CircRNA (Ribonucleic Acid) and application thereof in anti-influenza virus

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to circRNA and application thereof in resisting influenza viruses. According to the circRNADRACO disclosed by the invention, the broad-spectrum antiviral protein DRACO can be expressed in a body, the expressed protein is long in continuous level, good in stability and good in biological safety, and the problems that a traditional DRACO drug is difficult to enter cells and poor in stability are solved. Meanwhile, the circRNADRACO can be combined with long dsRNA to activate caspase, trigger cascade reaction of cell apoptosis, cause cell apoptosis and inhibit replication of influenza viruses including influenza A viruses, so that the effect of effectively preventing and treating the influenza viruses is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a circRNA and its application in resisting influenza virus. Background Art

[0002] In recent years, new and emerging infectious diseases have occurred frequently, among which infectious diseases caused by viruses are still an important type of disease that seriously endangers global life and health. Viruses can quickly adapt to new hosts, have strong infectivity and survival ability in the body, and their genomes are often unstable and highly variable. Traditional vaccine and antibody drug development cycles are relatively long, require large investments, and are highly dependent on the viral genome sequence, and have very limited protective effects against diseases caused by highly mutated viruses. Therefore, the development of broad-spectrum antiviral drugs is necessary and urgent.

[0003] DRACO (double-stranded RNA activated caspase oligomerizer) is a dsRNA-activated caspase oligomer that activates two natural cellular processes by integrating dsRNA detection in the interferon pathway and apoptosis signaling molecules in the apoptosis pathway, exerting a broad-spectrum antiviral effect. Some studies have shown that after almost all viruses infect cells, the virus will produce long dsRNA (>21-23 bp) during replication and transcription in the infected cells, while uninfected mammalian cells generally do not produce long dsRNA, and the natural immune defense process will use this difference to resist viral infection. Currently, DRACO has been proven to be non-toxic to 11 mammalian cells and has effective antiviral effects on 15 different types of RNA and DNA viruses. Since all different types of RNA and DNA viruses will produce long-chain dsRNA pathogen-associated molecular patterns (PAMPs) during infection and replication, DRACO should theoretically be effective against all viruses and is a potential broad-spectrum antiviral drug molecule.

[0004] DRACO is of great significance for antiviral treatment. For example, some studies have shown that DRACO is effective in a dose-dependent manner for MDCK cells infected with influenza A (H1N1) virus, and has the potential to be a new anti-H1N1 influenza virus therapeutic drug. Other studies have demonstrated that based on the design concept of DRACO, a DRACO protein designed according to the relevant gene sequence of pigs was constructed for the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), and it was proved that DRACO can significantly reduce the N protein of HP-PRRSV at the transcription and translation levels, showing strong antiviral activity, laying the foundation for the development of new drugs against porcine blue ear disease. However, the traditional DRACO protein drugs still have problems such as complex molecular structure, difficulty in cell entry, and poor stability. Summary of the invention

[0005] The object of the present invention is to provide a circRNA and its application in anti-influenza virus, wherein the circRNA can achieve stable expression of DRACO protein in body cells, effectively inhibit the replication of influenza A virus, and achieve the effect of preventing and treating influenza A virus.

[0006] The present invention provides a circRNA DRACO , the circRNA DRACO The nucleotide sequence is shown in SEQ ID NO.1.

[0007] The present invention also provides the circRNA described in the above technical solution DRACO Application in the preparation of medicines for preventing and treating influenza viruses.

[0008] The present invention also provides the circRNA described in the above technical solution DRACO Application in the preparation of anti-influenza virus vaccines.

[0009] Preferably, the influenza virus comprises influenza A virus.

[0010] Preferably, the influenza A virus includes influenza A (H1N1) virus and / or influenza A (H3N2) virus.

[0011] Preferably, the drug comprises a drug that inhibits influenza virus replication.

[0012] The present invention also provides a drug for preventing and treating influenza virus, wherein the active ingredient of the drug includes the circRNA described in the above technical solution. DRACO .

[0013] Preferably, the drug also includes pharmaceutically acceptable excipients.

[0014] The present invention also provides an anti-influenza virus vaccine, wherein the effective component of the vaccine comprises the circRNA described in the above technical solution. DRACO .

[0015] Preferably, the vaccine also includes a vaccine adjuvant.

[0016] Beneficial effects: The present invention provides a circRNA DRACO , the circRNA DRACO The nucleotide sequence is shown in SEQ ID NO.1, and the circRNA DRACO Application in the preparation of drugs for preventing and treating influenza viruses and in the preparation of vaccines against influenza viruses. The circRNA of the present invention DRACO The broad-spectrum antiviral protein DRACO can be expressed in vivo, and the expressed protein has a long-lasting level, good stability, and good biosafety, overcoming the problem of traditional DRACO drugs being difficult to enter cells and having poor stability. DRACO It can bind to long dsRNA, activate caspase, trigger a cascade reaction of cell apoptosis, cause cell apoptosis, inhibit the replication of influenza viruses including influenza A virus, and achieve the effect of effectively preventing and treating influenza viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 The protein expression results of the synthetic RNA in Example 1; Figure 2 For the mRNA in Example 1 DRACO and circRNA DRACO Effects on cell activity; Figure 3 For the mRNA in Example 1 DRACO and circRNA DRACO stability; Figure 4 is the circRNA in Example 1 DRACO Mediate apoptosis of cells containing dsRNA; Figure 5 is the circRNA transfection efficiency of MDCK cells in Example 1; Figure 6The effect of different infection multiplicity of infection of H1N1 influenza virus on MDCK cells in Example 1; Figure 7 The figure shows the effect of different infection multiplicity of infection of H3N2 influenza virus on MDCK cells in Example 1. DETAILED DESCRIPTION

[0019] The present invention provides a circRNA DRACO , the circRNA DRACO

[0020] As a nucleic acid drug, unlike mRNA, which still has problems such as poor stability, low efficiency, and innate immunogenicity limitations, circRNA is an emerging member of the non-RNA family and a covalently closed single-stranded transcript. Most of the circRNAs currently studied are generated by reverse splicing of the exon messenger RNA precursor (pre-mRNA). In this process, the intron sequences on both sides of the downstream splicing donor site and the upstream splicing acceptor site are close to each other, undergoing a transesterification reaction to form a 3', 5'-phosphodiester bond, and then forming a circular molecule through covalent connection. Unlike traditional linear RNA, circRNA does not have a 5' end cap and a 3' end poly (A) tail. This special structure protects it from degradation by nucleases and presents superior stability. Therefore, circRNA is a supplement and development of mRNA technology. Based on the above advantages, the present invention designs and constructs a circRNA that can successfully express the broad-spectrum antiviral protein DRACO in cells. DRACO , the DRACO protein can be successfully expressed in cells, bind to the virus to cause cell apoptosis, and inhibit the replication of influenza virus, proving the feasibility of converting the broad-spectrum antiviral protein DRACO into a circRNA drug form to exert an antiviral effect.

[0021] Based on the above advantages, the circRNA DRACO The technical scheme for preparing a drug for preventing and treating influenza virus and preparing an anti-influenza virus vaccine also belongs to the protection scope of the present invention. The influenza virus of the present invention preferably includes influenza A virus, and further preferably includes influenza A H1N1 virus and / or influenza A H3N2 virus. The drug of the present invention preferably includes a drug that inhibits influenza virus replication.

[0022] The present invention also provides a drug for preventing and treating influenza virus, wherein the active ingredient of the drug includes the circRNA described in the above technical solution. DRACO The drug of the present invention preferably also includes pharmaceutically acceptable excipients. The present invention has no special restrictions on the type and amount of the excipients, which can be conventionally selected according to preparation requirements.

[0023] The present invention also provides an anti-influenza virus vaccine, wherein the effective component of the vaccine comprises the circRNA described in the above technical solution. DRACO The vaccine of the present invention preferably also includes a vaccine adjuvant. The present invention has no particular limitation on the type and amount of the vaccine adjuvant, which can be conventionally selected according to preparation requirements.

[0024] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] Example 1 circRNA DRACO And its use in anti-influenza virus, the steps are as follows: 1 Experimental Materials 1.1 Cells, viruses and RNA sources circRNA was synthesized by Gisai Biotechnology Co., Ltd.; linear mRNA was synthesized by Jinan Protein Technology Co., Ltd. Influenza A (H1N1) virus (A / Puerto Rico / 8 / 1934) and influenza A (H3N2) virus (A / Port Chalmers / 1 / 73) were purchased from the American type culture collection (ATCC). HEK293T cells were purchased from Punosai Life Science Co., Ltd.; MDCK cells were preserved by Zhao Zhihu's research group at the Institute of Bioengineering, Academy of Military Medical Sciences.

[0026] 1.2 Main reagents APAF1 antibody, Caspase-9 inhibitor Z-LEHD-FMK, and Pan Caspase inhibitor Z-VAD-FMK were purchased from Abcam Biotechnology Company; GSPure ® Circ-eGFP was purchased from Geneseed Biotechnology Co., Ltd.; mouse anti-β-Actin monoclonal antibody was purchased from MBL Biotechnology Co., Ltd.; Caspase-Glo ® 3 / 7 Detection Kit, CellTiter 96 ® AQueous single solution cell proliferation detection kit was purchased from Promega Biotechnology Company; poly(I:C) and TPCK were purchased from Sigma Biotechnology Company; AlamarBlue ™ Cell viability detection reagents and Lipofectamine MessengerMAX were purchased from Thermo Fisher Scientific; FastPure Cell / TissueTotal RNA Isolation Kit V2 was purchased from Novazon Biotechnology; DMEM high-glucose basal medium and fetal bovine serum were purchased from Prosper Biotechnology; Hepes-Tris 10% high-resolution precast gel was purchased from Yisheng Biotechnology; Omni-Easy ™ Fast protein loading buffer, Omni-ECL ™The ultrasensitive chemiluminescence detection kit was purchased from Yazyme Biotechnology Co., Ltd.; Escherichia coli competent cells DH5α were purchased from Tiangen Biotechnology Co., Ltd., TransScript ® All-in-One First-Strand cDNA SynthesisSuperMix for qPCR, PerfectStart ® Visual Green qPCR SuperMix was purchased from Quanshijin Biotechnology Co., Ltd.

[0027] 2 Experimental methods 2.1 Sequence design and RNA preparation Using the amino acid sequence of the recombinant protein DRACO as a template, sequence optimization and mRNA synthesis were performed at Suzhou Nearshore Protein Technology Co., Ltd. DRACO The optimized coding sequence of the recombinant protein DRACO is shown in SEQ ID NO.3, mRNA DRACO The sequence is composed of the sequence shown in SEQ ID NO.3 and the framework protein, and other framework sequences required for expression are provided and added by Nearshore Protein Technology Co., Ltd., and those skilled in the art can synthesize mRNA at Nearshore Protein Technology Co., Ltd. based on the content disclosed in this application. DRACO The optimized coding region sequence was sent to Gisai Biotechnology Co., Ltd. as a template to synthesize circRNA DRACO The specific nucleotide sequence is shown in SEQ ID NO.1.

[0028] The amino acid sequence of recombinant protein DRACO is as SEQ ID As shown in NO.2, specifically: MAGDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKNAAAKLAVEILNKEKKAVSPLLLTTTNSSEGLSMGNYIGLINRIAQKKRLTVNYEQCASGVHGPEGFHYKCKM GQKEYSIGTGSTKQEAKQLAAKLAYLQILSEETSVKSDYLSSGSGGGSGMDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPTQQQRAAMLIKMILKKDNDSYVSFYNALLHEGYKDLAALLHDGIPVVSSS*; The optimized coding region sequence of the recombinant protein DRACO is shown in SEQ ID NO.3, specifically 5'--3'; 2.2 Transfection HEK293T cells or MDCK cells with good growth status were inoculated into 12-well plates so that the cell confluence reached about 70-80% before transfection. DRACODenature at 70°C for 3 min in a PCR instrument and place on ice for 2 min. According to the instructions, use Lipofectamine MessengerMAX to transfect RNA samples into HEK293T or MDCK cells, culture for the corresponding time according to the experimental plan, and collect lysed cells and supernatant for subsequent detection.

[0029] 2.3 Cell viability assay Using CellTiter96 ® Single solution cell proliferation detection kit (MTS method), 37℃ water bath for 10 min to completely melt; add 20 μL reagent to 100 μL culture medium in each well of 96-well plate; incubate at 37℃, 5% CO2 incubator for 1-4 h; read the absorbance value at 490 nm to calculate cell activity.

[0030] Using AlamarBlue ™ Cell viability detection reagent: add 10 μL of LamarBlue reagent to 90 μL of culture medium in each well of a 96-well plate and incubate at 37°C in a 5% CO2 incubator for 1-4 h; record the absorbance values ​​at 570 nm and 600 nm to calculate cell activity.

[0031] 2.4 qPCR Total RNA from cell samples was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2. ® Reverse transcription was performed using All-in-One First-Strand cDNA Synthesis SuperMix for qPCR reagent, incubated at 42°C for 15 min, and heated at 85°C for 5 sec to obtain cDNA.

[0032] The cDNA template obtained by reverse transcription was used for qPCR, DRACO-Forward: 5'-AGCGTGAAGTCCGATTACCT-3'; DRACO-Reverse: 5'-TCTCTTCCTCGCTGATGGTC-3'. The reaction program was set as 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 sec, 60℃ annealing and extension for 1 min, for a total of 40 cycles.

[0033] 2.5 Cell protein extraction Collect the cell pellet, add an appropriate amount of RIPA mixture (1 mL RIPA + 10 μL PI (100×) + 10 μL pmsf (1 mM)) to the sample, mix well by pipetting, and incubate at 4°C for 10-15 min. Centrifuge at 4°C for 15 min, take the supernatant, and measure the protein concentration. Add the mixture into Omni-Easy ™ Rapid protein loading buffer (5×) was used, and the sample was loaded after cooling in a 100°C water bath for 10 min.

[0034] 2.6 Western Blot After the boiled sample was cooled, it was centrifuged at 5000 r / min for 3 min. The supernatant sample solution with 100 μg protein content was added to Hepes-Tris 10% high-resolution precast gel for SDS-PAGE electrophoresis. The protein on the gel was transferred to a 0.45 μm PVDF membrane by 300 mA constant current electrophoresis for 100 min; 5% skim milk was blocked for 1 h. The primary antibody was incubated overnight at 4°C and washed 3 times with TBST, 10 min / time. The secondary antibody was incubated at room temperature and washed 5 times with TBST, 8 min / time. The chemiluminescent detection reagent was mixed in equal volumes and evenly added to the membrane to observe the target band.

[0035] 2.7 Cell apoptosis assay Caspase-Glo ® 3 / 7 Test Kit reagents are balanced to room temperature and Caspase-Glo ® 3 / 7 Buffer is poured into Caspase-Glo ® 3 / 7 freeze-dried substrate, invert and mix until completely dissolved; add 100 μL of mixed reagent to 100 μL of culture medium per well of a 96-well plate equilibrated to room temperature; gently shake at 300-500 rpm for 30 sec, incubate at room temperature for 30 min-3 h; after incubation, transfer to an opaque all-white 96-well plate and read the fluorescence value of the sample.

[0036] 2.8 Hemagglutination titer (HA) Take 10 mL of fresh chicken blood, wash twice with PBS containing penicillin (100 U / mL) and streptomycin (0.1 mg / mL), centrifuge at 1000 rpm and 4℃ for 5 min, discard the supernatant, add 20 mL of PBS to resuspend, and take 1 mL to dilute 10 times for subsequent experiments. On a V-shaped 96-well plate, dilute 2-fold to the eleventh well, and the twelfth well is a virus-free control. Add 50 μL of the prepared chicken blood red blood cells to each well of the virus dilution solution, gently shake to mix, let stand for about 30 min, and observe the results.

[0037] 2.9 Virus titer determination Using standard TCID50 Virus titration was performed using the tissue cell half-infectious dose (WID) method. MDCK cells were inoculated in 96-well plates for 24 h, washed twice with PBS, and 100 μL of DMEM virus diluent (serum-free) containing 1 μL / mL TPCK was added to each well for 10-fold serial dilutions. The plates were adsorbed for 1 h, the virus solution was discarded, the plates were washed with PBS, and the plates were cultured for 2-4 days to observe the CPE results.

[0038] 3 Experimental results and analysis 3.1 circRNA DRACO Expression of DRACO protein Lipofectamine MessengerMAX transfection reagent was used to verify the transfection efficiency of circRNA in HEK293T cells. ® Circ-eGFP was transfected into cells, and the transfection efficiency of HEK293T cells was observed 24 h later. Figure 1 As shown, A is HEK293T transfected with circRNA eGFP Bright field image; B is HEK293T transfected with circRNA eGFP Dark field image; C is mRNA DRACO and circRNA DRACO Protein expression. Figure 1 As can be seen from Figures A and B, the efficiency of HEK293T cell transfection with circRNA is very high, about 90% or more.

[0039] Western blotting was used to observe the untransfected control group and transfected mRNA DRACO and transfection of circRNA DRACO The protein expression levels of the three groups ( Figure 1 C), the results show that the synthetically constructed mRNA DRACO and circRNA DRACO Can effectively express DRACO protein in cells.

[0040] 3.2 Cell viability assay The mRNA of different quality was determined by MTS method and Alamar Blue method. DRACO and circRNA DRACO Effect of transfection on the activity of HEK293T cells. Figure 2 As shown, A is the detection of mRNA by MTS method and Alamar Blue method DRACOA is the cytotoxicity of circRNA DRACO transfection on HEK293T cells; B is the MTS method and Alamar Blue method to detect the cytotoxicity of circRNA DRACO transfection on HEK293T cells. The data are expressed as the mean ± SD of 3 independent experiments, and the error bars are standard deviations. Figure 2 It can be concluded that compared with the untransfected control group, the transfection of 0.5 μg, 1 μg, 1.5 μg, and 2.0 μg of mRNA DRACO and circRNA DRACO There was no significant effect on cell activity, indicating that the compounds had no cytotoxicity and good biosafety.

[0041] 3.3 CircRNA stability detection mRNA DRACO and circRNA DRACO After transfection into HEK293T cells, cells were collected at five time points, namely 12 h, 24 h, 36 h, 48 h, and 72 h, and mRNA was evaluated by qPCR. DRACO and circRNA DRACO The RNA levels were Figure 3 As shown, A is mRNA DRACO and circRNA DRACO Comparison of stability at the transcription level; B is mRNA DRACO The sustained level of expressed protein; C is circRNA DRACO The data are presented as the mean ± SD of three independent experiments, with error bars representing standard deviation and ns representing P >0.05,* P <0.05,** P <0.01. Figure 3 It can be concluded from A that circRNA DRACO The overall degradation rate is slower than that of mRNA DRACO More stable.

[0042] mRNA DRACO and circRNA DRACO After transfection into HEK293T cells, HEK293T cells were collected 1-8 days after transfection, and mRNA was detected by Western blotting. DRACO and circRNA DRACO The protein expression level of Figure 3 From B to C, we can conclude that mRNA DRACO The expressed DRACO protein band gradually weakened with time and gradually degraded on the eighth day, while the circRNA DRACO The expressed DRACO protein still had a relatively obvious band on the eighth day, indicating that circRNADRACO The expressed protein lasts longer. This experiment proves that circRNA DRACO The transcriptional stability is good, and the expressed DRACO protein is sustained for a long time, suggesting that circRNA DRACO There are advantages in preventing viral diseases.

[0043] 3.4 Cell apoptosis assay circRNA DRACO , poly (I: C) (polyinosinic acid-polycytidylic acid, CAS No. 42424-50-0) and circRNA DRACO The cells were transfected with poly (I:C) and Caspase-Glo 3 / 7 to detect apoptosis signals. Figure 4 As shown, compared with the untreated control group, circRNA DRACO Co-transfection with poly (I: C) can induce cell apoptosis, and the addition of Pan-caspase inhibitor Z-VAD-FMK and caspase 9 inhibitor Z-LEHD-FMK can inhibit DRACO-mediated cell apoptosis in the presence of dsRNA, proving that circRNA DRACO The DRACO protein expressed after transfection into cells can bind to double-stranded RNA analogs, effectively mediating apoptosis of cells containing dsRNA, providing a basis for the next step of antiviral experiments.

[0044] 3.5 Validation of circRNA DRACO Inhibits the replication of influenza A (H1N1) virus Lipofectamine MessengerMAX transfection reagent was used to verify the transfection efficiency of circRNA in MDCK cells. ® Circ-eGFP was transfected into cells, and the transfection efficiency of MDCK cells was observed after 24 h. Figure 5 , where A is MDCK transfected circRNA eGFP Bright field image; B is MDCK transfected with circRNA eGFP Dark field image.

[0045] Depend on Figure 5 It can be concluded that the efficiency of circRNA transfection in MDCK cells is about 50-60%, which is lower than that of HEK293T cells. This shows that circRNA can be transfected into cells and translate proteins, but the amount of protein expressed by circRNA in cells may be affected by multiple factors such as transfection efficiency and cell state.

[0046] Then, the inoculation amount of influenza A (H1N1) virus (A / Puerto Rico / 8 / 1934) was determined. MDCK cells were inoculated in 12-well plates to a density of about 70-90% after cell attachment. The next day, the cells were inoculated with MOI=0.01 and MOI=0.1 respectively. Cell growth was observed after 48 hours. Figure 6 As shown, A is the growth of cells in the control group that were not infected with the virus; B is the CPE of MDCK cells when the multiplicity of infection was MOI=0.01; C is the CPE of MDCK cells when the multiplicity of infection was MOI=0.1; D is the circRNA DRACO Hemagglutination titer to prevent influenza A (H1N1) virus; E is circRNA DRACO Hemagglutination titer for treating influenza A (H1N1) virus; F is circRNA DRACO TCID for prevention of influenza A (H1N1) virus 50 titer; G is circRNA DRACO TCID for the treatment of influenza A (H1N1) virus 50 Titer. Data are presented as mean ± SD of 3 independent experiments, error bars are standard deviation, * P <0.05, *** P <0.001.

[0047] Depend on Figure 6 From A to C, we can conclude that 48 hours after infection, almost all cells with MOI=0.1 died, and too high an inoculation dose was not conducive to the experiment, so MOI=0.01 was selected for subsequent experiments.

[0048] MDCK cells were seeded in 12-well plates to a cell density of about 60% after cell attachment. CircRNA was first transfected DRACO 72 hours after transfection, influenza A (H1N1) virus was inoculated at a multiplicity of infection (MOI) of 0.01. 72 hours after infection, the cell supernatant was collected and 50 μL was taken for hemagglutination titer determination. Figure 6 D and F show that the untreated infected group and circRNA DRACO In the infection group 72 hours after transfection, the hemagglutination titer of both groups was 1:4. Take 100 μL of cell supernatant and make ten-fold serial dilutions, add them to 96-well plates respectively, and culture them for 3-5 days. Observe the occurrence of CPE at each dilution under an inverted microscope, and calculate the virus titer according to the Reed-Muench method. Transfect circRNA 72 hours before H1N1 influenza virus infection DRACO , which can effectively inhibit the replication of influenza virus by 0.68 TCID 50 Titer, at this time, only the virus titer of the infected group is transfected with circRNADRACO Group titer 10 0.68 times (about 4.78 times), indicating that circRNA DRACO It can effectively inhibit the replication of influenza A (H1N1) virus and prevent influenza A (H1N1) virus infection.

[0049] Depend on Figure 6 From E and G in Figure 1, we can conclude that the treatment group was first infected with the virus and then transfected with circRNA 6 h, 24 h, and 48 h later. DRACO , 72 h after infection, the virus was collected for hemagglutination titer determination, and the results showed that the hemagglutination titer of the untreated control group was 1:4; 6 h after infection, the circRNA was transfected DRACO , the hemagglutination titer was 1:2; circRNA was transfected 24 h after infection DRACO , the hemagglutination titer was 1:2; circRNA was transfected 48 h after infection DRACO , the hemagglutination titer was 1:4. After MDCK was infected with influenza A (H1N1) virus, the circRNA was transfected at 6 h and 24 h in the treatment group DRACO The hemagglutination titers of the two groups were different from those of the control group, and the virus titers of the samples were further measured. DRACO , inhibiting 1.05 TCID 50 At this time, the virus titer of the virus-infected group was 10 times that of the transfected group 6 hours later. 1.05 times (about 11.22 times); circRNA was transfected 24 hours after infection DRACO , inhibiting 0.92 TCID 50 At this time, the virus titer of the virus-infected group was 10 of the titer of the transfected group 24 hours later. 0.92 times (about 8.32 times); circRNA was transfected 48 hours after infection DRACO , inhibiting 0.54 TCID 50 At this time, the virus titer of the virus-infected group was 10 times that of the transfected group 48 hours later. 0.54 times (about 3.48 times), proving that after infection with H1N1 virus, circRNA was transfected within 48 h. DRACO It can effectively inhibit the replication of influenza virus and has potential therapeutic effect.

[0050] 3.6 Validation of circRNA DRACO Inhibits the replication of influenza A(H3N2) virus First, the inoculation dose of influenza A (H3N2) virus (A / Port Chalmers / 1 / 73) strain was determined. MDCK cells were inoculated in a 12-well plate so that the density of cells after attachment was about 70-90%. The next day, the virus was inoculated at MOI=0.01 and MOI=0.1, respectively.

[0051] The results are as follows Figure 7 As shown in A to C, A is the growth of cells in the control group that were not infected with the virus; B is the CPE of MDCK cells when the multiplicity of infection was MOI=0.01; C is the CPE of MDCK cells when the multiplicity of infection was MOI=0.1. It can be seen that after observing the cell growth 48 hours later, about half of the cells with MOI=0.1 died, and too high a virus dose was not conducive to the experiment, so MOI=0.01 was selected for subsequent experiments.

[0052] MDCK cells were seeded in 12-well plates to a cell density of about 60% after cell attachment. CircRNA was first transfected DRACO 72 hours after transfection, influenza A (H3N2) virus was inoculated at a multiplicity of infection (MOI) of 0.01. 72 hours after infection, the supernatant was collected and 50 μL was taken for hemagglutination titer determination. Figure 7 As shown in D, the untreated infected group and circRNA DRACO 72 hours after transfection, the hemagglutination titer of the two groups was 1:8. Take 100 μL of sample virus solution and make ten-fold serial dilutions, add them to 96-well plates, culture for 3-5 days, observe the CPE occurrence of each dilution under an inverted microscope, and calculate the virus titer according to the Reed-Muench method. Figure 7 As shown in F, where D is circRNA DRACO Hemagglutination titer to prevent influenza A(H3N2) virus; F is circRNA DRACO TCID for prevention of influenza A(H3N2) virus 50 Titer.

[0053] Depend on Figure 7 D and F in Figure 1 show that circRNA was transfected 72 h before influenza A (H3N2) virus infection. DRACO , inhibiting 0.31 TCID 50 Titer, at this time, only the virus titer of the infected group is transfected with circRNA DRACO Group titer 10 0.31 times (about 2.04 times), indicating that circRNA DRACO It can inhibit the replication of H3N2 influenza virus, but the preventive effect is not significant.

[0054] The treatment group was first infected with the virus, and then transfected with circRNA 6 h, 24 h, and 48 h later. DRACO The results showed that the hemagglutination titer of the untreated infection group was 1:16; circRNA was transfected 6 h after infection DRACO , the hemagglutination titer was 1:8; circRNA was transfected 24 h after infection DRACO , the hemagglutination titer was 1:16; circRNA was transfected 48 h after infection DRACO , the hemagglutination titer was 1:16. After influenza A H3N2 virus infected MDCK, only the treatment group transfected with circRNA at 6 h DRACO The hemagglutination titer of the group was different from that of the control group. The virus titer of the samples was further measured. The results were as follows Figure 7 As shown in E and G, E is a circRNA DRACO Hemagglutination titer for treating influenza A (H3N2) virus; G is circRNA DRACO TCID for the treatment of influenza A(H3N2) virus 50 Titer, in Figure 7 The data are presented as the mean ± SD of three independent experiments, with error bars representing standard deviation and ns representing P >0.05, * P <0.05, ** P <0.01, *** P <0.001.

[0055] Depend on Figure 7 E and G show that circRNA was transfected 6 h after infection with H3N2 influenza virus. DRACO , inhibiting 1.05 TCID 50 At this time, the virus titer of the virus-infected group was 10 times that of the transfected group 6 hours later. 1.13 times (about 13.49 times); circRNA was transfected 24 hours after infection DRACO , inhibiting 0.92 TCID 50 At this time, the virus titer of the virus-infected group was 10 of the titer of the transfected group 24 hours later. 0.85 times (about 7.08 times); circRNA was transfected 48 hours after infection DRACO , inhibiting 0.54 TCID 50 At this time, the virus titer of the virus-infected group was 10 times that of the transfected group 48 hours later. 0.57 times (about 3.72 times), proving that after infection with influenza A (H3N2) virus, circRNA was transfected within 48 h. DRACO It can effectively inhibit the replication of influenza virus, indicating that circRNA DRACO It has a therapeutic effect on influenza A (H3N2) virus infection.

[0056] From the above examples, it can be concluded that the present invention designs and constructs a circRNA that can successfully express the broad-spectrum antiviral protein DRACO in cells. DRACO . And transfect 0.5-2 μg of circRNA DRACO There is no significant effect on cell activity, indicating that circRNA DRACO The biosafety of circRNA is good. In addition, the experimental results of qPCR proved that circRNA DRACO The transcriptional level is stable, and Western Blot results show that circRNA DRACO The expressed protein level is long-lasting, suggesting that circRNA DRACO It has advantages in preventing viral diseases. Further apoptosis experiments were used to investigate circRNA DRACO The results showed that circRNA DRACO It can effectively mediate apoptosis of cells containing dsRNA, and this apoptosis can be eliminated under the action of caspase-9 and Pan-caspase inhibitors, proving that circRNA DRACO The DRACO protein expressed in cells can bind to long dsRNA, activate caspase, trigger a cascade of apoptosis, and thus kill the cells.

[0057] circRNA DRACO The results of in vitro antiviral experiments showed that circRNA was transfected within 72 h before and 48 h after influenza A (H1N1) virus infection. DRACO , which can effectively inhibit viral replication; circRNA was transfected within 48 h after influenza A (H3N2) virus infection DRACO It can effectively inhibit viral replication, but circRNA was transfected 72 h before influenza A (H3N2) virus infection DRACO It can inhibit viral replication, but the effect is not significant. DRACO It has better prevention and treatment effects on H1N1 influenza and better treatment effects on H3N2 influenza, but the prevention effect is general. This difference in the activity of inhibiting viral replication may be related to the two different types of virus infection, the type and level of dsRNA produced during replication, etc., but the specific reasons need further study. However, as far as the results of this invention are concerned, circRNA DRACO The ability to successfully express DRACO protein in cells, bind to the virus to cause cell apoptosis, and inhibit the replication of influenza A virus proves the feasibility of converting the broad-spectrum antiviral protein DRACO into a circRNA drug form to exert an antiviral effect.

[0058] In general, the present invention successfully designed and constructed a circRNA that can express the broad-spectrum antiviral protein DRACO in vivo, and can be successfully used to inhibit the replication of influenza A virus. It not only expands the ideas for the in vitro application research and development of circRNA, but also demonstrates its potential as a platform for the development of drugs to prevent or treat viral infections. At the same time, it provides more possibilities for the development of antiviral drugs and lays a foundation for their further research.

[0059] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A circRNA DRACO , characterized in that, The circRNA DRACO The nucleotide sequence is shown in SEQ ID NO.

1.

2. The circRNA according to claim 1 DRACO Application in the preparation of medicines for preventing and treating influenza viruses.

3. The circRNA according to claim 1 DRACO Application in the preparation of anti-influenza virus vaccines.

4. The use according to claim 2 or 3, characterized in that: The influenza virus includes influenza A virus.

5. The use according to claim 4, characterized in that: The influenza A virus includes influenza A (H1N1) virus and / or influenza A (H3N2) virus.

6. The use according to claim 2, characterized in that: The drugs include drugs that inhibit influenza virus replication.

7. A drug for preventing and treating influenza virus, characterized in that: The active ingredient of the drug includes the circRNA according to claim 1 DRACO .

8. The drug according to claim 7, characterized in that The medicine also includes pharmaceutically acceptable excipients.

9. A vaccine against influenza virus, characterized in that: The effective ingredient of the vaccine includes the circRNA according to claim 1 DRACO .

10. The vaccine according to claim 9, characterized in that The vaccine also includes a vaccine adjuvant.

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