Application of FTO gene or protein as target spot in screening and preparing anti-canine parvovirus drug

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of an FTO gene or protein as a target spot in screening and preparing canine parvovirus resisting drugs. The invention provides an application of a host cell FTO gene as a target spot in screening or preparing a drug for inhibiting canine parvovirus replication, wherein the nucleotide sequence of the FTO gene is shown as SEQ ID NO: 1. The invention also provides application of the host cell FTO protein as a target spot in screening or preparing a medicine for inhibiting canine parvovirus replication, wherein the amino acid sequence of the FTO protein is shown as SEQ ID NO: 2. By silencing the FTO gene of the host cell or inhibiting the FTO protein of the host cell, the replication level of the CPV in the cell is obviously reduced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of FTO gene or protein as a target in screening and preparing anti-canine parvovirus drugs. Background Art

[0002] Canine parvovirus (CPV) is one of the most serious pathogens of canines and belongs to the genus Protoparvovirus of the family Parvoviridae. CPV is a non-enveloped linear single-stranded DNA (ssDNA) virus with a genome length of 5323 nucleotides. The virus has many hosts, mutates rapidly, and spreads widely. Even in areas of intensive vaccination, CPV infection is still one of the causes of acute gastroenteritis and death in young and puppies. CPV infection can cause vomiting, fever, diarrhea, and dehydration in dogs, with a high morbidity and mortality rate. In the face of CPV infection, there is currently no widely recognized specific antiviral drug, so it is particularly important to find new treatment strategies.

[0003] Fat mass and obesity-associated protein (FTO) has been identified as being associated with obesity and weight gain and is highly expressed in the brain and muscle. So far, there have been no reports on FTO as a target for anti-canine parvovirus drugs. Summary of the invention

[0004] The inventors accidentally discovered in the experiment that after silencing the FTO gene of the host cell or inhibiting the FTO protein of the host cell, the replication level of canine parvovirus (CPV) in the host cell was significantly reduced.

[0005] Based on the above research results, the present invention provides the use of the host cell FTO gene as a target in screening or preparing a drug for inhibiting the replication of canine parvovirus, wherein the nucleotide sequence of the FTO gene is shown in SEQ ID NO:1.

[0006] Also provided is the use of an FTO gene inhibitor in the preparation of a drug for inhibiting the replication of canine parvovirus, wherein the nucleotide sequence of the FTO gene is shown in SEQ ID NO: 1.

[0007] In the above application, the FTO gene inhibitor may be a small interfering RNA (siRNA).

[0008] In the above application, the nucleotide sequence of the small interfering RNA is any one of SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0009] Also provided is the use of a host cell FTO protein as a target in screening or preparing a drug for inhibiting the replication of canine parvovirus, wherein the amino acid sequence of the FTO protein is shown in SEQ ID NO:2.

[0010] Also provided is the use of an FTO protein inhibitor in the preparation of a drug for inhibiting the replication of canine parvovirus, wherein the amino acid sequence of the FTO protein is shown in SEQ ID NO:2.

[0011] Also provided is a drug for inhibiting the replication of canine parvovirus, the active ingredient of which is a FTO gene inhibitor, and the nucleotide sequence of the FTO gene is shown in SEQ ID NO:1.

[0012] In the above-mentioned drugs, the FTO gene inhibitor may be small interfering RNA (siRNA).

[0013] In the above-mentioned drug, the nucleotide sequence of the small interfering RNA is any one of the groups selected from SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0014] Also provided is another drug for inhibiting the replication of canine parvovirus, the active ingredient of which is a FTO protein inhibitor, and the amino acid sequence of the FTO protein is shown in SEQ ID NO:2.

[0015] The FTO gene silencing experiment showed that compared with the replication level of canine parvovirus (CPV) in the control group cells, the replication level of CPV in FTO gene silenced cells (si-220, si-431 and si-1120) was significantly (P<0.05) lower ( Figure 3 ). FTO protein inhibition experiments have shown that after treating host cells with FTO protein inhibitors (meclofenac sodium, Dac51, FB23), the replication level of CPV in host cells was significantly reduced (P<0.0001) ( Figure 4 ).

[0016] In summary, the present invention significantly reduces the replication level of CPV in cells by silencing the FTO gene of host cells or inhibiting the FTO protein of host cells. Therefore, the FTO gene and FTO protein of host cells can be used as targets for anti-canine parvovirus drugs, and have good application prospects in the screening and preparation of anti-canine parvovirus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The relative expression level of FTO gene in cells is shown; NC represents F81 cells transfected with negative control siRNA (control group); si-220, si-431 and si-1120 represent F81 cells transfected with FTO gene siRNA; ** represents P < 0.0001.

[0018] Figure 2 The expression level of FTO protein in cells is shown; A: Western Blot experimental results of FTO protein in cells, with β-actin as the internal reference protein; B: Grayscale statistical graph of the bands in A, the data are the ratio of the grayscale of the FTO band to the grayscale of the β-actin band, and the control group is used as a reference for normalization; NC represents F81 cells transfected with negative control siRNA (control group); si-220, si-431 and si-1120 represent F81 cells transfected with FTO gene siRNA; ** represents P<0.01; ** represents P<0.001.

[0019] Figure 3 The figure shows the replication level of canine parvovirus (CPV) in host cells after infection; A: Western Blot experimental results of VP2 protein of CPV virus, with β-actin as the internal reference protein; B: Grayscale statistical graph of the bands in A, the data are the ratio of the grayscale of VP2 band to the grayscale of β-actin band, and the control group is used as a reference for normalization; NC represents F81 cells transfected with negative control siRNA (control group); si-220, si-431 and si-1120 represent F81 cells transfected with FTO gene siRNA; * represents P<0.05; ** represents P<0.01; ** represents P<0.001.

[0020] Figure 4The expression level of VP2 protein and the number of viral copies in host cells after canine parvovirus (CPV) infection are shown; A and D: Western Blot results of VP2 protein in F81 cells treated with 50 μM and 100 μM meclofenac sodium (MA) after CPV infection (A) and RT-qPCR absolute quantification results of VP2 gene (D); B and E: Western Blot results of VP2 protein in F81 cells treated with 2.5 μM and 5 μM Dac51 after CPV infection (B) and RT-qPCR absolute quantification results of VP2 gene (E); C and F: Western Blot results of VP2 protein in F81 cells treated with 30 μM and 60 μM F23 after CPV infection (C) and RT-qPCR absolute quantification results of VP2 gene (F); DMSO indicates F81 cells treated with DMSO (control group); ** indicates P < 0.0001.

[0021] Sequence Description

[0022] SEQ ID NO: 1 Coding sequence (CDS) of FTO gene;

[0023] SEQ ID NO:2 Amino acid sequence of FTO protein;

[0024] SEQ ID NO:3: the positive strand sequence of the small interfering RNA (FTO-Felis catus-220) targeting the FTO gene;

[0025] SEQ ID NO: 4 Antisense strand sequence of small interfering RNA (FTO-Felis catus-220) against FTO gene;

[0026] SEQ ID NO:5: the positive strand sequence of the small interfering RNA (FTO-Felis catus-431) targeting the FTO gene;

[0027] SEQ ID NO:6 Antisense strand sequence of small interfering RNA (FTO-Felis catus-431) against FTO gene;

[0028] SEQ ID NO:7: positive strand sequence of small interfering RNA (FTO-Felis catus-1120) targeting FTO gene;

[0029] SEQ ID NO:8 Antisense strand sequence of small interfering RNA (FTO-Felis catus-1120) against FTO gene;

[0030] SEQ ID NO:9 coding sequence (CDS) of β-actin gene;

[0031] SEQ ID NO: 10 Forward primer sequence of FTO gene;

[0032] SEQ ID NO:11 reverse primer sequence of FTO gene;

[0033] SEQ ID NO: 12 Forward primer sequence of β-actin gene;

[0034] SEQ ID NO:13 reverse primer sequence of β-actin gene;

[0035] SEQ ID NO: 14 Coding sequence (CDS) of the VP2 gene of canine parvovirus New CPV-2a strain SD6;

[0036] SEQ ID NO:15 Forward primer sequence of VP2 gene;

[0037] SEQ ID NO:16 Reverse primer sequence of VP2 gene. DETAILED DESCRIPTION

[0038] The following implementation plans are provided:

[0039] 1. Use of the host cell FTO gene as a target in screening or preparing a drug for inhibiting the replication of canine parvovirus, wherein the nucleotide sequence of the FTO gene is shown in SEQ ID NO: 1.

[0040] 2. Use of an FTO gene inhibitor in the preparation of a drug for inhibiting canine parvovirus replication, wherein the nucleotide sequence of the FTO gene is shown in SEQ ID NO: 1.

[0041] 3. The use described in embodiment 2, wherein the FTO gene inhibitor is small interfering RNA.

[0042] 4. The use described in embodiment 3, wherein the nucleotide sequence of the small interfering RNA is as shown in SEQ ID NO:3 and SEQ ID NO:4.

[0043] 5. The use described in embodiment 3, wherein the nucleotide sequence of the small interfering RNA is as shown in SEQ ID NO:5 and SEQ ID NO:6.

[0044] 6. The use described in embodiment 3, wherein the nucleotide sequence of the small interfering RNA is as shown in SEQ ID NO:7 and SEQ ID NO:8.

[0045] 7. Use of host cell FTO protein as a target in screening or preparing drugs for inhibiting canine parvovirus replication, wherein the amino acid sequence of the FTO protein is shown in SEQ ID NO: 2.

[0046] 8. Use of an FTO protein inhibitor in the preparation of a drug for inhibiting canine parvovirus replication, wherein the amino acid sequence of the FTO protein is shown in SEQ ID NO: 2.

[0047] 9. A drug for inhibiting the replication of canine parvovirus, wherein the active ingredient is an FTO gene inhibitor, and the nucleotide sequence of the FTO gene is shown in SEQ ID NO: 1.

[0048] 10. The drug according to embodiment 9, wherein the FTO gene inhibitor is a small interfering RNA.

[0049] 11. The drug according to embodiment 10, wherein the nucleotide sequence of the small interfering RNA is as shown in SEQ ID NO:3 and SEQ ID NO:4.

[0050] 12. The drug according to embodiment 10, wherein the nucleotide sequence of the small interfering RNA is as shown in SEQ ID NO:5 and SEQ ID NO:6.

[0051] 13. The drug according to embodiment 10, wherein the nucleotide sequence of the small interfering RNA is as shown in SEQ ID NO:7 and SEQ ID NO:8.

[0052] 14. A drug for inhibiting the replication of canine parvovirus, wherein the active ingredient is a FTO protein inhibitor, and the amino acid sequence of the FTO protein is shown in SEQ ID NO: 2.

[0053] The present invention will be further described below in conjunction with the examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0054] If not otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure. If not otherwise specified, the experimental methods and conditions used in the following examples are all conventional experimental methods and conditions in the art, and reference can be made to relevant experimental manuals, known documents or manufacturer specifications. Unless otherwise defined, the meanings of all technical and scientific terms used herein are the same as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0055] The cat kidney cells (F81 cells) used in the following examples were preserved by the Animal Husbandry and Veterinary Research Institute of Beijing Academy of Agriculture and Forestry Sciences. The cell recovery and passage methods are as follows, wherein the complete medium used is 1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (GIBCO), 100 U / ml penicillin and 0.1 mg / mL streptomycin.

[0056] Cell recovery: Preheat the complete culture medium in a 37°C water bath. Take out the frozen F81 cells from liquid nitrogen, quickly place them in a 37°C water bath, and shake until dissolved. Transfer the dissolved F81 cell cryopreserved solution to the preheated complete culture medium, shake gently, and place in a 37°C, 5% CO2 incubator for culture. The next day, replace the culture medium in the cell culture medium with fresh complete culture medium.

[0057] Cell passaging: F81 cells are passaged when they grow to 80% to 90% of the bottom area of ​​the culture flask. Aspirate the culture medium, rinse the cells with PBS solution, and remove the residual culture medium and floating cells. After adding trypsin, place the cells in a 37°C, 5% CO2 incubator for 3 minutes for cell digestion, and then add an equal volume of complete culture medium to terminate the digestion. Transfer the mixture to a centrifuge tube and centrifuge at 1200r / min for 3 minutes. Discard the upper layer of liquid, leaving the cell clumps, resuspend the cells with complete culture medium, and culture them in a 37°C, 5% CO2 incubator.

[0058] The canine parvovirus (CPV) used in the following examples is the New CPV-2a strain SD6 (297Ala, 426Asn), which was isolated and identified by Yang Bing's research group at the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences, and is recorded in "Hongzhuan Zhou, XiaSu, Lulu Lin, Jin Zhang, Qi Qi, Fangfang Guo, Fuzhou Xu, Bing Yang. Inhibitory Effects of Antiviral Drug Candidates on Canine Parvovirus in F81 cells [J]. Viruses, 2019, 11 (8): 742-742." The GenBank accession number of the VP2 gene of this strain is MN101724, and its coding sequence is shown in SEQ ID NO: 14.

[0059] The main reagents and consumables used in the following examples: negative control siRNA (Shanghai Jima Pharmaceutical Technology Co., Ltd., catalog number A06001), RPMI 1640 medium (Gibco), 0.05% trypsin-EDTA (Gibco), fetal bovine serum (FBS) (Gibco), dimethyl sulfoxide (DMSO) (MERCK, cell culture grade), penicillin-streptomycin solution (100×) (Gibco), phosphate buffered saline (PBS) (biosharp), Culture medium (Gibco), RNAiMAX (Thermo Scientific), Trizol (Invitrogen), total RNA extraction kit RNeasy Mini Kit (QIAGEN), FastKing cDNA first-strand synthesis kit (genomic DNA removed) (TianGenBiotech, Beijing, China), SuperReal fluorescence quantitative premix reagent enhanced version (SYBR GREEN) (TianGenBiotech, Beijing, China), Mammalian Total Protein Extraction Kit (Beijing Quanshijin Biotechnology), BCA protein concentration determination kit (Beijing Quanshijin Biotechnology), PVDF membrane (MERCK), Western primary antibody diluent (TOYOBO), Western secondary antibody diluent (TOYOBO), prestained protein molecular weight standard Thermo Scientific PageRuler (Thermo Scientific), SuperSignal TM West Pico PLUS chemiluminescent substrate (ThermoScientific), meclofenac sodium (MA) (Selleck), FB23 (Selleck), Dac51 (Selleck).

[0060] The main instruments used in the following examples are: cell culture incubator (Thermo Scientific), real-time fluorescence quantitative PCR instrument (BIO-RAD, CFX Connect TM Real-Time System), SDS-PAGE electrophoresis tank (BIO-RAD, Mini-PROTEAN Tetra), ultra-micro spectrophotometer (Thermo Scientific, NANODROP ONE), and biological safety cabinet (Haier, HFsafe1500LC).

[0061] Example 1

[0062] Silencing the FTO gene in host cells significantly reduced the replication level of canine parvovirus (CPV) in cells

[0063] 1.1 Design and synthesis of FTO gene siRNA

[0064] The coding sequence (CDS) of the FTO gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein (FTO protein) encoded by it is shown in SEQ ID NO: 2. Three small interfering RNAs (siRNAs) were designed based on the FTO gene sequence and named FTO-Felis catus-220, FTO-Felis catus-431 and FTO-Felis catus-1120, respectively, and their nucleotide sequences are as follows:

[0065] FTO-Felis catus-220-F:5'-GCAAAGAGAAGUCGAAUAUTT-3'(SEQ ID NO:3)

[0066] FTO-Felis catus-220-R:5'-AUAUUCGACUUCUCUUUGCTT-3'(SEQ ID NO:4)

[0067] FTO-Felis catus-431-F:5'-GCUGAAAUAGCGGCCUGCUUTT-3'(SEQ ID NO:5)

[0068] FTO-Felis catus-431-R:5'-AAGCAGCCGCUAUUUCAGCTT-3'(SEQ ID NO:6)

[0069] FTO-Felis catus-1120-F:5'-GCCCGCGGUUCUGAAACAATT-3'(SEQ ID NO:7)

[0070] FTO-Felis catus-1120-R:5'-UUGUUUCAGAACCGCGGGCTT-3'(SEQ ID NO:8)

[0071] Wherein F represents the sense strand and R represents the antisense strand. Suzhou Jima Company was commissioned to synthesize the above siRNA.

[0072] 1.2 FTO gene siRNA transfection cells

[0073] FTO-Felis catus-220, FTO-Felis catus-431, FTO-Felis catus-1120 and negative control siRNA (5'-UUCUCCGAACGUGUCACGUTT-3', 5'-ACGUGACACGUUCGGAGAATT-3') (Shanghai Jima Pharmaceutical Technology Co., Ltd., Cat. No. A06001) were used to transfect cat kidney cells (F81 cells) respectively. The negative control siRNA is a universal negative control, has no homology with the sequence of the target gene, is used to control the transfection process, and has no effect on the cells.

[0074] The transfection method was as follows: F81 cells in good growth condition and in the logarithmic growth phase were digested and resuspended in 1640 medium (Gibco, Grand Island, NY, USA) containing 2% fetal bovine serum (FBS) to finally dilute to 3.3×10 5 cells. 1.7 mL of cell suspension was evenly spread into a 6-well plate. 150 μL 5 μL of culture medium dilution RNAiMAX transfection reagent. Use Dilute siRNA (60 pmol) in culture medium. RNAiMAX transfection reagent was mixed in a volume ratio of 1:1 and incubated at room temperature for 5 minutes to obtain siRNA-liposome complexes. siRNA-liposome complexes were added to the cell culture medium in a 6-well plate, 300 μL per well, and the mixture was gently shaken and placed in a 37°C, 5% CO2 incubator for culture. After 6 hours, the cell culture medium was replaced with 1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (GIBCO), 100 U / ml penicillin and 0.1 mg / mL streptomycin, and the cells were continued to be cultured. Cell samples were collected after 48 hours for detection of FTO gene silencing efficiency and FTO protein expression levels.

[0075] The transfected cells were obtained: si-220 (cells transfected with FTO-Felis catus-220), si-431 (cells transfected with FTO-Felis catus-431), si-1120 (cells transfected with FTO-Felis catus-1120) and control group (cells transfected with negative control siRNA).

[0076] 1.3 Detection of FTO gene silencing efficiency

[0077] The relative expression of FTO gene in si-220, si-431 and si-1120 cells was detected by real-time fluorescence quantitative PCR (RT-qPCR) to evaluate the efficiency of FTO gene silencing. Total RNA in si-220, si-431, si-1120 and control group cells was extracted using RNeasy Mini Kit (QIAGEN), and RNA was reverse transcribed into cDNA using FastKing cDNA First Chain Synthesis Kit (TianGen Biotech). Using cDNA as a template, real-time fluorescence quantitative PCR was performed using the specific primers FTO-F / FTO-R of the FTO gene. The gene β-actin encoding β-actin was used as an internal reference gene. The coding sequence of the β-actin gene is shown in SEQ ID NO:9, and its specific primers are actin-F / actin-R.

[0078] The nucleotide sequences of the primers are as follows:

[0079] FTO-F: 5'-AGGTGGCTCTGCAGAATGTC-3' (SEQ ID NO: 10)

[0080] FTO-R: 5'-GCCACTCGAACTCAACCTCA-3' (SEQ ID NO: 11)

[0081] actin-F: 5'-CATGTACGTGGCCATCCAGGC-3' (SEQ ID NO: 12)

[0082] actin-R: 5'-CTCCTTGATGTCACGCACAAT-3' (SEQ ID NO: 13)

[0083] The quantitative PCR system was: 2×SuperReal PreMix Plus (SYBR Green) (TianGen Biotech), 10 μL; forward primer (10 μM), 0.8 μL; reverse primer (10 μM), 0.8 μL; cDNA template, 2 μL; RNase-free ddH2O, 6.4 μL. The quantitative PCR program was: (95°C for 3 minutes) for 1 cycle; (95°C for 10 seconds, 60°C for 30 seconds) for 40 cycles.

[0084] The experiment was set up with three biological replicates. GraphPad Prism was used to perform statistical analysis on the RT-qPCR results, and the 2-ΔΔCT method was used to calculate the relative expression of the FTO gene. The data analysis results are shown as mean ± standard deviation (mean ± SD). After the three biological data obtained were in accordance with the normal distribution, one-way ANOVA was used to compare the data between multiple groups; the significant differences were: *P<0.05; **P<0.01; **P<0.001; **P<0.0001. P<0.05 was considered statistically significant.

[0085] The results are as follows Figure 1 As shown, compared with the control group (NC), the relative expression levels of FTO gene in si-220, si-431 and si-1120 cells were significantly (P<0.0001) decreased.

[0086] 1.4 Detection of FTO protein expression level

[0087] The expression level of FTO protein in si-220, si-431 and si-1120 cells was detected by Western Blot. Mammalian Total Protein Extraction Kit (Beijing Quanshijin Biotechnology) was used to extract the total protein of si-220, si-431, si-1120 and control group cells, and the protein concentration was determined using BCA protein concentration determination kit (Beijing Quanshijin Biotechnology). The extracted proteins were subjected to SDS-PAGE and then transferred to PVDF membrane (MERCK). 5% skim milk was prepared with 1×TBST. The PVDF membrane was blocked with 5% skim milk at room temperature for 1 hour. Rabbit anti-FTO antibody (ABclonal, catalog number A1438) was diluted with primary antibody diluent (TOYOBO) at 1:1000 as the primary antibody. Mouse anti-β-actin antibody (ABclonal, catalog number AC004) was diluted with primary antibody diluent (TOYOBO) at 1:6600 as the primary antibody. The primary antibody was placed in the incubation box, the PVDF membrane containing the target band was cut, and it was placed in the corresponding primary antibody, and incubated overnight at 4°C with shaking. The next day, wash the PVDF membrane with 1×TBST for 3 times, 10 minutes each time. Dilute goat anti-rabbit HRP GoatAnti-Rabbit IgG (H+L) (ABclonal, catalog number AS014) with secondary antibody diluent (TOYOBO) at 1:5300 as the secondary antibody. Dilute goat anti-mouse HRP GoatAnti-Mouse IgG (H+L) (SeraCare, 5450-0011) with secondary antibody diluent (TOYOBO) at 1:6600 as the secondary antibody. Place the secondary antibody in the incubation box, place the PVDF membrane incubated with the primary antibody in the corresponding secondary antibody, and incubate at room temperature for 1 hour. Wash the PVDF membrane with 1×TBST for 3 times, 10 minutes each time. Evenly cover the surface of the PVDF membrane with chemiluminescent developer and develop after incubation for 2 minutes.

[0088] The experiment was set up with three biological replicates. ImageJ was used to analyze the grayscale of the developed bands, and then GraphPad Prism was used to perform statistical analysis of the data. The data analysis results are shown as mean ± standard deviation (mean ± SD). After the three biological data obtained were in accordance with the normal distribution, one-way ANOVA was used to compare the data between multiple groups; the significant differences were: *P<0.05; **P<0.01; **P<0.001; **P<0.0001. P<0.05 was considered statistically significant.

[0089] The results are as follows Figure 2 As shown, compared with the control group (NC), the expression level of FTO protein in si-220, si-431 and si-1120 cells was significantly (P<0.001) decreased.

[0090] 1.5 Canine parvovirus (CPV) infection experiment

[0091] 48 hours after F81 cells were transfected with siRNA, the pre-diluted New CPV-2a strain SD6 virus solution was added to si-220, si-431, si-1120 and control group cells with an infection multiplicity (MOI) of 0.1. The cells were then cultured in a 37°C, 5% CO2 incubator. After 24 hours, cell samples were taken for Western Blot experiments to detect the replication level of CPV virus in cells after silencing of the host cell FTO gene.

[0092] use Mammalian Total Protein Extraction Kit (Beijing Quanshijin Biotechnology) was used to extract the total protein of si-220, si-431, si-1120 and control group cells 24 hours after CPV infection, and the protein concentration was determined using the BCA protein concentration assay kit (Beijing Quanshijin Biotechnology). The extracted proteins were subjected to SDS-PAGE and then transferred to a PVDF membrane (MERCK). 5% skim milk was prepared with 1×TBST. The PVDF membrane was blocked with 5% skim milk at room temperature for 1 hour. Mouse anti-VP2 monoclonal antibody (INGENASA, Spain, M.15.CPV.I5F8) was diluted 1:100 with primary antibody diluent (TOYOBO) as the primary antibody. Mouse anti-β-actin antibody was diluted 1:6600 with primary antibody diluent (TOYOBO) as the primary antibody. The primary antibody was placed in the incubation box, the PVDF membrane containing the target band was cut, and it was placed in the corresponding primary antibody, and incubated overnight at 4°C with shaking. The next day, wash the PVDF membrane with 1×TBST for 3 times, 10 minutes each time. Dilute goat anti-mouse HRP GoatAnti-Mouse IgG (H+L) (SeraCare, 5450-0011) with secondary antibody diluent (TOYOBO) at 1:6600 as the secondary antibody. Place the secondary antibody in the incubation box, place the PVDF membrane incubated with the primary antibody in the secondary antibody, and incubate at room temperature for 1 hour. Wash the PVDF membrane with 1×TBST for 3 times, 10 minutes each time. Evenly cover the chemiluminescent developer on the surface of the PVDF membrane and develop after incubation for 2 minutes.

[0093] The experiment was set up with three biological replicates. ImageJ was used to analyze the grayscale of the developed bands, and then GraphPad Prism was used to perform statistical analysis of the data. The data analysis results are shown as mean ± standard deviation (mean ± SD). After the three biological data obtained were in accordance with the normal distribution, one-way ANOVA was used to compare the data between multiple groups; the significant differences were: *P<0.05; **P<0.01; **P<0.001; **P<0.0001. P<0.05 was considered statistically significant.

[0094] The results are as follows Figure 3 As shown, the replication level of CPV in si-220, si-431 and si-1120 cells was significantly (P<0.05) lower than that in control (NC) cells. Therefore, inhibition of the FTO gene in host cells can significantly reduce CPV replication in host cells.

[0095] Example 2

[0096] Inhibition of host cell FTO protein significantly reduces the replication level of canine parvovirus (CPV) in cells

[0097] 2.1 FTO protein inhibition experiment

[0098] Meclofenamate Sodium (MA) (CAS: 6385-02-0), FB23 (CAS: 2243736-35-6) and Dac51 (CAS: 2243944-92-3) are all FTO protein inhibitors purchased from Selleck. DMSO (MERCK, cell culture grade) was used to dissolve meclofenamate sodium (MA), FB23 and Dac51 respectively to prepare a mother solution with a concentration of 10mM. 100μL of the mother solution was transferred to 300μL of maintenance medium to prepare a 2.5mM stock solution. The maintenance medium is a 1640 medium (Gibco, Grand Island, NY, USA) containing 2% fetal bovine serum (GIBCO), 100U / ml penicillin and 0.1mg / mL streptomycin. The 2.5mM stock solution was gradiently diluted to obtain working solutions with concentrations of 1250μM, 250μM, 125μM, and 62.5μM. The working solution was added into the culture medium of cat kidney cells (F81 cells) to treat the cells with drugs.

[0099] MA treatment group: F81 cells were treated with meclofenac sodium (MA), and the final concentrations of MA were set to 50 μM and 100 μM, respectively.

[0100] Dac51 treatment group: F81 cells were treated with Dac51, and the final concentrations of Dac51 were set to 2.5 μM and 5 μM, respectively.

[0101] FB23 treatment group: F81 cells were treated with FB23, and the final concentrations of FB23 were 30 μM and 60 μM, respectively.

[0102] Control group: F81 cells were treated with DMSO to a final concentration of 0.1%.

[0103] After 1 hour of treatment, the cells in each group were infected with canine parvovirus New CPV-2a strain SD6 at a multiplicity of infection (MOI) of 0.1. The cells in each group were collected 24 hours after CPV infection and used for real-time fluorescence quantitative PCR and protein immunoblotting (Western Blot) experiments.

[0104] 2.2 Detection of viral VP2 gene copy number

[0105] Absolute quantitative PCR was used to evaluate the replication of CPV in cells after the host cell FTO protein was inhibited. Total DNA was isolated from CPV-infected F81 cells using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The isolated total DNA was used as a template to amplify a 119 bp fragment of the VP2 gene (SEQ ID NO: 14) using CPV-specific primers VP2-F / VP2-R. The nucleotide sequences of the primers are as follows:

[0106] VP2-F:5'-CAAATAGAGCATTGGGCTTACC-3'(SEQ ID NO:15)

[0107] VP2-R: 5'-TCCCATTTGAGTTACACCACG-3' (SEQ ID NO: 16)

[0108] The obtained 119 bp fragment was cloned into pMD18-T (Takara, Shiga, Japan) to obtain the recombinant plasmid pMD-VP2S. The concentration of the pMD-VP2S plasmid was accurately measured, and the molar concentration of the plasmid was calculated based on the plasmid size (number of base pairs) and the concentration of the plasmid. The pMD-VP2S plasmid was serially diluted 10-fold to obtain a concentration of 10 6 , 10 5 , 10 4 , 10 3 , 10 2Copies / μL of standard plasmid solution. Fluorescence quantitative PCR was performed on the standard plasmid solution using CPV-specific primers VP2-F / VP2-R. The PCR instrument was CFX Connect TM Real-time Fluorescence Quantitative PCR Detection System (Bio-Rad, CA, USA). Each 20 μL quantitative PCR reaction system contained 10 μL of 2×SuperReal PreMix Plus (SYBRGreen) (TianGen Biotech, Beijing, China), 0.2 μM forward primer, 0.2 μM reverse primer, and 1 μL standard plasmid solution. The quantitative PCR program was: (95°C for 3 minutes) 1 cycle; (95°C for 5 seconds, 60°C for 30 seconds) 45 cycles. After the reaction, the standard curve was generated by the instrument supporting software Bio-Rad CFX Maestro 2.2.

[0109] Total DNA was extracted from each group of cells collected in 2.1 above (MA-treated group, Dac51-treated group, FB23-treated group, and control group) using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Fluorescence quantitative PCR was performed on the obtained total cell DNA using CPV-specific primers VP2-F / VP2-R. The PCR instrument was CFX Connect TM Real-time Fluorescence Quantitative PCR Detection System (Bio-Rad, CA, USA). Each 20 μL quantitative PCR reaction system contained 10 μL of 2×SuperReal PreMix Plus (SYBR Green) (TianGen Biotech, Beijing, China), 0.2 μM forward primer, 0.2 μM reverse primer, and 1 μL of total cell DNA solution. The quantitative PCR program was: (95°C for 3 minutes) 1 cycle; (95°C for 5 seconds, 60°C for 30 seconds) 45 cycles. After the reaction, the CPV virus copy number was calculated using the above standard curve by the instrument supporting software Bio-Rad CFX Maestro2.2. The experiment was set up with three biological replicates. The data analysis results are shown as mean ± standard deviation (mean ± SD). After the biological data obtained from the three experiments were in accordance with normal distribution, one-way ANOVA was used to compare the data among multiple groups; the significant differences were as follows: *P<0.05; **P<0.01; **P<0.001; **P<0.0001. P<0.05 was considered statistically significant.

[0110] The results showed that meclofenac sodium (MA) at concentrations of 50 μM and 100 μM could significantly inhibit CPV replication ( Figure 4 D); Dac51 treatment concentrations of 2.5 μM and 5 μM can significantly inhibit CPV replication ( Figure 4 E); FB23 treatment concentrations of 30 μM and 60 μM can significantly inhibit CPV replication ( Figure 4 F). This proves that inhibiting the FTO protein in host cells can significantly reduce CPV replication in host cells.

[0111] 2.3 Detection of viral VP2 protein expression level

[0112] The VP2 protein expression levels of the cells in the MA-treated group, Dac51-treated group, FB23-treated group and control group 24 hours after CPV infection were detected by Western Blot experiment.

[0113] use Mammalian Total Protein Extraction Kit (Beijing Quanshijin Biotechnology) was used to extract total protein from cells in the MA treatment group, Dac51 treatment group, FB23 treatment group and control group, and the protein concentration was determined using the BCA protein concentration assay kit (Beijing Quanshijin Biotechnology). The extracted proteins were subjected to SDS-PAGE and then transferred to a PVDF membrane (MERCK). 5% skim milk was prepared with 1×TBST. The PVDF membrane was blocked with 5% skim milk at room temperature for 1 hour. Mouse anti-VP2 monoclonal antibody (INGENASA, Spain, M.15.CPV.I5F8) was diluted 1:100 with primary antibody diluent (TOYOBO) as the primary antibody. Mouse anti-β-actin antibody was diluted 1:6600 with primary antibody diluent (TOYOBO) as the primary antibody. The primary antibody was placed in the incubation box, the PVDF membrane containing the target band was cut, and it was placed in the corresponding primary antibody, and incubated overnight at 4°C with shaking. The next day, wash the PVDF membrane with 1×TBST for 3 times, 10 minutes each time. Dilute goat anti-mouse HRP GoatAnti-Mouse IgG (H+L) (SeraCare, 5450-0011) with secondary antibody diluent (TOYOBO) at 1:6600 as the secondary antibody. Place the secondary antibody in the incubation box, place the PVDF membrane incubated with the primary antibody in the secondary antibody, and incubate at room temperature for 1 hour. Wash the PVDF membrane with 1×TBST for 3 times, 10 minutes each time. Evenly cover the chemiluminescent developer on the surface of the PVDF membrane and develop after incubation for 2 minutes.

[0114] The experiment was set up with three biological replicates. ImageJ was used to analyze the grayscale of the developed bands, and then GraphPad Prism was used to perform statistical analysis of the data. The data analysis results are shown as mean ± standard deviation (mean ± SD). After the three biological data obtained were in accordance with the normal distribution, one-way ANOVA was used to compare the data between multiple groups; the significant differences were: *P<0.05; **P<0.01; **P<0.001; **P<0.0001. P<0.05 was considered statistically significant.

[0115] The results showed that the expression level of viral VP2 protein was significantly reduced when the treatment concentration of meclofenac sodium (MA) was 50 μM, and the viral VP2 protein was almost not expressed when the treatment concentration was 100 μM ( Figure 4 A); When the concentration of Dac51 was 2.5 μM and 5 μM, the viral VP2 protein was almost not expressed ( Figure 4 B); When the concentration of FB23 was 30μM and 60μM, the viral VP2 protein was almost not expressed ( Figure 4 C). This further proves that inhibiting FTO protein in host cells can significantly reduce CPV replication in host cells.

Claims

1. Use of the host cell FTO gene as a target in screening or preparing a drug for inhibiting the replication of canine parvovirus, wherein the nucleotide sequence of the FTO gene is shown in SEQ ID NO:

1.

2. Use of an FTO gene inhibitor in the preparation of a drug for inhibiting canine parvovirus replication, wherein the nucleotide sequence of the FTO gene is shown in SEQ ID NO:

1.

3. The use according to claim 2, characterized in that: The FTO gene inhibitor is small interfering RNA.

4. The use according to claim 3, characterized in that: The nucleotide sequence of the small interfering RNA is any one of SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:

8.

5. Use of host cell FTO protein as a target in screening or preparing drugs for inhibiting canine parvovirus replication, wherein the amino acid sequence of the FTO protein is shown in SEQ ID NO:

2.

6. Use of an FTO protein inhibitor in the preparation of a drug for inhibiting canine parvovirus replication, wherein the amino acid sequence of the FTO protein is shown in SEQ ID NO:

2.

7. A drug for inhibiting the replication of canine parvovirus, wherein the active ingredient is an FTO gene inhibitor, and the nucleotide sequence of the FTO gene is shown in SEQ ID NO:

1.

8. The drug according to claim 7, characterized in that The FTO gene inhibitor is small interfering RNA.

9. The drug according to claim 8, characterized in that The nucleotide sequence of the small interfering RNA is any one of SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:

8.

10. A drug for inhibiting the replication of canine parvovirus, wherein the active ingredient is a FTO protein inhibitor, and the amino acid sequence of the FTO protein is shown in SEQ ID NO: 2.

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