Application of alpha-adduction protein ADD1 in animal anti-influenza

Abstract

The invention discloses an application of alpha-adduction protein ADD1 in animal anti-influenza. An in-vivo experiment verifies that the proliferation level of IAV can be remarkably reduced by inhibiting the expression of ADD1 in a mouse, and the survival rate of an infected mouse is remarkably improved. Further research shows that ADD1 has high conservative property in different species, including poultry DF1 cells and porcine PK-15 cells. Experiments show that the proliferation of IAV in the cells can be effectively inhibited by knocking out ADD1. The key effect of ADD1 in the IAV infection process is defined for the first time, it is revealed that ADD1 can serve as a potential target for research and development of anti-influenza virus drugs, a new thought is provided for research and development of anti-influenza virus drugs, and important application value is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and, in particular, relates to the application of α - adducin ADD1 in avian anti - influenza. Background Art

[0002] Influenza A Virus (IAV) is a single - stranded negative - sense RNA virus with strong transmission ability and high mutability. It can not only spread among poultry but also infect humans through cross - species transmission, posing a serious threat to the livestock and poultry breeding industry and human health. IAV infection can cause symptoms such as acute respiratory syndrome, decreased egg production, and multi - organ damage in poultry. It has strong transmissibility and high lethality, often resulting in large - scale death of poultry and bringing huge economic losses to the breeding industry. In addition, avian influenza virus can also infect humans. The outbreaks caused by H5N1 in 1997 and H7N9 in 2013 led to hundreds of human infections with a mortality rate of over 50%, posing a great threat to human health and public health security. IAV is extremely prone to mutation, which may lead to the failure of drugs and vaccines targeting viral proteins. At present, the prevention and control measures for avian influenza mainly rely on vaccination, biosafety prevention and control, and emergency culling. However, these methods have obvious limitations in dealing with the rapid mutation of the virus. In contrast, antiviral strategies targeting host factors are more stable and less affected by virus mutation. Developing antiviral strategies targeting host factors can not only improve the prevention and control efficiency but also reduce the risk of vaccine or drug failure caused by virus mutation.

[0003] ADD1 is one of the members of the adducin protein family, encoded by the ADD1 gene on avian chromosome 4. Its protein structure includes an N - terminal head domain, a neck domain, and a C - terminal tail domain. ADD1 is a known actin - binding protein with the following three actin - regulating functions: (1) It can bind to the barbed end of actin filaments, thereby inhibiting the incorporation of actin monomers; (2) It can bundle actin filaments; (3) It can recruit spectrin through its C - terminal domain and participate in the assembly of the spectrin - actin membrane skeleton network, maintaining the mechanical connection and dynamic remodeling between the cell membrane and the cytoskeleton.

[0004] Studies have shown that ADD1 is a phosphorylation substrate of multiple protein kinases (such as PKA, PKC, Rho kinase, etc.), and its function is jointly regulated by phosphorylation modification and calmodulin. Phosphorylated ADD1 not only regulates the assembly of the cytoskeleton but also participates in various cellular processes such as cell - cell contact, cell migration, and pseudopod formation. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a novel prevention and control strategy against influenza virus infection. By targeting the host factor ADD1, the limitations of existing prevention and control means in dealing with the rapid mutation of the virus are overcome.

[0006] The technical solution of the present invention is: the application of an inhibitor of ADD1 gene or protein in the preparation of a drug against influenza A virus.

[0007] Further, the inhibitor is any one of the following:

[0008] (a) A substance that silences the ADD1 gene;

[0009] (b) A substance that inhibits the activity of ADD1 protein;

[0010] (c) A substance that targets and knocks out the ADD1 gene.

[0011] Further, the substance that silences the ADD1 gene is siRNA or shRNA that targets and silences the ADD1 gene.

[0012] Further, the substance that inhibits the activity of ADD1 protein is a specific small molecule compound or an ADD1 protein antibody that can bind to the ADD1 protein and inhibit its activity.

[0013] Further, the substance that targets and knocks out the ADD1 gene is the CRISPR / Cas editing system.

[0014] A method for constructing an anti-influenza A virus cell model, by knocking out the ADD1 gene of the cell with the CRISPR / Cas editing system or transfecting the cell with an interfering RNA that silences the ADD1 gene, thereby obtaining a cell model with inhibited ADD1 gene expression and enhanced anti-influenza A virus ability.

[0015] A method for constructing an anti-influenza A virus animal model, by knocking out the ADD1 gene of the animal with the CRISPR / Cas editing system or transfecting the animal with an interfering RNA that silences the ADD1 gene, thereby obtaining an animal model with inhibited ADD1 gene expression and enhanced anti-influenza A virus ability.

[0016] sgRNA, the nucleotide sequence is as shown in SEQ ID No.5 or SEQ ID No.6.

[0017] The application of the above-mentioned sgRNA in the preparation of a drug against influenza A virus.

[0018] The ADD1 gene is conserved in multiple species. Its mRNA sequence accession number in pigs (Sus scrofa) is XM_013978492.2 in the NCBI database, and the amino acid sequence encoding the corresponding protein is SEQ ID No. 2; its mRNA sequence accession number in chickens (Gallus gallus) is NM_001079730.3, and the amino acid sequence of the encoded protein is SEQ ID No. 4.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] It is first demonstrated that ADD1 is a key host factor during IAV infection, clarifying its important role in virus replication. Through cell-level and animal model experiments, it is confirmed that inhibiting the expression or function of ADD1 can significantly reduce the proliferation level of IAV, showing good antiviral effects. Further research shows that ADD1 is highly conserved in different species, and knocking out this gene can effectively inhibit the replication of IAV in porcine PK-15 cells and avian DF1 cells. Based on the above findings, the present invention proposes an antiviral intervention strategy targeting ADD1, which has a solid biological basis and application potential. It not only provides a new target for the development of anti-influenza drugs, but also provides an effective means for the prevention and control of avian and swine influenza, especially suitable for important host animals such as pigs and poultry, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Construction and verification of the LentiCRISPR v2-sgADD1 plasmid; (A) Sequencing to identify whether the sgRNA targeting the avian ADD1 gene is cloned into the LentiCRISPR v2 vector; (B) Sequencing to identify whether the sgRNA targeting the porcine ADD1 gene is cloned into the LentiCRISPR v2 vector;

[0022] Figure 2 : Construction and verification of ADD1-knockout PK-15 and DF1 monoclonal cell lines; (A) Western blot to detect the expression level of ADD1 protein in DF1 cells; (B) Western blot to detect the expression level of ADD1 protein in PK-15 cells;

[0023] Figure 3 : ADD1 knockout inhibits the proliferation of IAV in DF1 cells. ***P<0.001, mean±SD (n = 3);

[0024] Figure 4: Knockout of ADD1 inhibits the proliferation of IAV from different host sources. **P < 0.01; ***P < 0.001; ****P < 0.0001, mean ± SD (n = 3);

[0025] Figure 5 : Western blot and qRT-PCR were used to detect the silencing efficiency of ADD1 in mouse lungs. si-NC: mice in the control siRNA treatment group; siADD1-1 / siADD1-2: two different siADD1 treatment groups of mice;

[0026] Figure 6 : Silencing of ADD1 alleviates the weight loss of mice caused by IAV infection. *P < 0.05; **P < 0.01, mean ± SEM (n = 10);

[0027] Figure 7 : Silencing of ADD1 increases the survival rate of IAV-infected mice. ****P < 0.0001, mean ± SD (n = 10);

[0028] Figure 8 : Silencing of ADD1 reduces the virus titer in the lungs of IAV-infected mice. *P < 0.05; **P < 0.01, mean ± SD (n = 3). Specific implementation manners

[0029] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from commercial channels unless otherwise specified.

[0030] Example 1: Construction and verification of ADD1-knockout PK-15 and DF1 monoclonal cell lines

[0031] The mRNA sequence of the ADD1 gene in pigs is the NCBI database accession number XM_013978492.2, the corresponding CDS sequence is shown in SEQ ID No.1, and the encoded protein sequence is shown in SEQ ID No.2; the mRNA sequence in chickens is NM_001079730.3, the corresponding CDS sequence is shown in SEQ ID No.3, and the encoded protein sequence is shown in SEQ ID No.4.

[0032] 1.1 Construction of sgRNA recombinant plasmid

[0033] (1) sgRNA design: Specific sgRNA sequences were designed for avian and porcine ADD1 genes, and BsmBI restriction sites were added at both ends for subsequent cloning. The sgRNA sequence for the avian ADD1 gene is 5’-ACACCAGTGAATGATCTCAG-3’ (SEQ ID No.5), and the sgRNA sequence for the porcine ADD1 gene is 5’-ACCCAAACAGATCCGCCAGT-3’ (SEQ ID No.6).

[0034] (2) sgRNA annealing: The synthesized sgRNA primers were diluted to 100 μM. 1 μL of each upstream and downstream primer was taken and mixed with 8 μL of ddH2O. The annealing program was 37°C for 30 min; 95°C for 5 min (-0.1°C / s); 25°C for 1 min. The annealed product was diluted 200-fold and stored at 4°C for later use.

[0035] (3) Enzyme digestion of the vector: The LentiCRISPR v2 (Addgene, 49535) vector was digested with BsmBI enzyme at 55°C for 30 min, and then the target fragment was separated by electrophoresis on a 1% agarose gel and recovered and purified. The sgRNA annealing product obtained in (2) was mixed with the lentiviral digested vector, and T4 ligase was used for ligation in a 16°C water bath for 1 - 2 h.

[0036] (4) The ligation product was added to 30 - 50 μL of DH5α competent cells for plasmid transformation. Single colonies were picked and subjected to sequencing identification.

[0037] (5) The bacterial solution with correct sequencing identification was cultured on a large scale, and the plasmid was extracted according to the operation manual of the Omega endotoxin-free plasmid extraction kit (D6950 - 02).

[0038] The results are as Figure 1 shown in A and B below. The sgRNAs targeting avian and porcine ADD1 genes were successfully ligated to the LentiCRISPR v2 vector, indicating the successful construction of the LentiCRISPR v2-sgADD1 plasmid.

[0039] 1.2 Construction of ADD1 knockout monoclonal cell lines

[0040] (1) According to Beyotime's Lipo8000 TMOperating instructions for transfection reagent (C0533): Co-transfect HEK293T cells with auxiliary plasmids pMD2.G (Addgene, 12259), psPAX2 (Addgene, 12260) and lentiviral plasmid LentiCRISPR v2-sgADD1 at a ratio of 1:2:3 to package lentivirus. 72 hours after transfection, collect the lentiviral supernatant and store it at -80 °C.

[0041] (2) Seed PK-15 cells (porcine kidney cells) and DF1 cells (chicken embryo fibroblasts) into two wells of a 6-well cell culture plate, with one well as the experimental group and the other as the control group.

[0042] (3) When the cell confluence rate reaches 20% - 30%, infect the cells with the lentivirus collected in (1): Add 1 mL of the lentiviral supernatant targeting the avian ADD1 gene to DF1 cells and 1 mL of the lentiviral supernatant targeting the porcine ADD1 gene to PK-15 cells. Subsequently, add 1 mL of DMEM (Biochannel, BC-M-005) medium containing 10% FBS (KEL Biotech, KC001-01) to each well. And add polybrene (GLPBIO, GC19206) at a final concentration of 8 μg / mL to incubate the cells.

[0043] (4) 12 hours after lentiviral infection, repeat the above step (3) for the second lentiviral infection.

[0044] (5) 72 hours after lentiviral infection, add puromycin (MCE, HY-K1057) at a concentration of 1.5 μg / mL to the cells for drug pressure screening.

[0045] (6) After the cells grow to a confluent monolayer, pick monoclonal cell lines by the limited dilution method.

[0046] 1.3 Detection of ADD1 knockout efficiency

[0047] (1) Digest the confluent monolayer of monoclonal cells with trypsin (Biochannel, BC-CE-005), collect the digested cells into an EP tube, wash the cells once with PBS, lyse the cells on ice for 30 minutes with mammalian protein extraction reagent (CWBIO, CW0889M), centrifuge at 12000 r / min at 4 °C for 10 minutes, collect the supernatant, add 5× protein loading buffer and mix well, and heat at 100 °C for 10 minutes in a metal bath.

[0048] (2) Perform SDS gel electrophoresis on the protein samples prepared in (1) above.

[0049] (3) After the electrophoresis is completed, carefully peel off the gel for membrane transfer.

[0050] (4) Antibody incubation and signal detection:

[0051] a. After the membrane transfer is completed, take out the NC membrane and place it in TBST containing 1% BSA (BioFroxx, 4240GR500), and shake it on a shaker for blocking for 1 - 2 h;

[0052] b. After blocking, add the corresponding primary antibodies: mouse monoclonal anti - GAPDH (Proteintech, 60004 - 1 - Ig) and rabbit polyclonal anti - ADD1 (Proteintech, 10791 - 1 - AP), and shake and incubate for 2 h;

[0053] c. Collect the primary antibodies and wash the NC membrane 5 times with TBST;

[0054] d. Add the corresponding secondary antibodies: HRP - labeled goat anti - rabbit (Biodragon, BF03008) and HRP - labeled goat anti - mouse (Biodragon, BF03001), and shake and incubate for 1 h;

[0055] e. Wash the NC membrane 5 times with TBST;

[0056] f. Use the ECL color development kit from Thermo for color development and reasonably adjust the exposure time.

[0057] The results are as Figure 2 shown in A and B below. In ADD1 - knockout DF1 cells and PK - 15 cells, the expression of ADD1 protein could not be detected, but the expression of ADD1 protein could be detected in wild - type control cells, indicating that ADD1 was successfully knocked out and the monoclonal cell line with ADD1 knockout was successfully constructed.

[0058] Example 2: ADD1 knockout inhibits the proliferation of IAV in DF1 cells

[0059] (1) Seed an equal amount of wild - type DF1 cells and ADD1 - knockout DF1 cells into a 12 - well plate. After the cells grow to confluence, wash the cells twice with serum - free DMEM, and infect the DF1 cells with A / duck / Sheyang / 1 / 2005 (YS / H5N1) at 0.01 MOI. After incubating in a 37°C CO2 incubator for 1 h, wash the cells twice with serum - free DMEM, and add DMEM medium containing 1% penicillin - streptomycin (GENOMBIO, GNM15140 - 1) and the corresponding concentration of TPCK, and incubate in a 37°C CO2 incubator. Collect the cell supernatants at 12, 24, and 36 h after infection and store them at - 80°C.

[0060] (2) TCID 50Experimental determination of virus titer:

[0061] a. Inoculate MDCK cells into a 96-well plate;

[0062] b. After the cells in the 96-well plate grow into a monolayer, perform a 10-fold serial dilution of the cell supernatant collected in (1). After dilution, inoculate the diluted virus into MDCK cells according to the virus infection steps in (1) of Example 2. Incubate in a 37 °C CO2 incubator for 72 h;

[0063] c. After 72 h, transfer the cell supernatant in the 96-well plate to a U-shaped hemagglutination plate, and determine whether influenza virus exists in this well through a hemagglutination experiment;

[0064] d. Use the Reed-Muench method to calculate the TCID of the virus 50 .

[0065] The results are as Figure 3 shown. Knocking out ADD1 on DF1 cells can significantly inhibit the proliferation of IAV.

[0066] Example 3:

[0067] Knockout of ADD1 inhibits the proliferation of IAV from different hosts

[0068] Inoculate swine influenza virus A / swine / Hubei / 221 / 2016 (HuB / H1N1), swine-origin influenza virus isolated from humans (A / Hunan / 42443 / 2015, HuN / H1N1), human influenza virus (A / Puerto Rico / 8-SV14 / 1934, PR8 / H1N1), and avian influenza virus (A / chicken / Hubei / 115 / 2016, 115 / H9N2) into wild-type and ADD1-knockout PK-15 cells at 0.01 MOI according to the virus inoculation steps described in (1) of Example 2, and collect the cell supernatant at 12, 24, and 36 h after infection. Determine the virus titer according to the experimental method described in (2) of Example 2.

[0069] The results are as Figure 4 shown. Knocking out ADD1 on PK-15 cells can significantly inhibit the proliferation of IAV from different hosts.

[0070] Example 4: Silencing ADD1 can reduce the mortality of mice infected with IAV

[0071] 4.1 Verification of the silencing effect of ADD1 siRNA in mice

[0072] Ten female SPF-grade BALB / c mice, 6 - 8 weeks old and weighing 18 - 21 g, were prepared and randomly divided into two groups (Group A and Group B). After inhalation anesthesia with ether, 50 μL (5 nM) of cholesterol-conjugated 2'-Ome-modified si-NC was instilled intranasally into each mouse in Group A, and 50 μL (5 nM) of cholesterol-conjugated 2'-Ome-modified siADD1 was instilled into each mouse in Group B. 72 h after nasal instillation, the mice were sacrificed and dissected, and the lungs were extracted and homogenized in 0.8 mL of PBS containing 1% penicillin-streptomycin. The homogenate was centrifuged at 12,000 r / min for 20 min at 4°C, and the supernatant was collected to detect the gene silencing effect of siADD1 by Western blot and qRT-PCR.

[0073] The results are as Figure 5 shown that the siRNA targeting ADD1 can significantly down-regulate the protein and mRNA expression levels of ADD1 in the lungs of mice.

[0074] 4.2 Silencing ADD1 reduces the viral titer in the lungs of mice and improves the survival rate of mice

[0075] (1) Fifty-four 6 - 8-week-old female BALB / c SPF mice were prepared and randomly divided into three groups (Group A, Group B, and Group C). Group A served as the PBS negative control group, and Group B and Group C were instilled with 50 μL (5 nM) of cholesterol-conjugated 2'-Ome-modified si-NC and siADD1, respectively. The instillation was performed intranasally under ether anesthesia 1 day before and 1 day after infection.

[0076] (2) Mice in Group A were instilled intranasally with 50 μL of PBS on day 0 as a mock infection control, and mice in Group B and Group C were instilled with 30 PFU of HuN / H1N1 (50 μL), respectively. The body weight changes of the mice were recorded daily within two weeks after infection, and their survival status was observed.

[0077] (3) On the 3rd and 5th days after infection, 3 mice were randomly sacrificed from each group. The lungs of the mice were excised and homogenized in 0.8 mL of PBS, centrifuged at 12,000 r / min for 20 min at 4°C, and the supernatant was collected for detecting the viral titer.

[0078] The results are as Figure 6 、 7 and Figure 8 shown that the siRNA targeting ADD1 can reduce the viral titer in the lungs of mice, alleviate the weight loss of mice caused by IAV infection, and significantly improve the survival rate of mice.

Claims

1. Use of an inhibitor of ADD1 gene or protein in the preparation of a drug against influenza A virus.

2. The application according to claim 1, wherein The inhibitor is any one of the following: (a) A substance that silences the ADD1 gene; (b) A substance that inhibits the activity of ADD1 protein; (c) A substance that targets and knocks out the ADD1 gene.

3. The application according to claim 2, wherein The substance that silences the ADD1 gene is siRNA or shRNA that targets and silences the ADD1 gene.

4. The application according to claim 2, characterized in that, The substance that inhibits the activity of ADD1 protein is a specific small molecule compound or an ADD1 protein antibody that can bind to the ADD1 protein and inhibit its activity.

5. The application according to claim 2, characterized in that The substance that targets and knocks out the ADD1 gene is the CRISPR / Cas editing system.

6. A method for constructing an anti-influenza A virus cell model, characterized in that, Knock out the ADD1 gene of cells by the CRISPR / Cas editing system or transfect cells with interfering RNA that silences the ADD1 gene, thereby obtaining a cell model with inhibited ADD1 gene expression and enhanced anti-influenza A virus ability.

7. A method for constructing an animal model resistant to influenza A virus, characterized in that, Knock out the ADD1 gene of animals by the CRISPR / Cas editing system or transfect animals with interfering RNA that silences the ADD1 gene, thereby obtaining an animal model with inhibited ADD1 gene expression and enhanced anti-influenza A virus ability.

8. sgRNA, with the nucleotide sequence shown in SEQ ID No.5 or SEQ ID No.

6.

9. Use of the sgRNA according to claim 8 in the preparation of a drug against influenza A virus.

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