Application of RBM6 in animal anti-influenza

Abstract

The invention discloses application of RBM6 in animal anti-influenza, and belongs to the field of animal breeding for disease resistance, after RBM6 gene is knocked out in a PK-15 cell in a targeted manner through a CRISPR / Cas9 knockout technology, IAV proliferation can be inhibited, and IAV infection can be resisted; meanwhile, after the RBM6 is knocked out in the mouse in a targeted mode through a CRISPR / Cas9 knockout technology, IAV replication virus titer can be inhibited, and therefore weight loss and death caused by IAV infection are resisted. The invention provides a new target for developing a novel vaccine drug for effectively and safely preventing and treating IAV, also provides a new candidate target gene for breeding research of anti-IAV, and has a wide application prospect.

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 infectious disease caused by influenza A virus (IAV), which can be transmitted between humans and animals, causing acute respiratory diseases in animals and humans. Among animal influenza viruses, especially the outbreak of highly pathogenic avian influenza, the poultry farming industry has suffered huge losses. Since 1959, highly pathogenic avian influenza of H5 and H7 subtypes has broken out in poultry or wild birds in many countries, resulting in a large number of deaths. In addition, avian influenza viruses can also infect humans. The H5N1 and H7N9 avian influenza viruses that broke out in 1997 and 2013, respectively, caused a large number of infections in the population and had an extremely high mortality rate. However, due to the continuous recombination and mutation of influenza viruses and the enhancement of influenza virus resistance, the prevention and treatment effects of traditional vaccines and antiviral drugs are becoming increasingly limited. Therefore, there is an urgent need to develop a new method for effectively regulating the disease resistance of animals, especially birds, against influenza viruses. At the same time, due to the unclear study of the interaction mechanism between IAV and the host and the lack of target genes for disease-resistant breeding, the development of drugs targeting IAV and the progress of disease-resistant breeding have been seriously restricted.

[0003] RBM6 is a protein containing an RNA-binding motif (RRM) and belongs to the RNA-binding protein family. RRM is a conserved domain that can bind to RNA molecules and participate in the processing, transport and stability regulation of RNA. RBM6 mainly binds to RNA through its RRM domain and regulates the expression and stability of various RNA molecules.

[0004] Recent studies have shown that RBM6 plays an important role in the occurrence and development of a variety of diseases. In cancer, RBM6 can affect the proliferation, differentiation and apoptosis of tumor cells by interacting with mRNA associated with oncogenes or tumor suppressor genes. In addition, RBM6 is also involved in the occurrence and development of other diseases such as neurodegenerative diseases and cardiovascular diseases. Because RBM6 plays an important role in a variety of diseases, it 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 the expression of the RBM6 gene 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 that targets and knocks out 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 composed of CasRx, Cpf1, Cas9, Cas13a, Cas13b, Cas13c or other proteins or their expression vectors and corresponding gRNA or its expression vector.

[0010] Application of a substance that inhibits the activity of RBM6 protein 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 the RBM6 protein is an antibody or a small molecule inhibitor targeting the 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 by 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 targeted knockout of RBM6 in PK-15 cells through CRISPR / Cas9 knockout technology, and found for the first time that RBM6 knockout cells can resist IAV infection by inhibiting the proliferation of IAV; at the same time, the applicant targeted knockout of RBM6 in mice through CRISPR / Cas9 knockout technology, and found for the first time that RBM6 knockout mice can resist weight loss and death caused by IAV infection by inhibiting IAV replication virus titer. This gene can be used as a new antiviral target for IAV. The present invention provides a new target for the development of a new vaccine drug that effectively and safely prevents and treats IAV, and also provides a new candidate target gene for anti-IAV breeding research, which has 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 detected the expression of RBM6 protein 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] Fig. 9 : The effect of RBM6 knockout in mice on the changes in mouse mortality caused by IAV infection.

[0026] Fig.10 :The effect of RBM6 knockout in mice on the 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 line

[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 oligo sgRNA synthesized by the company to 100nM, take 1μL of each complementary sequence and add 8μLddH2O 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 sequencing is correct, the bacterial culture is expanded and the plasmid is extracted according to the operating instructions of the Omega Plasmid Extraction 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 line

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

[0040] (2) Porcine kidney PK-15 cells stably expressing Cas9 protein (PK-15-Cas9) 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% dual antibody.

[0041] (3) When the cell density reaches 20% to 30%, add sgRNA lentivirus targeting the RBM6 gene and DMEM 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 the cells are cultured until a confluent monolayer is formed. 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) The culture was continued for about 14 days until the single cells grew into single cell colonies. The cells of the single cell colonies were digested with trypsin and subcultured into 24-well cell culture plates to obtain monoclonal gene knockout cell lines.

[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), the 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: pre-denaturation at 95°C for 5 min, cycle reaction was denaturation at 95°C for 20 s, annealing at 60°C for 20 s, extension at 72°C for 1 min, a total of 35 cycles, and final extension at 72°C for 5 min. The PCR product was 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, thereby failing to produce RBM6 protein.

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

[0056] (1) Preparation of protein samples

[0057] After the PK-15-Cas9 cells and RBM6-KO monoclonal cell lines have grown to full size, the culture medium is aspirated and washed 1-2 times with pre-cooled PBS. After scraping the cells, add an appropriate amount of protein lysis buffer (1 μL of protease inhibitor protease inhibitor cocktail (Sigma, P8340) is added to every 100 μL RRIPA lysis buffer), lyse on ice for 30 minutes, centrifuge at 12000 rpm for 10 minutes, aspirate the supernatant of the lysis product into a 1.5 mL centrifuge tube, add the corresponding volume of 5×SDS-PAGE loading buffer, boil at 100°C for 10 minutes, and store 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 detection found 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 Using CCK-8 to detect the activity of RBM6-KO monoclonal cell lines

[0065] (1) The same number and appropriate amount of experimental group cells and control group cells were seeded into 96-well plates, with 8 technical replicates in each group. The cells were cultured 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, measure the absorbance at 450 nm using an enzyme reader.

[0068] (4) 24h and 36h after cell inoculation, perform the same procedure 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 activity.

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

[0072] The RBM6-KO cell line and the control cell line were inoculated into a 12-well plate. After the cells were fully covered with a monolayer, the culture medium was discarded and washed twice with DMEM. At the same time, influenza viruses of different species were infected at MOI = 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, 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 TCID 50 The virus 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) Inoculate an appropriate amount of MDCK cells into a 96-well cell culture plate.

[0075] (3) When the cells are fully covered with a monolayer, wash the cells twice with serum-free DMEM, then inoculate each dilution of the virus solution into one column (8 wells) of a 96-well cell plate, 100 μL per well, and replace with a maintenance solution containing 0.3% TPCK after 1 hour. Culture the cells in a 37°C CO2 incubator.

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

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

[0078] The results are as follows Figure 6 As shown, 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 MOI of 0.01 and 0.1, respectively, to detect the ability of RBM6 knockout cells to resist IAV-induced cell death. The same number of RBM6-KO and control cells were inoculated in 12-well plates, and cell growth and proliferation were observed under a microscope 72 hours after IAV infection. Figure 7 As shown in the figure, 72 hours after IAV infection, the cells in the control group had severe cytopathic effects and a large number of cell deaths; compared with the cells in the control group, the survival rate of the RBM6 knockout cells was significantly increased under the infection conditions of IAV MOI of 0.01 and 0.1. The experimental results show that knocking out RBM6 can resist 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. CRISPR / Cas9 technology was used to design sgRNA, and RBM6 gene knockout (RBM6) was obtained by applying 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 nasal drops as the experimental group; at the same time, mice were anesthetized with ether and an equal volume of PBS was added as the control group.

[0083] 4.1 Knockout of RBM6 in mice inhibits weight loss and death caused by IAV infection

[0084] RBM6 + / - After wild-type mice were infected with influenza virus (A / Hunan / 42443 / 2015), the mental state of the mice was observed every day, and the weight and survival rate of the mice were recorded for 14 consecutive days. Figure 8 and Fig. 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 the RBM6 + / -In mice, IAV infection initially caused weight loss, but the weight gradually recovered on the 9th day after infection, and the final survival rate of mice was 40%. The experimental results showed that knocking out RBM6 in mice inhibited weight loss and death caused by IAV infection.

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

[0086] In RBM6 + / - On the 3rd and 5th day after wild-type mice were infected with IAV, 3 mice were taken from each of the 4 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, and the supernatant was taken and TCID 50 The test measures the viral titer of IAV in lung tissue. Fig.10 As shown, RBM6 was expressed on days 3 and 5 after IAV infection. + / - The virus 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 titer of IAV replication.

Claims

1. Use of a substance that inhibits the expression of the RBM6 gene in the preparation of a drug for treating or preventing diseases caused by influenza A virus infection.

2. The use according to claim 1, characterized in that: The substance that inhibits the expression of the RBM6 gene in cells is an RNA interference fragment targeting the RBM6 gene or a gene editing vector that targets and knocks out the RBM6 gene.

3. The use according to claim 2, characterized in that: The RNA interference fragment is dsRNA, shRNA or siRNA.

4. The use according to claim 2, characterized in that: The gene editing vector is a CRISPR / Cas editing system.

5. Use of substances that inhibit the activity of RBM6 protein in the preparation of drugs for treating or preventing diseases caused by influenza A virus infection.

6. The use according to claim 5, characterized in that: The substance that inhibits the activity of the RBM6 protein is an antibody or a small molecule inhibitor targeting the RBM6 protein.

7. A method for constructing an anti-influenza A virus animal model or cell model, characterized in that: The RBM6 gene of animals or cells is knocked out through gene editing technology to obtain an animal model or cell model with improved resistance to influenza A virus.

8. The construction method according to claim 7, characterized in that: The animal is a mouse, and the cells are pig-derived kidney cells.

9. The construction method according to claim 7 or 8, characterized in that: The improved resistance refers to inhibiting the proliferation of influenza A virus and resisting weight loss or death caused by influenza A virus infection.

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