: Guangzhou
State: Guangdong
Country: China
PostalCode：510730
PhoneNumber：(0)13302207795
FaxNumber：020-37617478
EmailAddress：chengducd@21cn.com
＜110〉OrganizationName: Guangzhou Top Genomics Ltd.
Application?Project
-------------------
＜120〉Title: be used to prevent or treat target small-interfering RNA preparation of hepatitis C and preparation method thereof
<130>AppFileReference：0
<140>CurrentAppNumber：
<141>CurrentFilingDate：____-__-__
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cacaacauug?guaaauugat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA1
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA1
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
caacauuggu?aaauugacut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA2
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA2
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agaucuggga?auacgauaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA3
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA3
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ccaugauggu?ggugucaaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA4
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA4
HCV-siRNA.WorkFile
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
acggagugga?ugugguuaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA5
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA5
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cucuacggau?aggaguuuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA6
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA6
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
caggacuugc?agucugucat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA7
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA7
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
aggacuugca?gucugucaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA8
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA8
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ggacuugcag?ucugucaaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA9
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA9
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cuugcagucu?gucaaaggut?t 21
HCV-siRNA.WorkFile
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA10
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA10
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ugcagucugu?caaaggugat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA11
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA11
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ucacaacauu?gguaaauugt?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA12
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA12
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gucacaacau?ugguaaauut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA13
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA13
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agucacaaca?uugguaaaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA14
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA14
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
aagucacaac?auugguaaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA15
SequenceDescription：
HCV-siRNA.WorkFile
Custom?Codon
------------
Sequence?Name：HCV-siRNA15
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
caagucacaa?cauugguaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA16
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA16
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
guaacaugug?aggguguuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA17
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA17
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gccuucaagu?aacaugugat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA18
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA18
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ggaggaacau?gauguuauct?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA19
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA19
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
acacauugga?ggaacaugat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA20
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA20
Sequence
HCV-siRNA.WorkFile
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agugagucau?cagaaucaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA21
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA21
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
aagugaguca?ucagaaucat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA22
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA22
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gaagugaguc?aucagaauct?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA23
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA23
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agaagugagu?caucagaaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA24
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA24
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
aagaagugag?ucaucagaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA25
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA25
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gaagaaguga?gucaucagat?t 21
<212>Type：DNA/RNA
HCV-siRNA.WorkFile
<211>Length：21
SequenceName：HCV-siRNA26
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA26
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agaagaagug?agucaucagt?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA27
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA27
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gagaagaagu?gagucaucat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA28
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA28
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ggagaagaag?ugagucauct?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA29
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA29
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
auggagaaga?agugagucat t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA30
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA30
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gaguaacuau?ggagugaaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA31
SequenceDescription：
Custom?Codon
HCV-siRNA.WorkFile
------------
Sequence?Name：HCV-siRNA31
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ccuggagagu?aacuauggat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA32
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA32
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ucaccuggag?aguaacuaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA33
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA33
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cucaccugga?gaguaacuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA34
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA34
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gaucucaccu?ggagaguaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA35
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA35
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agccaccauu?aaagaaggat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA36
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA36
Sequence
--------
HCV-siRNA.WorkFile
＜213〉OrganismName: people's operation
<400>PreSequenceString：
uggagccacc?auuaaagaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA37
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA37
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agaagaacac?gaggaaggat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA38
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA38
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cgcagaagaa?cacgaggaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA39
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA39
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gcgcagaaga?acacgaggat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA40
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA40
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agaguaccag?accuacaaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA41
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA41
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
aagaguacca?gaccuacaat?t 21
<212>Type：DNA/RNA
<211>Length：21
HCV-siRNA.WorkFile
SequenceName：HCV-siRNA42
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA42
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
caagaguacc?agaccuacat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA43
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA43
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gucaagagua?ccagaccuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA44
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA44
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
caaggucaag?aguaccagat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA45
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA45
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gacaagguca?agaguaccat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA46
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA46
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gugacaaggu?caagaguact?t 21
<212>Type：DNA
<211>Length：21
SequenceName：HCV-siRNA47
SequenceDescription：
Custom?Codon
------------
HCV-siRNA.WorkFile
Sequence?Name：HCV-siRNA47
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ggugacaagg?ucaagaguat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA48
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA48
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ccucggcccu?ggugauaaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA49
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA49
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cgcauguaag?gaggaugaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA50
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA50
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
uuggugaugu?caaagauuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA51
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA51
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ggaguuuggu?gaugucaaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA52
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA52
Sequence
--------
＜213〉OrganismName: artificial sequence
HCV-siRNA.WorkFile
<400>PreSequenceString：
cgcacuaaca?ugcaugcaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA53
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA53
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cuuccgcacu?aacaugcaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA54
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA54
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cgaccuuccg?cacuaacaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA55
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA55
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gcgaccuucc?gcacuaacat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA56
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA56
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cagcgaccuu?ccgcacuaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA57
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA57
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ggccauuugg?acauaaugat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA58
HCV-siRNA.WorkFile
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA58
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
aaggccauuu?ggacauaaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA59
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA59
Sequence
-------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gaaggccauu?uggacauaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA60
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA60
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
guaagauggu?uguaaaugut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA61
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA61
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cacccgaaga?gcccuucaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA62
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA62
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ccacccgaag?agcccuucat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA63
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA63
HCV-siRNA.WorkFile
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ccggcacuuu?aguacucuut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA64
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA64
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
auaugcagcc?ggcacuuuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA65
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA65
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ggagucacaa?accuguagat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA66
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA66
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ggacaaccgu?ccucuuucut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA67
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA67
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cguccucuuu?cuccguggat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA68
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA68
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
HCV-siRNA.WorkFile
guccucuuuc?uccguggagt?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA69
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA69
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ccucuuucuc?cguggaggut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA70
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA70
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gcaccaccgg?agggacguat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA71
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA71
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ucgaaagagu?ccaggacuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA72
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA72
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
uccgacucca?cgcgggugat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA73
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA73
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cgacuccacg?cgggugaugt?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA74
SequenceDescription：
HCV-siRNA.WorkFile
Custom?Codon
------------
Sequence?Name：HCV-siRNA74
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
acuccacgcg?ggugauguut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA75
SequenceDescription：
Custom?Codon
-------------
Sequence?Name：HCV-siRNA75
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agacaacugg?cuagcugaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA76
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA76
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
uacccgcguu?ggcacgagat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA77
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA77
Sequence
---------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
acccgcguug?gcacgagaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA78
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA78
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cccgcguugg?cacgagaaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA79
SequenceDescription：
Custom?Codon
-------------
Sequence?Name：HCV-siRNA79
HCV-siRNA.WorkFile
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
caggucuuga?agucagucat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA80
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA80
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ggacccacgu?gccgacgcat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA81
SequenceDescription：
Custom?Codon
-------------
Sequence?Name：HCV-siRNA81
Sequence
---------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gcaggaagua?gauugaccat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA82
SequenceDescription：
Custom?Codon
-------------
Sequence?Name：HCV-siRNA82
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ggaaguagau?ugaccaggut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA83
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA83
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gcgaugccgg?cgcccacgat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA84
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA84
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cgaugccggc?gcccacgaat?t 21
HCV-siRNA.WorkFile
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA85
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA85
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gaugccggcg?cccacgaaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA86
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA86
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ggcgcccacg?aaagccgaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA87
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA87
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
augccaucaa?ugaugcuaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA88
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA88
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ugccaucaau?gaugcuauut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA89
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA89
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agcgcuuucu?gcuugaacut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA90
SequenceDescription：
HCV-siRNA.WorkFile
Custom?Codon
------------
Sequence?Name：HCV-siRNA90
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
acucuuccau?uucaucgaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA91
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA91
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ucuuccauuu?caucgaacut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA92
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA92
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
uccauuucau?cgaacuccut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA93
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA93
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
uuucaucgaa?cuccugguat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA94
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA94
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ucaucgaacu?ccugguagat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA95
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA95
Sequence
HCV-siRNA.WorkFile
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ucgaacuccu?gguagagaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA96
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA96
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gccucccgga?caagauaaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA97
SequenceDescription：
Custom?Codon
Sequence?Name：HCV-siRNA97
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agauaauccu?acccacaaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA98
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA98
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ugcaugccau?gauguauuut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA99
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA99
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
augccaugau?guauuuggut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA100
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA100
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gccaugaugu?auuugguuat?t 21
<212>Type：DNA/RNA
HCV-siRNA.WorkFile
<211>Length：21
SequenceName：HCV-siRNA101
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA101
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
guaugagaca?cuuccacaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA102
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA102
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
uaugagacac?uuccacauut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA103
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA103
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
augagacacu?uccacauuut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA104
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA104
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ugagacacuu?ccacauuugt?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA105
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA105
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gagacacuuc?cacauuugat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA106
SequenceDescription：
Custom?Codon
HCV-siRNA.WorkFile
------------
Sequence?Name：HCV-siRNA106
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agacacuucc?acauuugaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA107
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA107
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cgcacaccgu?ggcuugguat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA108
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA108
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gcacaccgug?gcuugguaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA109
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA109
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
accguggcuu?gguaugcuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA110
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA110
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agcggguuga?uccaagaaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA111
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA111
Sequence
--------
HCV-siRNA.WorkFile
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ccaaaucucc?aggcauugat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA112
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA112
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gcccaaaucu?ccaggcauut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA113
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA113
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
acgcccaaau?cuccaggcat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA114
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA114
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
acccaacacu?acucggcuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA115
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA115
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
uucgcgaccc?aacacuacut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA116
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA116
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ggccuuucgc?gacccaacat?t 21
<212>Type：DNA/RNA
<211>Length：21
HCV-siRNA.WorkFi1e
SequenceName：HCV-siRNA117
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA117
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cuaucaggca?guaccacaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA118
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA118
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agcacccuau?caggcaguat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA119
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA119
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
acucgcaagc?acccuaucat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA120
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA120
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ugaugcacgg?ucuacgagat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA121
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA121
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
caugaugcac?ggucuacgat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA122
SequenceDescription：
Custom?Codon
------------
HCV-siRNA.WorkFile
Sequence?Name：HCV-siRNA122
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gcucaugaug?cacggucuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA123
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA123
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gauuugugcu?caugaugcat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA124
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA124
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
auacccucgu?ugccauagat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA125
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA125
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
acccaaauua?cgcgaccuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA126
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA126
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ugaccuuacc?caaauuacgt?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA127
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA127
Sequence
--------
＜213〉OrganismName: artificial sequence
HCV-siRNA.WorkFile
<400>PreSequenceString：
augaccuuac?ccaaauuact?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA128
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA128
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gaugaccuua?cccaaauuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA129
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA129
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cgaugaccuu?acccaaauut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA130
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA130
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ucgaugaccu?uacccaaaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA131
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA131
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
aucgaugacc?uuacccaaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA132
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA132
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agaggaagau?agagaaagat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA133
HCV-siRNA.WorkFile
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA133
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ucaagaccuu?agcccaguut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA134
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA134
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
caaucaagac?cuuagcccat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA135
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA135
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ucacaaucaa?gaccuuagct?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA136
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA136
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
aucacaauca?agaccuuagt?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA137
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA137
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
caucacaauc?aagaccuuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA138
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA138
HCV-siRNA.WorkFile
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gcaucacaau?caagaccuut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA139
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA139
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
guagcaucac?aaucaagact?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA140
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA140
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
aguagcauca?caaucaagat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA141
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA141
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agaguagcau?cacaaucaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA142
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA142
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
aagaguagca?ucacaaucat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA143
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA143
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
HCV-siRNA.WorkFile
caaagaguag?caucacaaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA144
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA144
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gcaaagagua?gcaucacaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA145
Sequenceuescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA145
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ccagcaaaga?guagcaucat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA146
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA146
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ucagagcggu?ccuguugaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA147
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA147
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
caguuuggag?ggagucauut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA148
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA148
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ccaguuugga?gggagucaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA149
SequenceDescription：
HCV-siRNA.WorkFile
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ugcuauucau?ccauguacat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA155
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA155
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ggugcuauuc?auccauguat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA156
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA156
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agccgcauuu?gguguaagut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA157
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA157
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
guagucaacu?agacaccuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA158
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA158
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gccaaagucu?guauggguat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA159
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA159
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
guagugccaa?agucuguaut?t 21
HCV-siRNA.WorkFile
Custom?Codon
------------
Sequence?Name：HCV-siRNA149
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cccaguuugg?agggagucat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA150
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA150
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
aaccugugug?cguagaacat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA151
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA151
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ugaaccugug?ugcguagaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA152
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA152
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cugcgacgcg?gguacaauat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA153
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA153
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
caauacacug?ggccacacat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA154
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA154
HCV-siRNA.WorkFile
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA160
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA160
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ugaccuugaa?gaugguaaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA161
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA161
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cugaccuuga?agaugguaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA162
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA162
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
ccugaccuug?aagaugguat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA163
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA163
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
uccugaccuu?gaagauggut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA164
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA164
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
acauccugac?cuugaagaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA165
SequenceDescription：
HCV-siRNA.WorkFile
Custom?Codon
------------
Sequence?Name：HCV-siRNA165
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
uacauccuga?ccuugaagat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA166
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA166
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
guacauccug?accuugaagt?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA167
SequenceDescription：
Custom?Codon
------------
Sequence?Name.HCV-siRNA167
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cguacauccu?gaccuugaat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA168
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA168
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
acguacaucc?ugaccuugat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA169
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA169
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
uacucccauu?ugauuacgat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA170
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA170
Sequence
HCV-siRNA.WorkFile
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
acauacuccc?auuugauuat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA171
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA171
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
aacauacucc?cauuugauut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA172
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA172
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
gaacauacuc?ccauuugaut?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA173
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA173
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
agaacauacu?cccauuugat?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA174
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA174
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
cagaacauac?ucccauuugt?t 21
<212>Type：DNA/RNA
<211>Length：21
SequenceName：HCV-siRNA175
SequenceDescription：
Custom?Codon
------------
Sequence?Name：HCV-siRNA175
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
CCKKHHHKKH?KKHGGLLALA?LHHLALLAHH?LALAL 35
<212>Type：PRT
HCV-siRNA.WorkFile
<211>Length：35
SequenceName: nucleic acid binding molecule 1
SequenceDescription：
Sequence
--------
＜213〉OrganismName: artificial sequence
<400>PreSequenceString：
KKALLALALH?HLALLAHHLA?LALKKA 26
<212>Type：PRT
<211>Length：26
SequenceName: nucleic acid binding molecule 2
SequenceDescription：

Claims (10)

1. a siRNA preparation that is used to prevent or treat hepatitis C is characterized in that being made up of the nucleic acid importing carrier of siRNA, liver cell targeting; The nucleic acid of described liver cell targeting imports carrier and is made up of liver cell targeting molecule and/or nucleic acid binding molecule.
2. the siRNA that is used to prevent or treat hepatitis C according to claim 1, it is characterized in that described siRNA can be made up of the single siRNA that acts on a target sequence, also can form by a plurality of siRNAs of the target sequence on a plurality of target sequences that act on a gene or a plurality of gene; Described target sequence is the rna gene group sequence of hepatitis C virus, or the receptor CD81 or the Fas gene of the hepatitis C virus of the sequence of morbidity host's self endogenous gene such as human liver cell, can be by being made up of a sequence in the sequence table at least.
3. the siRNA preparation that is used to prevent or treat hepatitis C according to claim 1 and 2 is characterized in that described siRNA can be the double-stranded RNA of the 21-23 base pair of chemosynthesis; Also can be carrier or the double-stranded RNA of expressing the 21-23 base pair of framework expression, described carrier or expression framework are rna plymerase iii promoter and the expression of rna plymerase iii terminator regulation and control siRNA in mammalian cell, and the rna plymerase iii promoter comprises the U6 promoter and the people H1 promoter in people source or Mus source.
4. the siRNA preparation that is used to prevent or treat hepatitis C according to claim 1 is characterized in that large protein or middle albumen or small protein or part S gene order expressed proteins that S gene regions that described liver cell targeting molecule is a hepatitis B virus is expressed; Described nucleic acid binding molecule is to be rich in basic amino acid and amphoteric amino acids molecule, sequence signature is KKALLALALHHLALLAHHLALALKKA or CCKKHHHKKHKKHGGLLALALHHLALLAHHLALAL, the nucleic acid binding molecule can be that linear molecule also can be the molecule (sequence signature is seen Fig. 1) of ramiform, and liver cell targeting molecule and nucleic acid binding molecule can prepare respectively and also can connect into fusion rotein by 2-10 GG or other aminoacid and form; It can be the polymine (PEI) that Polyethylene Glycol (PEG) is modified.
5. the siRNA preparation that is used to prevent or treat hepatitis C according to claim 4, it is characterized in that: liver cell targeting molecule or nucleic acid binding molecule can be synthetic by chemical method, also can express in yeast cells or mammalian cell or insect baculovirus.
6. the siRNA preparation that is used to prevent or treat hepatitis C according to claim 5, it is characterized in that: the described production system of expressing in yeast cells comprises yeast cells and Yeast expression carrier, wherein yeast cells can be Pichia sp. cell, brewing yeast cell, and Yeast expression carrier is pICZ α or pYES2/CT or other Yeast expression carrier; The mammalian cell expression production system comprises people's Chinese hamster ovary celI and mammalian cell exogenous gene expression carrier; The insect cell expression production system comprises insect cell and rhabdovirus expression vector.
7. the described preparation method that is used to prevent or treat the siRNA preparation of hepatitis C of claim 1, it is characterized in that: the targeting proteins of recombinant production is behind ultracentrifugation or column purification, dialysis back lyophilizing, be dissolved in the pH5-7PBS solution and 30-100: the siRNA of 1 ratio mixes, use the electricity of 2-4mm to swash cup, under the condition of 220V and 500-1500uF, handle, make siRNA and targeting proteins form complex, can be used for the interior and in-vitro transfection of body of the hepatocellular siRNA of targeting.
8. the described preparation method that is used to prevent or treat the siRNA preparation of hepatitis C of claim 1, it is characterized in that: the fusion molecule of targeting proteins and nucleic acid binding molecule is behind ultracentrifugation or column purification, dialysis back lyophilizing, being dissolved in pH is in the PBS solution of 5-7, with 30-150: the siRNA of 1 ratio mixes, make siRNA and fusion molecule form complex, can be used for the interior and in-vitro transfection of body of the hepatocellular siRNA of targeting.
9. the described preparation method that is used to prevent or treat the siRNA preparation of hepatitis C of claim 1, it is characterized in that: the ramiform nucleic acid binding molecule of chemosynthesis and siRNA press 10: 1 mixed, after leaving standstill certain hour, form complex, mix with targeting proteins again, finally form targeting proteins/ramiform nucleic acid binding molecule/siRNA complex, can be used for the interior and in-vitro transfection of body of the hepatocellular siRNA of targeting.
10. the described preparation method that is used to prevent or treat the siRNA preparation of hepatitis C of claim 1, it is characterized in that: get an amount of lactosylation or glycosyl galactose albumen and be dissolved in dimethyl sulfoxide (DMSO) adding TEA, under the protection of nitrogen, stirred 1 minute, PEG (PEG3400-N-hydroxy succinimide) is with 2-5: 1 part by weight adds, at room temperature stirred 4 hours, after organic solvent is evaporated completely, obtain the PEG of lactosylation or glycosyl galactose albumen chelating; The PEG of lactosylation for preparing or glycosyl galactose albumen chelating is dissolved in DMSO, adds the TEA of 4-8 times of volume, stirs, and add PEI under stirring condition, and adding PEI quality is 20-40 a times of PEG, reaction overnight under the stirring condition; The lactosylation that obtains or the PEG-PEI of glycosyl galactose albumen chelating and PEI are dissolved in 5mM HEPES buffer by 1: 1 mixed, and it is 8 5mM HEPES buffer that synthetic siRNA also is dissolved in pH value; Both mix by 2: 1 mass ratio, and vibration mixing 30 seconds obtains the hepatocellular siRNA preparation of targeting.
CNB2004100515625A 2004-09-22 2004-09-22 Targeted small interference RNA formulation for preventing and treating Hepatitis C and its preparation method Expired - Fee Related CN100344331C (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101305095B (en) * 2005-09-12 2012-09-26 索马根尼科斯公司 Inhibition of viral gene expression using small interfering RNA
CN103351597A (en) * 2007-11-07 2013-10-16 犹他大学研究基金会 Cleavable modifications to reducible poly(amido ethylenimine)s to enhance nucleotide delivery
CN108888777A (en) * 2018-06-19 2018-11-27 徐州医科大学 A kind of galactolipin-PEG- inositol-PEI genophore and its compound formed with gene and application
WO2021121166A1 (en) * 2019-12-17 2021-06-24 南京大学 Multi-targeted sirna for treating cancers

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1427008A (en) * 2001-12-14 2003-07-02 殷冬生 Method of designing and selecting natural siRNA as gene medicine and medicine formulation
CN1320113C (en) * 2003-06-05 2007-06-06 复旦大学 Method for preparing small interference RNA molecule by using coli bacillus fermentation
CN1217700C (en) * 2003-07-08 2005-09-07 中国医学科学院血液学研究所 Multi-medicine medicine-resistant RNA interference medicine for resisting tumor
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

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101305095B (en) * 2005-09-12 2012-09-26 索马根尼科斯公司 Inhibition of viral gene expression using small interfering RNA
CN102827841A (en) * 2005-09-12 2012-12-19 索马根尼科斯公司 Inhibition of viral gene expression using small interfering RNA
CN103351597A (en) * 2007-11-07 2013-10-16 犹他大学研究基金会 Cleavable modifications to reducible poly(amido ethylenimine)s to enhance nucleotide delivery
CN108888777A (en) * 2018-06-19 2018-11-27 徐州医科大学 A kind of galactolipin-PEG- inositol-PEI genophore and its compound formed with gene and application
WO2021121166A1 (en) * 2019-12-17 2021-06-24 南京大学 Multi-targeted sirna for treating cancers

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