CN104107437B - A kind of RNA being used for the treatment of hepatitis B disturbs composition and method of making the same - Google Patents

Abstract

The invention discloses an RNA interference composition for treating hepatitis B virus and a preparation method thereof. The RNA interference composition for treating hepatitis B virus of the present invention has a very significant inhibitory effect on the DNA and antigen of hepatitis B virus in the liver and blood, has good specificity, and has good specificity and is effective on liver, blood and other tissues. The side effect is small; the preparation method of the present invention is simple and easy, and can be used not only in the preparation of the composition of the present invention, but also in the preparation of other nucleic acid compositions, and has a wide range of applications.

Description

A RNA interference composition for treating hepatitis B virus and its preparation method

technical field

The invention belongs to the field of hepatitis B drugs, and in particular relates to an RNA interference composition for treating viral hepatitis B and a preparation method thereof.

Background technique

It is estimated that the population infected with hepatitis B virus (HBV) is as high as 2 billion people - one-third of the world's population, and more than 3.5 million people will become chronically infected. According to reports, 15-40% of patients with HBV infection will develop cirrhosis, liver failure or liver cancer (HCC), and 500,000 to 1.2 million people die from HBV infection every year. The prevalence of HBV in the United States is estimated to be about 0.4%. However, voluntary sampling data suggest that prevalence in certain foreign-born minority groups exceeds 15%. During the 1990s, the number of HBV-related diagnosis outpatients and hospitalizations increased severalfold. Likewise, total hospitalization costs are estimated to have increased from $357 million in 1990 to $1.5 billion in 2003 and then held at $1.3 billion.

There are about 120 million hepatitis B virus carriers in China, which seriously plague people's life, work, and physical and mental health. Every year, about 300,000 people die from liver failure, cirrhosis, and liver cancer caused by hepatitis B virus infection. At present, the main drugs on the market are interferon and nucleoside (acid) drugs. Their common characteristics are that the treatment is not complete, the toxicity is high, the virus is easy to develop drug resistance to them, and there is no cure for drug-resistant strains. An entirely new approach to treatment is urgently needed. The successful research and development of "Aggway" will not only have huge economic value, but also have huge scientific and social value.

The ultimate goal of treating HBV is to inhibit or eliminate HBV, alleviate or stop the liver damage caused by HBV infection, and prevent the development of liver failure and liver cancer. The most important short- and medium-term treatment goal is to maximize HBV DNA suppression rate. However, complete eradication of HBV is difficult because it integrates into the host genome, generating cccDNA that continues as a potential relapse propensity. Pegylated interferon alfa-2a (PEG-IFN-alpha) and interferon alfa-2a (IFN-alpha), nucleosides (lamivudine, entecavir, and telbivudine) and nucleotide analogues Drugs (adefovir and tenofovir) are commonly used in the FDA-approved anti-HBV drug market. The main disadvantage of interferon therapy is its significant side effects, which limit its long-term use. It is often ineffective in patients with decompensated cirrhosis and normal transaminases. Furthermore, only one-third of patients responded to PEG-IFN-α antivirals. Although nucleoside (acid) analogues inhibit HBV replication and reduce hepatic necroinflammation, they cannot completely eradicate the virus. In addition, after drug discontinuation, rebound of viremia was observed in most patients. In addition, long-term treatment produces drug-resistant HBV strains, leading to treatment failure.

Contents of the invention

The purpose of the present invention is to overcome the defects of the prior art and provide an RNA interference composition for treating viral hepatitis B.

Another object of the present invention is to provide a method for preparing the above-mentioned RNA interference composition for treating viral hepatitis B.

Technical scheme of the present invention is as follows:

An RNA interference composition for treating hepatitis B virus, characterized in that: comprising one or a mixture of the following siRNA sequence groups:

First siRNA sequence set:

One justice chain, the sequence is:

5'-GGGUUUUUCUUGUUGACAAdTdTUAUAGGGUUUUUCUUGUUGACAAdTdT-3'

Two antisense strands, the sequence is: 5'-UUGUCAACAAGAAAAACCCCG-3';

Second siRNA sequence set:

Two sense strands, the sequence is: 5'-GGGUUUUUCUUGUUGACAAdTdT-3'

One antisense strand, the sequence is:

5'-UUGUCAACAAGAAAAACCCCGUAUAUUGUCAACAAGAAAAACCCCG-3';

Third siRNA sequence set:

One justice chain, the sequence is:

5'-CCGUGUGCACUUCGCUUCAdTdTUAUACCGUGUGCACUUCGCUUCAdTdT-3'

Two antisense strands, the sequence is: 5'-UGAAGCGAAGUGCACACGGUC-3';

Fourth siRNA sequence set:

Two sense strands, the sequence is: 5'-CCGUGUGCACUUCGCUUCAdTdT-3'

One antisense strand, the sequence is:

5'-UGAAGCGAAGUGCACACGGUCUAUAUGAAGCGAAGUGCACACGGUC-3';

Fifth siRNA sequence set:

One justice chain, the sequence is:

5'-mCCGmUGUGmCACUUCGCUUmCAdTdTUAUUmCCGmUGUGmCACUUCGCUUmCAdTdT-3'

Two antisense strands, the sequence is: 5'-UGAAGCGAAGUGmCAmCACGGUC-3';

Sixth siRNA sequence set:

Two sense strands, the sequence is: 5'-mCCGmUGUGmCACUUCGCUUmCAdTdT-3'

One antisense strand, the sequence is:

5'-UGAAGCGAAGUGmCAmCACGGUCuauaUGAAGCGAAGUGmCAmCACGGUC-3';

Seventh siRNA sequence set:

One justice chain, the sequence is:

5'-mCCGmUGUGmCACUUCGCUUmCAdTdTUAUUmCCGmUGUGmCACUUCGCUUmCAdTdT-3'

Two antisense strands, the sequence is: 5'-UGAAGCGAAGUGCACACGGUC-3';

Eighth siRNA sequence set:

Two sense strands, the sequence is: 5'-CCGUGUGCACUUCGCUUCAdTdT-3'

One antisense strand, the sequence is:

5'-UGAAGCGAAGUGmCAmCACGGUCuauaUGAAGCGAAGUGmCAmCACGGUC-3';

Ninth siRNA sequence set:

One justice chain, the sequence is:

5'-mGGGUUUUUUCUUGUUGAmCAAdTdTUAUAmCCGmUGUGmCACUUCGCUUmCAdTdT-3'

Two antisense strands, the sequence is: 5'-UGAAGCGAAGUGmCAmCACGGUC-3';

Tenth siRNA sequence set:

The sequence of the sense strand is: 5'-mCCGmUGUGmCACUUCGCUUmCAdTdT-3'

The sequence of the antisense strand is: 5'-UGAAGCGAAGUGmCAmCACGGUC-3';

Eleventh siRNA sequence set:

One justice chain, the sequence is:

5'-GGGUUUUUCUUGUUGACAAdTdTUAUACCGUGUGCACUUCGCUUCAdTdT-3'

Two antisense strands, the sequences are: 5'-UGAAGCGAAGUGCACACGGUC-3' and 5'-UUGUCAACAAGAAAAACCCCG-3';

Twelfth siRNA sequence set:

The sequence of the sense strand is: 5'-CCGUGUGCACUUCGCUUCAdTdT-3'

The sequence of the antisense strand is: 5'-UGAAGCGAAGUGCACACGGUC-3';

Thirteenth siRNA sequence set:

One justice chain, the sequence is:

5'-mGGGUUUUUUCUUGUUGAmCAAdTdTUAUAmCCGmUGUGmCACUUCGCUUmCAdTdT-3'

Two antisense strands, the sequences are: 5'-UGAAGCGAAGUGCACACGGUC-3' and 5'-UUGUCAACAAGAAAAACCCCG-3';

The above-mentioned bases with m in front indicate 2'-O-methylated RNA bases, dT is thymidine, and others are RNA bases.

In a preferred embodiment of the present invention, also comprise the component of following weight ratio:

Wherein the structural formula of the B1C1 compound is as follows:

The weight ratio of the sum of the weights of the above components to one or the mixture of the siRNA sequence groups is 2-10:1.

In a preferred embodiment of the present invention, the cationic lipid is DOTMA, the phospholipid is phosphatidylethanolamine, and the lipid-polyethylene glycol is cholesterol polyethylene glycol.

Another technical solution of the present invention is as follows:

A kind of preparation method of above-mentioned RNA interference composition for treating hepatitis B, it is characterized in that: comprise the steps:

(1) Dissolving one or a combination of the siRNA sequence groups in water or water containing 9% sucrose to obtain solution 1;

(2) Weigh the following components by weight, and dissolve these components in alcohol to obtain solution 2:

Wherein the structural formula of the B1C1 compound is as follows:

(3) After mixing solution 1 and solution 2 evenly, carry out vacuum filtration at room temperature to gradually evaporate the alcohol;

(4) After most of the alcohol evaporates, homogenize to obtain a suspension;

(5) Freeze-drying the above suspension to form a dried product to obtain the RNA interference composition for treating viral hepatitis B.

In a preferred embodiment of the present invention, the cationic lipid is DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane, trimethylammonium chloride), the phospholipid is phosphatidylethanolamine, and the lipid Quality-polyethylene glycol is cholesterol polyethylene glycol.

In a preferred embodiment of the present invention, the weight ratio of the weight sum of the components in the step (2) to one group or mixture of the siRNA sequence groups is 2-10:1, and the solution one The volume ratio of solution two is 1:1-0.2.

In a preferred embodiment of the present invention, in the step (4), the particle size in the homogenized suspension is 5-250 nm.

The beneficial effects of the present invention are:

1. The RNA interference composition for treating hepatitis B virus of the present invention has a very significant inhibitory effect on hepatitis B viral DNA and antigens in the liver and blood;

2. The specificity of the RNA interference composition for the treatment of viral hepatitis B of the present invention is good;

3. The RNA interference composition for the treatment of viral hepatitis B of the present invention has little side effects on liver, blood and other tissues;

4. The preparation method of the present invention is simple and easy, and can be used not only in the preparation of the composition of the present invention, but also in the preparation of other nucleic acid compositions, and has a wide range of applications.

Description of drawings

Fig. 1 is the experimental result figure among the embodiment of the present invention 3;

Fig. 2 is the southern blot experimental result figure of the mouse liver HBV DNA on the 17th day after the start of administration of Example 4 of the present invention;

Fig. 3 is the comparison result figure of the semi-quantitative PCR experiment of the mouse liver HBV DNA on the 17th day after the start of administration of Example 4 of the present invention;

Fig. 4 is the qRT-PCR experimental comparison result figure of mouse liver HBcAg RNA on the 17th day after the start of administration of Example 4 of the present invention;

Fig. 5 is the comparison result figure of the qRT-PCR experiment of mouse liver HBsAg RNA on the 17th day after the start of administration of Example 4 of the present invention;

Fig. 6 is the comparison result figure of the qRT-PCR experiment of the mouse liver ApoB RNA on the 17th day after the start of administration of Example 4 of the present invention;

Fig. 7 is the comparison result graph of the mouse blood HBeAg content before the administration of Example 4 of the present invention;

Fig. 8 is a comparison result diagram of mouse blood HBeAg content on the 17th day after the start of administration of Example 4 of the present invention;

Fig. 9 is a comparison result diagram of mouse blood HBsAg content on the 17th day after the start of administration of Example 4 of the present invention;

Fig. 10 is a comparison result diagram of mouse blood albumin content after the administration of Example 4 of the present invention;

Fig. 11 is the comparison result figure of the blood alkaline phosphatase content of the mice that started administration of Example 4 of the present invention;

Fig. 12 is a comparison result diagram of blood alanine aminotransferase (ALT) content in mice after the start of administration of Example 4 of the present invention;

Fig. 13 is a comparison result diagram of mouse blood amylase content after the start of administration of Example 4 of the present invention;

Fig. 14 is a comparison result diagram of the total bilirubin content in mouse blood after the start of administration of Example 4 of the present invention;

Figure 15 is a comparison result diagram of the blood urea nitrogen content in the blood of mice after the start of administration of Example 4 of the present invention;

Fig. 16 is a comparison result diagram of blood calcium content in mice after the administration of Example 4 of the present invention;

Fig. 17 is a comparison result diagram of the blood phosphorus content of mice after the administration of Example 4 of the present invention;

Fig. 18 is a comparison result diagram of blood creatinine content in mice after the start of administration of Example 4 of the present invention;

Figure 19 is a comparison result diagram of blood glucose content in mice after the start of administration of Example 4 of the present invention;

Fig. 20 is a comparison result diagram of blood sodium content in mice after the administration of Example 4 of the present invention;

Figure 21 is a comparison result diagram of the blood potassium content of mice after the start of administration of Example 4 of the present invention;

Figure 22 is a comparison result diagram of the total protein content in blood of mice after the start of administration in Example 4 of the present invention;

Fig. 23 is a graph showing comparison results of blood globulin levels in mice after the start of administration in Example 4 of the present invention.

Detailed ways

The technical solutions of the present invention will be further illustrated and described below through specific embodiments in conjunction with the accompanying drawings.

Example 1

Compound B1C1 is N,N-bis(3-aminopropyl)methylamine

(N,N-Bis(3-aminopropyl)methylamine) and epoxy compounds are obtained by reacting in a glass bottle equipped with a stirring bar at 90°C without solvent. The reaction time is 24-72 hours at 90°C. Its N,N-bis(3-aminopropyl)methylamine and epoxyalkyl compound (alkyl(C12-C14) glycidyl ether, Alkyl(C12-C14)glycidyl ether[68609-97-2], Sigma company) in the reaction ratio (molar ratio) is 1:4. The results were known to be confirmed by thin layer chromatography (TLC) and only one major product was present in the reaction mixture. The product of the reactant can be applied after being purified;

The structural formula of the B1C1 compound is as follows:

Example 2

An RNA interference composition for treating hepatitis B virus, comprising one of the following siRNA sequence groups:

First siRNA sequence set:

One justice chain, the sequence is:

5'-GGGUUUUUCUUGUUGACAAdTdTUAUAGGGUUUUUCUUGUUGACAAdTdT-3'

Two antisense strands, the sequence is: 5'-UUGUCAACAAGAAAAACCCCG-3';

Second siRNA sequence set:

Two sense strands, the sequence is: 5'-GGGUUUUUCUUGUUGACAAdTdT-3'

One antisense strand, the sequence is:

5'-UUGUCAACAAGAAAAACCCCGUAUAUUGUCAACAAGAAAAACCCCG-3';

Third siRNA sequence set:

One justice chain, the sequence is:

5'-CCGUGUGCACUUCGCUUCAdTdTUAUACCGUGUGCACUUCGCUUCAdTdT-3'

Two antisense strands, the sequence is: 5'-UGAAGCGAAGUGCACACGGUC-3';

Fourth siRNA sequence set:

Two sense strands, the sequence is: 5'-CCGUGUGCACUUCGCUUCAdTdT-3'

One antisense strand, the sequence is:

5'-UGAAGCGAAGUGCACACGGUCUAUAUGAAGCGAAGUGCACACGGUC-3';

Fifth siRNA sequence set:

One justice chain, the sequence is:

5'-mCCGmUGUGmCACUUCGCUUmCAdTdTUAUUmCCGmUGUGmCACUUCGCUUmCAdTdT-3'

Two antisense strands, the sequence is: 5'-UGAAGCGAAGUGmCAmCACGGUC-3';

Sixth siRNA sequence set:

Two sense strands, the sequence is: 5'-mCCGmUGUGmCACUUCGCUUmCAdTdT-3'

One antisense strand, the sequence is:

5'-UGAAGCGAAGUGmCAmCACGGUCuauaUGAAGCGAAGUGmCAmCACGGUC-3';

Seventh siRNA sequence set:

One justice chain, the sequence is:

5'-mCCGmUGUGmCACUUCGCUUmCAdTdTUAUUmCCGmUGUGmCACUUCGCUUmCAdTdT-3'

Two antisense strands, the sequence is: 5'-UGAAGCGAAGUGCACACGGUC-3';

Eighth siRNA sequence set:

Two sense strands, the sequence is: 5'-CCGUGUGCACUUCGCUUCAdTdT-3'

One antisense strand, the sequence is:

5'-UGAAGCGAAGUGmCAmCACGGUCuauaUGAAGCGAAGUGmCAmCACGGUC-3';

Ninth siRNA sequence set:

One justice chain, the sequence is:

5'-mGGGUUUUUUCUUGUUGAmCAAdTdTUAUAmCCGmUGUGmCACUUCGCUUmCAdTdT-3'

Two antisense strands, the sequence is: 5'-UGAAGCGAAGUGmCAmCACGGUC-3';

Tenth siRNA sequence set:

The sequence of the sense strand is: 5'-mCCGmUGUGmCACUUCGCUUmCAdTdT-3'

The sequence of the antisense strand is: 5'-UGAAGCGAAGUGmCAmCACGGUC-3';

Eleventh siRNA sequence set:

One justice chain, the sequence is:

5'-GGGUUUUUCUUGUUGACAAdTdTUAUACCGUGUGCACUUCGCUUCAdTdT-3'

Two antisense strands, the sequences are: 5'-UGAAGCGAAGUGCACACGGUC-3' and 5'-UUGUCAACAAGAAAAACCCCG-3';

Twelfth siRNA sequence set:

The sequence of the sense strand is: 5'-CCGUGUGCACUUCGCUUCAdTdT-3'

The sequence of the antisense strand is: 5'-UGAAGCGAAGUGCACACGGUC-3';

Thirteenth siRNA sequence set:

One justice chain, the sequence is:

5'-mGGGUUUUUUCUUGUUGAmCAAdTdTUAUAmCCGmUGUGmCACUUCGCUUmCAdTdT-3'

Two antisense strands, the sequences are: 5'-UGAAGCGAAGUGCACACGGUC-3' and 5'-UUGUCAACAAGAAAAACCCCG-3';

The above-mentioned bases with m in front represent 2'-O-methylated RNA bases, and dT is thymidine;

The RNA interference composition also includes components in the following weight ratios:

The weight ratio of the sum of the weights of the above components to one or the mixture of the siRNA sequence groups is 2-10:1.

The above-mentioned preparation method of the RNA interference composition for the treatment of hepatitis B comprises the following steps:

(1) Dissolving one of the siRNA sequence groups in water containing 0.9% water and 5% sucrose to obtain solution 1;

(2) Weigh the following components by weight, and dissolve these components in alcohol to obtain solution 2:

The weight ratio of the weight sum of the components in step (2) to one group or mixture of the siRNA sequence group is 2-10:1; the volume ratio of solution 1 to solution 2 after preparation is 1:1- 0.2

(3) After mixing solution 1 and solution 2 evenly, carry out vacuum filtration at room temperature to gradually evaporate the alcohol;

(4) After most of the alcohol evaporates, homogenize to obtain a suspension, and the particle size in the homogenized suspension is 5-250nm;

(5) Freeze-drying the above suspension to form a white or slightly yellow dry product, thus obtaining the RNA interference composition for treating viral hepatitis B.

Before the above-mentioned RNA interference composition is administered, it only needs to be added with an appropriate amount of water for injection, shaken or dissolved into a suspension. This preparation method can also be generally used as a preparation method for other nucleic acid drugs.

Example 3

The siRNA sequence groups used for the treatment of viral hepatitis B in the first to thirteenth siRNA sequence groups in Example 1 were numbered 1 to 13 respectively, and the following cell experiments were carried out respectively;

Cell experiments:

(1) Cell transfection: HepG2.2.15 cells were seeded in 96-well culture plates and cultured overnight at 37°C in a carbon dioxide incubator (5% carbon dioxide), so that the cloning rate of the cells used for transfection the next morning About 40%, 0.5 μL siRNA (1 μM) was diluted with 10 μL DMEM, and 0.4 μL RFect (Agave Pharma, Inc. Seattle), a commercially available nucleic acid cell transfection aid) was diluted with 10 μL DMEM and mixed with Let stand at room temperature for 5 minutes; vortex the diluted siRNA and RFect for 10 seconds, and let stand at room temperature for 20 minutes to obtain the transfection complex; Add 20 μL of the transfection complex respectively, and culture the cells at 37°C in a carbon dioxide incubator (5% carbon dioxide);

(2) Extraction of mRNA: One to two days after transfection, wash the cells once with 100 μL PBS, then add 100 μL lysis buffer (Turbocapture kit, Qiagen) to lyse; transfer 80 μL lysate to a 96-well mRNA capture plate, store on ice Incubate at room temperature for 1 hour; pour off the lysate, wash the capture plate with buffer, 100 μL per well; then add 80 μL of elution buffer to each well, and incubate at 65 °C for 5 minutes; The elution solution containing mRNA was transferred to a new 96-well transparent plate;

(3) Take 3 μL of the above-mentioned isolated mRNA for real-time RT-PCR. The kit used is SYBR Green one-step real-time RT-PCR kit (SensiMix one-step SYBR Green kit, Bioline). Specifically, the above-mentioned mRNA was mixed with 11 μL master Mix (including 7 μL of 2x master mix (including reverse transcriptase), 1 μL of forward and reverse primers (6 μM), 0.3 μL of 50X SYBR green and 2.7 μL of water); the reverse transcription reaction program is as follows: 42°C for 30 minutes, 95°C 15 minutes to start Taq polymerase, the reaction thermal cycle is as follows: 95°C for 15 seconds, 60°C for 30 seconds, 72°C for 20 seconds, and the gene expression changes were analyzed using the ΔΔCt method;

The experimental results are shown in Figure 1, the first to the twelfth siRNA sequence group compared with the untransfected group and the control siRNA transfected group, all significantly reduced the amount of HBV core antigen RNA and HBV surface antigen RNA in the cell, because The results of the thirteenth group and the twelfth group are not much different, so the experimental results of the thirteenth group are not shown.

Example 4

All procedures used in the conducted animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) and complied with applicable local, state and federal regulations. The RNA interference compositions corresponding to the first to thirteenth siRNA sequence groups were respectively prepared according to the method in Example 1, and the following animal experiments were carried out. The mice used in the animal experiments were HBV transgenic mice.

Animal experiments were divided into five groups:

ApoB group, negative siRNA group (siRNA corresponding to the ApoB gene, which is a gene completely unrelated to HBV in the liver);

HBV1 group (optional one of the RNA interference compositions of the first to thirteenth siRNA sequence groups above);

HBV2 group (an optional other one of the RNA interference compositions of the first to thirteenth siRNA sequence groups above);

ADV group, the positive control group (the anti-hepatitis B drug adefovir dipivoxil currently being used clinically);

Placebo group (sucrose solution only).

The dosage of the above-mentioned ApoB group, siRNA composition and placebo group was 2 mg/kg body weight, a total of three injections, each time separated by seven days (the first injection was 0 days, a total of 14 days), at the last injection Three days later (the 17th day), the blood was collected and the animals were killed, and the hepatitis virus inhibition indexes and blood biochemical indexes of the tissue were measured; in the ADV group, the usage of adefovir dipivoxil was 10 mg/kg body weight per day, orally for 14 days, After three days (the 17th day), blood and tissue were taken to determine its hepatitis virus suppression and blood biochemical indicators;

Among them, the analysis of liver HBV DNA was carried out by southern blot and semi-quantitative PCR, and the analysis of liver HBV RNA was also carried out by semi-quantitative PCR:

The specific steps of southern blot are as follows: the liver tissue is homogenized in the lysate. Liver tissue (approximately 0.1 g) was homogenized with a grinding pestle in a solution containing lysis buffer (1 mM EDTA, 10 mM Tris, 10 mM NaCl, 0.5% SDS, proteinase K). For DNA extraction, the homogenate was incubated at 55° C. for 12 hours, and then an equal volume of phenol (Phenol) was added. Samples were mixed and centrifuged at 12,000 xg for 10 minutes. , then chloroform was added to the supernatant and centrifuged again. The DNA was then precipitated out with NaCl and ethanol. Dissolve the precipitated DNA pellet in TE buffer (pH 8.0) containing ribonuclease A. A certain amount of DNA (usually containing 40 μg DNA) was digested with HindIII digestive enzyme (NewEngland Biolabs, MA) at 37°C It has been proved that the HBV gene sequence does not contain the HindIII endon site in 3 hours. Digested DNA was re-extracted and separated by 1% TAE agarose gel electrophoresis. DNA is then transferred to a BioDyne B positively charged nylon membrane. DNA was immobilized on the membrane after UV irradiation. Hybridization was probed using [32P]CTP-labeled, HaeIII-digested HBV genome cloned into the pBluescript plasmid. Hybridization was performed overnight at 60°C in a solution containing 10% PEG-8000, 0.05 M sodium phosphate, 0.33 mg/ml salmon sperm DNA, 7% SDS. The radioactive signal was determined using a phosphorimaging method (Optiquant) and the density of radioactive bands was determined;

The specific steps of semi-quantitative PCR of liver HBV DNA and liver HBV RNA are as follows: Real-time PCR is used, the primers used are HBV3 forward primer ATAAAACGCCGCAGACACATC, HBV3 reverse primer AACCTCCAATCACTCACCAACC, and the probe is HBV3Taq-manprobe

[6～FAM]-AGCGATAACCAGGACAAGTTGGAGGACA-[BHQ1a～6FAM]; the reaction system is 25μL, including 12.5μL FullVelocityTM QPCR master mix, 0.375μL diluted reference dye (1:500), 0.25μL StratascriptTM RT/RNase block enzyme mixture and 0.5μL FullVelocity™ Enzyme. The reaction program was: 2 min at 95 °C, 40 cycles of 10 s at 95 °C, 30 s at 60 °C. Determined using the standard curve method.

Blood biochemical indicators were analyzed using commercially available ELISA kits (International Immunodiagnostics, Foster City, CA) corresponding to the required biochemical indicators.

Experimental results and conclusions: As shown in Figure 2 to Figure 5, and Figure 7 to Figure 10, both the HBV1 group and the HBV2 group have significant effects on HBV DNA in liver tissue, HBsAg and HBeAg in blood, and HBsAg and HBcAg in liver tissue. Very significant inhibitory effect, adefovir dipivoxil has significant inhibitory effect on HBV DNA in liver tissue, but has no effect on HBsAg, HBeAg in blood and HBsAg, HBcAg in liver tissue; as shown in Figure 6 It was shown that the HBV1 group and the HBV2 group had no effect on the RNA amount of the ApoB gene in the liver, and their specificity was good; less side effects.

The hepatitis B inhibitory effects of HBV1 and HBV2 (including viral DNA, blood and liver tissue antigens) are very significant.

The above is only a preferred embodiment of the present invention, so the scope of the present invention cannot be limited accordingly, that is, equivalent changes and modifications made according to the patent scope of the present invention and the content of the specification should still be covered by the present invention In the range.

Claims (7)

1. the RNA being used for the treatment of hepatitis B disturbs a compositions, it is characterized in that: siRNA sequence group is as follows:

Positive-sense strand one, sequence is:

5’-GGGUUUUUCUUGUUGACAAdTdTUAUACCGUGUGCACUUCGCUUCAdTdT-3’

Antisense strand two, sequence is respectively: 5 '-UGAAGCGAAGUGCACACGGUC-3 ' and

5’-UUGUCAACAAGAAAAACCCCG-3’；

RNA base before above-mentioned adds that 2 '-O-methylates or the chemical modification of fluorine atom, to increase stability; DT is thymidine, and other are RNA base.

2. a kind of RNA being used for the treatment of hepatitis B as described in claim 1 disturbs compositions, it is characterized in that: the component also comprising following weight ratio:

Cation lipid 0.5-3 weight portion

B1C1 compound 1-10 weight portion

Phosphatidase 0-5 weight portion

Cholesterol 0-5 weight portion

Lipid-Polyethylene Glycol 2-24 weight portion;

The structural formula of wherein said B1C1 compound is as follows:

The weight of said components and be 2-10:1 with the weight ratio of described siRNA sequence group.

3. a kind of RNA being used for the treatment of hepatitis B disturbs compositions as claimed in claim 2, and it is characterized in that: described cation lipid is DOTMA, described phospholipid is PHOSPHATIDYL ETHANOLAMINE, and described lipid-Polyethylene Glycol is cholesterol Polyethylene Glycol.

4. the RNA being used for the treatment of hepatitis B according to claim 2 disturbs a preparation method for compositions, it is characterized in that: comprise the steps:

(1) described siRNA sequence group is dissolved in water or containing in the water of 9% sucrose, obtains solution one;

(2) take following component by weight, and these components dissolved obtained solution two in ethanol:

Cation lipid 0.5-3 weight portion

B1C1 compound 1-10 weight portion

Phosphatidase 0-5 weight portion

Cholesterol 0-5 weight portion

Lipid-Polyethylene Glycol 2-24 weight portion;

The structural formula of wherein said B1C1 compound is as follows:

(3) after mutually being mixed homogeneously with solution two by solution one, under room temperature, carry out vacuum filtration, ethanol is wherein evaporated gradually;

(4) after most vaporized alcohol, carry out homogenizing, obtain suspension, then high pressure homogenize;

(5) by above-mentioned suspension lyophilizing, form dry thing, the RNA being used for the treatment of hepatitis B described in obtaining disturbs compositions.

5. a kind of RNA being used for the treatment of hepatitis B disturbs the preparation method of compositions as claimed in claim 4, it is characterized in that: described cation lipid is DOTMA, described phospholipid is PHOSPHATIDYL ETHANOLAMINE, and described lipid-Polyethylene Glycol is cholesterol Polyethylene Glycol.

6. a kind of RNA being used for the treatment of hepatitis B disturbs the preparation method of compositions as claimed in claim 5, it is characterized in that: the weight of the component in described step (2) and be 2-10:1 with the weight ratio of described siRNA sequence set, described solution one is 1:1-0.2 with the volume ratio of solution two.

7. a kind of RNA being used for the treatment of hepatitis B disturbs the preparation method of compositions as claimed in claim 4, and it is characterized in that: in described step (4), the granular size in the suspension after homogenizing is 5-250nm.