CN1257284C - Method of blocking expression of hepatitis B virus - Google Patents

Method of blocking expression of hepatitis B virus Download PDF

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CN1257284C
CN1257284C CNB03119222XA CN03119222A CN1257284C CN 1257284 C CN1257284 C CN 1257284C CN B03119222X A CNB03119222X A CN B03119222XA CN 03119222 A CN03119222 A CN 03119222A CN 1257284 C CN1257284 C CN 1257284C
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sidna
positive
sense strand
antisense strand
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CN1526818A (en
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裴瑞卿
赵昕
汪芳迅
郭永
尹鸿瑛
程京
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BOAO BIOCHIP Co Ltd BEIJING
Tsinghua University
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Priority to CNB03119222XA priority Critical patent/CN1257284C/en
Priority to PCT/CN2003/000718 priority patent/WO2004078181A1/en
Priority to AU2003258452A priority patent/AU2003258452A1/en
Priority to KR1020057016557A priority patent/KR20060029597A/en
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    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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Abstract

The present invention discloses a method for blocking the expression of hepatitis B viruses, the aim of which is to block the expression of the hepatitis B viruses by using the method. The method for blocking the expression of hepatitis B viruses provided by the present invention uses an RNAi technology to block the expression of the hepatitis B viruses (HBV) in human liver cells. Specifically, four groups of siDNA sequences are provided, and plasmid siDNA-1, siDNA-2, siDNA-3 and siDNA-4-pBS/U6-GFP containing the four groups of siDNA sequences and a preparing method of the plasmid are provided. The present invention uses the RNAi technology for blocking the expression of the hepatitis B viruses, and has an important function for treating the hepatitis B.

Description

The method that a kind of extracorporeal blocking hepatitis B virus is expressed
Technical field
The present invention relates to the method that a kind of extracorporeal blocking hepatitis B virus is expressed in the genetically engineered field.
Background technology
Hepatitis B is infected by hepatitis B virus (HBV) and causes, is the most general a kind of in the disease of viral infection, is a kind of universal disease.Up to 20%, estimate to have in the world wide HBV carrier of 2-3 hundred million at South East Asia and parts of Africa infection rate.Chronic HBV has the sickness rate of 1-10% in the adult, higher among the children, and infection rate is 100% in the baby that HBeAg male mother is given birth to.
HBV is that a kind of diameter is the small-sized hepadnavirus of 42nm (hepatotropic DNA virus), is made up of the bag quilt and the core of outside.Surface antigen is the main component of HBV bag quilt, and as the leading indicator of diagnosing HBV to infect.The core of HBV virus is made up of cAg (HBcAg), archaeal dna polymerase/ThermoScript II and viral genome.HBV genome size is 3,200bp, and its portion gene and coded product thereof are as shown in table 1.
Table 1, HBV genome portion gene and coded product thereof
Gene Function
Pre-Surface1 Coded surface antigen HBsAg, the diagnosis leading indicator
Pre-Surface2
Surface
Pre-Core Coding e antigen (HBeAg) serum index
Core Coding cAg (HBcAg), nuclear structure albumen
The P-gene Coding DNA polysaccharase/ThermoScript II, virus replication
The X-gene Coding x antigen (HBxAg), function is uncertain
The infection of hepatitis B virus (HBV) is the major cause that causes chronic hepatitis, liver cirrhosis and be converted into liver cancer.More deep to the research that HBV infects, this comprises the acute and chronically infected research at HBV.At present the methods of treatment that in some patient, HBV is infected mainly be with interferon-' alpha ' and-β, or share also available gene therapy with synthetic antiviral or other medicines.But the genome of the HBV in different patients (comprising acute and chronic viral hepatitis B patient) source has sudden change largely, and this gene difference has brought very big difficulty for the treatment of hepatitis B virus.
Recent years is emerging, the RNA that particularly uses in the antiviral research of Mammals disturbs (RNAi) technology can efficiently block the expression of certain specific gene, and its achievement in research is as the research (Lee to blocking-up HIV expression aspect, N, S., Dohjima, T., Bauer, G., Li, H., Li, M.J., Ehsani, A., Dalvaterra, P.and Rossi, J. (2002) Nature Biotechnology, 19,500-505) and the another kind of research (Kapadia that causes the HCV of liver cirrhosis, S.B., Brideau-Andersen, A.and Chisari, F.V. (2003) Proc.Natl.Acad.Sci.USA, 100,2014-2018) grade has caused huge repercussion in the RNAi field.
RNAi is a kind of antiviral mechanism of finding in plant, nematode (Caenorhabditis elegans) and fruit bat (Drosophila) at first, the gene silencing mechanism (PTGS) after promptly transcribing.It is the phenomenon that is induced by double-stranded RNA (dsRNA).In this process, 1) dsRNA is siRNA (Cullen, B.R. (2002) Nat.Immunol.3,597-599 by the double-stranded RNA that the enzyme of a kind of RNaseIII of being similar to (being known as Dicer) is cut into 21-23nt (nucleotide); Hannon, G.J. (2002) Nature 418,244-251; Tuschl, T. (2001) Chembiochem 2,239-245; Sharp, P.A. (2001) Genes Dev.15,485-490; Moss, E.G. (2001) Curr.Biol.11, R772-R775); 2) these siRNA small molecules and a kind of protein complex are also referred to as RNA-inductive reticent mixture (RNA induced silercing complex, RISC) specificity combination; 3) RISC directly is incorporated on the siRNA nucleotide sequence homologous mRNA position therewith (for example HBV transcribes on the mRNA that the back forms); 4) purpose mRNA is being cut off in the centre with 21-23nt homology part, thus degraded and lose function (Cullen, B.R. (2002) Nat.Immunol.3,597-599; Hannon, G.J. (2002) Nature 418,244-251; Tuschl, T. (2001) Chembiochem 2,239-245; Sharp, P.A. (2001) Genes Dev.15,485-490; Moss, E.G. (2001) Curr.Biol.11, R772-R775).The maximum characteristics of RNAi effect are the gene orders of its very single-minded inhibition and its sequence homology, and other irrelevant sequence is had no effect.In recent years, the RNAi technology is being widely used in particularly because the disease that virus causes is called as " a kind of brand-new, breathtaking new technology " (Song, E., Lee, S.K., Wang, J., Ince, N., Ouyang, N, Min, J., Chen, J., Shankar, P., and Lieberman, J. (2003) Nature Medicine; 10.1038/nm828).
RNAi exists (Cullen, B.R. (2002) Nat.Immunol.3,597-599 equally in animal body; Yu, J.Y., DeRuiter, S.L.and Turner, D.L. (2002) Proc.Natl.Acad.Sci.USA 99,6047-6052; Brummelkamp, T.R., Bernards, R.and Agami, R. (2002) Science 296,550-553; Sui, G., Soohoo, C., Affarel, B., Gay, F., Y.andForrester, W.C. (2002) Proc.Natl.Acad Sci.USA 99,5515-5520), for example it can block HCV (hepatitis C virus), HIV-1 (human immunodeficiency virus-1), FHV (flock house virus), Rous sarcoma virus, the expression (Kapadia of virus such as dengue virus and poliovirus, S.B., Brideau-Andersen, A.and Chisari, F.V. (2003) Proc.Natl.Acad.Sci.USA, 100,2014-2018), but mostly these viruses are RNA viruses, and HBV is a double-stranded DNA virus.
Summary of the invention
The purpose of this invention is to provide the method that a kind of inhibition of hepatitis b virus is expressed.
The method that inhibition of hepatitis b virus provided by the invention is expressed is to use the expression of RNAi technology inhibition of hepatitis b virus HBV in human liver cell.
The invention provides four groups of siDNA sequences:
1)siDNA-1
SEQ ID № I (positive-sense strand 1a): 5 ' GGCTGCTATGCCTCATCTTCTA 3 '
SEQ ID № II (antisense strand 1b): 5 ' AGCTTAGAAGATGAGGCATAGCAGCC 3 '
SEQ ID № III (positive-sense strand 2a): 5 ' AGCTTAGAAGATGAGGCATAGCAGCCCTTTTTG 3 '
SEQ ID № IV (antisense strand 2b): 5 ' AATTCAAAAAGGGCTGCTATGCCTCATCTTCTA 3 '
2)siDNA-2
SEQ ID № V (positive-sense strand 1a): 5 ' GGCCTATGGGAGTGGGCCTCAA 3 '
SEQ ID № VI (antisense strand 1b): 5 ' AGCTTTGAGGCCCACTCCCATAGGCC 3 '
SEQ ID № VII (positive-sense strand 2a): 5 ' AGCTTTGAGGCCCACTCCCATAGGCCCTTTTTG 3 '
SEQ ID № VIII (antisense strand 2b): 5 ' AATTCAAAAAGGGCCTATGGGAGTGGGCCTCAA 3 '
3)siDNA-3
SEQ ID № IX (positive-sense strand 1a): 5 ' GGAAGCCTCCAAGCTGTGCCTA 3 '
SEQ ID № X (antisense strand 1b): 5 ' AGCTTAGGCACAGCTTGGAGGCTTCC 3 '
SEQ ID № XI (positive-sense strand 2a): 5 ' AGCTTAGGCACAGCTTGGAGGCTTCCCTTTTTG 3 '
SEQ ID № XII (antisense strand 2b): 5 ' AATTCAAAAAGGGAAGCCTCCAAGCTGTGCCTA 3 '
4)siDNA-4
SEQ ID № XIII (positive-sense strand 1a): 5 ' GGAAGAAGAACTCCCTCGCCTA 3 '
SEQ ID № XIV (antisense strand 1b): 5 ' AGCTTAGGCGAGGGAGTTCTTCTTCC 3 '
SEQ ID № XV (positive-sense strand 2a): 5 ' AGCTTAGGCGAGGGAGTTCTTCTTCCCTTTTTG 3 '
SEQ ID № XVI (antisense strand 2b): 5 ' AATTCAAAAAGGGAAGAAGAACTCCCTCGCCTA 3 '
The plasmid and the clone that comprise above-mentioned four groups of DNA all belong to protection scope of the present invention.Concrete plasmid is siDNA-1 ,-2 ,-3 and-4-pBS/U6-GFP.
Plasmid siDNA-1 ,-2 ,-3 and-universal architecture of 4-pBS/U6-GFP as shown in Figure 2.
Plasmid siDNA-1,-2,-3 and-construction process of 4-pBS/U6-GFP is: will be connected by the HindIII restriction enzyme site of design in sequence between the positive-sense strand 1a/ antisense strand 1b of siDNA-1, siDNA-2, siDNA-3, siDNA-4 and the positive-sense strand 2a/ antisense strand 2b respectively, the segment after the connection is inserted into respectively between the restriction enzyme site of ApaI in the pBS/U6 plasmid and EcoRI.GFP (green fluorescent protein, green fluorescence protein) segment is inserted on 3 ' the terminal SmaI site of siDNA.
The invention provides four kinds of siDNA nucleotide sequences, and on the basis of plasmid pBS/U6, transform plasmid as can express GFP plasmid, compare, can be used as better siDNA transfection mark with cotransfection.
The stable cell lines that contains siDNA of the present invention's screening will provide indispensable experiment material to next step research of infecting with RNAi technology treatment HBV, also belong to protection scope of the present invention.And will in the treatment of hepatitis B virus, play an important role.
Description of drawings
Fig. 1 is used to clone the pBS/U6 plasmid map of siDNA
Fig. 2 plasmid siDNA-1 ,-2 ,-3 and-the universal architecture collection of illustrative plates of 4-pBS/U6-GFP
Fig. 3 is to visible light after the HepG2.2.15 cellular segregation that contains fluorescence-siDNA and fluorescence microscope figure through FACS (Fluorescence-Activated Cell Sorting)
Fig. 4 is the relative concentration and the time relation curve of the HBV surface antigen that produced through different siDNA cells transfected
Fig. 5 is the relative concentration histogram of the HBV surface antigen that different cell produced
Embodiment
Material
Cell and plasmid
1, the structure of plasmid is that the host carries out with E.coli DH5 α;
2, the HBV positive cell is HepG2.2.15;
3, the plasmid that is used to clone siDNA is pBS/U6, and its collection of illustrative plates is seen accompanying drawing 1;
4, GFP (green fluorescent protein, green fluorescence protein) cassette is that the clone comes out from the pCR3.1-Uni of InvitrogenLife Technology, it comprise the functional zone (753bp) of GFP and both sides thereof from P CMVTo the 1739bp between " BGH polyadenylation ", before the GFP sequence, added NLS (nucleic-location-site) sequence area simultaneously.
The design of embodiment 1, siDNA (small interfering DNA)
From GenBank TMThe dna sequence dna that obtains the HBV of different sources amounts to 19 covers, its GenBank TMSequence number sees Table 2:
Table 2, be used to design the HBV sequence (GenBank of siDNA TMSequence number)
Sequence NO. GenBank NO.
Sequence 1 AB076679
Sequence 2 AB076678
Sequence 3 AY090461
Sequence 4 AY090460
Sequence 5 AY090459
Sequence 6 AY090458
Sequence 7 AY090457
Sequence 8 AY090456
Sequence 9 AY090455
Sequence 10 AY090454
Sequence 11 AY090453
Sequence 12 AY090452
Sequence 13 E10905
Sequence 14 NC 003977
Sequence 15 AB074756
Sequence 16 AB074755
Sequence 17 AB064316
Sequence 18 AB064315
Sequence 19 AB064314
Dna sequence dna to above-mentioned 19 cover HBV carries out homology analysis, with its total conserved regions (length is greater than 19nt) design siDNA.Designed 4 cover siDNA altogether, principle of design is:
1, natural U6 promotor comprises 3 G, transcribes from then on initially, and these three G are transformed in RNAi makes up, as the part of siRNA.In the pBS/U6 carrier, after the U6 promotor, followed by an ApaI (GGGCCC) site.With ApaI pBS/U6 carrier enzyme is cut digestion, be cut into flush end with Klenow or T4-DNA polysaccharase, also stay next G, therefore for 2 G are put back to, first oligo (oligo 1a) should be with " GG " beginning, and second oligo (being complementary oligo 1b) should finish with " CC ".
2, in second couple of oligo, oligo 2a should have " CCC ", should be " TTTTT " (being used for Pol III Transcription Termination) and an EcoRI restriction enzyme site (being used for the oligo subclone to carrier) after " CCC ".In oligo 2b, need " GGG " and EcoR I restriction enzyme site.
3, between two couples of oligo, connect, so all four oligo need the HindIII recognition sequence with HindIII.
4, determine not have among the designed oligo EcoRI site and Hind III site,, select other restriction enzyme site to carry out subclone if having.Be the form that is used for making up the oligo of each RNAi carrier below:
Oligo 1a 5’GG….A 3’
Oligo 1b 3’CC….TTCGA 5’
Oligo 2a 5’AGCTT….CCC TTTTT G 3’
Oligo 2b 3’A….GGG AAAAA CITAA 5’
Oligo 1a wherein, 1b reverse complemental, Oligo 2a, 2b reverse complemental; The genomic dna of 1a and 2b (with homologous sequence in the genome of need blocking-up) is identical, and the sequence of the genomic dna of 2a and 1b is identical.The final like this siDNA that obtains is one and oppositely repeats completely that this RNA that transcribes out will be folded into hairpin structure, can work as siRNA well.The HBV conserved sequence that is used to design siDNA is as shown in table 3:
Table 3, be used to design the HBV conserved sequence of siDNA
Be used to design the HBV sequence conserved regions of siDNA nt The protein-coding region * at sequence place
1 (404)TTC CTC TTC ATC CTG CTG CTA TGC CTC ATC TTC TT* 35 P-1and HBsAg
2 (640)CCT ATG GGA GTG GGC CTC AG 20 P-1and HBsAg
3 (1866)TTC AAG CCT CCA AGC TGT GCC TTG G 25 HBeAgCore
4 (2373)GAA GAA GAA CTC CCT CGC CTC GCA GAC G 28 HBeAgCore and P-2
* P: polysaccharase 1-1626 (P-1) and 2310-3215 (P-2); HBsAg: surface antigen (LHBS, MHBS, HBsAg) 1-838and 2851-3215; HBeAgCore:1817-2455; X-protein:1377-1841.The position of numeral Nucleotide in genome.
Designed siDNA is:
1)siDNA-1
SEQ ID № 1 (positive-sense strand 1a): 5 ' GGCTGCTATGCCTCATCTTCTA 3 '
SEQ ID № 2 (antisense strand 1b): 5 ' AGCTTAGAAGATGAGGCATAGCAGCC 3 '
SEQ ID № 3 (positive-sense strand 2a): 5 ' AGCTTAGAAGATGAGGCATAGCAGCCCTTTTTG 3 '
SEQ ID № 4 (antisense strand 2b): 5 ' AATTCAAAAAGGGCTGCTATGCCTCATCTTCTA 3 '
2)siDNA-2
SEQ ID № 5 (positive-sense strand 1a): 5 ' GGCCTATGGGAGTGGGCCTCAA 3 '
SEQ ID № 6 (antisense strand 1b): 5 ' AGCTTTGAGGCCCACTCCCATAGGCC 3 '
SEQ ID № 7 (positive-sense strand 2a): 5 ' AGCTTTGAGGCCCACTCCCATAGGCCCTTTTTG 3 '
SEQ ID № 8 (antisense strand 2b): 5 ' AATTCAAAAAGGGCCTATGGGAGTGGGCCTCAA 3 '
3)siDNA-3
SEQ ID № 9 (positive-sense strand 1a): 5 ' GGAAGCCTCCAAGCTGTGCCTA 3 '
SEQ ID № 10 (antisense strand 1b): 5 ' AGCTTAGGCACAGCTTGGAGGCTTCC 3 '
SEQ ID № 11 (positive-sense strand 2a): 5 ' AGCTTAGGCACAGCTTGGAGGCTTCCCTTTTTG 3 '
SEQ ID № 12 (antisense strand 2b): 5 ' AATTCAAAAAGGGAAGCCTCCAAGCTGTGCCTA 3 '
4)siDNA-4
SEQ ID № 13 (positive-sense strand 1a): 5 ' GGAAGAAGAACTCCCTCGCCTA 3 '
SEQ ID № 14 (antisense strand 1b): 5 ' AGCTTAGGCGAGGGAGTTCTTCTTCC 3 '
SEQ ID № 15 (positive-sense strand 2a): 5 ' AGCTTAGGCGAGGGAGTTCTTCTTCCC TTTTTG 3 '
SEQ ID № 16 (antisense strand 2b): 5 ' AATTCAAAAAGGGAAGAAGAACTCCCTCGCCTA 3 '
The structure of embodiment 2, plasmid
To be connected by the HindIII restriction enzyme site of design in sequence between the positive-sense strand 1a/ antisense strand 1b of siDNA-1, siDNA-2, siDNA-3, siDNA-4 and the positive-sense strand 2a/ antisense strand 2b respectively, the segment after the connection is inserted between the restriction enzyme site of ApaI in the pBS/U6 plasmid and EcoRI.GFP (green fluorescent protein, greenfluorescence protein) segment is inserted on 3 ' the terminal SmaI site of siDNA, through determined dna sequence, the siDNA segment that proof is cloned is correctly inserted, and can be used for the transfection human liver cell is HepG2.2.15 (structure of plasmid is seen accompanying drawing 2).The host who is used for plasmid clone is E.coliDH5 α, and substratum is for adding the LB substratum of 0.1g/lAmpicillin (penicillin (Sigma)).
The transfection of embodiment 3, siDNA-pBS/U6-GFP
1.HBV the male human liver cell is DMEM (GIBCO) high glucose medium of HepG2.2.15 with interpolation 10%FBS, is containing 5%CO 237 ℃ of incubators in leave standstill cultivation.
2.siDNA-pBS/U6-GFP plasmid transfection is finished with Effectene Transfection Reagent (QIAGEN) test kit, concrete operations are as follows:
(1) preceding 24 hours inoculation about 0.5-2 * 10 on six orifice plates of the DMEM substratum that contains 10% serum (FBS) of transfection 5Individual HBV male human liver cell is the HepG2.2.15 cell, 37 ℃ be cultured to transfection before;
(2) the observation of cell growth is normal before the transfection, and density is between the 40-80% saturation ratio;
(3) in the 1.5ml centrifuge tube TE of 0.4 μ g siDNA-pBS/U6-GFP plasmid DNA (concentration is not less than 0.1 μ g/ μ l) with pH7-8 dissolved, add Buffer EC to final volume 100 μ l, add 3.2 μ l Enhancer, vibrator vibrated for 1 second;
(4) room temperature (15-25 ℃) was placed 2-5 minute, and is centrifugal a little, and the drop on the tube wall is got rid of;
(5) add 10 μ l Effectene Transfection Reagent, with liquid-transfering gun pressure-vaccum 5 times up and down;
(6) room temperature is placed 5-10 minute to form the transfection complex body;
(7) during placing 5-10 minute, be that the suction of HepG2.2.15 cell culture supernatant is gone with male human liver cell in 6 orifice plates, wash cell once with 2ml PBS, add the 1.6ml fresh culture;
(8) add 600 μ l substratum in the transfection complex body, with liquid-transfering gun pressure-vaccum 2 times up and down, transfection composite is added drop-wise to rapidly on the cell of six orifice plates, mix gently, 37 ℃ are continued to cultivate;
(9) if cytotoxicity discarded substratum in 6-18 hour after transfection, with the PBS flushing once, change fresh culture.
Embodiment 4, FACS (Fluorescence-Activated Cell Sorting) sorting cells and Fluirescence observation
Cell before and after the transfection is with LEICA MPS60 (Solms, Germany) fluorescence microscope and gather image.Through cells transfected visible significantly green fluorescence under fluorescent microscope.HepG2.2.15 cell through the siDNA-pBS/U6-GFP transfection is used FACSDiva instrument (Becton Dickinson Bioscience after 48h cultivates, IOWA, USA) separation has the cell (cell that promptly has siDNA) of GFP fluorescence and collects, and continues to cultivate.The result shows that the FACS technology can come out the cell sorting that has GFP (promptly having siDNA) effectively as shown in Figure 3.
The relative concentration and the time relation of the HBV surface antigen that embodiment 5, the different siDNA cells transfected of process are produced
With HBV male human liver cell is that HepG2.2.15 and the HBV male human liver cell that only contains pBS/U6-GFP (not containing siDNA) plasmid are HepG2.2.15 cell cell in contrast.With the plasmid transfection HBV male human liver cell that contains different siDNA is the HepG2.2.15 cell, after the 48h cultivation, whether contain fluorescence according to cell, to contain the cellular segregation of fluorescence and collect with the FACS technology, continue to cultivate, simultaneously with the control cells of cultivating, at different incubation times, get culture supernatant, with " hepatitis B surface antigen is measured test kit S-01 " (Ying Kexin wound, Xiamen, China) the ELISA method that provides according to producer is measured the HBV surface antigen HBsAg content in the supernatant liquor, to measure the inhibition degree that different siRNA express HBV.
The result shows the amount of the HBsAg that different cell produces as shown in Figure 4, increases along with the prolongation of incubation time.Two control cells wherein: it is close that HBV male human liver cell is that HepG2.2.15 and the HBV male human liver cell that only contains pBS/U6 (not containing siDNA) plasmid are that sAg that the HepG2.2.15 cell is produced increases degree, and calculate in contrast, the inhibiting rate of siRNA; And compare with control cells through different siDNA (siDNA-1 ,-2 and-4)-pBS/U6-GFP cells transfected, the speed that produces HBsAg obviously slows down, and generation also has obvious decline in various degree.This has illustrated that different siRNA have in various degree restraining effect to the expression of HBV surface antigen.Among Fig. 4, institute is the time of cell cultures after FACS separates the target time; HepG2.2.15 be do not pass through any plasmid transfection, HBV male stable cell lines; PBS/U6 does not contain HepG2.2.15 cell siDNA, process pBS/U6-GFP transfection; SiRNA-1 ,-2 ,-4 respectively representative through siDNA-1 ,-2 and-the HepG2.2.15 cell of 4-pBS/U6-GFP plasmid transfection.
The inhibition efficient that embodiment 6, different siRNA express the HBV surface antigen
With the HepG2.2.15 cell of different siDNA transfections, separate back cultivation 120h through FACS, the cell harvesting that will contain fluorescence with the FACS technology, with the HBV male human liver cell of cultivating 120h be that HepG2.2.15 and the HBV male human liver cell that only contains pBS/U6 (not containing siDNA) plasmid of cultivating 120h are that the HepG2.2.15 cell is measured HBV surface antigen HBsAg content jointly, measuring method is with embodiment 5.The result as shown in Figure 5, among the figure, HepG2.2.15 be do not pass through any plasmid transfection, HBV male stable cell lines; PBS/U6 does not contain HepG2.2.15 cell siDNA, process pBS/U6-GFP transfection; SiRNA-1 ,-2 ,-3 ,-4 respectively representative through siDNA-1 ,-2 ,-3 and-the HepG2.2.15 cell of 4-pBS/U6-GFP plasmid transfection.
The result of Fig. 5 is as shown in table 4, shows, siRNA-1 can reach 80% to the inhibiting rate of HBV surface antigen, is 60% for siRNA-3 secondly; SiRNA-2 and-4 also has the obvious suppression effect to the generation of HBsAg respectively, and its inhibiting rate is respectively 21.9% and 39%.Wherein with do not contain siDNA, through the pBS/U6-GFP cells transfected in contrast.
The inhibition efficient that table 4, different siRNA express the HBV surface antigen.
pBS/U6 siDNA-1 siDNA-2 siDNA-3 siDNA-4
Inhibiting rate (%) 0 80±8.8 21.9±1.1 60* 39.0±0.041
* exclude Figure of description 4,5.
Sequence table
<160>16
<210>1
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
ggctgctatg cctcatcttc ta 22
<210>2
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
agcttagaag atgaggcata gcagcc 26
<210>3
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
agcttagaag atgaggcata gcagcccttt ttg 33
<210>4
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
aattcaaaaa gggctgctat gcctcatctt cta 33
<210>5
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
ggcctatggg agtgggcctc aa 22
<210>6
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
agctttgagg cccactccca taggcc 26
<210>7
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>7
agctttgagg cccactccca taggcccttt ttg 33
<210>8
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>8
aattcaaaaa gggcctatgg gagtgggcct caa 33
<210>9
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>9
ggaagcctcc aagctgtgcc ta 22
<210>10
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>10
agcttaggca cagcttggag gcttcc 26
<210>11
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>11
agcttaggca cagcttggag gcttcccttt ttg 33
<210>12
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>12
aattcaaaaa gggaagcctc caagctgtgc cta 33
<210>13
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>13
ggaagaagaa ctccctcgcc ta 22
<210>14
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>14
agcttaggcg agggagttct tcttcc 26
<210>15
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>15
agcttaggcg agggagttct tcttcccttt ttg 33
<210>16
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>16
aattcaaaaa gggaagaaga actccctcgc cta 33

Claims (7)

1, a kind of method of extracorporeal blocking hepatitis B virus expression is to use the expression of RNAi method inhibition of hepatitis b virus in human liver cell; SiDNA in the described RNAi method is one of following four kinds of situations:
1) the SEQ ID № 1 in the sequence table, SEQ ID № 2, SEQ ID № 3 and SEQ ID № 4; Wherein, SEQ ID № 1 and 3 is a positive-sense strand, and SEQ ID № 2 and 4 is an antisense strand;
2) the SEQ ID № 5 in the sequence table, SEQ ID № 6, SEQ ID № 7 and SEQ ID № 8; Wherein, SEQ ID № 5 and 7 is a positive-sense strand, and SEQ ID № 6 and 8 is an antisense strand;
3) the SEQ ID № 9 in the sequence table, SEQ ID № 10, SEQ ID № 11 and SEQ ID № 12; Wherein, SEQ ID № 9 and 11 is a positive-sense strand, and SEQ ID № 10 and 12 is an antisense strand;
4) the SEQ ID № 13 in the sequence table, SEQ ID № 14, SEQ ID № 15 and SEQ ID № 16; Wherein, SEQ ID № 13 and 15 is a positive-sense strand, and SEQ ID № 14 and 16 is an antisense strand.
2, a kind of plasmid that comprises siDNA; Described siDNA is one of following four kinds of situations:
1) the SEQ ID № 1 in the sequence table, SEQ ID № 2, SEQ ID № 3 and SEQ ID № 4; Wherein, SEQ ID № 1 and 3 is a positive-sense strand, and SEQ ID № 2 and 4 is an antisense strand;
2) the SEQ ID № 5 in the sequence table, SEQ ID № 6, SEQ ID № 7 and SEQ ID № 8; Wherein, SEQ ID № 5 and 7 is a positive-sense strand, and SEQ ID № 6 and 8 is an antisense strand;
3) the SEQ ID № 9 in the sequence table, SEQ ID № 10, SEQ ID № 11 and SEQ ID № 12; Wherein, SEQ ID № 9 and 11 is a positive-sense strand, and SEQ ID № 10 and 12 is an antisense strand;
4) the SEQ ID № 13 in the sequence table, SEQ ID № 14, SEQ ID № 15 and SEQ ID № 16; Wherein, SEQ ID № 13 and 15 is a positive-sense strand, and SEQ ID № 14 and 16 is an antisense strand.
3, plasmid according to claim 2 is characterized in that: described plasmid has physical map as shown in Figure 2.
4, a kind of method that makes up plasmid: the siDNA sequence is connected by the HindIII restriction enzyme site of design in sequence, and the segment after the connection is inserted between the restriction enzyme site of ApaI in as shown in Figure 1 the pBS/U6 plasmid and EcoRI, obtains the purpose plasmid; Described siDNA is one of following four kinds of situations:
1) the SEQ ID № 1 in the sequence table, SEQ ID № 2, SEQ ID № 3 and SEQ ID № 4; Wherein, SEQ ID № 1 and 3 is a positive-sense strand, and SEQ ID № 2 and 4 is an antisense strand;
2) the SEQ ID № 5 in the sequence table, SEQ ID № 6, SEQ ID № 7 and SEQ ID № 8; Wherein, SEQ ID № 5 and 7 is a positive-sense strand, and SEQ ID № 6 and 8 is an antisense strand;
3) the SEQ ID № 9 in the sequence table, SEQ ID № 10, SEQ ID № 11 and SEQ ID № 12; Wherein, SEQ ID № 9 and 11 is a positive-sense strand, and SEQ ID № 10 and 12 is an antisense strand;
4) the SEQ ID № 13 in the sequence table, SEQ ID № 14, SEQ ID № 15 and SEQ ID № 16; Wherein, SEQ ID № 13 and 15 is a positive-sense strand, and SEQ ID № 14 and 16 is an antisense strand.
5, method according to claim 4 is characterized in that: described method comprises that also the encoding gene with green fluorescent protein is inserted into the step on 3 ' the terminal SmaI site of siDNA.
6, a kind of clone that comprises claim 2 or 3 described plasmids.
CNB03119222XA 2003-03-05 2003-03-05 Method of blocking expression of hepatitis B virus Expired - Fee Related CN1257284C (en)

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