CN1257284C - Method for blocking expression of hepatitis B virus in vitro - Google Patents

Abstract

The invention discloses a method for blocking the expression of hepatitis B virus in vitro. The purpose is to block the expression of hepatitis B virus by this method. The method for blocking the expression of hepatitis B virus provided by the invention is to block the expression of hepatitis B virus HBV in human liver cells by using RNAi technology. Specifically provides four groups of siDNA sequences, plasmids containing the four siDNA sequences and a construction method thereof. The invention uses RNAi technology to block the expression of hepatitis B virus, and plays an important role in the treatment of hepatitis B.

Description

A method for blocking the expression of hepatitis B virus in vitro

technical field

The invention relates to a method for blocking the expression of hepatitis B virus in vitro in the field of genetic engineering.

Background technique

Hepatitis B, caused by hepatitis B virus (HBV) infection, is the most common type of viral infectious disease and a worldwide distribution disease. In Southeast Asia and parts of Africa, the infection rate is as high as 20%, and it is estimated that there are 200-300 million HBV carriers worldwide. Chronic HBV has an incidence of 1-10% in adults, higher in children and 100% of infants born to HBeAg-positive mothers.

HBV is a small hepatotropic DNA virus (hepatotropic DNA virus) with a diameter of 42nm, which consists of an outer coat and a core. Surface antigen is the main component of HBV coating and serves as the main indicator for diagnosis of HBV infection. The core of HBV virus consists of core antigen (HBcAg), DNA polymerase/reverse transcriptase and viral genome. The size of the HBV genome is 3,200bp, and some genes and their encoded products are shown in Table 1.

Table 1. Partial genes of HBV genome and their encoded products Gene Function Pre-Surface1 Encoded surface antigen HBsAg, the main indicator of diagnosis Pre-Surface2 Surface Pre-Core Encoded e-antigen (HBeAg) serum indicators Core Encodes core antigen (HBcAg), nuclear structural protein P-gene Encodes DNA polymerase/reverse transcriptase, viral replication X-gene Encodes x antigen (HBxAg), function undetermined

Hepatitis B virus (HBV) infection is the main cause of chronic hepatitis, liver cirrhosis and transformation into liver cancer. Research on HBV infection has been relatively in-depth, including research on acute and chronic HBV infection. At present, the treatment method for HBV infection in some patients is mainly to use interferon-α and -β, or combined with synthetic antiviral drugs or other drugs, and gene therapy can also be used. However, the genomes of HBV from different patients (including acute and chronic hepatitis B patients) have a greater degree of mutation, and this genetic difference has brought great difficulties to the treatment of hepatitis B virus.

In recent years, the emerging RNA interference (RNAi) technology, especially used in mammalian anti-virus research, can efficiently block the expression of a specific gene, and its research results, such as research on blocking the expression of HIV (Lee, 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 another type of HCV that causes liver cirrhosis (Kapadia, S.B., Brideau-Andersen, A.and Chisari, F.V. (2003) Proc.Natl.Acad.Sci.USA, 100, 2014-2018) etc. have been in RNAi The field has aroused great repercussions.

RNAi was originally discovered in plants, nematodes (Caenorhabditis elegans) and fruit flies (Drosophila) as an antiviral mechanism, namely post-transcriptional gene silencing mechanism (PTGS). It is a phenomenon induced by double-stranded RNA (dsRNA). In this process, 1) dsRNA is cut into 21-23nt (nucleotide) double-stranded RNA by an enzyme (called Dicer) similar to RNaseIII, namely siRNA (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); 2) These small siRNA molecules bind specifically to a protein complex, also known as the RNA-induced silencing complex (RISC); 3) RISC directly binds to On the mRNA site homologous to the siRNA nucleic acid sequence (for example, on the mRNA formed after HBV transcription); 4) the target mRNA is cut in the middle of the 21-23nt homologous part, thereby degrading and losing 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 biggest feature of RNAi is that it very specifically inhibits the gene sequence homologous to its sequence, and has no effect on other irrelevant sequences. In recent years, RNAi technology is being widely used especially in diseases caused by viruses, and is called "a brand-new and exciting 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 also exists in animals (Cullen, B.R. (2002) Nat.Immunol.3, 597-599; 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. and Forrester, 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 The expression of viruses such as sarcoma virus, dengue virus and poliovirus (Kapadia, S.B., Brideau-Andersen, A.and Chisari, F.V. (2003) Proc.Natl.Acad.Sci.USA, 100, 2014-2018), but these viruses are more is an RNA virus, while HBV is a double-stranded DNA virus.

Contents of the invention

The purpose of the present invention is to provide a method for blocking the expression of hepatitis B virus.

The method for blocking the expression of hepatitis B virus provided by the invention is to use RNAi technology to block the expression of hepatitis B virus HBV in human liver cells.

The present invention provides four groups of siDNA sequences:

1) siDNA-1

SEQ ID № I (sense strand 1a): 5'GGCTGCTATGCCTCATCTTCTA 3'

SEQ ID № II (antisense strand 1b): 5'AGCTTAGAAGATGAGGCATAGCAGCC 3'

SEQ ID № III (sense strand 2a): 5'AGCTTAGAAGATGAGGCATAGCAGCCCTTTTTG 3'

SEQ ID № IV (antisense strand 2b): 5'AATTCAAAAAGGGCTGCTATGCCTCATCTTCTA 3'

2) siDNA-2

SEQ ID № V (sense strand 1a): 5'GGCCTATGGGAGTGGGCCTCAA 3'

SEQ ID № VI (antisense strand 1b): 5'AGCTTTGAGGCCCACTCCCATAGGCC 3'

SEQ ID № VII (sense strand 2a): 5'AGCTTTGAGGCCCACTCCCATAGGCCCTTTTTG 3'

SEQ ID № VIII (antisense strand 2b): 5'AATTCAAAAAGGGCCTATGGGAGTGGGCCTCAA 3'

3) siDNA-3

SEQ ID № IX (sense strand 1a): 5'GGAAGCCTCCAAGCTGTGCCTA 3'

SEQ ID № X (antisense strand 1b): 5'AGCTTAGGCACAGCTTGGAGGCTTCC 3'

SEQ ID № XI (sense strand 2a): 5'AGCTTAGGCACAGCTTGGAGGCTTCCCTTTTTG 3'

SEQ ID № XII (antisense strand 2b): 5'AATTCAAAAAGGGAAGCCTCCAAGCTGTGCCTA 3'

4) siDNA-4

SEQ ID № XIII (sense strand 1a): 5'GGAAGAAGAACTCCCTCGCCTA 3'

SEQ ID № XIV (antisense strand 1b): 5'AGCTTAGGCGAGGGAGTTCTTCTTCC 3'

SEQ ID № XV (justice strand 2a): 5'AGCTTAGGCGAGGGAGTTTCTTCTTCCCTTTTTG 3'

SEQ ID № XVI (antisense strand 2b): 5'AATTCAAAAAGGGAAGAAGAACTCCCTCGCCTA 3'

Plasmids and cell lines comprising the above four sets of DNA all belong to the protection scope of the present invention. Specific plasmids are siDNA-1, -2, -3 and -4-pBS/U6-GFP.

The general structure of plasmid siDNA-1, -2, -3 and -4-pBS/U6-GFP is shown in Figure 2.

The construction method of plasmid siDNA-1, -2, -3 and -4-pBS/U6-GFP is as follows: the sense strand 1a/antisense strand 1b of siDNA-1, siDNA-2, siDNA-3, siDNA-4 The sense strand 2a/antisense strand 2b are connected by the HindIII restriction site designed in the sequence, and the ligated fragments are respectively inserted between the ApaI and EcoRI restriction sites in the pBS/U6 plasmid. The GFP (green fluorescent protein, green fluorescence protein) fragment was inserted into the SmaI site at the 3' end of the siDNA.

The invention provides four siDNA nucleotide sequences, and on the basis of the plasmid pBS/U6, the plasmid is transformed into a plasmid capable of expressing GFP, which can be used as a better siDNA transfection marker compared with co-transfection.

The stable cell line containing siDNA screened by the present invention will provide indispensable experimental materials for further research on the treatment of HBV infection with RNAi technology, and also belongs to the protection scope of the present invention. And will play an important role in the treatment of hepatitis B virus.

Description of drawings

Figure 1 Map of pBS/U6 plasmid used for cloning siDNA

Figure 2 The general structure map of plasmid siDNA-1, -2, -3 and -4-pBS/U6-GFP

Figure 3 is the visible light and fluorescence microscope observation pictures of HepG2.2.15 cells containing fluorescence-siDNA separated by FACS (Fluorescence-Activated Cell Sorting)

Figure 4 is the relationship between the relative concentration and time of the HBV surface antigen produced by cells transfected with different siDNA

Figure 5 is a histogram of the relative concentrations of HBV surface antigens produced by different cells

Detailed ways

Material

cells and plasmids

1. The plasmid was constructed using E.coli DH5α as the host;

2. The HBV positive cell line is HepG2.2.15;

3. The plasmid used for cloning siDNA is pBS/U6, and its map is shown in Figure 1;

4. GFP (green fluorescence protein, green fluorescence protein) cassette is cloned from pCR3.1-Uni of InvitrogenLife Technology, which includes the functional region (753bp) of GFP and the line from P _CMV to "BGH polyadenylation" on both sides 1739bp between them, and the NLS (nucleic-location-site) sequence region was added before the GFP sequence.

The design of embodiment 1, siDNA (small interfering DNA)

A total of 19 sets of HBV DNA sequences from different sources were obtained from GenBank ^TM , and their GenBank ^TM serial numbers are shown in Table 2:

Table 2. HBV sequence (GenBank ^TM number) used to design siDNA 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

Homology analysis was carried out on the DNA sequences of the above 19 sets of HBV, and siDNA was designed based on their common conserved regions (length greater than 19 nt). A total of 4 sets of siDNA were designed, and the design principles were as follows:

1. The natural U6 promoter contains 3 Gs, from which transcription starts. These 3 Gs are modified in RNAi construction as part of siRNA. In the pBS/U6 vector, an ApaI (GGGCCC) site follows immediately after the U6 promoter. Digest the pBS/U6 vector with ApaI, cut the blunt end with Klenow or T4-DNA polymerase, and leave a G, so in order to put the 2 G back, the first oligo (oligo 1a) should be "GG ", the second oligo (i.e. complementary oligo 1b) should end with "CC".

2. In the second pair of oligos, oligo 2a should have "CCC", followed by "TTTTT" (for Pol III transcription termination) and an EcoRI restriction site (for subcloning the oligo into the vector middle). In oligo 2b, "GGG" and EcoR I restriction sites are required.

3. Between two pairs of oligos, HindIII is used to connect, so all four oligos need to have HindIII recognition sequences.

4. Make sure that there are no EcoRI sites and Hind III sites in the designed oligo. If there are, select other restriction sites for subcloning. The following is the format of the oligo used to construct each RNAi vector:

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’

Among them, Oligo 1a, 1b are reverse complementary, and Oligo 2a, 2b are reverse complementary; the genomic DNA of 1a and 2b (with the homologous sequence in the genome to be blocked) is the same, and the genomic DNA of 2a and 1b has the same sequence. The resulting siDNA is a complete inverted repeat, and the transcribed RNA will be folded into a hairpin structure, which can function well as siRNA. The HBV conserved sequences used to design siDNA are shown in Table 3:

Table 3. HBV conserved sequences used to design siDNA Conserved region of HBV sequence for designing siDNA nt The protein coding region where the sequence is located* 1 (404)TTC CTC TTC ATC CTG CTG CTA TGC CTC ATC TTC TT* 35 P-1 and HBsAg 2 (640)CCT ATG GGA GTG GGC CTC AG 20 P-1 and 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: polymerase 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. Numbers indicate the position of the nucleotide in the genome.

The designed siDNA is:

1) siDNA-1

SEQ ID № 1 (sense strand 1a): 5'GGCTGCTATGCCTCATCTTCTA 3'

SEQ ID № 2 (antisense strand 1b): 5'AGCTTAGAAGATGAGGCATAGCAGCC 3'

SEQ ID № 3 (sense strand 2a): 5'AGCTTAGAAGATGAGGCATAGCAGCCCTTTTTG 3'

SEQ ID № 4 (antisense strand 2b): 5'AATTCAAAAAGGGCTGCTATGCCTCATCTTCTA 3'

2) siDNA-2

SEQ ID № 5 (sense strand 1a): 5'GGCCTATGGGAGTGGGCCTCAA 3'

SEQ ID № 6 (antisense strand 1b): 5'AGCTTTGAGGCCCACTCCCATAGGCC 3'

SEQ ID № 7 (justice strand 2a): 5'AGCTTTGAGGCCCACTCCCATAGGCCCTTTTTG 3'

SEQ ID № 8 (antisense strand 2b): 5'AATTCAAAAAGGGCCTATGGGAGTGGGCCTCAA 3'

3) siDNA-3

SEQ ID № 9 (sense strand 1a): 5'GGAAGCCTCCAAGCTGTGCCTA 3'

SEQ ID № 10 (antisense strand 1b): 5'AGCTTAGGCACAGCTTGGAGGCTTCC 3'

SEQ ID № 11 (sense strand 2a): 5'AGCTTAGGCACAGCTTGGAGGCTTCCCTTTTTG 3'

SEQ ID № 12 (antisense strand 2b): 5'AATTCAAAAAGGGAAGCCTCCAAGCTGTGCCTA 3'

4) siDNA-4

SEQ ID № 13 (sense strand 1a): 5'GGAAGAAGAACTCCCTCGCCTA 3'

SEQ ID № 14 (antisense strand 1b): 5'AGCTTAGGCGAGGGAGTTCTTCTTCC 3'

SEQ ID № 15 (Justice Strand 2a): 5'AGCTTAGGCGAGGGAGTTTCTTCTTCCC TTTTTG 3'

SEQ ID № 16 (antisense strand 2b): 5'AATTCAAAAAGGGAAGAAGAACTCCCTCGCCTA 3'

Embodiment 2, construction of plasmid

Connect the sense strand 1a/antisense strand 1b and sense strand 2a/antisense strand 2b of siDNA-1, siDNA-2, siDNA-3, and siDNA-4 respectively by the HindIII restriction site designed in the sequence, The ligated fragment was inserted between the ApaI and EcoRI restriction sites in the pBS/U6 plasmid. The GFP (green fluorescent protein, green fluorescence protein) fragment was inserted into the SmaI site at the 3' end of the siDNA. After DNA sequence determination, it was proved that the cloned siDNA fragment had been inserted correctly and could be used to transfect the human liver cell line HepG2.2.15( The structure of the plasmid is shown in Figure 2). The host used for plasmid cloning was E.coliDH5α, and the medium was LB medium supplemented with 0.1 g/l Ampicillin (penicillin (Sigma)).

Embodiment 3, transfection of siDNA-pBS/U6-GFP

1. The HBV-positive human liver cell line HepG2.2.15 was cultured statically in a 37° C. incubator containing 5% CO ₂ with DMEM (GIBCO) high-glucose medium supplemented with 10% FBS.

2. The siDNA-pBS/U6-GFP plasmid transfection was completed with the Effectene Transfection Reagent (QIAGEN) kit, the specific operation is as follows:

(1) 24 hours before transfection, inoculate about 0.5-2× ¹⁰⁵ HBV-positive human liver cell line HepG2.2.15 cells on a six-well plate containing DMEM medium containing 10% serum (FBS), and cultivate them at 37°C until Before transfection;

(2) Observe that the cells grow normally before transfection, and the density is between 40-80% saturation;

(3) In a 1.5ml centrifuge tube, dissolve 0.4μg siDNA-pBS/U6-GFP plasmid DNA (concentration not lower than 0.1μg/μl) with pH7-8 TE, add Buffer EC to a final volume of 100μl, add 3.2μl Enhancer, the oscillator oscillates for 1 second;

(4) Place at room temperature (15-25°C) for 2-5 minutes, centrifuge slightly, and shake off the liquid droplets on the tube wall;

(5) Add 10 μl Effectene Transfection Reagent, blow up and down with a pipette 5 times;

(6) Place at room temperature for 5-10 minutes to form a transfection complex;

(7) During the period of 5-10 minutes, the positive human liver cell line HepG2.2.15 cell culture supernatant in the 6-well plate was sucked off, the cells were washed once with 2ml PBS, and 1.6ml fresh medium was added;

(8) Add 600 μl medium to the transfection complex, blow up and down twice with a pipette gun, quickly drop the transfection complex onto the cells in the six-well plate, mix gently, and continue culturing at 37°C;

(9) If cytotoxicity occurs, discard the medium 6-18 hours after transfection, wash once with PBS, and replace with fresh medium.

Example 4, FACS (Fluorescence-Activated Cell Sorting) sorting cells and fluorescence observation

Cells before and after transfection were observed with a LEICA MPS60 (Solms, Germany) fluorescence microscope and images were collected. The transfected cells showed obvious green fluorescence under the fluorescence microscope. The HepG2.2.15 cells transfected with siDNA-pBS/U6-GFP were cultured for 48 h, and the cells with GFP fluorescence (i.e., cells with siDNA) were separated and collected with FACSDiva instrument (Becton Dickinson Bioscience, IOWA, USA). Continue to cultivate. The results are shown in FIG. 3 , indicating that the FACS technique can effectively sort out the cells with GFP (that is, with siDNA).

Example 5, the relationship between the relative concentration and time of the HBV surface antigen produced by cells transfected with different siDNA

The HBV-positive human liver cell line HepG2.2.15 and the HBV-positive human liver cell line HepG2.2.15 containing only pBS/U6-GFP (without siDNA) plasmid were used as control cells. Transfect the HBV-positive human liver cell line HepG2.2.15 cells with plasmids containing different siDNAs. After 48 hours of culture, according to whether the cells contain fluorescence, use FACS technology to separate and collect the cells containing fluorescence, and continue to cultivate. Simultaneously with the culture Take the culture supernatant together with the control cells at different culture times, and use the "Hepatitis B surface antigen assay kit S-01" (Intech Xinchuang, Xiamen, China) to measure the supernatant in the supernatant according to the ELISA method provided by the manufacturer. The content of HBV surface antigen HBsAg was determined to determine the degree of inhibition of different siRNAs on HBV expression.

The results are shown in Figure 4, indicating that the amount of HBsAg produced by different cells increases with the prolongation of culture time. Two of the control cells: the HBV-positive human liver cell line HepG2.2.15 and the HBV-positive human liver cell line HepG2.2.15 cells containing only pBS/U6 (without siDNA) plasmids produced similar sAg increases, and served as As a control, the inhibition rate of siRNA was calculated; and the cells transfected with different siDNA (siDNA-1, -2 and -4)-pBS/U6-GFP compared with the control cells, the speed of producing HBsAg was obviously slowed down, and the production amount was also reduced. Significant declines to varying degrees. This shows that different siRNAs have different inhibitory effects on the expression of HBV surface antigens. In Figure 4, the marked time is the time of cell culture after FACS separation; HepG2.2.15 is a HBV-positive stable cell line without any plasmid transfection; GFP-transfected HepG2.2.15 cells; siRNA-1, -2, -4 represent HepG2.2.15 cells transfected with siDNA-1, -2 and -4-pBS/U6-GFP plasmids, respectively.

Embodiment 6, the inhibitory efficiency of different siRNA to HBV surface antigen expression

HepG2.2.15 cells transfected with different siDNAs were separated by FACS and cultured for 120 hours. Cells containing fluorescence were collected by FACS technology. They were compared with the HBV-positive human liver cell line HepG2.2.15 cultured for 120 hours and pBS-only cells cultured for 120 hours. The HBV-positive human liver cell line HepG2.2.15 cells of the /U6 (without siDNA) plasmid were used to jointly measure the HBV surface antigen HBsAg content, and the determination method was the same as in Example 5. The results are shown in Figure 5. In the figure, HepG2.2.15 is a HBV-positive stable cell line without any plasmid transfection; pBS/U6 is HepG2.2.15 transfected with pBS/U6-GFP without siDNA Cells; siRNA-1, -2, -3, -4 represent HepG2.2.15 cells transfected with siDNA-1, -2, -3 and -4-pBS/U6-GFP plasmids, respectively.

The result of Fig. 5 is shown in table 4, shows, and the inhibitory rate of siRNA-1 to HBV surface antigen can reach 80%, is 60% for siRNA-3 secondly; Inhibition, the inhibition rate was 21.9% and 39%. Cells transfected with pBS/U6-GFP without siDNA were used as control.

Table 4. Inhibition efficiency of different siRNAs on HBV surface antigen expression. pBS/U6 siDNA-1 siDNA-2 siDNA-3 siDNA-4 Inhibition rate(%) 0 80±8.8 21.9±1.1 60* 39.0±0.041

* Not included in Figures 4 and 5 of the manual.

Sequence Listing

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<213> Artificial sequence

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agcttagaag atgaggcata gcagcc 26

<210>3

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<212>DNA

<213> Artificial sequence

<220>

<223>

<400>3

agcttagaag atgaggcata gcagcccttt ttg 33

<210>4

<211>33

<212>DNA

<213> Artificial sequence

<220>

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<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

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<212>DNA

<213> Artificial sequence

<220>

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<400>6

agctttgagg cccactccca taggcc 26

<210>7

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<213> Artificial sequence

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agctttgagg cccactccca taggcccttt ttg 33

<210>8

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<213> Artificial sequence

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ggaagaagaa ctccctcgcc ta 22

<210>14

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agcttaggcg agggagttct tcttcc 26

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Claims (7)

1. A method for blocking the expression of hepatitis B virus in vitro is to use the RNAi method to block the expression of hepatitis B virus in human hepatocytes; the siDNA in the RNAi method is one of the following four situations: 1) SEQ ID № 1, SEQ ID № 2, SEQ ID № 3 and SEQ ID № 4 in the sequence listing; wherein, SEQ ID № 1 and 3 are sense strands, and SEQ ID № 2 and 4 are antisense strands; 2) SEQ ID № 5, SEQ ID № 6, SEQ ID № 7 and SEQ ID № 8 in the sequence listing; wherein, SEQ ID № 5 and 7 are sense strands, and SEQ ID № 6 and 8 are antisense strands; 3) SEQ ID № 9, SEQ ID № 10, SEQ ID № 11 and SEQ ID № 12 in the sequence listing; wherein, SEQ ID № 9 and 11 are sense strands, and SEQ ID № 10 and 12 are antisense strands; 4) SEQ ID № 13, SEQ ID № 14, SEQ ID № 15 and SEQ ID № 16 in the sequence listing; wherein, SEQ ID № 13 and 15 are sense strands, and SEQ ID № 14 and 16 are antisense strands. 2. A plasmid comprising siDNA; the siDNA is one of the following four situations: 1) SEQ ID № 1, SEQ ID № 2, SEQ ID № 3 and SEQ ID № 4 in the sequence listing; wherein, SEQ ID № 1 and 3 are sense strands, and SEQ ID № 2 and 4 are antisense strands; 2) SEQ ID № 5, SEQ ID № 6, SEQ ID № 7 and SEQ ID № 8 in the sequence listing; wherein, SEQ ID № 5 and 7 are sense strands, and SEQ ID № 6 and 8 are antisense strands; 3) SEQ ID № 9, SEQ ID № 10, SEQ ID № 11 and SEQ ID № 12 in the sequence listing; wherein, SEQ ID № 9 and 11 are sense strands, and SEQ ID № 10 and 12 are antisense strands; 4) SEQ ID № 13, SEQ ID № 14, SEQ ID № 15 and SEQ ID № 16 in the sequence listing; wherein, SEQ ID № 13 and 15 are sense strands, and SEQ ID № 14 and 16 are antisense strands. 3. The plasmid according to claim 2, characterized in that: the plasmid has a physical map as shown in FIG. 2 . 4. A method for constructing a plasmid: the siDNA sequence is connected by the HindIII restriction site designed in the sequence, and the ligated fragment is inserted into the restriction site of ApaI and EcoRI in the pBS/U6 plasmid as shown in Figure 1 Between the points, the target plasmid is obtained; the siDNA is one of the following four situations: 1) SEQ ID № 1, SEQ ID № 2, SEQ ID № 3 and SEQ ID № 4 in the sequence listing; wherein, SEQ ID № 1 and 3 are sense strands, and SEQ ID № 2 and 4 are antisense strands; 2) SEQ ID № 5, SEQ ID № 6, SEQ ID № 7 and SEQ ID № 8 in the sequence listing; wherein, SEQ ID № 5 and 7 are sense strands, and SEQ ID № 6 and 8 are antisense strands; 3) SEQ ID № 9, SEQ ID № 10, SEQ ID № 11 and SEQ ID № 12 in the sequence listing; wherein, SEQ ID № 9 and 11 are sense strands, and SEQ ID № 10 and 12 are antisense strands; 4) SEQ ID № 13, SEQ ID № 14, SEQ ID № 15 and SEQ ID № 16 in the sequence listing; wherein, SEQ ID № 13 and 15 are sense strands, and SEQ ID № 14 and 16 are antisense strands. 5. The method according to claim 4, characterized in that: the method further comprises the step of inserting the coding gene of green fluorescent protein into the SmaI site at the 3' end of the siDNA. 6. A cell line comprising the plasmid of claim 2 or 3.

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