CN101322847A - Anti-hepatitis b virus gene medicament based on siRNA pool interference and mediated by complex carrier - Google Patents

Abstract

The invention discloses a siRNA interference-based anti-HBV gene-based drug mediated by complex carriers. Based on the characteristic of HBV and expression products thereof, active epitope protein in HBsAg expressed by HBV is biologically linked with cationic liposome and the epitope protein specially binds with hepatic cell surface receptors with the help of the HBsAg to induce RNAi to enter hepatic cells; meanwhile, the meltability of bilayers of the cationic liposome and the cytolipin is used for increasing the efficiency of an siRNA expressed carrier entering into the hepatic cells. The drug of the invention can improve the effectiveness of the siRNA, biological stability in the human body, safety and effective targeting delivery and can be used in the clinical treatment.

Description

Anti-hepatitis b virus gene medicament based on the interferential mediated by complex carrier in siRNA pond

Technical field

The present invention relates to a kind of RNAi anti-hepatic-B virus medicine, promptly in the hepatitis B surface antigen of expressing with hepatitis B virus effectively epi-position albumen and cationic-liposome biological chain fetch mediate rna i and enter medicine in the hepatocyte, the present invention is specifically related to the anti-hepatitis b virus gene medicament based on the interferential mediated by complex carrier in siRNA pond.

Background technology

The persistent infection of hepatitis B virus (HBV) is the important health problem in the whole world.The whole world has 3.5 hundred million the infecteds approximately at present, wherein 75% is distributed in the Asian-Pacific area.1992-1995 whole nation viral hepatitis seroepidemiological survey shows, population of China hepatitis B virus infection rate is 57.6%, the hepatitis B virus carrying rate is 9.75%, calculate that in view of the above the whole nation has 6.9 hundred million people once to infect hepatitis B virus, wherein 1.2 hundred million people carry hepatitis B virus for a long time, estimate that according to the expert there are existing trouble chronic viral hepatitis B patient 2,000 ten thousand people in the whole nation at present.In the infectious disease of China's statutory report, the morbidity number of hepatitis B and sickness rate are high always for many years occupies the forefront.Hepatitis B causes heavy financial burden for patient, family, society, bring the influence that can not be ignored to socio-economic development, being the major reason that many families drive into poverty by medical crises, back into poverty by medical crises, simultaneously also causing a series of social problems, is China present stage one of the most outstanding public health problem.Therefore, work out a kind of novel effective hepatitis B virus resisting medicine and will bring huge social and economic benefit.

The antiviral therapy that control HBV infects mainly adopts interferon and nucleoside analog, this two classes medicine is present clinical Therapeutic Method commonly used, but these medicines are to the antiviral curative effect of chronic hepatitis B defectiveness all, the lasting response rate of high dose recombinant interferon only is 30%, and clinical side effects is bigger; Though the nucleoside analog antiviral activity is stronger, virus replication bounces fast after the drug withdrawal, and causes the multidrug resistant disease strain after the prolonged application, and these all might bring out increasing the weight of of original hepatitis.

RNA disturbs (RNA interference in recent years, RNAi) discovery of technology, for new approach has been opened up in the treatment of chronic HBV infection, its principle is the short dsrna by 19-24 nucleotide fragments, be siRNA (short interference RNA, siRNA) bring out specificity cutting or Degradation to endogenous in the host cell slurry or exogenous rna, thereby suppress the expression of genes of interest, be PTGS (post transcriptionalgene silencing, PTGS).RNAi has high efficiency and high specific, be widely used in the gene functional research of plant, fungus, anthelmintic and lower animal and higher mammal, because the immense value that the RNAi technology demonstrates, ten big sciences that are chosen as 2001 and 2002 by " Science " magazine one of are achieved, and obtain the Nobel Prize in 2006 years simultaneously.RNA disturbs and can be regarded as a kind of and the similar defense mechanism of immune system, and preliminary experimental result shows that this imagination might come true.But, can hinder HIV in time multiplexed cell system as P24, Vif, nef, tat or rev with some gene of siRNA HIV (human immunodeficiency virus) inhibiting (HIV).Also have by RNA and disturb the report of each viroid of inhibition, as ridge Sui poliovirus, human papillomavirus, hepatitis B virus and hepatitis C virus in time multiplexed cell system.A large amount of experiment in vitro research datas shown this technology in gene functional research application prospect and new drug development in obtain great potential.Except that using artificial synthetic little interferential RNA, people have successfully used hair clip shape RNA or have generated little interferential RNA sample transcript with plasmid DNA or virus as carrier in cell, suppress exogenous specifically or endogenous gene at mammal or people's cell inner expression.

Existing scholar disturbs with RNA preliminary research has been carried out in the effect of hepatitis B virus both at home and abroad.And a series of researchs have reported that the RNA perturbation technique all significantly suppresses protein expression and the dna replication dna of HBV in testing in vivo and in vitro, have shown that this technology has great application prospect in the treatment chronic hepatitis B.But RNA disturbs being extensive use of and also needing to solve some critical problems clinically: as more effective design, intravital biological stability, safety and efficient targeting transmission etc.Mostly be at present to adopt direct injection or utilize liposome, but effective epi-position albumen and cationic-liposome biological chain fetch the research that mediate rna i enters in the hepatocyte and do not appear in the newspapers as yet in the hepatitis B surface antigen of expressing with hepatitis B virus with in synthetic siRNA or the siRNA expression vector transfered cell.

Summary of the invention

The objective of the invention is: a kind of anti-hepatitis b virus gene medicament based on the interferential mediated by complex carrier in siRNA pond is provided, the complex carrier of this medicine can improve the effectiveness of siRNA, intravital biological stability, safety and efficient targeting transmission, realize the clinical practice of siRNA, improve the effectiveness of clinical practice.

The present invention is that the technical scheme that addresses the above problem employing is: according to the characteristics of hepatitis B virus and expression product thereof, with effective epi-position albumen and cationic-liposome bio-link in the hepatitis B surface antigen of hepatitis B virus expression, combine with surface of hepatocytes receptor generation specificity by hepatitis B surface antigen epi-position albumen, mediation siRNA enters in the hepatocyte, utilize the meltable of cationic-liposome and cytolipin bilayer simultaneously, strengthen the siRNA expression vector and enter the interior efficient of hepatocyte.

Step of the present invention is as follows:

1, siRNA is synthetic;

2, at the structure of each gene regions of HBV (S, C, P, X) siRNA and siRNA pond expression vector;

3, with effective epi-position albumen and cationic-liposome bio-link in the hepatitis B surface antigen of hepatitis B virus expression, mediate rna i enters in the hepatocyte.

Key of the present invention is in the hbs antigen cell transmission of the RNAi that effective epi-position is protein mediated: determine in the hbs antigen the effectively proteic aminoacid sequence of epi-position, synthetic hbs antigen epi-position albumen is with effective epi-position albumen and cationic-liposome bio-link in the synthetic hbs antigen.

Effectively the epi-position protein sequence is as follows in the hbs antigen:

cag?gcg?ggg?ttt?ttc?ttg?ttg?aca?aga?atc?ctc?aca?ata?cca?cag?agt?cta?gac?tcg?Q

A G F F L L T R I L T I P Q S L D S

Synthetic by American AB I company.

Cationic-liposome composed as follows:

DC-chol (3 β-(N-(N ', N '-dimethyl aminoethyl)) the carbamate cholesterol)/DOPE (two oleoyl phosphoethanolamines)=1: 1 (mol ratio), provide by Sigma company.

The bio-link of effective epi-position albumen and cationic-liposome is synthetic by American AB I company in the synthetic hbs antigen.

The siRNA sequence is as follows:

SiRNA-S1: positive-sense strand (5 '-GUGGUGGACUUCUCUCAAUTT-3 ')

Antisense strand (5 '-AUUGAGAGAAGUCCACCACTT-3 ')

SiRNA-S2: positive-sense strand (5 '-AACCUCCAAUCACUCACCAACTT-3 ')

Antisense strand (5 '-GUUGGUGAGUGAUUGGAGGUUTT-3 ')

SiRNA-S3: positive-sense strand (5 '-GCCUCAUCUUCUUGUUGGUTT-3 ')

Antisense strand (5 '-ACCAACAAGAAGAUGAGGCTT-3 ')

SiRNA-S4: positive-sense strand (5 '-GGUAUGUUGCCCGUUUGUCTT-3 ')

Antisense strand (5 '-GACAAACGGGCAACAUACCTT-3 ')

The present invention has following advantage:

(1) effective epi-position albumen of HBsAg can combine with surface of hepatocytes receptor generation specificity;

(2) cationic-liposome and cytolipin bilayer have good blending;

(3) effective epi-position albumen and cationic-liposome bio-link can increase the targeting effect that siRNA pond expression vector cell transmits among the HBsAg;

(4) siRNA pond expression vector has biological stability and safety preferably in vivo;

(5) siRNA pond expression vector has stronger gene silencing effect to HBV in vivo;

(6) can reduce the level of emiocytosis HBsAg behind the shRNA plasmid transfection.

Description of drawings

Fig. 1 is the structure at each gene regions of HBV (S, C, P, X) siRNA and siRNA pond expression vector;

Fig. 2 is the concrete transfer mode figure of siRNA complex carrier of the present invention.

The siRNAs of Fig. 3 chemosynthesis is transfected into the inhibitory action of HBsAg being expressed behind the HepG2.2.15 cell with the complex carrier carrier systems.(A) mRNA level (B) protein level.HBsAgmRNA and HBsAg protein level detect with real-time RT-PCR and ELISA method respectively after 72 hours in transfection.

The HepG2.2.15 cell is advanced in the transfection of Fig. 4 .shRNA expression plasmid.Transcribing back 72 hours respectively with ELISA, real-time RT-PCR with detect PCR pHBV-S1 in real time, pHBV-S2, pHBV-S4, HBsAg gene silencing effect (A) albumen of pHBV-2S and pHBV-3S plasmid, (B) RNA and (C) DNA.

Fig. 5. plasmid-encoded shRNA in the HepG2.2.15 cell to the RNAi interference effect of HBsAg.The cell kind is in 24 orifice plates, with 1mg/ hole shRNA expression plasmid transfection.After the transfection 72 hours, cell fixation, HBsAg immunostaining, microscopic examination.These results show and the expression of Quality Control specimen comparison HBsAg is eased down to varying level.

Fig. 6 .shRNA expression plasmid transfection HepG2.2.15 cell detected the IFN-γ that discharges in the culture medium after 72 hours with the ELISA method.The IFN-γ concentration change of HepG2.2.15 emiocytosis is not obvious behind the result the has shown transfection shRNA expression plasmid, illustrates and does not induce IFN to reply.

The specific embodiment

Below in conjunction with instantiation, further detailed elaboration the present invention, outstanding substantial characteristics of the present invention and good effect can be embodied from following example, but they are not that the present invention is made any restriction.

Material

PsiSTRIKE ^TMPlasmid kit is available from Promega company, antibiotic G418 is available from Sigma-Aldrich company, bovine albumin serum (BSA) (composition V, purity＞98%) available from USB company, DMEM (Dulbecco ' s modified eagle ' s medium), benzylpenicillin (5000 units/ml), Trypsin-EDTA, Trizol, DNase I, HBsAg epi-position albumen-cationic-liposome complex carrier, PureLink synthetic by ABI company ^TMViral RNA/DNA test kit, Mus monoclonal anti-HBsAg and Alexa Fluor 488 sheep anti-mouse iggs are available from Invitrogen company, restricted enzyme (PstI, EcoRI, ClaI, BsrGI, SgfI and BglII.) available from New England Biolabs company, reverse transcriptase is available from Applied Biosystems company, HBsAg ELISA detection kit is available from Abazyme company, and siRNAs and primer are synthetic by Integrated DNA Technologies company.

The siRNA design is with synthetic

At hepatitis B HBsAg zones of different design siRNA (Gene Bank Accession#NM_U95551), see Table 1, these siRNA are 19-21nt and 2-nt deoxyribonucleotide and at the genomic conservative S of ayw HBV district, the design of these siRNA with reference to Ambion (http://www.ambion.com/techl ib/misc/siRNA_finder.html) and Invitrogen (https: //rnaidesigner.invitrogen.com/rnaiexpress/design.do), the specificity of all siRNA sequences is by BLAST search (www.ncbi.nlm.nih.gov) checking, and all s iRNA are synthetic by Integrated DNATechnologies company.

The target site of table 1si RNA sequence and HBV

The structure of shRNA expression plasmid

By discerning different siRNA sequences, select three ordered sequences to be converted into shRNA sequence at the different target regions of HBsAg, see Table 2A, use psiSTRIKE ^TMConstruction expression is anti--pHBV-S1 of HBsAg, and pHBV-S2, the shRNA expression vector of pHBV-S4 and Quality Control, it comprises a U6RNA polymerase promoter.In order to make up shRNA pond expression plasmid pHBV-2S and pHBV-3S, a synthetic sequence positive-sense strand (5 '-ACCGGA ATT CCG GAT ATC GAT GTA CAG CGG CCG CGA TCG CGA C-3) and the antisense strand (5 '-TGC AGT CGC GAT CGC GGC CGC TGT ACA TCG ATA TCCGGA ATT C-3 ') that comprises the sequence of a plurality of restricted enzyme inserts psiSTRIKE ^TMPlasmid is referred to as the pEsiST carrier; Use EcoRI and ClaI digestion pEsiST and comprise a large amount of restriction endonuclease sites, the positive-sense strand of shRNA-S1 ' (5 '-AAT TCG TGG TGG ACT TCT CTC AAT CTT CCT GTC AATTGA GAG AAG TCC ACC ACAT-3 ') and antisense strand (5 '-CGAT GT GGT GGA CTT CTC TCA ATT GAC AGG AAG ATT GAG AGA AGT CCA CCA CG-3 ') annealing is cloned into the EcoRI-ClaI site of pEsiST carrier, is referred to as pshRNA-S1 ' plasmid; Use SgfI and BglII digestion pshRNA-S1 ' plasmid, the positive-sense strand of shRNA-S4 ' (5 '-CGC GGT ATG TTG CCC GTT TGT CCT TCC TGT CAG ACA AAC GGG CAA CAT ACC TTT TTA-3 ') and antisense strand (5 '-GATCT AAA AAG GTA TGT TGC CCG TTT GTC TGA CAG GAA GGA CAA ACG GGC AAC ATACC GCGAT-3 ') annealing are cloned into the SgfI-BglII site of pshRNA-S1 ' plasmid, be referred to as the pHBV-2S plasmid, shRNA-S4 ' end comprises a TTTTT and stretches generation pol III termination signal.Use BsrGI and SgfI digestion pHBV-2S plasmid then, the positive-sense strand of shRNA-S2 ' (5 '-GTACA AAC CTC CAA TCA CTC ACC AAC CTT CCT GTC AGT TGG TGA GTG ATT GGA GGT T GCGAT-3 ') and antisense strand (5 '-CGC AAC CTC CAA TCACTC ACC AAC TGA CAG GAA GGT TGG TGA GTG ATT GGA GGT TT-3 ') annealing are cloned into the BsrGI-SgfI site of pHBV-2S plasmid, are referred to as the pHBV-3S vector plasmid; So 2-3 shRNA (seeing Table 2B) is inserted into psiSTRIKE ^TM(see figure 1) produces pHBV-2S and pHBV-3S plasmid vector in the carrier.

Table 2 is at the sequence of the shRNA of the different target regions of HBsAg

The structure of cell delivery system

Effectively the epi-position protein sequence is as follows in the hepatitis B surface antigen that hepatitis B virus is expressed:

cag?gcg?ggg?ttt?ttc?ttg?ttg?aca?aga?atc?ctc?aca?ata?cca?cag?agt?cta?gac?tcg?Q

A G F F L L T R I L T I P Q S L D S

Cationic-liposome composed as follows:

DC-chol (3 β-(N-(N ', N '-dimethyl aminoethyl)) the carbamate cholesterol)/DOPE (two oleoyl phosphoethanolamines)=1: 1 (mol ratio), provide by Sigma company.

The bio-link of synthetic HBsAg albumen and cationic-liposome is synthetic by American AB I company.

Cell culture and transfection

HepG2.2.15 cell line is in the DMEM culture fluid of 4% hyclone and 330 μ g/ml and antibiotic G418, and 37 ℃, 5%CO2 are cultivated in the humidification couveuse, preceding 24 hours of transfection, and cell is with every hole 1 * 10 on 24 well culture plates ⁵Density divide kind.

Effective epi-position protein sequence and the formed complex carrier of cationic-liposome bio-link are with the back concentration transfectional cell with 40-80nmol/L of ratio parcel of 1 μ g/1 μ g in the siRNA plasmid vector usefulness hepatitis B surface antigen, equally, the shRNA expression vector also with behind the complex carrier parcel respectively with the concentration transfectional cell in 0.5 μ g, 1 μ g, 2 μ g/ holes, if blank and transfection reagent contrast, respectively do three multiple holes, after the transfection 72 hours, harvesting is further analyzed.

The detection of granular size and ζ potential energy

Plasmid and complex liposome are diluted to 250 μ l with the glucose of 5%w/v respectively, then DNA are joined in the complex liposome, in room temperature effect 45 minutes.Change the blending ratio of lipid/plasmid, detect granular size and ζ potential energy in 25 ℃ respectively by dynamic light scattering method with Malvern Zetasizer.

HBV DNA in fluorescence quantitative PCR detection HBsAg mRNA and the culture medium

Respectively with siRNA or shRNA expression plasmid transfection HepG2.2.15 cell, with the cell trypsinization of 72h after the transfection, centrifugal collection, resuspended, the centrifugal collection of reuse PBS, extract total DNA with the DNA extraction test kit in strict accordance with explanation, with HBV DNA and the HBsAg mRNA in real-time fluorescence quantitative PCR and the real-time RT-PCR detection culture medium.

Auele Specific Primer at HBV S gene is a positive-sense strand: 5 '-GAT TCC TAG GAC CCCTTC TC-3 ', and antisense strand: 5 '-GGA GGA CAG GAG GTT GGT GA-3 ', the condition of PCR in real time is as follows: 50 ℃, 2min; 95 ℃, 10min; 95 ℃, 15s; 54 ℃, 1min, * 40cycles; 72 ℃, 5min.

Immunofluorescence detects

With shRNA expression plasmid transfection HepG2.2.15 cell after after the transfection 72 hours, with 3% formalin fixed 15min, wash 3 times with DPBS, with 0.1%Briji98/PBS effect 2min, wash 2 times with DPBS, with 20% sheep blood serum sealing 30min, with monoclonal mouse-anti-HBsAg antibody as first antibody and Cy3-sheep anti-mouse igg as second antibody room temperature reaction 1h, film-making, microscopic examination.

The result

The gene silencing effect of siRNA sequence

Select HBsAg as target gene, detect the ability of the reticent HBV gene expression of siRNA.The relatively gene silencing effect (seeing Table 1) of the siRNA of 4 different 19-21bp in four different genes districts of targeting HBsAg.These siRNA separately or 4 mixing with mixed carrier parcel transfection HepG2.2.15 cell.As shown in Figure 2, siRNA is that sequence-specific and dosage are dependent to the gene silencing of HBsAg, and 3 kinds of siRNAs have caused the reduction significantly of HBsAg mRNA level, and contrast siRNA is to the almost not effect of gene expression of HBsAg.Act on specimen with siRNA-S1, siRNA-S2, siRNA-S3 and siRNA-S4 with the dosage of 40nmol/L and can make that HBsAg mRNA expression reduces by 40.9,13.41,17.4and 40.16%.When siRNA-S1, siRNA-S2, siRNA-S3 and siRNA-S4 dosage are increased to 80nmol/L, can increase the gene silencing effect of HBsAg, be respectively 71.77,58.79,54.49and 84.69%, as Fig. 3 A.

With the ELISA method detect transfection after 3 days HBsAg be secreted into level in the culture medium.After siRNA-S1, siRNA-S2, siRNA-S3 and siRNA-S4 dosage effect, be secreted into that the HBsAg level has been lowered 39.67,31.79,30.55 and 42.29% respectively in the culture medium with 40nmol/L; When effect dosage was increased to 80nmol/L, the concentration of HBsAg reduced significantly in the culture medium, is respectively 58.72,55.03,46.01 and 61.71%, as Fig. 3 B; SiRNA-S4 has shown the highest gene silencing level in these several siRNA, when transfection dosage reached 80nmol/L, the mRNA of HBsAg and protein level be lowered 84.69% and 61.71% respectively.Other siRNA shows as the inhibitory action of varying level to the expression of HBsAg.The expression no change of siRNA matched group HBsAg illustrates that siRNA has sequence-specific to the inhibitory action of HBsAg.

The structure of shRNA expression plasmid

By discerning different siRNA sequences, select three highly active siRNA sequences to be converted into the shRNA sequence of the different target regions of targeting HBsAg, these sequences comprise the required unique restriction site of clone at 5 ' and 3 ' end, and, then these sequence clones are gone into psiSTRIKE in the TTTTT sequence (seeing Table 2A) that can produce pol III termination signal that has of positive-sense strand ^TM, listed as Fig. 1.PHBV-S1, pHBV-S2, pHBV-S4 produce the dna fragmentation of 2 about 3047bp and 1370bp with digestion with restriction enzyme.PHBV-2S and pHBV-3S carrier are differentiated with EcoRI and Bgl II enzymic digestion.The length of pHBV-2S and pHBV-3S product is respectively 118bp and 163bp, and DNA sequence result shows that the fragment of insertion has desired sequence.

ShRNA expresses the excretory effect of HBsAg

In order to estimate of the influence of siRNA pond, use psiSTRIKE to HBV biocycle ^TMCarrier is directly synthetic a large amount of siRNA in HepG2.2.15 cell line.PHBV-S 1 behind clone, amplification and the purification, pHBV-S2, pHBV-S4, pHBV-2S and pHBV-3S, the mixture of formation plasmid complex carrier is by dynamic light scattering technology for detection granular size and ζ potential energy.The mean size of the compound particles of these plasmid complex carriers is 200-400nm, and ζ potential energy is 20-30Mv.Detection is advanced the effect of the shRNA expression plasmid of HepG2.2.15 cell to the HBsAg gene silencing with the concentration transfection in 0.5,1 and 2 μ g/ holes, and each sequence has all obtained a large amount of HBsAg gene silencing effect (see figure 4)s.Compare the concentration (seeing Fig. 4 A) of the HBsAg in the inhibition culture medium that shRNA expression plasmid transfection HepG2.2.15 cell can be a large amount of with contrast shRNA expression plasmid transfection group.Compare with the plasmid of the single shRNA of coding, coding shows the reticent effect of more effective HBsAg at 3 shRNA plasmids of HBsAg gene zones of different.

We have detected the total RNA that extracts in the HepG2.2.15 cell of transfection with real-time RT-PCR, and the result has proved conclusively the plasmid of three shRNAs of coding (pHBV-3S), can make the concentration of HBsAgmRNA reduce 63.2-87.7% (seeing Fig. 4 B).

Detected the HBV DNA in the culture medium with PCR in real time, shown in Fig. 3 C, when the HepG2.2.15 cell transfecting shRNA plasmid of coding targeting HBsAg can be a large amount of the level of its HBV DNA of reduction.When with the coding 3 shRNAs (pHBV-3S) plasmid transfection the time suppression ratio be about 85%.

HBsAg express immunofluorescence analysis

At the HepG2.2.15 cell transfecting coding after 72 hours, use microscopic examination after the cellular immunization dyeing at the shRNA plasmid of HBsAg.The painted fluorescence intensity of HBsAg is followed successively by: HBV-S1＞HBV-S2＞HBV-S4＞HBV-2S＞HBV-3S (see figure 5).The cell HBsAg level with the shRNA plasmid transfection that illustrates is reduced in various degree.The cell of pHBV-2S and pHBV-3S plasmid transfection shows low fluorescence, and the low expression of HBsAg is described.

The inductive IFN of shRNA replys

Whether induced IFN to reply in order to detect shRNA, after 72 hours, detect the IFN-γ that discharges in the culture medium with the ELISA method, as shown in Figure 6 at shRNA expression plasmid transfection HepG2.2.15 cell, back IFN-γ concentration with the transfection of shRNA expression plasmid does not increase, and illustrates and does not induce IFN to reply.

Above result shows in the hepatitis B surface antigen that hepatitis B virus expresses that effectively epi-position protein sequence and cationic-liposome bio-link transmit system as the cell of s iRNA expression vector, compare with present commonly used single cation carrier system, the gene transmission efficiency is higher, non-target position effect is little, the nonspecific immune response effect is little, meet the effect of the anti-hepatitis B virus of expection imagination, can be used for clinical.

Claims (4)

1, based on the anti-hepatitis b virus gene medicament of the interferential mediated by complex carrier in siRNA pond, it is characterized in that: this medicine is according to the characteristics of hepatitis B virus and expression product thereof, with effective epi-position albumen and cationic-liposome bio-link in the hepatitis B surface antigen of hepatitis B virus expression, combine with surface of hepatocytes receptor generation specificity by hepatitis B surface antigen epi-position albumen, mediate rna i enters in the hepatocyte, utilize the meltable of cationic-liposome and cytolipin bilayer simultaneously, strengthen the siRNA expression vector and enter the interior efficient of hepatocyte.

2, the anti-hepatitis b virus gene medicament based on the interferential mediated by complex carrier in siRNA pond according to claim 1 is characterized in that: effectively the epi-position protein sequence is as follows in the hbs antigen:

cag?gcg?ggg?ttt?ttc?ttg?ttg?aca?aga?atc?ctc?aca?ata?cca?cag?agt?cta?gac?tcg

Q A G F F L L T R I L T I P Q S L D S。

3. the anti-hepatitis b virus gene medicament based on the interferential mediated by complex carrier in siRNA pond according to claim 1 is characterized in that: cationic-liposome composed as follows:

DC-chol (3 β-(N-(N ', N '-dimethyl aminoethyl)) the carbamate cholesterol)/DOPE (two oleoyl phosphoethanolamines)=1: 1 (mol ratio).

4. the anti-hepatitis b virus gene medicament based on the interferential mediated by complex carrier in siRNA pond according to claim 1 is characterized in that: the siRNA sequence is as follows:

SiRNA-S1: positive-sense strand (5 '-GUGGUGGACUUCUCUCAAUTT-3 ')

Antisense strand (5 '-AUUGAGAGAAGUCCACCACTT-3 ')

SiRNA-S2: positive-sense strand (5 '-AACCUCCAAUCACUCACCAACTT-3 ')

Antisense strand (5 '-GUUGGUGAGUGAUUGGAGGUUTT-3 ')

SiRNA-S3: positive-sense strand (5 '-GCCUCAUCUUCUUGUUGGUTT-3 ')

Antisense strand (5 '-ACCAACAAGAAGAUGAGGCTT-3 ')

SiRNA-S4: positive-sense strand (5 '-GGUAUGUUGCCCGUUUGUCTT-3 ')

Antisense strand (5 '-GACAAACGGGCAACAUACCTT-3 ').

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