Application of STK11 in animal anti-influenza

Abstract

The invention discloses application of STK11 in animal anti-influenza, STK11 is knocked out in a targeted mode in NPTr cells through a CRISPR / Cas9 knockout technology, and it is found for the first time that the cells with the STK11 knocked out can inhibit IAV replication so as to resist IAV infection; meanwhile, the expression of endogenous STK11 is down-regulated in the mouse through an RNA interference technology, and the situation that the mouse with the knocked-down STK11 can inhibit IAV replication so as to improve the conditions of weight loss and death of the mouse caused by IAV infection is found for the first time. The gene can be used as a novel antiviral target of IAV. The invention provides a new target for developing a novel 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-resistant breeding, and particularly to the application of serine / threonine kinase 11 (STK11) in animal anti-influenza. Background Art

[0002] Influenza A virus (IAV) can infect a variety of mammals and birds. Among them, avian influenza virus, as a highly contagious pathogen, uses wild waterfowl and poultry as natural hosts and has the potential to cross-species infect mammals. In the past century, avian influenza virus has directly caused four human influenza pandemics, seriously threatening global public health security and causing significant economic losses to the poultry farming industry. In recent years, sporadic human infection events caused by highly pathogenic avian influenza such as H5 and H7 have occurred frequently and have a high fatality rate, highlighting their potential pandemic risk. IAV is extremely prone to mutation and recombination, resulting in the loss of efficacy of drugs targeting viral proteins and the reduction of vaccine effectiveness. Therefore, finding drugs that target host factors for anti-influenza, especially anti-avian influenza, has become an urgent need at present. However, there is still a lack of systematic analysis of the molecular mechanism of IAV replication, especially how host factors regulate the virus life cycle. Identifying key host factors that the virus depends on for replication can not only reveal the molecular basis of its cross-species transmission, but also provide a theoretical basis for developing broad-spectrum antiviral strategies targeting host-virus interactions and host-targeted disease-resistant breeding, which has important scientific value for preventing and controlling the epidemic caused by IAV, especially avian influenza virus, and ensuring global public health security.

[0003] STK11, also known as LKB1 (liver kinase B1), is a highly conserved serine / threonine kinase encoded by the STK11 gene located on the short arm of chromosome 19. The full-length STK11 protein consists of 433 amino acids, including an N-terminal domain with a nuclear localization signal, a central domain with kinase catalytic activity, and a C-terminal domain with regulatory functions. STK11 is a constitutively active master kinase that forms a heterotrimeric complex with the pseudokinase STRAD and the scaffold protein MO25, and this interaction causes it to translocate from the nucleus to the cytoplasm. In the cytoplasm, STK11 phosphorylates AMPK and 12 other AMPK-related kinases, including MARK-1 / 2 / 3 / 4, NUAK1 / 2, SIK-1 / 2 / 3, BRSK-1 / 2, and SNAK, and plays an important role in processes such as cellular energy regulation, cell metabolism, cell polarity, and cellular damage response. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide the application of STK11 in the prevention and control of influenza virus, especially avian influenza virus.

[0005] The technical solution of the present invention is: the use of an inhibitor of STK11 gene or protein in the preparation of a drug against influenza A virus.

[0006] Further, the inhibitor is any one of the following substances:

[0007] (a) a substance that silences the STK11 gene;

[0008] (b) a substance that targets and knocks out the STK11 gene;

[0009] (c) a substance that inhibits the activity of STK11 protein.

[0010] Further, the substance that silences the STK11 gene is siRNA, shRNA, dsRNA or miRNA that targets and silences the STK11 gene.

[0011] Further, the substance that targets and knocks out the STK11 gene is the CRISPR / Cas editing system.

[0012] Further, in the CRISPR / Cas editing system, the target sequence of sgRNA is as shown in SEQ ID No.1.

[0013] Further, the substance that inhibits the activity of STK11 protein is a specific small molecule compound or antibody that can bind to STK11 protein and inhibit its activity.

[0014] A method for constructing a cell model against influenza A virus, in which the STK11 gene of the cell is knocked out by the CRISPR / Cas editing system or the interfering RNA that silences the STK11 gene is transfected into the cell, so as to obtain a cell model with inhibited STK11 gene expression and enhanced resistance to influenza A virus.

[0015] A method for constructing an animal model against influenza A virus, in which the STK11 gene of the animal is knocked out by the CRISPR / Cas editing system or the interfering RNA that silences the STK11 gene is transfected into the animal, so as to obtain an animal model with inhibited STK11 gene expression and enhanced resistance to influenza A virus.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The applicant used CRISPR / Cas9 gene knockout technology to target and knockout STK11 in NPTr cells, and for the first time found that cells with STK11 knockout could inhibit the replication of IAV and thus resist IAV infection. At the same time, by using RNA interference technology to down-regulate the expression of endogenous STK11 in mice, it was first found that mice with STK11 knockdown could improve the weight loss and death of mice caused by IAV infection by inhibiting IAV replication. This gene can be used as a new antiviral target for IAV. The present invention provides a new target for the development of new drugs for effectively and safely preventing and treating IAV, and also provides a new candidate target gene for breeding research against IAV, with broad application prospects. Description of the Drawings

[0018] Figure 1 : Construction and verification of a lentiviral vector targeting the porcine STK11 gene; (A) sgRNA sequence targeting the porcine STK11 gene; (B) Sequencing identification of successful construction of the sgRNA lentiviral vector.

[0019] Figure 2 : Construction and verification of the NPTr-Cas9-STK11 monoclonal cell line; (A) Determination of the knockout of the STK11 gene in NPTr-Cas9-STK11 knockout cells at the genomic level; (B) Detection of the expression of STK11 protein in NPTr-Cas9-STK11 knockout cells by Western Blot; (C) Detection of the effect of STK11 knockout on cell viability by CCK-8.

[0020] Figure 3 : Effect of STK11 knockout in NPTr cells on IAV replication. **P<0.01; ***P<0.001; ****P<0.0001, mean±SD(n = 3).

[0021] Figure 4 : Effect of STK11 knockout in NPTr cells on cell death caused by IAV infection.

[0022] Figure 5 : Effect of down-regulating the expression of STK11 in mice using siRNA on the weight change of mice caused by IAV infection.

[0023] Figure 6 : Effect of down-regulating the expression of STK11 in mice using siRNA on the mortality change of mice caused by IAV infection.

[0024] Figure 7:Effect of siRNA-mediated downregulation of STK11 expression on viral titer in mouse lung tissues infected with IAV. **P < 0.01; ***P < 0.001, mean ± SD (n = 3). Detailed implementation methods

[0025] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the test materials used in the following examples are all purchased from commercial channels.

[0026] Example 1:

[0027] Construction and verification of STK11 knockout cell line

[0028] The NCBI number of the mRNA of the target gene STK11 is 100512213, and the corresponding CDS sequence is shown in SEQ ID No. 2, encoding the protein shown in SEQ ID No. 3.

[0029] 1.1 Design of sgRNA targeting STK11 and construction of expression vector

[0030] (1) Design of sgRNA: Determine the gene sequence encoding STK11, design and synthesize sgRNA targeting the coding sequence of STK11, and the target sequence of sgRNA is 5'-AGGCCATTGGCAATCTCAGG-3' (SEQ ID No. 1), see Figure 1 A in.

[0031] (2) Dilute the synthesized sgRNA to 100 μM, take 1 μL of each complementary sequence, add 8 μL of ddH2O and mix. Then place the mixture in a PCR instrument for annealing 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.

[0032] (3) Digest the LentiGuide-Puro vector with BsmbI, react at 55°C for 30 min, add DNA loading buffer to the digestion product, and then perform electrophoresis separation to recover the band with a larger molecular weight (about 8,000 bp).

[0033] (4) Mix 1 μL of gRNA diluted 200 times, 50 ng of digested vector and 5 μL of Solution I, and make up to a 10 μL system with ddH2O, and perform ligation in a 16°C water bath for 1 - 2 h.

[0034] (5) Transform the ligation product into DH5α, pick a single colony for sequencing verification.

[0035] (6) Expand the colonies with correct sequencing and extract the plasmids according to the operation manual of the plasmid miniprep kit (D6950-02) from Omega. The results are as Figure 1 shown in B below. The sgRNA targeting the STK11 gene was successfully ligated to the LentiGuide-Puro vector, indicating that the recombinant plasmid of sgRNA targeting STK11 was successfully constructed, and it was named LentiGuide-Puro-sgSTK11.

[0036] 1.2 Construction of STK11 monoclonal knockout cell line

[0037] (1) Package the sgRNA lentivirus targeting the STK11 gene using a three-plasmid packaging system. According to the operation manual of the Lipo8000 TM transfection reagent (C0533) from Beyotime, co-transfect HEK293T cells with the helper plasmid pMD2.G (Addgene, 12259), psPAX2 (Addgene, 12260) and the target plasmid LentiGuide-Puro-sgSTK11 to package the lentivirus.

[0038] (2) Seed two wells of a 6-well plate with NPTr cells (NPTr-Cas9) stably expressing Cas9 protein. One well serves as the experimental group and the other as the control group, and culture them in DMEM complete medium containing 10% FBS and 1% double antibody.

[0039] (3) When the cell density reaches about 30%, add the sgRNA lentivirus targeting the STK11 gene (diluted 1:1 in DMEM) and DMEM medium to the experimental and control wells respectively, and add polybrene at a final concentration of 8 μg / mL, and continue to culture.

[0040] (4) After 12 h of lentivirus infection, replace it with fresh DMEM complete medium and continue to culture.

[0041] (5) When the cell confluence reaches 80%-90%, perform drug pressure screening using puromycin (Puromycin, Puro) at a concentration of 1.5 μg / mL. After all the control group cells die, replace the experimental group with complete medium containing 0.5 μg / mL Puro and continue to culture until the cells no longer die to obtain a polyclonal knockout cell line.

[0042] (6) Seed the above polyclonal knockout cells into a 96-well cell plate by limited dilution method.

[0043] (7) Continue to culture for about 14 days. Wait until a single cell grows into a single cell colony. Digest the cells of the single cell colony with trypsin and passage them into a 24-well cell culture plate to obtain a monoclonal gene knockout cell line, named STK11-KO cells.

[0044] 1.3 Verify the expression and cell viability of STK11 in STK11-KO cells

[0045] 1.3.1 Detect the knockout of the STK11 gene in STK11-KO cells at the genomic level

[0046] (1) Extraction of cell genomic DNA

[0047] Operate according to the operation manual of the Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304) of TIANGEN Company to extract the genomic DNA of STK11-KO and control group NPTr-Cas9 cells.

[0048] (2) Using the above genomic DNA as a template, PCR amplify the fragment containing the sgRNA target sequence in the STK11 genome and perform sequencing. The sequencing primers are: STK11-F: 5’-CAGGGCATCGTGCACAAGG-3’ and STK11-R: 5’-TCACAGACGCCACTCAGATCTG-3’.

[0049] The PCR amplification program is as follows: pre-denaturation at 95°C for 5 min, the cycling reaction is denaturation at 95°C for 20 s, annealing at 60°C for 20 s, extension at 72°C for 1 min, for a total of 35 cycles, and finally extension at 72°C for 5 min.

[0050] The sequencing results are as Figure 2 shown in A below. A 17-bp deletion that can cause frameshift mutation occurred near the sgRNA target sequence in the STK11 genome.

[0051] 1.3.2 Detect the knockout of STK11 in STK11-KO cells at the protein level

[0052] (1) Preparation of protein samples

[0053] Collect NPTr-Cas9 and STK11-KO cell samples, add an appropriate amount of mammalian protein extraction reagent (add Protease Inhibitor Cocktail in advance according to 1:99), lyse on ice for 30 min, then centrifuge at 12000 rpm for 10 min, aspirate the supernatant into a 1.5 mL centrifuge tube, add the corresponding volume of 5×SDS-PAGE loading buffer, boil at 100°C for 10 min, and store at -20°C.

[0054] (2) SDS-PAGE electrophoresis

[0055] Use the Yamei PAGE Gel Rapid Preparation Kit (PG112) to prepare the gel according to the instructions. Perform protein electrophoresis separation on the above samples.

[0056] (3) Transfer the protein from the gel to the NC membrane, and then place the NC membrane in TBST containing 1% BSA for blocking;

[0057] (4) After the blocking is completed, incubate with STK11 rabbit polyclonal antibody (Proteintech, 10746-1-AP) and GAPDH mouse monoclonal antibody (Proteintech, 60004-1-Ig) for the primary antibody.

[0058] (5) Subsequently, incubate with the corresponding HRP-conjugated goat anti-rabbit IgG (ABclonal, AS028) and HRP-conjugated goat anti-mouse IgG (ABclonal, AS071) for the secondary antibody.

[0059] (6) Use the ECL luminescence method for protein detection.

[0060] The results are as Figure 2 shown in B below. Compared with NPTr-Cas9 cells, the STK11 protein is completely not expressed in STK11-KO cells, indicating that the STK11 gene has been successfully knocked out in this cell line.

[0061] 1.3.3 Detect the effect of knocking out STK11 on cell viability using CCK-8

[0062] (1) Seed the same number and appropriate amount of NPTr-Cas9 and STK11-KO cells into 96-well plates, with 8 replicates for each group of cells, and continue to culture in an incubator at 37°C with 5% CO2.

[0063] (2) At 12, 24, and 36 h after cell seeding, add 10 μL of CCK-8 reagent to each well and continue to incubate in the incubator for 1-2 h.

[0064] (3) After the incubation is completed, measure the absorbance at 450 nm using a microplate reader.

[0065] (5) Perform calculation and processing according to the calculation formula.

[0066] The experimental results are as Figure 2 shown in C below. Knocking out STK11 has no significant effect on cell viability.

[0067] Example 2:

[0068] Knocking out STK11 inhibits the replication of IAV

[0069] STK11-KO and NPTr-Cas9 cells were seeded in 12-well plates. After the cells reached confluence, the culture medium was discarded and the cells were washed twice with DMEM. Subsequently, the cells were infected with influenza viruses from different species at an MOI of 0.01, namely A / swine / Hubei / 221 / 2016 (HuB / H1N1), A / swine / Henan / F26 / 2017 (F26 / H1N1), A / Puerto Rico / 8-SV14 / 1934 (PR8 / H1N1), and A / chicken / Shanghai / SC197 / 2013 (SH13 / H9N2). After incubation at 37 °C for 1 h, the supernatant was discarded, and the cells were washed twice with DMEM. Then, the medium containing 0.1% TPCK was added, and the cells were further cultured in an incubator at 37 °C. At 12, 24, and 36 h after virus infection, the cell supernatants were collected, and the virus titer of IAV in the supernatant was measured by TCID 50 The specific method is as follows:

[0070] (1) An appropriate amount of MDCK cells was seeded onto a 96-well cell culture plate. When the cells reached confluence, the cells were washed twice with serum-free DMEM.

[0071] (2) The collected virus solution was serially diluted 10-fold to an appropriate concentration.

[0072] (3) The virus solution of each dilution was inoculated into one vertical row (8 wells) of a 96-well cell plate, 100 μL per well. Then, the plate was incubated in a 37 °C CO2 incubator. After 1 h, the virus solution in each well was discarded, and the cells were washed twice with DMEM. The maintenance medium containing 0.3% TPCK was added, and the cells were cultured in a 37 °C CO2 incubator.

[0073] (4) After 72 h, the cell supernatant in the 96-well plate was transferred to a U-shaped hemagglutination plate. Whether influenza virus existed in the well was determined by hemagglutination test, and the TCID of the virus was calculated using the Reed-Muench method 50 .

[0074] The results are as Figure 3 shown. Knockout of STK11 significantly inhibited the proliferation of IAV from different species.

[0075] Example 3:

[0076] Knockout of STK11 can resist IAV-induced cell death

[0077] IAV infection can cause cytopathic effects and even induce cell death. NPTr-Cas9 and STK11-KO cells were infected with HuB-H1N1 at MOIs of 0.01 and 0.1 respectively, and the ability of cells to resist IAV-induced cell death after knocking out STK11 was detected. The same number of NPTr-Cas9 and STK11-KO cells were seeded in 12-well plates. After the cells grew to confluence, they were infected with IAV. At 72 h post-infection, the cell survival was observed. The results were as Figure 4 shown. Almost all NPTr-Cas9 cells died, while more than 90% and 80% of STK11-KO cells survived under the infection conditions with IAV MOIs of 0.01 and 0.1 respectively. The results indicated that knocking out STK11 could resist IAV-induced cell death.

[0078] Example 4:

[0079] Inhibition of IAV replication after silencing STK11 in mice mediated by siRNA

[0080] Using RNA interference technology, cholesterol-conjugated 2'-Ome-modified siRNA specifically targeting murine STK11 was designed. Forty-eight 6-week-old female BALB / c SPF mice were prepared and evenly divided into 3 groups. One day before and one day after infection, the three groups of mice were anesthetized with ether and then instilled with PBS, cholesterol-conjugated 2'-Ome-modified siControl, and siSTK11 by nasal drip respectively. On day 0, the mice in the siControl and siSTK11 groups were infected with 30 PFU of IAV (A / Hunan / 42443 / 2015) by nasal drip, and the PBS group was mock-infected.

[0081] 4.1 Inhibition of weight loss and death caused by IAV infection after silencing STK11 in mice

[0082] The body weight changes of the mice were recorded daily within two weeks from the day of infection, and the survival of the mice was observed. The results were as Figure 5 and Figure 6 shown. Silencing STK11 in mice significantly improved the weight loss of infected mice and also increased the survival rate of mice by 20%.

[0083] 4.2 Inhibition of IAV replication virus titer after silencing STK11 in mice

[0084] On days 3 and 5, 3 mice from each group were sacrificed, and their lungs were removed, added with 0.8 ml of PBS containing 1% double antibodies for dilution and homogenization. The homogenate was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was taken to measure the virus titer of IAV in the lung tissue by TCID 50 assay. As Figure 7As shown, significantly inhibiting the virus titer of IAV replication after silencing STK11 in mice.

Claims

Use of an inhibitor of STK11 gene or protein in the preparation of a drug against influenza A virus.

2. The use according to claim 1, wherein The inhibitor is any one of the following substances: (a) A substance that silences the STK11 gene; (b) A substance that targets and knocks out the STK11 gene; (c) A substance that inhibits the activity of STK11 protein.

3. The use according to claim 2, characterized in that, The substance that silences the STK11 gene is siRNA, shRNA, dsRNA or miRNA that targets and silences the STK11 gene.

4. The use according to claim 2, characterized in that, The substance that targets and knocks out the STK11 gene is the CRISPR / Cas editing system.

5. The use according to claim 4, characterized in that, In the CRISPR / Cas editing system, the sgRNA target sequence is as shown in SEQ ID No.

1.

6. The use according to claim 2, characterized in that, The substance that inhibits the activity of STK11 protein is a specific small molecule compound or antibody that can bind to STK11 protein and inhibit its activity.

7. A method for constructing an anti-influenza A virus cell model, characterized in that, Knock out the STK11 gene of cells by the CRISPR / Cas editing system or transfect cells with interfering RNA that silences the STK11 gene, thereby obtaining a cell model with inhibited STK11 gene expression and enhanced anti-influenza A virus ability.

8. A method for constructing an animal model resistant to influenza A virus, characterized in that, Knock out the STK11 gene of animals by the CRISPR / Cas editing system or transfect animals with interfering RNA that silences the STK11 gene, thereby obtaining an animal model with inhibited STK11 gene expression and enhanced anti-influenza A virus ability.

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