CN100447243C - HBV-specific interference target site gene and its siRNA and its application in anti-HBV infection - Google Patents

Abstract

The invention provides a hepatitis B virus (HBV) specific interference target site gene, siRNA produced by the gene and the application of the gene and siRNA in the preparation of therapeutic drugs for resisting HBV infection. The HBV target gene screened by the present invention will produce siRNA effector molecules that can specifically recognize and degrade HBV after being expressed by the vector, no matter whether the expression siRNA recombinant vector is used for direct intravenous injection, or artificially synthesized siRNA is injected into B In patients with hepatitis, it can produce highly effective and specific anti-HBV effects. The HBV targeting siRNA screened by the present invention has the following characteristics: high specificity, high efficiency, persistence and safety, and the present invention is expected to provide a new method for the treatment of HBV infection.

Description

HBV-specific interference target site gene and its siRNA and its application in anti-HBV infection

technical field

The present invention relates to gene therapy in the field of biotechnology, in particular to the construction of hepatitis B virus (hepatitis Bvirus, HBV) specific interference target site gene, siRNA (small interfering RNA, small interfering RNA) generated by the above-mentioned gene and the resulting Application of the above-mentioned gene and siRNA in the preparation of therapeutic drugs for anti-HBV infection.

Background technique

HBV is a hepadnavirus, a circular DNA virus containing a partial double strand. The long strand of viral DNA is the negative strand, and the short strand is the positive strand. The HBV genome is compact and exquisite, with a size of about 3.2kb. Persistent HBV infection is one of the current serious global health problems and the most common cause of chronic liver disease. At present, there are about 350 million chronic HBV-infected patients in the world, and nearly 1 million people die from chronic liver diseases related to HBV infection every year. my country is a high-incidence area of hepatitis B. Viral hepatitis, liver cirrhosis and liver cancer caused by persistent HBV infection seriously endanger the life and health of the people in our country: nearly 10% of the population is HBV carriers, and 3000 patients with current hepatitis B About 300,000 people die from hepatitis B and its related complications every year. Therefore, seeking an effective treatment method for hepatitis B has become a major issue to be solved urgently in the current medical field. Currently approved drugs to control HBV persistent infection mainly include interferon-α and nucleoside analogues such as lamivudine and adefovir, but the efficacy of antiviral treatments so far is not satisfactory: high-dose recombinant interferon The sustained response rate is only about 30%. Although nucleoside analogues such as lamivudine have strong antiviral activity, the rapid rebound of viral replication after drug withdrawal and the emergence of drug-resistant virus strains make the implementation of clinical antiviral programs facing great challenges. Finding a new anti-HBV treatment scheme with high efficiency, specificity and low toxicity has always been the unremitting goal of people.

RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing phenomenon mediated by double-stranded RNA (dsRNA), which widely exists in a variety of organisms. The essence of RNAi is a sequence-specific acquired immune response mediated by RNA, a primitive genome protection mechanism against foreign gene expression, and an organism's immune system at the genome level. The core function of RNAi is immunity against viral infection, maintaining the stability of transposons in the genome, clearing abnormal RNA, and participating in the regulation of gene expression. At present, RNAi has developed into a new type of gene blocking technology, which is used to efficiently and specifically shut down or reduce the expression of specific genes, and is widely used in new gene screening, gene function identification, and gene therapy. Positive progress has been made in the treatment of , showing very broad application prospects.

It has been confirmed that the mechanism of dsRNA-induced RNAi is that dsRNA in cells is degraded by Dicer-1 enzyme to produce siRNA, which recognizes and degrades mRNA homologous to siRNA with the participation of other activating enzymes. In mammalian cells, the introduction of 21nt siRNA successfully inhibited the expression of target genes in cells, while avoiding the non-specific effect of dsRNA activation of the interferon system.

Compared with the classic anti-HBV drugs interferon and nucleoside analogues, RNAi technology has many advantages in anti-HBV treatment: ①Specifically targeting viral transcripts to block viral replication, without activating non-specific cellular responses, adverse reactions Minimal; ②RNAi has high efficiency, and a small amount of siRNA can inhibit HBV mRNA and reduce viral expression products; ③There are many potential RNAi target sites in the HBV genome, which can provide hundreds of RNAi target sites, and choose conservative The regional site can effectively prevent the emergence of virus variants; ④The effect of RNAi does not depend on the replication of the virus, siRNA can play its role in inhibiting viral gene transcription and reducing protein expression levels when the virus is not actively replicating, and Lamy Furidin can only inhibit the replication activity of HBV, so it can be used as an adjuvant therapy for antiviral drug lamivudine.

Contents of the invention

The purpose of the present invention is to construct a novel molecule with strong specificity, high activity and continuous anti-HBV infection, and provide a kind of HBV-specific interference target site gene, siRNA generated by the transcription of the above-mentioned gene, and the preparation of the gene and siRNA. Application in therapeutic drugs against HBV infection.

Technical solution of the present invention is:

The HBV-specific interference target site gene has a sequence of one of Sequence Listing <400>1-5.

The siRNA produced by the transcription of the above-mentioned gene has a sequence of one of 6-15 in the sequence listing <400>.

The application of the above-mentioned gene in the preparation of therapeutic medicine for resisting HBV infection.

Application of the above siRNA in the preparation of therapeutic drugs for anti-HBV infection.

The present invention is based on the principle that siRNA can efficiently and specifically shut down or reduce the expression of specific genes, and uses siRNA produced by pSUPER vector (Brummelkamp TR, et al.Science 2002; 296:550-553) to achieve high-efficiency, specificity, and sustained efficacy against HBV. inhibition. The HBV target gene screened by the present invention will produce siRNA effector molecules that can specifically recognize and degrade HBV after being expressed by the vector, no matter whether the expression siRNA recombinant vector is used for direct intravenous injection, or artificially synthesized siRNA is injected into B In patients with hepatitis, it can produce highly effective and specific anti-HBV effects.

The HBV-targeted genes screened by the present invention have the following characteristics: ① High specificity—RNAi is a gene silencing mechanism at the post-transcriptional level, and the HBV-targeted siRNA can degrade the mRNA transcribed by HBV very specifically, while irrelevant genes do not Affected, it was found in the experiment that siRNA targeting EGFP (green fluorescent protein) did not have a significant impact on the expression of HBV genes; ② high efficiency - a relatively small amount of siRNA molecules can produce a strong RNAi effect and inhibit HBV mRNA 3. Persistence——HBV-specific siRNA can be expressed for a long time after vector transfection into cells, achieving long-term inhibition of HBV in vivo ; ④Safety—Since the 21nt siRNA is used, it will not activate the interferon system and produce non-specific effects, and the adverse reactions are minimal.

The invention is expected to provide a new method for the treatment of HBV infection.

Description of drawings

Figure 1 is a schematic diagram of pSUPER vector transcription to generate siRNA.

Figure 2 is an information map of RNA interference sites of the HBV gene and its transcripts.

Fig. 3 is a graph showing the double enzyme digestion identification results of the siRNA expression vector.

Figure 4 is a graph showing the results of cell cycle analysis of cell lines stably expressing siRNA.

Figure 5 is the growth curve of cells stably expressing siRNA.

Fig. 6 is a graph showing the degradation of HBV gene mRNA by siRNA.

Fig. 7 is a graph showing the degradation of HBV transcripts by siRNA.

Fig. 8 is a graph showing the inhibitory effect of siRNA on the secretion of HBsAg (A) and HBeAg (B) from cells.

Fig. 9 is a diagram (200×) of immunofluorescence detection results of siRNA inhibiting the expression of HBV antigen.

Figure 10 is a Western blot analysis diagram of the cell lysate.

Figure 11 is a graph showing the inhibitory effect of siRNA on culture supernatant (A) and intracellular (B) HBV DNA.

Figure 12 is a graph showing the sustained inhibition of HBsAg (A) and HBeAg (B) expression by siRNA.

Fig. 13 is a graph showing the inhibitory effect of siRNA on HBsAg in HBV transgenic mice.

Figure 14 is HE staining images of livers of HBV transgenic mice (A) and C57BL mice (B).

Figure 15 is a graph showing siRNA inhibiting the expression of HBsAg in the liver of transgenic mice

Figure 16 is a graph showing that siRNA inhibits the replication of HBV DNA in transgenic mice

Detailed ways

1. Construction of HBV-specific siRNA expression vector

(1) Strategies for generating siRNA from DNA transcription

Each of the designed oligonucleotide chains is 64nt, and the 5' end and 3' end respectively contain BglII and HindIII restriction sites, which are convenient for connection with the pSUPER vector. The 19nt coding sequence at the 5' end is homologous to the target gene, and another 19nt sequence is reverse complementary to it. A 9nt TTCAAGAGA spacer sequence forms a circular structure in between, and a transcription termination signal TTTTT is added at the end. The oligonucleotide chains with the above-mentioned structural features are annealed and phosphorylated to form double-stranded DNA, and then inserted into the pSUPER vector (see Figure 1). The pSUPER vector with the above structure can be transcribed to generate hairpin-shaped RNA (shRNA), which becomes siRNA after digestion with Dicer to inhibit gene expression.

(2) Selection of HBV gene interference sites and design of oligonucleotides

According to the HBV nucleotide sequence (U95551) reported in GenBank, on the siRNA design website http://wwwl.qiagen.com/Products/GeneSilencing/CustomSiRna/SiRnaDesigner.aspx , the length of the selected gene coding region starting with AA is 21 bases, and the sequence with G+C content of about 50% was used as a candidate siRNA target site. BLAST ( http://www.ncbi.nlm.nih.gov/blast/ ) homology analysis of the designed siRNA sequence confirmed that it was a HBV-specific sequence. Select the 1683-1701, 1580-1598, 671-689, 413-431, 2027-2045 nucleotide sequences of the HBV coding region, and design two 64nt oligonucleotide chains that can encode siRNA according to the requirements of the pSUPER vector. Both ends of the acid sequence contain BglII and Hind III restriction sites, which can be directly connected to the pSUPER vector that has been cut with the same restriction enzymes. In addition, the siRNA expression vector pSUPER-EGFP of enhanced green fluorescent protein (EGFP), which has nothing to do with the HBV sequence, was designed as a control. The information on the RNA interference sites for the HBV gene and its transcripts is shown in Figure 2.

(3) Double-stranded oligonucleotide cloned into pSUPER vector

①Annealing and phosphorylation of oligonucleotides

The synthesized single-stranded oligonucleotide was dissolved in H ₂ O, and its concentration was adjusted to 3 μg/μl. Take 1 μl of corresponding oligonucleotide synthesis fragments, add 48 μl of annealing buffer, incubate at 95°C for 5 minutes, incubate at 70°C for 10 minutes, and slowly cool to 4°C to obtain annealed double-stranded DNA; take 2 μl of annealed oligonucleotides, add 1 μl T4 PNK buffer, 1 μl 1mM ATP, 1 μl T4 PNK, 5 μl H ₂ O, phosphorylated in a water bath at 37°C for 30 minutes, and incubated at 70°C for 10 minutes to inactivate PNK.

② Ligation of double-stranded oligonucleotide and carrier

The annealed and phosphorylated oligonucleotides were ligated with the pSUPER vector digested by Bgl II and HindIII, transformed into DH5α competent cells, the clones were picked, shaken and cultured to extract the plasmid, and identified by EcoR I and Hind III double enzyme digestion , the positive plasmid cut out a band of about 300bp, while the size of the empty plasmid was 240bp (accompanying drawing 3). Sequence determination confirmed that the sequences of the recombinant plasmids were completely consistent with the designed sequences, and they were named pSUPER-HBV1, pSUPER-HBV2, pSUPER-HBV3, pSUPER-HBV4, pSUPER-HBV5 and pSUPER-EGFP.

2. siRNA inhibits gene expression and replication of HBV in cells

The siRNA expression vector with the correct sequence and the pTK-Hyg plasmid were co-transfected into HepG22.2.15 cells stably expressing HBV at a molar ratio of 10:1, and a stable monoclonal cell line was obtained by 200 μg/ml hygromycin resistance screening, and the introduction Effect of siRNA on cell cycle and cell growth rate; RT-PCR and Northern blot were used to detect the inhibition of HBV mRNA at the mRNA level; microparticle enzyme immunoassay (MEIA) was used to quantitatively detect the HBV mRNA in the culture supernatant at the protein level HBsAg and HBeAg content, immunofluorescent staining and Western blot to detect the expression of corresponding antigens in the cells; fluorescent quantitative PCR (FQ-PCR) to detect the replication of HBV DNA inside and outside the cells; and dynamically observe the long-term inhibitory effect of siRNA on HBV.

(1) siRNA has no obvious effect on the cell cycle

The selected monoclonal cells were digested and washed to make a single cell suspension, fixed and stained, the cell cycle was detected and the cell proliferation index was calculated. The results of flow cytometry analysis showed (accompanying drawing 4, table 1) stably expressing siRNA monoclonal cell line, cell cycle and proliferation index compared with contrast HBV-pSUPER there is no significant difference, shows that importing HBV-specific siRNA expression vector has great effect on HepG2 2.2.15 The growth of cells has no obvious effect.

Table 1 Cell cycle and proliferation index (PI) of cell lines stably expressing siRNA

(2) siRNA has no significant effect on cell growth curve

Inoculate the screened monoclonal cells in 7 96-well plates according to 3×10 ³ /100 μl/well. When culturing for 1, 2, 3, 4, 5, 6, and 7 days, take 1 96-well plate for each, discard and culture Add 20 μl of 5 mg/ml MTT to each well, continue culturing for 4 hours, then suck off the culture solution, add 150 μl DMSO to each well, shake for 10 minutes to dissolve the crystals, and measure the absorbance at 490 nm with an enzyme-linked immunosorbent assay. The growth curve of each cell was drawn with the culture time as the abscissa and the absorbance value as the ordinate. It was found that the growth rate of cells stably expressing siRNA was similar to that of control cells, and there was no significant difference between the groups (Fig. 5)

(3) siRNA degradation of HBV mRNA in HepG2 2.2.15 cells

According to the gene sequence of HBV, RT-PCR primers P1 and P2 for amplifying HBV 356-714, and primers G1 and G2 for amplifying glyceraldehyde phosphate dehydrogenase (GAPDH) gene were designed. Extract the total RNA of the monoclonal cells screened out, reverse transcribe to generate cDNA, and use the corresponding primers to perform PCR amplification of HBV mRNA according to the following parameters: 94°C for 4 minutes; 94°C for 30sec, 55°C for 30sec, 72°C for 45sec, total 25 cycles; 72°C extension for 5 min. The results showed that the band of the RT-PCR product of cells in the interference group at the 358bp position was significantly darker than that of the control band, and the HBV mRNA expression products of transfected pSUPER-EGFP and pSUPER cells had no significant change compared with HepG2 2.2.15 cells, suggesting that The expression of HBV mRNA was significantly inhibited; while the internal reference GAPDH mRNA could be amplified by RT-PCR to produce a 250bp fragment in each cell, and there was no significant difference in the expression between each group (accompanying drawing 6), showing that siRNA has an effect on HepG2 2.2.15 The degradation of HBV mRNA in cells is efficient and specific.

P1 5’-CAA CTT GTC CTG GTT ATC GC-3’

P2 5’-AAG CCC TAC GAA CCA CTG AA-3’

G1 5’-CAA CGG ATT TGG TCG TAT TGG G-3’

G2 5’-CCT GGA AGA TGG TGA TGG GAT T-3’

(4) Degradation effect of siRNA on HBV transcript

The total RNA of monoclonal cells extracted and screened was subjected to denaturing gel electrophoresis, transmembrane transfer, and hybridization with α- ^32P -labeled probes. The results of autoradiography showed that HBV 3.5kb, 2.4kb, 2.4kb, The 2.1kb transcript band was clear, while the corresponding band of the cells in the interference group was significantly weakened, and the HBV transcript band in the cells with obvious inhibitory effect was basically invisible (Fig. The degradation of HBV RNA in the medium is efficient and specific.

(5) Inhibitory effect of siRNA on secretion of HBsAg and HBeAg in cell culture supernatant

The HBsAg and HBeAg contents of 3×10 ⁵ selected cells were cultured under double resistance conditions for 48h and 72h by microparticle enzyme immunoassay (MEIA). The results showed that both HBsAg and HBeAg in the culture supernatant were significantly inhibited, and pSUPER-HBV2 had the strongest inhibitory effect, and its inhibition rates on HBsAg and HBeAg in the supernatant of cells cultured for 48 hours were 97% and 87%, respectively. The inhibition rates of antigens were 98% and 88%; pSUPER-HBV5 was the second most effective. Its inhibition rates for HBsAg and HBeAg in the supernatant of cells cultured for 48 hours were 87.4% and 77%, respectively, and the inhibition rates of corresponding antigens for 72 hours were 90% and 80.8%; other carriers also have a good inhibitory effect on the secretion of HBsAg and HBeAg, the inhibition efficiency of HBsAg is 60.2% (pSUPER-HBV 3, 72h) to 91.7% (pSUPER-HBV1, 72h), the inhibition of HBeAg Efficiencies range from 53.8% to 64.4%. The above results were significantly different from the corresponding pSUPER control group (P<0.05), but there was no significant difference between pSUPER-EGFP and the control group (Fig. 8). It suggested that the inhibitory effect of siRNA was efficient and specific.

(6) siRNA inhibits the expression of HBsAg and HBcAg in cells

Immunofluorescence staining was performed on the screened monoclonal cells, and the results showed that the red fluorescent-labeled virus antigens in the cells were significantly reduced, and the expression of HBsAg and HBcAg could not be detected in the pSUPER-HBV2 and pSUPER-HBV5 transfected cells, while the pSUPER- The expression of the antigen in EGFP and the control group did not decrease significantly (Fig. 9), indicating that siRNA can efficiently and specifically inhibit the synthesis of intracellular HBV antigen.

(7) Western blot detection of intracellular HBsAg

The Western blot detection result that cell lysate is carried out shows, the expression of HBsAg in the cell expressing HBV-specific siRNA is obviously down-regulated, and the expression of antigen in pSUPER-EGFP and control group has no obvious reduction (accompanying drawing 10), shows that siRNA does It can efficiently and specifically inhibit the synthesis of intracellular HBV antigens.

(8) Inhibitory effect of siRNA on HBV DNA

Take 3×10 ⁵ cell culture supernatant for 72 hours and 5 μl of cellular genomic DNA at a concentration of 1.0ng/μl and add them to the prepared PCR reaction tubes respectively. At the same time, set up a negative control and a positive quantitative gradient control, mix well and place in PE7700 automatic fluorescence Detector, amplify according to the following conditions: pre-denaturation at 93°C for 2min, 93°C for 45s, 55°C for 60s, first do 10 cycles, then do 30 cycles at 93°C for 30s, 55°C for 45s. After the reaction, the copy number of HBV was calculated automatically by computer analysis. The results of fluorescent quantitative PCR (fluorogenic quantitative PCR, FQ-PCR) showed that siRNA can reduce the copy number of HBV DNA inside and outside cells, among which pSUPER-HBV2 and pSUPER-HBV5 can reduce the HBV DNA in the culture supernatant by 2 orders of magnitude, and the inhibition rate is as high as 99.7% and 98.5%, and the inhibition rates of the remaining pSUPER-HBV1, pSUPER-HBV4 and pSUPER-HBV3 on HBV DNA in the culture supernatant were 95.7%, 92.3% and 62.9% (Fig. 11A). The results of the FQ-PCR detection of the genomic DNA of the cells were similar to those obtained in the culture supernatant, and the inhibition rates of pSUPER-HBV(1-5) to HBV DNA in the cells were 81.1%, 82.8%, 63.8%, 74.5% and 82.1% (Figure 11B). The above FQ-PCR results show that siRNA can inhibit the replication of HBV DNA in cells.

(9) siRNA continuously inhibits the expression of HBV antigen

The selected monoclonal cells were continuously subcultured in MEM medium containing 10% FBS, 200 μg/ml G418 and 200 μg/ml hygromycin, and the cells were regularly inoculated into 6-well plates with 3 × 10 ⁵ in 3 duplicate wells. The cells were cultured in DMEM containing the same concentration of G418 and hygromycin for 72 hours under the same conditions, and the culture supernatants of each period were collected for the detection of HBsAg and HBeAg. The long-lasting and dynamic observation of HBV antigen expression found that the siRNA transcribed by the vector can continuously and efficiently inhibit the expression of HBV genes: pSUPER-HBV2 and pSUPER-HBV5 with better inhibitory effect can not suppress HBsAg in the culture process of up to 10 months. Almost remained unchanged, only HBeAg slightly increased; the results of pSUPER-HBV1 and pSUPER-HBV4 were similar (Fig. Impure related.

3. Inhibitory effect of siRNA on HBV in transgenic mice

(1) Treatment of HBV transgenic mice

Clean grade C57BL/6J-HBV transgenic mice (6-8 weeks old, body weight 15-20g) were purchased from the Department of Experimental Animal Science, Peking University Health Science Center. Twenty-four HBV transgenic mice were randomly divided into 3 groups: pSUPER-HBV2 group, pSUPER-HBV5 group and pSUPER group, with 8 mice in each group.

Administration by hydrodynamics-based transfection: Prepare saline solution (1.5-2.0ml) according to 10% of the body weight of HBV transgenic mice, and take 40 μg of plasmid DNA from different groups and dissolve them in corresponding volume of saline solution middle. Disinfect the tail of the mouse fixed outside the mousetrap, puncture the tail vein with the smallest scalp needle, and inject the liquid into the mouse quickly (<5 seconds) after the blood returns. On the 4th and 7th day after the injection, 4 mice in each group were treated respectively, blood samples were collected for the detection of experimental indicators, and liver tissue slices were taken for HE or immunohistochemical staining.

(2) Inhibitory effect of siRNA on HBsAg in HBV transgenic mice

The pSUPER-HBV2, pSUPER-HBV5 with better anti-HBV effect and the control vector pSUPER were injected into mice by hydraulic transfection method. The results of HBsAg determination in serum showed that on the 4th and 7th day after injection, pSUPER-HBV2 had no effect on mice. The inhibition rates of HBsAg were 59.3% and 60.3%, respectively, and the inhibition rates of pSUPER-HBV5 were 26% and 40%. It shows that the anti-HBV effect of pSUPER-HBV2 in vivo is better than that of pSUPER-HBV5, and the result on the 7th day is slightly better than the result on the 4th day (Fig. 13).

(3) siRNA inhibits the expression of HBsAg in the liver of transgenic mice

There were no visible lesions in the tissues and organs of C57BL/6J-HBV transgenic mice. Under the light microscope, the hepatocytes of C57BL/6J-HBV transgenic mice were diffusely swollen, and the cytoplasm of the hepatocytes was uniformly stained with eosin (ground-glass degeneration). Nuclear pyknosis was deeply stained, eosinophilic bodies were seen in some liver cytoplasm, bile duct epithelial cells were irregularly arranged, and eosinophilic substances in the cytoplasm increased (Fig. 14). There were no obvious pathological changes in liver tissue of C57BL mice.

The liver of HBV transgenic mice introduced with siRNA was fixed, embedded in paraffin, and the sections were dewaxed and hydrated for immunohistochemical staining. As a result, it was observed that there were a large number of brown in the hepatic cytoplasm of HBV transgenic mice in the control group (injected with pSUPER plasmid) granules (HBsAg), while the brown granules in the hepatocytes of HBV transgenic mice injected with 40 μg recombinant plasmid 7d were significantly reduced, and the proportion of stained positive cells decreased. The expression level of HBsAg in the liver tissue of the HBV transgenic mice in the pSUPER-HBV2 treatment group was close to the staining results of the normal C57BL mice ( FIG. 15 ).

(4) siRNA inhibits the replication of HBV DNA in transgenic mice

The transgenic mouse serum was taken to detect HBV DNA by FQ-PCR. The FQ-PCR results showed that the inhibition rates of pSUPER-HBV2 on HBV DNA in mice were 67.6% and 78.3% on the 4th and 7th days, and the inhibition rates of pSUPER-HBV5 60.3% and 73% (Figure 16). It shows that the siRNA transcribed by the vector can inhibit the replication of HBV DNA in transgenic mice.

(5) Effect of siRNA on serum alanine aminotransferase (ALT) in transgenic mice

The liver is rich in alanine aminotransferase (alanine aminotransferase, ALT). When the liver is damaged, ALT can be released into the serum, resulting in high ALT. Therefore, the detection of serum ALT can reflect the damage of the liver.

After injecting the siRNA expression vector, the ALT content in the serum of the mice was measured by the rate method on a 7070 automatic biochemical analyzer. The results showed that the ALT levels in the mice were compared with those in the control group on the 4th and 7th day after injection of pSUPER-HBV2 and pSUPER-HBV5. There was no significant difference (Table 2). It shows that the siRNA transcribed by the carrier will not cause damage to the liver when used in vivo.

The influence of table 2 siRNA on serum ALT of transgenic mice (x±s, n=4)

sequence listing

<110> Yang Angang

<120> HBV-specific interference target site gene and its siRNA and its application in anti-HBV infection

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Claims (4)

1, HBV specificity interference target point gene, its sequence are sequence table＜400〉2 sequence.

2, produced or synthetic siRNA through carrier by the gene of claim 1, its sequence is sequence table＜400〉sequence of one of 8-9.

3, gene as claimed in claim 1 is used for the application of the medicine of anti HBV infecting in preparation.

4, siRNA as claimed in claim 2 is used for the application of the medicine of anti HBV infecting in preparation.

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