Application of RBM6 in animal anti-influenza

Abstract

The present invention discloses the application of RBM6 in animal influenza resistance, belonging to the field of animal disease resistance breeding. Targeted knockout of the RBM6 gene in PK-15 cells using CRISPR / Cas9 technology can inhibit the proliferation of IAVs, thereby resisting IAV infection. At the same time, targeted knockout of RBM6 in mice using CRISPR / Cas9 technology can inhibit IAV replication and viral titer, thereby resisting weight loss and death caused by IAV infection. This invention provides a new target for the development of new vaccines and drugs that are effective and safe for the prevention and treatment of IAVs, and also provides a new candidate target gene for anti-IAV breeding research, with broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of animal disease resistance breeding, and in particular to the application of RBM6 in animal resistance to influenza. Background Art

[0002] Influenza is a zoonotic disease caused by influenza A virus (IAV). It can be transmitted between humans and animals, causing acute respiratory illness in both animals and humans. Among animal influenza viruses, outbreaks of highly pathogenic avian influenza (HPAI), particularly H5 and H7 subtypes, have caused significant losses to the poultry industry. Since 1959, outbreaks of HPAI, H5N1 and H7N9, have occurred in poultry and wild birds in many countries, resulting in massive deaths. Avian influenza viruses can also infect humans. The H5N1 and H7N9 avian influenza viruses that emerged in 1997 and 2013, respectively, have caused widespread human infection and extremely high mortality rates. However, due to the continuous recombination and mutation of influenza viruses and the increasing drug resistance of influenza viruses, the effectiveness of traditional vaccines and antiviral drugs is becoming increasingly limited. Therefore, there is an urgent need to develop new and effective methods for regulating influenza virus resistance in animals, particularly birds. Furthermore, the lack of clear understanding of the mechanisms of IAV-host interaction and the lack of target genes for disease-resistant breeding have severely hampered the development of IAV-targeted drugs and the progress of disease-resistant breeding.

[0003] RBM6 is a protein that contains an RNA-binding motif (RRM) and belongs to the RNA-binding protein family. RRM is a conserved domain that binds to RNA molecules and is involved in regulating RNA processing, transport, and stability. RBM6 primarily binds to RNA through its RRM domain, regulating the expression and stability of various RNA molecules.

[0004] Recent studies have shown that RBM6 plays a crucial role in the development and progression of various diseases. In cancer, RBM6 can influence tumor cell proliferation, differentiation, and apoptosis by interacting with mRNAs associated with oncogenes or tumor suppressor genes. Furthermore, RBM6 is involved in the development and progression of other diseases, including neurodegenerative and cardiovascular diseases. Because of its crucial role in various diseases, RBM6 is considered a potential therapeutic target. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a new method for effectively regulating the disease resistance of animals to influenza A virus.

[0006] The technical solution of the present invention is: use of a substance that inhibits RBM6 gene expression in the preparation of a drug for treating or preventing diseases caused by influenza A virus infection.

[0007] Furthermore, the substance that inhibits the expression of the RBM6 gene in the cell is an RNA interference fragment targeting the RBM6 gene or a gene editing vector targeting the knockout of the RBM6 gene.

[0008] Furthermore, the RNA interference fragment is dsRNA, shRNA or siRNA.

[0009] Furthermore, the gene editing vector is a CRISPR / Cas editing system, such as a gene knockout system consisting of CasRx, Cpf1, Cas9, Cas13a, Cas13b, Cas13c or other proteins or their expression vectors and corresponding gRNA or its expression vector.

[0010] Use of a substance that inhibits RBM6 protein activity in the preparation of a drug for treating or preventing diseases caused by influenza A virus infection.

[0011] Furthermore, the substance that inhibits the activity of RBM6 protein is an antibody or small molecule inhibitor targeting RBM6 protein.

[0012] A method for constructing an animal model or cell model resistant to influenza A virus, wherein the RBM6 gene of the animal or cell is knocked out through gene editing technology, thereby obtaining an animal model or cell model with improved resistance to influenza A virus.

[0013] Furthermore, the animal is a mouse, and the cells are pig-derived kidney cells.

[0014] Furthermore, the improved resistance refers to inhibiting the proliferation of influenza A virus and resisting weight loss or death caused by influenza A virus infection.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The applicant used CRISPR / Cas9 knockout technology to target and knock out RBM6 in PK-15 cells, and for the first time found that RBM6-knockout cells can resist IAV infection by inhibiting IAV proliferation. At the same time, using CRISPR / Cas9 knockout technology to target and knock out RBM6 in mice, the applicant found for the first time that RBM6-knockout mice can resist IAV infection-induced weight loss and death by inhibiting IAV replication and viral titer. This gene can serve as a new antiviral target for IAV. The present invention provides a new target for the development of new vaccines and drugs that are effective and safe for the prevention and treatment of IAV, and also provides a new candidate target gene for anti-IAV breeding research, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Design of sgRNA targeting porcine RBM6 gene.

[0018] Figure 2 : Sequencing identification of plasmid.

[0019] Figure 3 : Gene level sequencing detected the RBM6 gene sequence in PK-15-Cas9-RBM6 cells.

[0020] Figure 4 : Western Blot detection of RBM6 protein expression in PK-15-Cas9-RBM6 cells.

[0021] Figure 5 :CCK-8 was used to detect the activity of cells after RBM6 knockout.

[0022] Figure 6 : Effect of RBM6 knockout in PK-15 cells on IAV replication.

[0023] Figure 7 : Effect of RBM6 knockout in PK-15 cells on cell death caused by IAV infection.

[0024] Figure 8 : Effect of RBM6 knockout in mice on body weight changes caused by IAV infection.

[0025] Figure 9 : The effect of RBM6 knockout in mice on the changes in mouse mortality caused by IAV infection.

[0026] Figure 10 : Effect of RBM6 knockout in mice on viral titer in mouse lung tissue caused by IAV infection. DETAILED DESCRIPTION

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0028] Example 1: Construction and verification of RBM6 knockout cell lines

[0029] The NCBI number of the mRNA of the target gene RBM6 gene is 100515053, the corresponding CDS sequence is shown in SEQ ID NO.1, and it encodes the protein shown in SEQ ID NO.2.

[0030] 1.1sgRNA design and expression vector construction

[0031] (1) sgRNA design: Select the RBM6 coding sequence targeted by the sgRNA. See the design of the targeting sgRNA for details. Figure 1, the sgRNA sequence was 5′-GCATGGTGACTATCGAGGAG-3′.

[0032] (2) After diluting the company's synthesized oligo sgRNA to 100nM, take 1μL of each complementary sequence and add 8μL ddH2O to mix.

[0033] (3) Place the mixture in a PCR instrument and anneal to obtain double-stranded gRNA. The annealing program is as follows: 37°C, 30 min; 95°C, 5 min; 95°C-25°C, -0.1°C / s; 25°C, 1 min.

[0034] (4) The Lenti-sgRNA-EGFP vector was digested with BbsI and reacted at 37°C for 30 min. The target fragment was recovered after nucleic acid gel electrophoresis, and the vector and sgRNA were connected using T4 ligase.

[0035] (5) The ligation product was transformed into DH5α, plated, and a single colony was picked for sequencing verification.

[0036] (6) After the colony is correctly identified by sequencing, the bacterial culture is expanded and the plasmid is extracted according to the operating instructions of the Omega Plasmid Miniprep Kit (D6950-02).

[0037] The results are as follows Figure 2 As shown, the sgRNA targeting the RBM6 gene was successfully connected to the vector, indicating that the sgRNA expression vector was successfully constructed.

[0038] 1.2 Construction of RBM6 knockout monoclonal cell lines

[0039] (1) Press Beyotime's Lipo8000 TM The sgRNA expression plasmid and the auxiliary plasmids pMD2.G (Addgene, 12259) and psPAX2 (Addgene, 12260) were co-transfected with the transfection reagent (C0533) according to the operating instructions to package the lentivirus.

[0040] (2) Porcine kidney cells PK-15 (PK-15-Cas9) stably expressing Cas9 protein were inoculated into two wells of a 6-well cell culture plate, one well as the experimental group and the other well as the control group, and cultured in DMEM medium containing 10% FBS and 1% double antibody.

[0041] (3) When the cell density reaches 20% to 30%, add sgRNA lentivirus targeting the RBM6 gene and DMEM culture medium containing 10% FBS and 1% double antibody, and add polybrene with a final concentration of 8 μg / mL to incubate the cells.

[0042] (4) 12 hours after lentivirus infection, repeat the lentivirus infection.

[0043] (5) After the cells are cultured to a confluent monolayer, they are transferred to a T25 cell culture flask and cultured until the cells are confluent monolayer. The fluorescence state of the cells is observed and the fluorescent cells are sorted using a flow cytometer.

[0044] (6) The cells obtained by flow cytometry sorting were inoculated into a 96-well cell plate by limiting dilution method to form single cell colonies.

[0045] (7) Continue culturing for about 14 days until the single cells grow into single cell colonies. Digest the cells of the single cell colonies with trypsin and transfer them to a 24-well cell culture plate to obtain a monoclonal gene knockout cell line.

[0046] 1.3 Verification of RBM6 expression and cell activity in RBM6-KO cell lines

[0047] 1.3.1 Detection of RBM6 gene editing in RBM6-KO monoclonal cells by genomic DNA sequencing

[0048] (1) Extraction of cell genomic DNA

[0049] According to the operating instructions of TIANGEN's blood / cell / tissue genomic DNA extraction kit (DP304), genomic DNA of the RBM6-KO monoclonal cell line and the control group PK-15-Cas9 cells was extracted.

[0050] (2) PCR amplification of the sequence near the sgRNA targeting RBM6 gene site and sent to the company for sequencing. The primer sequences are as follows:

[0051] RBM6-F: 5'-CAGAAGAATGTGCTGTGATT-3'

[0052] RBM6-R: 5'-GGAGGAGCATCCCTAACCTCT-3'.

[0053] The PCR program was set as follows: initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 20 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 1 minute, with a final extension at 72°C for 5 minutes. The PCR products were sent to the company for sequencing.

[0054] The sequencing results are as follows Figure 3 As shown, the RBM6-KO monoclonal cell line was found to have a 16-base deletion, resulting in a frameshift mutation in the RBM6 gene, and thus no RBM6 protein was produced.

[0055] 1.3.2 Detection of RBM6 expression in RBM6-KO cells using Western Blot

[0056] (1) Preparation of protein samples

[0057] After PK-15-Cas9 cells and RBM6-KO monoclonal cell lines have grown to confluence, the culture medium is aspirated and the cells are washed 1-2 times with pre-chilled PBS. After scraping the cells, an appropriate amount of protein lysis buffer is added (1 μL of protease inhibitor cocktail (Sigma, P8340) is added per 100 μL of RRIPA lysis buffer). Lyse on ice for 30 minutes, then centrifuge at 12,000 rpm for 10 minutes. The supernatant of the lysate is transferred to a 1.5 mL centrifuge tube, and the corresponding volume of 5× SDS-PAGE loading buffer is added. The cells are boiled at 100°C for 10 minutes and stored at -20°C.

[0058] (2) SDS-PAGE electrophoresis

[0059] (3) Western Blot analysis:

[0060] a) The primary antibodies were RBM6 rabbit polyclonal antibody (Proteintech, 14360-1-AP) and GAPDH mouse monoclonal antibody (Proteintech, 60004-1-Ig).

[0061] b) Secondary antibody incubation: HRP-conjugated goat anti-mouse IgG (ABclonal, AS071), HRP-conjugated goat anti-rabbit IgG (ABclonal, AS028);

[0062] c) Protein detection: ECL luminescence method was used.

[0063] The results are as follows Figure 4 As shown, Western Blot analysis revealed that the PK-15-Cas9 cells in the control group expressed RBM6 protein, but the RBM6-KO monoclonal cell line in the experimental group did not express RBM6 protein, indicating that the RBM6 gene had been successfully knocked out in this cell line.

[0064] 1.3.3 Detection of RBM6-KO monoclonal cell line activity using CCK-8

[0065] (1) Equal numbers of experimental and control cells were seeded into 96-well plates, with eight technical replicates per group. Culture was continued in a 5% CO2, 37°C incubator.

[0066] (2) 12 hours after cell inoculation, add 10 μL of CCK-8 reagent to each well and continue incubating in the incubator for 1-2 hours.

[0067] (3) After incubation, the absorbance at 450 nm was measured using a microplate reader.

[0068] (4) 24h and 36h after cell inoculation, perform the same steps as above.

[0069] (5) Perform calculations according to the formula.

[0070] The results of CCK-8 cell viability assay were as follows: Figure 5 As shown, knockout of RBM6 had no significant effect on cell viability.

[0071] Example 2: Knockout of RBM6 inhibits IAV replication

[0072] The RBM6-KO cell line and the control cell line were seeded into a 12-well plate. After the cells were confluent as a monolayer, the medium was discarded and the cells were washed twice with DMEM. At the same time, they were infected with influenza viruses of different species at an MOI of 0.01, namely A / swine / Hubei / 221 / 2016 (HuB / H1N1), A / swine / Henan / F26 / 2017 (F26 / H1N1), A / PuertoRico / 8-SV14 / 1934 (PR8 / H1N1) and A / chicken / Shanghai / SC197 / 2013 (SH13 / H9N2). After incubation at 37°C for 1 hour, the supernatant was discarded, the cells were washed twice with DMEM, and then cultured in a 37°C incubator with medium containing 0.1% TPCK. The cell supernatant was collected 12, 24, and 36 hours after virus infection and the TCID 50 The viral titer of IAV in the supernatant was determined by the test. The specific method is as follows:

[0073] (1) Dilute the collected virus solution 10-fold to an appropriate concentration.

[0074] (2) An appropriate amount of MDCK cells was inoculated into a 96-well cell culture plate.

[0075] (3) When the cells are confluent as a monolayer, wash the cells twice with serum-free DMEM. Then, inoculate one vertical column (8 wells) of a 96-well plate with 100 μL of each virus dilution per well. After 1 hour, replace the solution with maintenance medium containing 0.3% TPCK. Incubate the plate in a 37°C CO2 incubator.

[0076] (4) After 72 hours, the cell supernatant in the 96-well plate was transferred to a U-shaped hemagglutination plate, and the hemagglutination test was performed to determine whether the well contained influenza virus.

[0077] (5) Calculate the TCID of the virus using the Reed-Muench method 50 .

[0078] The results are as follows Figure 6 As shown in the figure, when RBM6 was knocked out, the proliferation of the four IAVs was significantly inhibited.

[0079] Example 3: RBM6 knockout cells are resistant to IAV-induced cell death

[0080] After a certain dose of IAV infects PK-15 cells, it will cause host cell pathology and even induce cell death. HuB-H1N1 was infected at an MOI of 0.01 and 0.1, respectively, to test the ability of RBM6 knockout cells to resist IAV-induced cell death. The same number of RBM6-KO and control cells were seeded in 12-well plates, and cell growth and proliferation were observed under a microscope 72 hours after IAV infection. Figure 7 As shown, 72 hours after IAV infection, cells in the control group showed severe cytopathic effects and massive cell death. Compared with the control group, RBM6 knockout cells showed significantly increased cell survival at both IAV infection MOIs of 0.01 and 0.1. These results demonstrate that knocking out RBM6 protects against IAV-induced cell death.

[0081] Example 4: Knockout of RBM6 can inhibit IAV-induced mouse death

[0082] RBM6 is located on chromosome 9 of mice. Using CRISPR / Cas9 technology, sgRNA was designed and RBM6 gene knockout (RBM6) was obtained by high-throughput electroporation of fertilized eggs combined with mouse subculture. + / - ) mice. + / - ) mice and wild-type mice were anesthetized with ether and infected with the same dose of influenza virus (A / Hunan / 42443 / 2015) by intranasal drops as the experimental group; at the same time, mice were anesthetized with ether and then dripped with an equal volume of PBS as the control group.

[0083] 4.1 RBM6 knockout in mice suppresses IAV infection-induced weight loss and mortality

[0084] RBM6 + / - After wild-type mice were infected with influenza virus (A / Hunan / 42443 / 2015), the mental state of the mice was observed daily, and the weight and survival rate of the mice were recorded for 14 consecutive days. Figure 8 and Figure 9 As shown, the weight of mice in the PBS control group gradually increased; when mice were infected with IAV, the weight of wild-type mice gradually decreased and all died on the 8th day after IAV infection; while in RBM6 + / -In mice, IAV infection initially causes weight loss, which gradually recovers by day 9, with a 40% survival rate. The results showed that knocking out RBM6 in mice suppressed IAV-induced weight loss and mortality.

[0085] 4.2 Knockout of RBM6 in mice inhibits IAV replication and viral titers

[0086] In RBM6 + / - On the 3rd and 5th day after wild-type mice were infected with IAV, three mice were taken from each of the four groups and killed. The lung tissues of the mice were taken and diluted with 0.8 ml of PBS containing 1% double antibody and homogenized. The homogenate was centrifuged at 12000 rpm for 10 min at 4 ° C. The supernatant was collected and analyzed by TCID 50 The test measures the viral titer of IAV in lung tissue. Figure 10 As shown, RBM6 was expressed on days 3 and 5 after IAV infection. + / - The viral titer in the lung tissue of mice was significantly lower than that of wild-type mice. The experimental results showed that knocking out RBM6 in mice inhibited the viral titer of IAV replication.

Claims

1. Use of an sgRNA targeting the RBM6 gene for knockout in the preparation of a drug for treating or preventing diseases caused by influenza A virus infection, wherein the sgRNA sequence is 5'-GCATGGTGACTATCGAGGAG-3'.

2. An anti-influenza A virus cell model, characterized in that: The RBM6 gene of porcine kidney cells was knocked out using a CRISPR / Cas9 editing vector, thereby obtaining a cell model with improved resistance to influenza A virus. The sgRNA sequence in the CRISPR / Cas9 editing vector was 5'-GCATGGTGACTATCGAGGAG-3'.

3. The anti-influenza A virus cell model according to claim 2, characterized in that: The improvement in resistance refers to the inhibition of the proliferation of influenza A virus.

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