Broad-spectrum anti-influenza A virus siRNA molecule as well as derivative and application thereof

Abstract

The invention discloses a broad-spectrum anti-influenza A virus siRNA molecule as well as a derivative and application thereof, and belongs to the technical field of biology. The name of the siRNA is siPB2-12 and / or siNP-4, and / or a derivative of the RNA molecule; one chain sequence of the siPB2-12 is SEQ ID No.1 in a sequence table, and the other chain sequence of the siPB2-12 is SEQ ID No.2 in the sequence table; one chain sequence of the siNP-4 is shown as SEQ ID No.5 in the sequence table, and the other chain sequence of the siNP-4 is shown as SEQ ID No.6 in the sequence table. The siPB2-12 and / or the siNP-4 and the derivative of the siPB2-12 and / or the siNP-4 provided by the invention can be used for effectively inhibiting H1N1, H3N2, H5N6, H7N9 and H9N2.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a broad-spectrum anti-influenza A virus siRNA molecule and its derivatives and applications. Background Art

[0002] Influenza virus, also known as influenza virus (IV), belongs to the Orthomyxoviridae family and is a single-stranded negative-sense RNA virus with an envelope. Influenza virus is the most common virus that causes respiratory infections in humans. Worldwide, influenza virus causes about 3 million to 5 million illnesses and 290,000 to 650,000 deaths each year, which shows the severity of its harm.

[0003] According to the difference in antigenicity of nucleocapside (NP) and matrix protein (M), influenza viruses can be divided into influenza A virus (IAV), influenza B virus (IBV), influenza C virus (ICV) and influenza D virus (IDV). Influenza A virus usually causes a global pandemic; influenza B virus generally only breaks out locally, but can also infect people widely and cause severe cases; influenza C virus only causes mild upper respiratory tract infection and does not cause a pandemic; currently influenza D virus only infects cattle and pigs, not humans.

[0004] Antigenic variation of influenza A virus enables the virus to escape the host immune response, and the host's immune response to sudden variants may be weakened or even ineffective, thus causing outbreaks of pandemic influenza and seasonal influenza. Due to the variation of influenza A virus, antigenic drift and antigenic shift occur in hemagglutinin (HA) and neuraminidase (NA), and a new influenza pandemic may break out in the population at any time. Antiviral drugs are a common means of defending against influenza virus infection, but there are few existing anti-influenza virus drugs, and research data show that the resistance rate of H1N1 subtype strains to oseltamivir is as high as 67%, 193 of 209 H3N2 subtype strains have developed resistance to adamantane, and highly pathogenic H5N1 avian influenza virus mutants resistant to oseltamivir have appeared. In addition, the most effective way to treat and prevent influenza virus is vaccination, but there is a significant lag in the development of vaccines to deal with sudden new influenza, and the antigenic drift and antigenic shift of the virus are an ever-changing challenge for available vaccines.

[0005] RNA interference (RNAi) is an important method in gene function research and innovative drug research. It is mainly mediated by small interfering RNA (siRNA) with the same length and reverse complement. siRNA is a double-stranded complementary RNA with 19 to 21 bp and a protruding base at the 3' end. It can be directly synthesized by chemical synthesis or cut by Dicer (a sequence-specific endonuclease) from dsRNA. siRNA-mediated gene silencing can destroy the integrity of the target protein mRNA by complementary pairing with the target RNA base, and inactivate the original target RNA in a highly precise manner, thereby inhibiting the expression of disease-related genes and producing a therapeutic effect. siRNA has the characteristics of high efficiency, strong gene targeting, short development cycle, rich candidate targets, high therapeutic efficiency, low drug toxicity and wide application fields. Therefore, it can be used as a tool for studying single genes in vivo and in vitro and a new idea for drug development. An article titled "Asymmetric RNA duplexes mediate RNA interference inmammalian cells" published in "Natrue Biotechnology" in December 2009 showed that asymmetric siRNA (aiRNA) is a better choice for gene silencing. The mechanism of action of aiRNA is the same as that of siRNA, but it usually contains a 15bp (or less than 15bp) sense strand, base protrusions at the 3' and 5' ends of the antisense strand, and an asymmetric structure in which the sense strand is shorter than the antisense strand. Compared with siRNA, aiRNA has many obvious advantages: ①RISC selects and combines with the antisense strand of aiRNA, while the sense strand does not participate in the entire interference process, so there is no off-target effect problem. At the same time, aiRNA reduces the probability of toxic side effects due to off-target effects by virtue of its superior targeting specificity and high efficiency; ②aiRNA drugs have high efficiency in silencing target genes, rapid onset, and long duration of action; ③Experiments have confirmed that aiRNA drugs can reduce or even eliminate the immunotoxicity of interferon response; ④aiRNA molecules are small and have less non-specific interferon response, which makes its delivery easier; ⑤aiRNA is short in length and does not require extensive chemical modification, which reduces the cost of drug research and development and synthesis. These advantages confirm that aiRNA is a better choice for gene silencing and a new hope for disease treatment.

[0006] In summary, considering the limitations of current anti-influenza virus strategies, new strategies for preventing and treating influenza viruses are urgently needed. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide siRNA that can resist multiple influenza subtypes. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] The present invention provides a double-stranded RNA molecule, a modified product thereof, or a pharmaceutically acceptable salt thereof, wherein the double-stranded RNA molecule is siPB2-12 and / or siNP-4, and / or a derivative of the RNA molecule; the sequence of one chain of the siPB2-12 is SEQ ID No.1 in the sequence list, and the sequence of the other chain is SEQ ID No.2 in the sequence list; the sequence of one chain of the siNP-4 is SEQ ID No.5 in the sequence list, and the sequence of the other chain is SEQ ID No.6 in the sequence list.

[0010] The derivative of siPB2-12 is a double-linked RNA molecule named aiPB2-12, and the derivative of siNP-4 is a double-linked RNA molecule named aiNP-4. The sequence of one chain of aiPB2-12 is SEQ ID No.3 in the sequence list, and the sequence of the other chain is SEQ ID No.4 in the sequence list. The sequence of one chain of aiNP-4 is SEQ ID No.7 in the sequence list, and the sequence of the other chain is SEQ ID No.8 in the sequence list.

[0011] Among them, SEQ ID No.1 consists of 19 ribonucleotides, SEQ ID No.2 consists of 19 ribonucleotides, the 1st to 19th ribonucleotides of SEQ ID No.1 are reverse complementary to the 1st to 19th ribonucleotides of SEQ ID No.2, and the double-stranded RNA formed by the two is named siPB2-12. SEQ ID No.3 consists of 15 ribonucleotides, SEQ ID No.4 consists of 19 ribonucleotides, the 1st to 15th ribonucleotides of SEQ ID No.3 are reverse complementary to the 1st to 15th ribonucleotides of SEQ ID The 2nd to 16th ribonucleotides of No.10 are reverse complementary, and the double-stranded RNA formed by the two is named siPB2-12. Among them, SEQ ID No.3 is the same as SEQ ID No.1 in terms of nucleotides 4 to 18, and SEQ ID No.4 is the same as SEQ ID No.2 in terms of target sites, except that the TT modification of SEQ ID No.2 is at the 3 end, and the TT modification of SEQ ID No.4 is at the 5 end. SEQ ID No.5 and SEQ ID No.6 are the same as SEQ ID No.1 and SEQ ID No.2. SEQ ID No.7 and SEQ ID No.8 are the same as SEQ ID No.3 and SEQ ID No.4.

[0012] The term "modification" refers to the product obtained by modifying the double-stranded RNA molecule. Various modification methods can be selected, including one or a combination of ribose modification, base modification and phosphate backbone modification. The term "pharmaceutically acceptable salt" refers to a salt that is suitable for contact with human and lower animal tissues without excessive toxicity, irritation, allergic reaction, etc., and is commensurate with a reasonable effect / risk ratio within the scope of reliable medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge, et al., J. Pharmaceutical Sciences, 1977, 66: 1 describes pharmaceutically acceptable salts in detail.

[0013] The present invention also provides a biological material containing the aforementioned double-stranded RNA molecule, wherein the biological material is a DNA molecule capable of producing the double-stranded RNA molecule, a recombinant vector containing the DNA molecule, or a recombinant microorganism containing the DNA molecule.

[0014] The present invention also provides an influenza virus inhibitor, which contains the aforementioned double-stranded RNA molecule, a modified substance thereof, or a pharmaceutically acceptable salt thereof.

[0015] The present invention also provides a drug for preventing and / or treating influenza, wherein the drug contains the aforementioned double-stranded RNA molecule, its modified substance, or its pharmaceutically acceptable salt.

[0016] The influenza virus inhibitor or the drug described herein is in the form of an aerosol, tablet, capsule, pellet, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, buccal tablet, suppository or lyophilized powder injection.

[0017] In the above, the active ingredients of the influenza virus inhibitor and the drug for preventing or / and treating influenza may be the double-stranded RNA molecule, its modified substance, or a pharmaceutically acceptable salt thereof. The active ingredients of the influenza virus inhibitor and the drug for preventing or / and treating influenza may also contain other substances, and those skilled in the art may determine the other substances according to the effects of the influenza virus inhibitor and the drug for preventing or / and treating influenza.

[0018] In the above, the influenza virus inhibitor and the drug for preventing or / and treating influenza may contain a suitable carrier or excipient in addition to the double-stranded RNA molecule, its modified substance, or its pharmaceutically acceptable salt. The carrier material here includes but is not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, water-soluble carrier materials are preferred. These materials can be made into a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the unit dosage form into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin, talc, etc.; binders, such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare a unit dosage form as a suppository, various carriers known in the art can be widely used. Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc.In order to prepare the unit dosage form into an injectable preparation, such as a solution, emulsion, freeze-dried powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injectable preparation. In addition, conventional cosolvents, buffers, pH regulators, etc. can also be added. In addition, if necessary, colorants, preservatives, spices, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparation. The above dosage form can be used for administration by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; cavity administration, such as rectal and vaginal; respiratory tract administration, such as nasal; mucosal administration. The above administration route is preferably injection.

[0019] The present invention also provides a use of the aforementioned double-stranded RNA molecule, its modified product, or its pharmaceutically acceptable salt, or the aforementioned biological material in preparing an influenza virus inhibitor or in inhibiting influenza virus.

[0020] Specifically, the influenza virus is H1NI, H3N2, H5N6 and / or H9N2 virus, and the influenza is caused by H1NI, H3N2, H5N6, H7N9 and / or H9N2 virus. The H1NI, H3N2, H5N6, H7N9 and / or H9N2 virus is specifically derived from A / Guangdong-Maonan / SWL1536 / 2019 (H1N1), A / Hong Kong / 2671 / 2019 (H3N2), A / Guangzhou / 39715 / 2014 (H5N6), A / Anhui / 1 / 2013 (H7N9), A / Guangxi / NN10.19T-NGS / 2018 (H9N2).

[0021] The present invention confirms through specific experiments that siPB2-12 designed according to the gene of influenza A gene, and aiPB2-12 designed based on siPB2-12, can effectively inhibit H1N1, H3N2, H5N6, H7N9 and H9N2, providing a new strategy for preventing and treating influenza virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1HA titer in the supernatant of MDCK cells infected with A / Guangdong-Maonan / SWL1536 / 2019 (H1N1). MDCK cells were transfected with siRNA targeting (a) PB2, (b) PB1, (c) PA, and (d) NP genes, and the hemagglutination titer in the supernatant was measured 48 hours after virus infection. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0023] Figure 2 Western blot analysis results of NP protein in the cell supernatant 48 hours after A / Guangdong-Maonan / SWL1536 / 2019 (H1N1) infection.

[0024] Figure 3 These are the results of Western blot analysis of intracellular HA protein and NP protein 48 hours after A / Guangdong-Maonan / SWL1536 / 2019 (H1N1) infection.

[0025] Figure 4 The HA titer of the supernatant of MDCK cells infected with different subtypes of influenza virus was measured. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0026] Figure 5 These are the results of Western blot analysis of NP protein in the cell supernatant 48 hours after infection with different subtypes of influenza virus.

[0027] Figure 6 The results of Western blot analysis of intracellular NP protein 48 hours after infection with different subtypes of influenza viruses.

[0028] Figure 7 The titer of influenza virus HA in the supernatant of cells with aiRNA interference was determined. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0029] Figure 8 This is the Western blot analysis result of influenza virus NP protein in the supernatant of aiRNA interference cells.

[0030] Fig. 9 This is the Western blot analysis result of aiRNA interfering with influenza virus NP protein in cells. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0032] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0033] Anti-NP antibody: A rabbit polyclonal antibody prepared by immunizing rabbits with the NP protein of A / WSN / 1993 (H1N1) (amino acid sequence number QDQ43414.1 on the NCBI website), which is described in the literature “Neddylation of M1 negatively regulates the replication of influenza A virus”.

[0034] Anti-HA antibody: The primary HA antibody is a polyclonal antibody derived from rabbits immunized with baculovirus-expressed H1 protein (amino acid sequence number ACQ76318.1 on the NCBI website), which was prepared and preserved by the inventor's laboratory and recorded in the document "Avian-to-Human Receptor-Binding Adaptation of Avian H7N9 Influenza Virus Hemagglutinin".

[0035] The following examples used GraphPad Prism 8 statistical software to process the data and used a Two-way ANOVA test. P < 0.05 indicated a significant difference.

[0036] Example 1: Initial screening of candidate siRNA molecules

[0037] Designed siRNA sequences for influenza A virus PB2, PB1, PA, NP genes. The siRNA molecules used in this example are shown in Table 1, and the aiRNA molecules are shown in Table 1. The siRNA sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0038] Table 1 Candidate siRNA molecules

[0039]

[0040] Positions 1-19 of the Sense strand of siPB2-12 correspond to SEQ ID No. 1, and positions 1-19 of the Sense strand of siPB2-12 correspond to SEQ ID No. 2.

[0041] The 1st to 19th positions of the Sense strand of siNP-4 correspond to SEQ ID No.5, and the 1st to 19th positions of the Sense strand of siNP-4 correspond to SEQ ID No.6.

[0042] 1. Experimental methods

[0043] (1) Preparation of A / Guangdong-Maonan / SWL1536 / 2019 (H1N1) virus

[0044] The experiments were conducted in the BSL-2 laboratory of the Institute of Microbiology, Chinese Academy of Sciences.

[0045] 100 μl of primary A / Guangdong-Maonan / SWL1536 / 2019(H1N1) virus was injected into the allantoic cavity of 10-day-old chicken embryos, sealed with wax, and cultured in a 37°C incubator for 72 hours. The growth status of the chicken embryos was observed every 12 hours. The allantoic fluid was harvested at 72 hours to determine the hemagglutination titer. The harvested allantoic fluid was the egg P1 generation A / Guangdong-Maonan / SWL 1536 / 2019(H1N1) virus.

[0046] (2) Cell culture and liposome transfection

[0047] MDCK cells were cultured at 2.5 × 10 6 / well density inoculated into 6-well cell culture plates, when the cells adhered to 70-80%, grouped as follows:

[0048] siPB2-1 group: according to siRNA transfection reagent (purchased from: Invitrogen TM, Catalog No.: 13778150) instructions, use siRNA transfection reagent Lipofectamine RNAiMAX to transfect siPB2-1 into MDCK cells, and the final content of siRNA in each well is 50nM. The experiment was repeated three times, with three replicate wells each time. After the cell culture plate was placed at 37°C and 5% CO2 for 24 hours, the culture medium was aspirated and washed with PBS 3 times. The cells were infected with A / Guangdong-Maonan / SWL1536 / 2019 (H1N1) virus at an MOI of 0.1 and incubated at 37°C for 1 hour. In order to promote viral infection, the cell culture plate was gently shaken every 15 minutes during incubation. After 1 hour, the virus dilution was aspirated, 500μl of virus maintenance solution (DMEM culture medium containing 2μg / mL trypsin, 100U / mL penicillin and 0.1mg / mL streptomycin) was added to each well, and the culture was continued at 37°C, and each index was detected according to the following operations:

[0049] ① Hemagglutination test: harvest cell supernatant at 12h, 24h, 36h, and 48h after virus infection, and use the influenza virus to agglutinate red blood cells to determine the virus hemagglutination titer. The specific method for determining the virus hemagglutination titer is as follows: take a 96-well V-shaped microplate and place it horizontally. Except for the first column, add 25μl PBS to each well. Add 50μl of the virus solution to be tested (the aforementioned cell supernatant) to the first well of each column. From the first well, dilute the virus solution to be tested to the 12th well, and discard 25μl from the last well. Then add 25μl of 1% chicken red blood cell suspension to each well, incubate at room temperature for 30min, and observe whether hemagglutination reaction occurs. HA titer (log2) = log2N, N is the dilution multiple of the virus solution to be tested.

[0050] ②Western-blot: 48 hours after virus infection, aspirate the culture medium to obtain the supernatant of cells infected with H1N1 strain after transfection with siPB2-1; wash the remaining adherent cells once with PBS, add 100μl cell lysis buffer (DAPI lysis buffer and protease inhibitor PMSF are prepared in a ratio of 100:1), place at 4℃ for 10min, use a pipette to blow the cells, transfer the obtained cell lysis buffer to a new 1.5ml EP tube as much as possible, and centrifuge at 4℃ and 12000rpm for 5min. Take the supernatant after centrifugation and mix it with SDS-PAGE Loading buffer (5×) at a ratio of 4:1. After a short centrifugation, boil the diluted cell sample at 100℃ for 5-10min and store it in a -80℃ refrigerator for later use. Western-blot was used to detect the expression of viral NP protein, HA protein and experimental internal reference β-actin protein. The antibodies used for detection included anti-NP antibody, anti-HA antibody and anti-β-actin antibody (purchased from: Proteintech, catalog number: 20536-1-AP).

[0051] siPB2-2 group, siPB2-11 group, siPB2-12 group, siPB1-1 group, siPB1-2 group, siPB1-3 group, siPB1-4 group, siPB1-5 group, siPB1-6 group, siPA-1 group, siPA-2 group, siPA-3 group, siPA-4 group, siNP-1 group, siNP-2 group, siNP-3 group, siNP-4 group, siNP-5 group, siNP-6 group, siNP-7 group and siNP-8 group: siPB2 -2, siPB2-11, siPB2-12, siPB1-1, siPB1-2, siPB1-3, siPB1-4, siPB1-5, siPB1-6, siPA-1, siPA-2, siPA-3, siPA-4, siNP-1, siNP-2, siNP-3, siNP-4, siNP-5, siNP-6, siNP-7 or siNP-8 replaced siPB2-1 in the siPB2-1 group, and the rest of the operations were the same as the siPB2-1 group.

[0052] siNC: siPB2-1 in the siPB2-1 group was replaced by siNC, and the rest of the operations were the same as those in the siPB2-1 group.

[0053] siGFP: siPB2-1 in the siPB2-1 group was replaced by siGFP, and the rest of the operations were the same as those in the siPB2-1 group.

[0054] MOCK: Lipofectamine RNAiMAX was used to replace the siRNA and transfection reagent complex in the siPB2-1 group, and the rest of the operations were the same as those in the siPB2-1 group.

[0055] Control (virus): DMEM medium was used to replace the siRNA transfection reagent Lipofectamine RNAiMAX and siPB2-1 in the siPB2-1 group, and the rest of the operations were the same as those in the siPB2-1 group.

[0056] 2. Results

[0057] (1) Determination of hemagglutination titer

[0058] Compared with the Control group, (a) the HA titer in the cell supernatant of the siPB2-1 and siPB2-11 transfected groups showed a downward trend, but there was no statistical significance; the HA titer in the cell supernatant of the siPB2-2 and siPB2-12 transfected groups was significantly reduced (P < 0.001 and P < 0.01, respectively), and the titer of three replicates in the siPB2-2 group was 2 1 , 2 1, 2 2 , siPB 2-12 group three replicates 2 2 , 2 1 , 2 3 (b) The HA titer in the cell supernatant of the siPB1-4 and siPB1-6 transfection groups showed a downward trend, but there was no statistical significance. (c) The HA titer in the cell supernatant of the siPA-1, siPA-3, and siPA-4 transfection groups showed a downward trend, but there was no statistical significance; the HA titer in the cell supernatant of the siPA-2 transfection group was significantly reduced (P < 0.01). (d) The HA titer in the cell supernatant of the siNP-2, siNP-3, siNP-4, siNP-5, siNP-6, siNP-7, and siNP-8 transfection groups showed a downward trend, but there was no statistical significance.

[0059] It can be seen that compared with the Control group, siPB2-2, siPB2-12, and siPA-2 transfection can significantly inhibit the replication of A / Guangdong-Maonan / SWL1536 / 2019 (H1N1) virus in MDCK cells ( Figure 1 ).

[0060] (2) Detection of NP protein in cell supernatant

[0061] Inhibitory effect = (gray value of control histone band - gray value of transfected histone band) / gray value of control histone band × 100%. Gray value statistical rules are as follows: gray value is obtained by analyzing protein bands in Western blot results using Image software.

[0062] At 48 h after virus infection, the expression of influenza virus NP protein in the cell supernatant was analyzed by Western blot (see Figure 2), siRNA has a certain inhibitory effect on the synthesis of influenza virus NP protein in cell supernatant. Compared with the control group, in the siRNA-PB2 transfection group, the inhibitory effects of siPB2-1, siPB2-2, and siPB2-11 on NP protein were 59%, 45%, and 49%, respectively, and the inhibitory effect of siPB2-12 was the best, reaching 78%; in the siRNA-PB1 transfection group, the inhibitory effects of siPB1-3 and siPB1-4 on NP protein were 14% and 20%, respectively, and the other siRNA-PB1 had no inhibitory effect or the inhibitory effect was not obvious; in the siRNA-PA transfection group, the inhibitory effects of siPA-2 and siPA-4 on NP protein were 23% and 12%, respectively, and the other siRNA-PA had no inhibitory effect or the inhibitory effect was not obvious; in the siRNA-NP transfection group, the inhibitory effect of siNP-4 on NP protein was 25%, and the other siRNA-NP had no inhibitory effect or the inhibitory effect was not obvious.

[0063] (3) Detection of intracellular NP protein and HA protein

[0064] The calculation method of the inhibitory effect refers to (2).

[0065] At 48 h after virus infection, the expression of influenza virus NP protein and HA protein in cells was analyzed by Western blot (see Figure 3), siRNA has a certain inhibitory effect on the synthesis of influenza virus NP protein and HA protein in cells. Compared with the Control group, in the siRNA-PB2 transfection group, the inhibitory effects of siPB2-1, siPB2-2, and siPB2-12 on NP protein were 27%, 21%, and 38%, respectively, and the inhibitory effects on HA protein were 62%, 54%, and 80%, respectively; in the siRNA-PB1 transfection group, the inhibitory effects of siPB1-1, siPB1-2, siPB1-3, siPB1-4, siPB1-5, and siPB1-6 on HA protein were 49%, 32%, 24%, 36%, 17%, and 10%, respectively, but there was no inhibitory effect or the inhibitory effect was not obvious on NA protein; in the siRNA-PA transfection group, siPA-1, siPA-2, siPA-3, and siPA- The inhibitory effects of siNP-2, siNP-3, siNP-4, siNP-5, siNP-6, siNP-7 and siNP-8 on NP protein were 46%, 56%, 73%, 60%, 55%, 49% and 47%, respectively, and the inhibitory effects of siNP-1, siNP-2, siNP-3, siNP-4, siNP-5, siNP-6, siNP-7 and siNP-8 on NP protein were 46%, 57%, 73%, 74%, 46%, 48%, 49% and 35%, respectively.

[0066] Example 2: Verification of siRNA interference function against different subtypes of influenza virus

[0067] A / Guangdong-Maonan / SWL1536 / 2019(H1N1): recorded in the document “The determination of haemagglutinin influenza antibodies in the Polish population in the epidemic season 2020 / 2021during the SARS-CoV-2pandemic”, the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0068] A / Hong Kong / 2671 / 2019(H3N2): recorded in the document “The determination of haemoglobin influenza antibodies in the Polish population in the epidemic season 2020 / 2021 during the SARS-CoV-2 pandemic”, the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0069] A / Guangzhou / 39715 / 2014(H5N6): recorded in the document “The complexity of humaninfected AIV H5N6 isolated from China”, the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0070] A / Anhui / 2013(H7N9): recorded in the document “Differences in the pathogenicity and inflammation responses induced by avian influenza A H7N9 virus infection in BALBc and C57BL6 mouse models”, the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0071] A / Guangxi / NN10.19T-NGS / 2018(H9N2): recorded in the document “Genetic, biological and epidemiological study on a cluster of H9N2 avian influenza virus infections among chick ens, a pet cat, and humans at a backyard farm in Guangxi, China”, which is available to the public from the Institute of Microbiology, Chinese Academy of Sciences. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0072] According to the results of Example 1, siPB2-12, siPB1-4, siPA-2 and siNP-4 from the candidate siRNA molecules in Example 1 were selected for broad-spectrum anti-influenza virus screening on A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 2013(H7N9) and A / Guangxi / NN 10.19T-NGS / 2018(H9N2) strains. The preparation methods of Kong / 2671 / 2019 (H3N2), A / Guangzhou / 39715 / 2014 (H5N6), A / Anhui / 2013 (H7N9) and A / Guangxi / NN10.19T-NGS / 2018 (H9N2) were all referred to Example 1. The siRNA molecule transfection and virus inoculation were referred to the experimental method shown in Example 1.

[0073] (1) Determination of hemagglutination titer

[0074] siPB2-12, siPB1-4, siPA-2, and siNP-4 were transfected respectively, and siRNA-NC, siRNA-GFP, and Control (infected with virus only) were used as negative control groups. 24 hours after transfection, MDCK cells were infected with influenza virus strains A / Guangd ong-Maonan / SWL1536 / 2019 (H1N1), A / Hong Kong / 2671 / 2019 (H3N2), A / Guangz hou / 39715 / 2014 (H5N6), A / Anhui / 1 / 2013 (H7N9), and A / Guangxi / NN10.19T-NGS / 2018 (H9N2), and the cell supernatant was harvested 48 hours after virus infection and the hemagglutination titer of the virus in the cell supernatant was detected (see Figure 4 ).

[0075] The results showed that compared with the Control group, siPB2-12 targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cell supernatants showed a significant decrease in HA titer (P<0.0001, P<0.001, P<0.001, and P<0.05, respectively); siPB1-4 targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong The HA titer in the supernatant of cells targeting A / Anhui / 1 / 2013(H7N9), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) was significantly decreased (P<0.01, P<0.0001, P<0.0001, P<0.01, and P<0.05, respectively). The HA titer in the supernatant of cells targeting A / Anhui / 1 / 2013(H7N9) was significantly decreased (P<0.01), and that in cells targeting A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) was significantly decreased (P<0.01, P<0.0001, P<0.0001, P<0.01, and P<0.05, respectively). There was a downward trend in the HATiter in the supernatant of A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells, but it was not statistically significant. titer was significantly decreased (P<0.01, P<0.001, P<0.001, P<0.01 and P<0.05, respectively).

[0076] When siPB2-12 targets A / Guangdong-Maonan / SWL1536 / 2019(H1N1), the titer of the three replicates in this group is 2 1 , 2 1 , 2 2When siPB2-12 targets A / Hong Kong / 2671 / 2019(H3N2), the titer of the three replicates in this group is 2 1 , 2 0 , 2 0 When siPB2-12 targeted A / Guangzhou / 39715 / 2014(H5N6), the titer of the three replicates in this group was 2 6 , 2 6 , 2 6 When siPB2-12 targeted A / Guangxi / NN10.19T-NGS / 2018(H9N2), the titer of the three replicates in this group was 2 0 , 2 0 , 2 0 .

[0077] When siPB1-4 targets A / Guangdong-Maonan / SWL1536 / 2019(H1N1), the titer of the three replicates in this group is 2 2 , 2 2 , 2 2 When siPB1-4 targets A / Hong Kong / 2671 / 2019(H3N2)A / Hong Kong / 2671 / 2019(H3N2), the titer of the three replicates in this group is 2 0 , 2 0 , 2 0 When siPB1-4 targeted A / Guangzhou / 39715 / 2014(H5N6), the titer of the three replicates in this group was 2 6 , 2 6 , 2 6 When siPB1-4 targeted A / Guangxi / NN10.19T-NGS / 2018(H9N2), the titer of the three replicates in this group was 2 0 , 2 0 , 2 0 .

[0078] When siNP-4 targeted A / Guangdong-Maonan / SWL1536 / 2019(H1N1), the titer of the three replicates in this group was 2 2 , 2 2 , 2 2 When siNP-4 targets A / Hong Kong / 2671 / 2019(H3N2)A / Hong Kong / 2671 / 2019(H3N2), the titer of the three replicates in this group is 2 1 , 2 0 , 20 When siNP-4 targeted A / Guangzhou / 39715 / 2014(H5N6), the titer of the three replicates in this group was 2 6 , 2 6 , 2 6 When siNP-4 targeted A / Guangxi / NN10.19T-NGS / 2018(H9N2), the titer of the three replicates in this group was 2 0 , 2 0 , 2 1 .

[0079] (2) Detection of NP protein in cell supernatant

[0080] At 48 h after virus infection, the expression of influenza virus NP protein in the cell supernatant was analyzed by Western blot (see Figure 5), siRNA has a certain inhibitory effect on the synthesis of influenza virus NP protein in the cell supernatant. Compared with the Control group, siPB2-12 targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), A / Guangxi / NN10.19T-NGS / 2018(H9N2) cell supernatant had an inhibitory effect of 61%, 48%, 46%, 31%, and 43%, respectively; siPB1-4 targeting A / Hong The inhibitory effects of NP protein in the supernatant of A / Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 44%, 53%, and 59%, respectively; siPA-2 targeted A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), A / Guangxi / The inhibitory effects of NP protein in the supernatant of NN10.19T-NGS / 2018(H9N2) cells were 62%, 40%, 36% and 20%, respectively; the inhibitory effects of siNP-4 targeting NP protein in the supernatant of A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9) and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 84%, 41%, 40% and 21%, respectively.

[0081] (3) Detection of intracellular NP protein

[0082] At 48 h after virus infection, the expression of influenza virus NP protein in cells was analyzed by Western blot (see Figure 6), siRNA has a certain inhibitory effect on the synthesis of influenza virus NP protein in cells. Compared with the Control group, siPB2-12 targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / HongKong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells had an inhibitory effect of 60%, 68%, 39%, and 65%, respectively; siPB1-4 targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong The inhibitory effects of siPA-2 targeting NP protein in A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6) and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 12%, 44%, 11% and 48%, respectively. The inhibitory effects of siPA-2 targeting NP protein in A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2) and A / Guangzhou / 39715 / 2014(H5N6) cells were 46%, 42% and 24%, respectively. The inhibitory effects of siNP-4 targeting NP protein in A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong The inhibitory effects of NP protein in Kong / 2671 / 2019 (H3N2) cells were 51% and 36%, respectively.

[0083] In summary, siPB2-12 and siNP-4 can widely inhibit H1, H3, H5, H7 and H9 subtype influenza viruses, and have a broad spectrum.

[0084] Example 3: Verification of the interference function of aiRNA on different subtypes of influenza viruses

[0085] After preliminary screening of candidate siRNA molecules, four siRNAs, siPB2-12, siPB1-4, siPA-2, and siNP-4, were selected to shorten their sense strands to 15 bp, as follows:

[0086] Table 2 aiPB2 molecules

[0087] siRNA Sensestrand(5'-3') Antisense strand (5'-3') aiPB2-12 CUCUAGCAUACUUAC TTAGUAAGUAUGCUAGAGUCC aiPB1-4 UGAUGGGCAUGUUCA TTUUGAACAUGCCCAUCAUCA aiPA-2 UGGUUCAACUCCUUC TTGGAAGGAGUUGAACCAAGA aiNP-4 UUUCUUCGGAGACAA TTAUUGUCUCCGAAGAAAUAA

[0088] Positions 1-15 of the sense strand of aiPB2-12 correspond to SEQ ID No.3, and positions 2-21 of the sense strand of aiPB2-12 correspond to SEQ ID No.4.

[0089] Positions 1-15 of the sense strand of aiNP-4 correspond to SEQ ID No.7, and positions 2-21 of the sense strand of aiNP-4 correspond to SEQ ID No.8.

[0090] The screening method is as follows:

[0091] 1. Preparation of A / Guangdong-Maonan / SWL1536 / 2019 (H1N1) virus

[0092] After MDCK cells were cultured in a culture flask until the monolayer was confluent, the primary A / Guangdong-Maonan / SWL1536 / 2019(H1N1) virus (MOI=0.1) was inoculated on the MDCK cells and incubated in a 35°C incubator for 72 hours. The cell culture flask was shaken to collect the virus supernatant and cell debris. After centrifugation, the supernatant was collected and stored in aliquots at -80°C and repeatedly frozen and thawed three times, which was the A / Guangdong-Maonan / SWL1536 / 2019(H1N1) virus.

[0093] 2. Cell culture and liposome transfection

[0094] MDCK cells were cultured at 2.5 × 10 6 / well density inoculated into 6-well cell culture plates, when the cells adhered to 70-80%, grouped as follows:

[0095] aiPB2-12 group: siRNA transfection reagent (purchased from Invitrogen TM, Catalog No.: 13778150) instructions, aiPB2-12 was transfected into MDCK cells with siRNA transfection reagent Lipofectamine RNAiMAX, and the final content of siRNA in each well was 50nM. The experiment was repeated three times, with three replicate wells each time. After the cell culture plate was placed at 37°C and 5% CO2 for 24 hours, the culture medium was aspirated and washed with PBS for 3 times by shaking. The cells were infected with A / Guangdong-Maonan / SWL1536 / 2019 (H1N1) virus at an MOI of 0.1 and incubated at 37°C for 1 hour. In order to promote viral infection, the cell culture plate was gently shaken every 15 minutes during incubation. After 1 hour, the virus dilution was aspirated, 500μl of virus maintenance solution (DMEM culture medium containing 2μg / mL trypsin, 100U / mL penicillin and 0.1mg / mL streptomycin) was added to each well, and the culture was continued at 37°C, and each index was detected according to the following operation:

[0096] ① Hemagglutination test: harvest cell supernatants at 12h, 24h, 36h, and 48h after virus infection, and use the influenza virus's property of agglutinating red blood cells to determine the virus' hemagglutination titer. The specific method for determining the virus' hemagglutination titer is as follows: take a 96-well V-shaped microplate and place it horizontally. Except for the first column, add 25μl of physiological saline to each well of the remaining wells. Add 50μl of the virus solution to be tested (the aforementioned cell supernatant) to the first well of each column. From the first well, dilute the virus solution to be tested to the 12th well, and discard 25μl from the last well. Then add 25μl of 1% chicken red blood cell suspension to each well, incubate at room temperature for 30min, and observe whether hemagglutination reaction occurs. HA titer (log2) = log2N, N is the dilution multiple of the virus solution to be tested.

[0097] ②Western-blot: 48 hours after virus infection, the culture medium was aspirated to obtain the supernatant of cells infected with H1N1 strain after transfection with siPB2-1; the remaining adherent cells were washed once with PBS, 100 μl of cell lysis buffer (DAPI lysis buffer and protease inhibitor PMSF were prepared in a ratio of 100:1) were added, and placed at 4°C for 10 minutes. The cells were blown with a pipette, and the obtained cell lysis buffer was transferred to a new 1.5 ml EP tube as much as possible, and centrifuged at 4°C and 12000 rpm for 5 minutes. The supernatant after centrifugation was mixed with SDS-PAGE Loading buffer (5×) at a ratio of 4:1. After a brief centrifugation, the diluted cell sample was boiled at 100°C for 5 to 10 minutes and stored in a refrigerator at -80°C for later use. The expression of viral NP protein, HA protein and experimental internal reference β-actin protein was detected by Western-blot. The antibodies used for detection included anti-NP antibody, anti-HA antibody and anti-β-actin antibody.

[0098] siPB2-12 group, aiPB1-4 group, siPB1-4 group, aiPA-2 group, siPA-2 group, aiNP-4 group or siNP-4 group: replace aiPB2-12 in the aiPB2-12 group with siRNA (or aiRNA) of siPB2-12, aiPB1-4, siPB1-4, aiPA-2, siPA-2 group, aiNP-4 or siNP-4, and the rest of the operations were the same as those of the aiPB2-12 group.

[0099] Control (virus) group: pure DMEM culture medium was used to replace the siRNA transfection reagents Lipofectamine RNAiMAX and aiPB2-12 in the siPB2-1 group, and the rest of the operations were the same as those in the aiPB2-12 group.

[0100] The experimental results are as follows:

[0101] (1) Determination of hemagglutination titer

[0102] siPB2-12 / aiPB2-12, siPB1-4 / aiPB1-4, siPA-2 / aiPA-2, siNP-4 / aiNP-4 were transfected respectively, and siRNA-GFP and Control (infected with virus only) were used as negative control groups. 24 hours after transfection, MDCK cells were infected with influenza virus strains A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2), and the cell supernatant was harvested 48 hours after virus infection and the hemagglutination titer of the virus in the cell supernatant was detected (see Figure 7The results showed that compared with the Control group, the HA titer in the supernatant of cells targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Anhui / 1 / 2013(H7N9) by aiPB2-12 was significantly reduced (P<0.0001, P<0.001, P<0.0001, and P<0.0001, respectively). The HA titer in the supernatant of cells targeting A / Guangxi / NN10.19T-NGS / 2018(H9N2) showed a downward trend, but it was not statistically significant. The HA titer in the supernatant of A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Anhui / 1 / 2013(H7N9) cells was significantly decreased (P<0.001, P<0.0001, and P<0.0001, respectively). The HA titer in the supernatant of A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells showed a downward trend, but it was not statistically significant. The HA titer in the supernatant of A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Anhui / 1 / 2013(H7N9) cells targeted by aiPA-2 was significantly decreased (P<0.001, P<0.0001, and P<0.0001, respectively). The titer of aiNP-4 targeting HA in the supernatant of A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells was significantly decreased (P<0.001, P<0.0001, and P<0.0001, respectively). The titer of HA in the supernatant of A / Guangdong-Maonan / SWL1536 / 2019(H1N1) and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells showed a downward trend, but it was not statistically significant. titer was significantly decreased (P<0.0001, P<0.01, P<0.0001, P<0.0001, P<0.01, respectively).

[0103] When aiPB2-12 targets A / Guangdong-Maonan / SWL1536 / 2019(H1N1), the titer of the three replicates in this group is 20 , 2 0 , 2 0 When aiPB2-12 targets A / Hong Kong / 2671 / 2019(H3N2), the titer of the three replicates in this group is 2 4 , 2 4 , 2 4 When aiPB2-12 targets A / Guangzhou / 39715 / 2014(H5N6), the titer of the three replicates in this group is 2 4 , 2 4 , 2 4 When aiPB2-12 targets A / Anhui / 1 / 2013(H7N9), the titer of the three replicates in this group is 2 5 , 2 5 , 2 5 When aiPB2-12 targeted A / Guangxi / NN10.19T-NGS / 2018(H9N2), the titer of the three replicates in this group was 2 1 , 2 1 , 2 2 .

[0104] When aiNP-4 targets A / Guangdong-Maonan / SWL1536 / 2019(H1N1), the titer of the three replicates in this group is 2 0 , 2 0 , 2 0 When aiNP-4 targets A / Hong Kong / 2671 / 2019(H3N2), the titer of the three replicates in this group is 2 4 , 2 4 , 2 4 When aiNP-4 targets A / Guangzhou / 39715 / 2014(H5N6), the titer of the three replicates in this group is 2 4 , 2 4 , 2 4 When aiNP-4 targets A / Anhui / 1 / 2013(H7N9), the titer of the three replicates in this group is 2 5 , 2 5 , 2 5 When aiNP-4 targeted A / Guangxi / NN10.19T-NGS / 2018(H9N2), the titer of the three replicates in this group was 2 1 , 2 1 , 2 0 .

[0105] It can be seen that compared with the Control group, aiPB2-12 and aiNP-4 transfection can significantly inhibit the replication of the virus in MDCK cells.

[0106] 2. Detection of NP protein in cell supernatant

[0107] At 48 h after virus infection, the expression of influenza virus NP protein in the cell supernatant was analyzed by Western blot (see Figure 8 ), aiRNA has a certain inhibitory effect on the synthesis of influenza virus NP protein in the cell supernatant. Compared with the Control group, aiPB2-12 targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), A / Guangxi / NN10.19T-NGS / 2018(H9N2) cell supernatants had an inhibitory effect of 38%, 85%, 64%, 41%, and 63%, respectively; aiPB1-4 targeting A / Hong The inhibitory effects of NP protein in the supernatant of A / Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 18%, 40%, 52%, and 34%, respectively; aiPA-2 targeted A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong The inhibitory effects of NP protein in the supernatant of A / Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 52%, 83%, 52%, 39%, and 35%, respectively; aiNP-4 targeted A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong The inhibitory effects of NP protein in the supernatants of Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 58%, 76%, 45%, 32%, and 30%, respectively.

[0108] 3. Detection of intracellular NP protein

[0109] At 48 h after virus infection, the expression of influenza virus NP protein in cells was analyzed by Western blot (see Fig. 9 ), siRNA has a certain inhibitory effect on the synthesis of influenza virus NP protein in cells. Compared with the Control group, aiPB2-12 targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), A / Guangxi / NN10.19T-NGS / 2018(H9N2) cell supernatants had inhibitory effects of 74%, 90%, 86%, and 40%, respectively; aiPB1-4 targeting A / Hong The inhibitory effects of NP protein in the supernatant of A / HongKong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 69%, 84%, 76%, and 73%, respectively. The inhibitory effects of aiNP-4 targeting NP protein in the supernatant of A / Anhui / 1 / 2013(H7N9) and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 47%, 66%, 77% and 73%, respectively; the inhibitory effects of aiNP-4 targeting NP protein in the supernatant of A / Anhui / 1 / 2013(H7N9) and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 78% and 41%, respectively.

[0110] The present invention designs siRNA targeting polymerase protein and nucleoprotein genes for influenza A virus H1, H3, H5, H7, and H9 subtypes. Through cell experiments, it is found that siPB2-12 and siNP-4 have antiviral activity for different subtypes of influenza virus. For siRNA with potential activity, this study designed its corresponding aiRNA, and through cell experiments, it was found that aiPB2-12 and aiNP-4 also have antiviral activity for different subtypes of influenza virus. The above research results show that siPB2-12, siNP-4 and their corresponding aiRNA have potential broad spectrum and specificity.

[0111] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A double-stranded RNA molecule, a modified product thereof, or a pharmaceutically acceptable salt thereof, characterized in that: The double-stranded RNA molecule is siPB2-12 and / or siNP-4, or / and a derivative of the RNA molecule; The sequence of one chain of the siPB2-12 is SEQ ID No. 1 in the sequence list, and the sequence of the other chain is SEQ ID No. 2 in the sequence list; The sequence of one chain of the siNP-4 is SEQ ID No. 5 in the sequence list, and the sequence of the other chain is SEQ ID No. 6 in the sequence list.

2. The double-stranded RNA molecule, its modified substance, or its pharmaceutically acceptable salt according to claim 1, characterized in that: The derivatives of the RNA molecule include derivatives of siPB2-12 and derivatives of siNP-4. The derivative of siPB2-12 is a double-linked RNA molecule named aiPB2-12, and the derivative of siNP-4 is a double-linked RNA molecule named aiNP-4. The sequence of one chain of aiPB2-12 is SEQ ID No. 3 in the sequence list, and the sequence of the other chain is SEQ ID No. 4 in the sequence list. The sequence of one chain of aiNP-4 is SEQ ID No. 7 in the sequence list, and the sequence of the other chain is SEQ ID No. 8 in the sequence list.

3. A biological material containing the double-stranded RNA molecule according to claim 1, wherein the biological material is a DNA molecule capable of producing the double-stranded RNA molecule, a recombinant vector containing the DNA molecule, or a recombinant microorganism containing the DNA molecule.

4. An influenza virus inhibitor, characterized in that The influenza virus inhibitor contains the double-stranded RNA molecule according to claim 1, a modified substance thereof, or a pharmaceutically acceptable salt thereof.

5. A drug for preventing and / or treating influenza, characterized in that: The drug contains the double-stranded RNA molecule according to claim 1, a modified substance thereof, or a pharmaceutically acceptable salt thereof.

6. The influenza virus inhibitor according to claim 4 or the drug according to claim 5, characterized in that: The influenza virus inhibitor or the drug is in the form of an aerosol, tablet, capsule, pellet, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, buccal tablet, suppository or lyophilized powder injection.

7. Use of the double-stranded RNA molecule according to claim 1 or 2, its modified product, or its pharmaceutically acceptable salt, or the biomaterial according to claim 3 in preparing an influenza virus inhibitor or in inhibiting influenza virus.

8. The influenza virus inhibitor according to claim 4 or 6 or the drug according to claim 5 or 6, characterized in that: The influenza virus is H1NI, H3N2, H5N6, H7N9 and / or H9N2 virus, and the influenza is caused by H1NI, H3N2, H5N6, H7N9 and / or H9N2 virus.

Images