African swine fever virus inhibitor

Abstract

The invention discloses a composition. The composition is a combination of any two or three of cyclosporine A, allipovir, allipovir and [MeIle] 4-cyclosporine. The invention also discloses the cyclosporin A, the allipovir, the allipovir, the [MeIle] 4-cyclosporin and the application of the composition in preparation of a preparation for inhibiting proliferation of the African swine fever virus. The medicine has an application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of pharmacy and relates to an African swine fever virus inhibitor. Background Art

[0002] African swine fever virus (ASFV) is the only member of the family Asfarviridae and the only known DNA arbovirus. The mortality rate of highly virulent infections is as high as 100%, seriously threatening the healthy development of the world's pig industry. The main transmission vectors of ASFV are domestic pigs, wild pigs, and soft ticks. The virus is divided into 24 genotypes and 8 serotypes, and genotypes I and II are the current epidemic strains. ASFV was first reported in Kenya, Africa in 1921, then spread to West Africa, and several years later, outbreaks occurred in Spain and Portugal respectively, and then spread to other European countries, South America, and the Caribbean region. Epidemiological investigations show that genotype II of ASFV is mainly prevalent in China, and genotypes I and I / II recombinant strains of ASFV also exist. With the widespread prevalence of ASF in China, new variant strains have continuously emerged, making the prevention and control situation of ASF more severe. African swine fever seriously endangers China's pig industry and causes huge losses to the development of China's economy and society. So far, no effective vaccines and antiviral drugs against ASF have been developed. The main reasons are the large genome of the virus and the complex structure of the virus particles. In addition, the virus immune escape mechanism and the host protective immune mechanism are still unclear. The P72 protein encoded by the B646L gene of African swine fever virus is the main component of the ASFV virus particle. It exists in the form of a homotrimer and accounts for 1 / 3 of the total mass of the ASFV virus particle, which is crucial for the formation of the African swine fever virus capsid and the assembly of virus particles. The P72 protein accumulates in host cells in the late stage of virus infection, but the stability regulation mechanism of the P72 protein in host cells is not clear. If the stability regulatory factors of the P72 protein can be found, there is hope to develop or utilize corresponding inhibitors to block the infection and replication of African swine fever virus. However, no specific inhibitors against the P72 protein have been found yet. Summary of the Invention

[0003] To solve the problems existing in the prior art, in the first aspect of the present invention, a composition is provided. The composition is Composition 1, Composition 2, Composition 3, or Composition 4;

[0004] The active ingredients of the Composition 1 include a first active substance and a second active substance;

[0005] The active ingredients of the Composition 2 include a second active substance and a third active substance;

[0006] The active ingredients of the Composition 3 include a first active substance and a third active substance;

[0007] The active ingredients of the composition 4 include a first active substance, a second active substance, and a third active substance;

[0008] The first active substance is cyclosporine A, a medicinal salt of cyclosporine A, or a combination thereof;

[0009] The second active substance is alisporivir, a medicinal salt of alisporivir, or a combination thereof;

[0010] The third active substance is [MeIle]4-cyclosporine, a medicinal salt of [MeIle]4-cyclosporine, or a combination thereof;

[0011] The structural formula of cyclosporine A is as follows:

[0012]

[0013] The structural formula of alisporivir is as follows:

[0014]

[0015] The structural formula of [MeIle]4-cyclosporine is as follows:

[0016]

[0017] In some embodiments, the molar ratio of any two active substances in the composition is 1:0.1 - 10.

[0018] In some embodiments, the medicinal salts of cyclosporine A include: hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurate, gluconate, fructuronate, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, citrate, fumarate, and ascorbate;

[0019] The medicinal salts of alisporivir include: hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurate, gluconate, fructuronate, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, citrate, fumarate, and ascorbate;

[0020] The pharmaceutical salts of [MeIle]4-cyclosporine include: the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurine, gluconate, fructonate, salicylate, nitrate, p-toluenesulfonate, mesylate, benzoate, citrate, lactate, citrate, fumarate and ascorbate of [MeIle]4-cyclosporine.

[0021] In some embodiments, the composition further contains pharmaceutically acceptable excipients.

[0022] The second aspect of the present invention provides an siRNA, which is any one, any combination of two or all three of the first siRNA, the second siRNA and the third siRNA;

[0023] The first siRNA contains RNAs with sequences shown in SEQ ID NO.14 and SEQ ID NO.15 respectively;

[0024] The second siRNA contains RNAs with sequences shown in SEQ ID NO.16 and SEQ ID NO.17 respectively;

[0025] The third siRNA contains RNAs with sequences shown in SEQ ID NO.18 and SEQ ID NO.19 respectively.

[0026] The third aspect of the present invention provides the use of a cyclophilin A inhibitor in the preparation of a preparation for preventing African swine fever, treating African swine fever, alleviating African swine fever or inhibiting the proliferation of African swine fever virus.

[0027] In some embodiments, the cyclophilin A inhibitor is selected from the composition described in the first aspect of the present invention, the first active substance in the composition described in the second aspect of the present invention, the second active substance in the composition described in the second aspect of the present invention, the third active substance in the composition described in the second aspect of the present invention, the siRNA targeting cyclophilin A, the shRNA targeting cyclophilin A, the sgRNA targeting cyclophilin, the antisense RNA targeting cyclophilin, the competitive inhibitor of African swine fever virus P72 protein binding to cyclophilin A, and the antibody or its derivative specifically binding to cyclophilin A;

[0028] Among them, the cyclophilin A-binding competitive inhibitor of African swine fever virus P72 protein can specifically bind to the African swine fever virus P72 protein and can block the amino acid residues at positions 139-142 of the African swine fever virus P72 protein; the cyclophilin A-binding competitive inhibitor of African swine fever virus P72 protein is selected from antibodies that specifically bind to the African swine fever virus P72 protein, antigen-binding fragments of antibodies that specifically bind to the African swine fever virus P72 protein, and fusion proteins containing antigen-binding fragments of antibodies that specifically bind to the African swine fever virus P72 protein;

[0029] The antibody or its derivative that specifically binds to cyclophilin A can specifically bind to cyclophilin A and can block the amino acid residues at positions 55, 60, 61, 63, 101, 113, 121, 122, and 126 of cyclophilin A. The antibody derivative that specifically binds to cyclophilin A is selected from antigen-binding fragments of antibodies that specifically bind to cyclophilin A and fusion proteins containing antigen-binding fragments of antibodies that specifically bind to cyclophilin A.

[0030] In some embodiments, the preparation is a drug or a feed additive; and / or

[0031] The siRNA targeting cyclophilin A is the siRNA described in the second aspect of the present invention.

[0032] In some embodiments, the mechanisms for preventing African swine fever, treating African swine fever, alleviating African swine fever, or inhibiting the proliferation of African swine fever virus include:

[0033] The cyclophilin A inhibitor hinders or weakens the binding of cyclophilin A to African swine fever virus P72 protein;

[0034] The cyclophilin A inhibitor reduces the stability of African swine fever virus P72 protein;

[0035] The cyclophilin A inhibitor hinders the correct folding of African swine fever virus P72 protein;

[0036] The cyclophilin A inhibitor promotes the degradation of African swine fever virus P72 protein;

[0037] The cyclophilin A inhibitor reduces the expression level of African swine fever virus P72 protein;

[0038] The cyclophilin A inhibitor interferes with the assembly of African swine fever virus particles.

[0039] In some embodiments, it is selected from the following A1, A2, and A3;

[0040] A1: The African swine fever virus strain is selected from GenBank accession number MK333180.1;

[0041] A2: The amino acid sequence of the African swine fever virus P72 protein is as shown in GenBank accession number QBH90570.1;

[0042] A3: The protein sequence of cyclophilin A is as shown in SEQ ID NO.1. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is the co - precipitation electrophoresis photograph of CypA and ASFV P72 protein in cells.

[0044] Figure 2 It is the co - precipitation electrophoresis photograph of CypA and ASFV P72 protein outside cells.

[0045] Figure 3 It is the co - localization fluorescence photograph of CypA and ASFV P72 protein in cells.

[0046] Figure 4 It is the prediction result of the complex structure of CypA and ASFV P72 protein.

[0047] Figure 5 It shows the effect of anti - P72 antibody on the interaction between CypA and ASFV P72 protein.

[0048] Figure 6 It shows the effect of CypA on P72 protein and ASFV infection.

[0049] Figure 7 It shows the interference of the cyclic peptide inhibitor of CypA on the interaction between CypA and P72.

[0050] Figure 8 It shows the degradation of P72 protein caused by the cyclic peptide inhibitor of CypA.

[0051] Figure 9 It shows the inhibition of ASFV infection and replication by the cyclic peptide inhibitor of CypA.

[0052] Figure 10 It shows the combined use of the cyclic peptide inhibitor of CypA.

[0053] Figure 11 It shows the effect of the cyclic peptide inhibitor of CypA on the expression of the ASFV transcriptome and the host inflammatory response. DETAILED DESCRIPTION OF THE INVENTION

[0054] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below in conjunction with the accompanying drawings.

[0055] The materials and instruments not described in the present invention are conventional materials and instruments in the art. The operation details not described in the present invention are conventional operations in the art. The software used in the present invention is operated by conventional methods with reference to the usage instructions provided by the software provider. The kits used in the present invention are operated by conventional methods with reference to the kit instruction manuals.

[0056] I. Experimental Drugs

[0057] Cyclosporine A, also known as cyclosporin A, code name CsA, was purchased from MCE, catalog number: #HY-B0579, CAS No.: 59865-13-3, and its molecular structure is as follows:

[0058]

[0059] Alisporivivr, also known as alisporivir, was purchased from MCE, catalog number: #HY-12559, CAS No.: 254435-95-5, and its molecular structure is as follows:

[0060]

[0061] NIM811, [MeIle]4-cyclosporine, was purchased from MCE, catalog number: #HY-P0025, CAS No.: 143205-42-9, and its molecular structure is as follows:

[0062]

[0063] The solvent for these three cyclic peptide inhibitors is DMSO. The inhibitors were dissolved in DMSO (purchased from Sigma) respectively and prepared into stock solutions with a concentration of 5 mM for standby.

[0064] II. Viruses and Cell Lines

[0065] The GenBank of the African swine fever virus wild-type strain Pig / HLJ / 2018 is MK333180.1.

[0066] African swine fever virus gene deletion strain (ASFV HLJ / 18-7GD strain, hereinafter referred to as HLJ / 18-7GD strain), which has deleted the EP402R gene and six genes located in the MGF360 / 505 region, was prepared and preserved by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The transformation process of the HLJ / 18-7GD strain is recorded in the patent document with the Chinese patent application number 201910348878.7. The corresponding name of the HLJ / 18-7GD strain in it is rASFVΔCD2V / 360-eGFP-mCherry strain, and the corresponding microorganism deposit number is CCTCC NO: V201924.

[0067] BK2258 cells are a kind of cells derived from boar kidney (Boar kidney cells), which were prepared and preserved by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The preparation method of BK2258 cells is recorded in the application documents of the Chinese patent with the application number CN202211137583.3.

[0068] Primary porcine alveolar macrophages (PAMs) were prepared and used by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences according to the conventional method.

[0069] Human embryonic kidney cells HEK293T were purchased from the Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection and cultured and used according to the conventional method.

[0070] Example 1: Detection of the interaction between CypA and ASFV P72 protein in host cells

[0071] (1) Preparation of Flag-P72 vector

[0072] The coding sequence of the B646L gene of the Pig / HLJ / 2018 strain is located at positions 103618-105558 of its genome, and the GenBank serial number of the P72 protein encoded by the B646L gene is QBH90570.1. All the P72 proteins described in the present invention refer to the P72 protein of African swine fever virus.

[0073] The genomic DNA of Pig / HLJ / 2018 strain was extracted using a viral genomic DNA extraction kit (purchased from Tiangen Biotech Co., Ltd.) according to the operating steps in the kit instructions. Then, primers F1 (SEQ ID NO.1) and R1 (SEQ ID NO.2) were used for amplification to obtain an amplification product, which was subjected to gel recovery. The gel-purified amplification product was subjected to homologous recombination with the pLenti-CMV-MCS-SV-BSD vector linearized with BamHⅠ and MluⅠ enzymes (modified and preserved by the laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The modification method was: inserting the Flag-tag coding sequence, SEQ ID NO.3, between the XbaⅠ and BamHⅠ restriction sites) according to the steps in the kit instructions. The recombinant vector was transformed into Escherichia coli DH5α strain, and the plasmid was extracted and verified by Sanger sequencing to confirm correct recombination. The coding sequence inserted in the recombinant vector was the same as the B646L gene coding sequence described above. Since the backbone vector contained a Flag-tag label, the recombinant vector expressed the P72 protein with a Flag-tag label (the recombinant vector was named Flag-P72).

[0074] F1: 5’-gacgacgatgacaagggatccggaATGGCATCAGGAGGAGC.

[0075] R1: 5’-ctggtcgacgcgtgggtttaaacTTAGGTACTGTAACGCAGCAC.

[0076] Flag-tag coding sequence: ATGGATTACAAGGACGACGATGACAAG.

[0077] (II) Preparation of B602L-His vector

[0078] The coding sequence of the B602L gene of Pig / HLJ / 2018 strain is located at positions 100716-102308 of its genome. The GenBank accession number of the pB602L protein encoded by the B602L gene is QBH90568.1.

[0079] Extract the genomic DNA of Pig / HLJ / 2018 strain using the operating steps in the viral genome extraction kit (purchased from Tiangen). Amplify using primers F2 (SEQ ID NO.4) and R2 (SEQ ID NO.5), obtain the amplification product and purify it. Use the homologous recombination kit (purchased from Nanjing Novoprotein Biological Co., Ltd.) to perform homologous recombination on the purified amplification product and the pCDNA3.0 vector (purchased from Invitrogen) linearized with KpnⅠ and XbaⅠ enzymes according to the steps in the kit instruction manual. Transform the recombinant vector into Escherichia coli DH5α strain, extract the plasmid and verify the correct recombination by Sanger sequencing. The coding sequence inserted in the recombinant vector is the same as the aforementioned B602L gene coding sequence. Since the recombinant vector expresses the pB602L protein with His-tag (name the recombinant vector B602L-His). When transfecting the Flag-P72 plasmid into cells, in order to ensure the correct folding of the P72 protein, it is necessary to co-transfect B602L-His at the same time.

[0080] F2: 5’-ctatagggagacccaagcttggtaccgccaccATGGCAGAATTTAATATTGATG AGCTTC.

[0081] R2: 5’-gacactatagaatagggccctctagaTTAatggtgatggtgatggtggccgctCAATTCTGCTTTTGTATATAAAATTTCTTTTTCG.

[0082] (III) Preparation of CypA-HA vector

[0083] Peptidylprolyl isomerase A (abbreviated as PPIA in English, also known as Cyclophilin A, abbreviated as CypA). The GenBank number of the porcine CypA gene is NM_214353.1 (its protein number is NP_999518).

[0084] The CypA protein sequence is as follows (SEQ ID NO.6):

[0085] MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGYKGSCFHRIIPGFMCQGGDFTRHNGTGGKSIYGEKFDDENFILKHTGPGILSMANAGPNTNGSQFFICTAKTEWLDGKHVVFGKVKEGMNIVEAMERFGSRNGKTSKKITIADCGQI。

[0086] Extract the total RNA of PAMs, reverse transcribe it into cDNA, and then amplify the cDNA with primers F3 (SEQ ID NO.7) and R3 (SEQ ID NO.8) to obtain the amplification product. Using a homologous recombination kit (purchased from Novoprotein Scientific Inc.), perform homologous recombination on the gel-purified amplification product and the pCDNA3.0-HA vector linearized with KpnⅠ and EcoRⅠ enzymes (modified and preserved by the laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The modification method is: insert a coding sequence of HA-tag between the XhoⅠ and XbaⅠ restriction sites, SEQ ID NO.9) according to the requirements of the instruction manual. Transform the recombinant vector into Escherichia coli DH5α strain, extract the plasmid and verify the correct recombination by Sanger sequencing. The coding sequence inserted in the recombinant vector is the same as the coding sequence of the aforementioned porcine CypA gene. Since the backbone vector contains an HA tag, the recombinant vector expresses the CypA protein with an HA tag (name the recombinant vector CypA-HA).

[0087] F3: 5’-ctatagggagacccaagcttggtaccgccaccATGGTTAACCCCACCGTCTTC TTC.

[0088] R3: 5’-cagtgtgatggatatctgcagaattcGATTTGTCCACAGTCAGCAATGGTG.

[0089] Coding sequence of HA-tag: TACCCCTACGACGTCCCCGACTACGCCTGATA A.

[0090] (IV) Detection of the intracellular interaction between CypA and ASFV P72 protein

[0091] Use the lipo293 transfection reagent (purchased from Beyotime Biotechnology Co., Ltd., Shanghai) to co-transfect HEK293T cells with Flag-P72, B602L-His plasmid and CypA-HA plasmid (1 μg each). The specific transfection steps refer to the lipo293 transfection instruction manual (#C0521) provided by Beyotime Biotechnology Co., Ltd. for operation.

[0092] After 48 hours of transfection, the cells were collected and lysed with Co-IP buffer; HA-CypA and Flag-P72 in the cell lysate supernatant were immunoprecipitated using anti-HA beads (purchased from Shanghai Lingyin Biotechnology Co., Ltd.). Mouse anti-β-actin antibody IgG (purchased from Nanjing Genscript Biotechnology Co., Ltd.), mouse anti-P72 protein antibody IgG (self-made in the laboratory), and mouse anti-HA antibody IgG (purchased from Shanghai Lingyin Biotechnology Co., Ltd.) were used as the primary antibodies respectively, and HRP-conjugated goat anti-mouse IgG (purchased from Jackson ImmunoResearch) was used as the secondary antibody. The immunoblotting experiment was carried out by ECL color development method.

[0093] Cells transfected only with Flag-P72 and B602L-His plasmids were used as negative controls.

[0094] The results are shown in Figure 1 , the upper part of the image is the result of co-precipitation using anti-HA beads, and the lower part of the image is the result of cell lysate enriched without using anti-HA beads. When enriching CypA-HA with anti-HA beads, Flag-P72 protein could also be detected simultaneously in the enrichment product; however, if CypA-HA was absent, strep-P72 could not be recruited by anti-HA beads. It is speculated that ASFV P72 protein can bind to CypA in host cells to form a protein complex.

[0095] Example 2: Detection of direct interaction between purified recombinant CypA and P72 in vitro.

[0096] (I) Preparation of GST-CypA vector

[0097] Using the CypA-HA plasmid as a template, PCR amplification was performed with primers F4 (SEQ ID NO.10) and R4 (SEQ ID NO.11) to obtain an amplification product. Using a homologous recombination kit (purchased from Novoprotein Scientific Inc.), the gel-purified amplification product was subjected to homologous recombination operation with the pGEX-4T-1 vector carrying GST linearized with BamHⅠ and NotⅠ enzymes (purchased from Cytiva) according to the requirements of the kit instructions. The recombinant vector was transformed into Escherichia coli DH5α strain, and the plasmid was extracted and verified by Sanger sequencing to be correctly recombined. The coding sequence inserted in the recombinant vector was the same as the aforementioned porcine CypA gene coding sequence. Since the backbone vector contains a GST tag, the recombinant vector expresses CypA protein carrying a GST tag (named GST-CypA).

[0098] F4: 5'-aatcggatctggttccgcgtggatccATGGTTAACCCCACCGTCTTCTTC。

[0099] R4: 5'-tcagtcagtcacgatgcggccgcTTAGATTTGTCCACAGTCAGCAATGGTG。

[0100] (II) Preparation of GST and GST-CypA recombinant proteins

[0101] The pGEX-4T-1 empty vector (which can express GST) and the GST-CypA vector were respectively transformed into Escherichia coli BL21(DE3) strain by heat shock. Single colonies were picked and cultured overnight in 2 mL of conventional LB medium. The overnight culture was inoculated into 1 L of conventional LB medium for scale-up culture for 2 - 3 hours. When the OD600 reached 0.6 - 0.8, 0.5 mM IPTG (purchased from Sangon Biotech, Shanghai) was used for induction expression (at 16 °C, overnight). The cells were collected by centrifugation and lysed, and then the lysate supernatant was collected by high-speed centrifugation. The GST and GST-CypA recombinant proteins were purified using GST 4FF agarose purification resin (purchased from Sangon Biotech, Shanghai) according to the operation manual.

[0102] (III) Preparation of trimeric strep-P72 recombinant protein

[0103] The trimeric p72 recombinant protein carrying the strep tag (named strep-P72) was purified from yeast cells (provided by Teacher Meng Geng of China Agricultural University). The preparation method of the Strep-P72 trimeric protein is described in the application documents of the Chinese patent application with the application number CN202110522777.4. For the preparation method, refer to Example 1 thereof.

[0104] (IV) Detection of the interaction between purified CypA and P72 in vitro

[0105] An appropriate amount of strep-P72 recombinant protein was separately mixed with GST and GST-CypA proteins in Co-IP buffer and incubated at 4°C for 2 hours. Then, strep-P72 was enriched using Strep-Tactin beads (purchased from Sangon Biotech, Shanghai). Finally, the enriched products were analyzed by immunoblotting. Mouse anti-GST antibody IgG (purchased from Shanghai Lingyin Biotechnology Co., Ltd.) and mouse anti-strep antibody IgG (purchased from Solarbio) were used as primary antibodies, and HRP-conjugated goat anti-mouse IgG (purchased from Jackson ImmunoResearch) was used as the secondary antibody. The immunoblotting experiment was performed using the ECL color development method. The specific operation steps can refer to the instruction manual of IBA-Lifesciences.

[0106] An appropriate amount of CypA recombinant protein (GST-CypA) was mixed with strep-P72 protein in Co-IP buffer, or CypA recombinant protein was separately mixed in Co-IP buffer as a control group (Ctrl group). It was incubated at 4°C for 2 hours, and then strep-P72 was enriched using Strep-Tactin beads (purchased from Sangon Biotech, Shanghai). Finally, the enriched products were analyzed by immunoblotting. Mouse anti-GST antibody IgG (purchased from Shanghai Lingyin Biotechnology Co., Ltd.) and mouse anti-strep antibody IgG (purchased from Solarbio) were used as primary antibodies, and HRP-conjugated goat anti-mouse IgG (purchased from Jackson ImmunoResearch) was used as the secondary antibody. The immunoblotting experiment was performed using the ECL color development method.

[0107] The results are as Figure 2 shown. In Figure 2 A, the upper figure is the result of co-precipitation, and the lower figure is the result of non-co-precipitation. Based on the enrichment of strep-P72, GST-CypA could be detected in the enriched products, but GST could not ( Figure 2 A), indicating that CypA could bind to P72 protein in vitro. In Figure 2 B, the upper figure is the result of co-precipitation, and the lower figure is the result of non-co-precipitation. In the absence of strep-P72, GST-CypA could not be recruited by Strep-Tactin beads alone, further indicating that CypA was recruited by beads through binding to P72 ( Figure 2 B). These in vitro binding experiment results demonstrated that there could be a direct interaction between P72 protein and CypA.

[0108] Example 3: Detection of co-localization of CypA protein and viral P72 protein in cells

[0109] PAMs cells were infected with wild-type ASFV (Pig / HLJ / 2018 strain) at an MOI of 1. After 24 hours of infection, the culture medium was discarded, and the cells were fixed with 4% PFA solution. The cells were permeabilized with 0.2% Triton X-100 and blocked with 1% BSA at room temperature. Then, the cells were incubated overnight at 4°C with mouse anti-P72 protein antibody IgG (diluted 1:200 by volume) (self-made in the laboratory) and rabbit anti-CypA protein antibody IgG (diluted 1:1000 by volume) (purchased from Proteintech). Subsequently, the cells were stained with AlexaFluor488-labeled goat anti-mouse IgG monoclonal antibody (green) (purchased from Invitrogen), AlexaFluor555-labeled goat anti-rabbit IgG monoclonal antibody (red) (purchased from Invitrogen), and DAPI (purchased from Sigma) (blue), and finally, the coverslips were mounted and confocal microscopy imaging was performed.

[0110] PAMs cells without virus infection were used as a control, and other treatments were performed in parallel.

[0111] The results are shown in Figure 3 . Compared with the non-infected group (Mock), the cells infected with ASFV significantly expressed the P72 protein, and the fluorescence signal of the P72 protein overlapped well with the signal of the host CypA protein, indicating that the two can co-localize in host cells.

[0112] Example 4: Structural prediction analysis of the binding of CypA to P72 using AlphaFold3

[0113] The structure of the human CypA protein has been resolved (PDB: 3K0N), but the structure of porcine CypA has not been resolved. In this invention, AlphaFold3 was used to predict the structure of porcine CypA (GenBank accession number NM_214353.1, numbered NP_999518), and a predicted structure of porcine CypA with a very high confidence level (pTM = 0.95) was obtained. This predicted structure is highly similar to the human CypA structure ( Figure 4 A). In addition, AlphaFold3 was used in this invention to predict the co-binding complex structure of porcine CypA and African swine fever P72 trimer, and a CypA-P72 complex structure with a very high confidence level was also obtained ( Figure 4B). The predicted structure of the complex shows that the substrate recognition region of CypA (mainly R55, F60, M61, Q63, A101, F113, W121, L122, and H126) is responsible for its binding to the P72 protein. Moreover, the predicted structure also shows that three CypA molecules respectively bind to the outer top regions of the three monomers in the P72 trimer.

[0114] Example 5: Detection of the effect of specific antibodies against P72 on the interaction between CypA and P72

[0115] In a previous research article on structural biology by the inventors of the present invention (Yu et al. p72 antigenic mapping reveals a potential supersite of vulnerability for African swine fever virus. Cell discovery 2024. PMID: 39080252), it was found that five anti-P72 monoclonal antibodies (B1, F11, C9, H3, and G6) derived from pigs can have different binding modes with the P72 protein ( Figure 5 A). Among them, antibodies B1, F11, C9, and H3 can all bind to the outer top region (mainly containing amino acid residues 139 - 142) and the central top region (amino acid residues 511 - 513) of the P72 protein ( Figure 5 A red dotted line region), which highly coincides with the predicted CypA binding region of P72 in the present invention as shown in Figure 4 . Thus, it is predicted that these four antibodies may affect the interaction between CypA and P72. In contrast, antibody G6 mainly binds to the central top position (amino acid residues 511 - 513) of P72 and does not bind to the outer top region (including amino acid residues 139 - 142) ( Figure 5 A); thus, it is speculated that antibody G6 does not affect or has a weak effect on the binding between CypA and P72.

[0116] The present invention tests the effect of these 5 P72 antibodies on the binding between CypA and P72 through in vitro binding experiments. Mix the strep-P72 recombinant protein with GST or GST-CpyA protein, and simultaneously add equal amounts of different mouse-derived P72 monoclonal antibody IgG (B1, F11, C9, H3, and G6) (self-made during the research operation of the aforementioned literature) or mouse anti-African swine fever virus P30 monoclonal antibody IgG (self-made). The material combination situation is shown in Figure 5The upper part of the B photo. Incubate at 4 °C for 2 hours. Then enrich strep-P72 with Strep-Tactin beads, and detect P72 and GST-CypA in the enriched product by immunoblotting (the specific method is the same as in Example 1). In addition, the content of antibody IgG was shown by Coomassie Brilliant Blue staining of SDS-PAGE gels processed in the same way in parallel.

[0117] The results are shown in Figure 5 B. The upper figure is the result of co-precipitation, and the lower figure is the result without co-precipitation. In the upper figure of co-precipitation, compared with the other four P72 antibodies, the content of GST-CpyA corresponding to G6 treatment is high while the content of IgG heavy chain is low, indicating that this antibody has little effect on the interaction between strep-P72 and GST-CypA; further, in the lower figure without co-precipitation enrichment treatment, GST-CypA is not washed off so its content is significantly higher than that of the co-precipitation-treated samples, and there is no obvious difference in the levels of the five antibodies, indicating that the amounts of proteins and antibodies added in each group of experiments are consistent. Most importantly, the addition of monoclonal antibodies B1, F11, C9, and H3 can effectively inhibit the binding of P72 protein to CypA protein, but the monoclonal antibody G6 cannot interfere with the interaction between P72 and CypA. This fully proves again that CypA and P72 can have a direct interaction, and the region that binds to CypA is indeed the outer top region of P72 (including amino acid residues 139-142), which is highly consistent with the AlphaFold3 prediction result. It can be reasonably expected that preparing a new antibody that binds to the outer top region of P72 (including amino acid residues 139-142) can interfere with the interaction between P72 and CypA and thus inhibit the proliferation of ASFV. Such antibodies are equivalent to P72-binding competitive inhibitors of CypA, and the binding of this type of competitive inhibitor to P72 can block amino acid residues 139-142 of P72, thereby blocking the binding of CypA to P72.

[0118] Example 6: Detection of the effect of CypA on P72 protein and ASFV infection

[0119] (1) Detection of the regulation of intracellular P72 protein level by CypA

[0120] The cultured wild-type (WT) HEK293T cell line (purchased from the Cell Bank of the Chinese Academy of Sciences) and the CypA knockout (CypA-KO) HEK293T cell line (self-made, obtained by knocking out the CypA gene in the HEK293T cell line through the CRISPR gene editing method) were digested into single cells respectively, passaged in equal amounts in 12-well plates, and cultured overnight. When the cells grew to 90% density, the plasmid Flag-P72 expressing P72 (the same as in Example 1) together with B602L-His (the same as in Example 1) were transfected into the two types of cells respectively, and the plasmid CypA-HA expressing CypA was co-transfected or not co-transfected at the same time. The non-transfected cells were used as negative controls respectively. 24 hours after transfection, the cells were lysed with 1X SDS loading buffer and denatured at high temperature to prepare samples, and analyzed by immunoblotting (the specific method was the same as in Example 1).

[0121] The results are shown in Figure 6 A. The protein level of P72 in CypA-KO cells was significantly lower than that in WT cells; overexpression of CypA could significantly promote the accumulation of P72 protein. This indicates that the high or low intracellular expression level of host CypA can positively regulate the protein level of P72. Therefore, CypA can increase the protein level of P72 in cells or improve the stability of P72 in cells.

[0122] (II) Detect the effect of CypA on ASFV infection

[0123] Three siRNAs targeting porcine CypA (siCypA-1, siCypA-2, and siCypA-3) were designed and synthesized, and non-targeting siRNA (NC) was used as a control. PAMs cells were transfected with each siRNA at a dose of 2 μg. After 48 hours of transfection, the cells were infected with the ASFV HLJ / 18-7GD strain at an MOI of 0.2. After 96 hours of infection, the infected supernatant was collected for nucleic acid extraction, and the relative content of the viral B646L gene fragment was detected by qPCR using primers F5 (SEQ ID NO.12) and R5 (SEQ ID NO.13). At the same time, samples were prepared from the infected cells. Immunoblotting experiments were performed using mouse anti-P72 antibody IgG (self-made), mouse anti-P30 antibody IgG (self-made), and mouse anti-β-actin antibody IgG (purchased from Nanjing Genscript Corporation) as primary antibodies, and HRP-conjugated goat anti-mouse IgG as the secondary antibody (purchased from Jackson ImmunoResearch), with ECL color development method; Immunoblotting experiments were also performed using rabbit anti-CypA protein antibody IgG (purchased from Proteintech) as the primary antibody and HRP-conjugated goat anti-rabbit IgG as the secondary antibody (purchased from Jackson ImmunoResearch) with the ECL color development method for analysis.

[0124] F5: 5’-CATGGGCAGCTTCAAACGTTTC.

[0125] R5: 5’-ACGGCGCCCTCTAAGGGT.

[0126] The results are shown in Figure 6 Figures B and 6C. Transfection of siRNAs targeting CypA in PAMs cells significantly reduced the protein level of CypA in the cells ( Figure 6 Figure B), indicating that all three synthesized siRNAs could effectively target CypA. More importantly, transfection of siCypA significantly reduced the production of ASFV P72 and P30 proteins ( Figure 6 Figure B), indicating that knockdown of CypA interfered with the replication of ASFV in cells. The PCR detection results further confirmed that knockdown of CypA significantly inhibited the accumulation of ASFV virus particles in the infected supernatant ( Figure 6 Figure C). These results suggest that reducing CypA significantly decreases the protein level of P72, which is not conducive to the infection and replication of ASFV.

[0127] The sequence of siCypA-1 is as follows:

[0128] siCypA-1-F (SEQ ID NO.14): GGUUCCUGCUUUCACAGAAUU.

[0129] siCypA-1-R (SEQ ID NO.15): UUCUGUGAAAGCAGGAACCUU。

[0130] The sequence of siCypA-2 is as follows:

[0131] siCypA-2-F (SEQ ID NO.16): GGGCAUGAAUAUUGUGGAAUU。

[0132] siCypA-2-R (SEQ ID NO.17): UUCCACAAUAUUCAUGCCCUU。

[0133] The sequence of siCypA-3 is as follows:

[0134] siCypA-3-F (SEQ ID NO.18): CUGACUGUGGACAAAUCUAUU。

[0135] siCypA-3-R (SEQ ID NO.19): UAGAUUUGUCCACAGUCAGUU。

[0136] The sequence of non-targeting siRNA is as follows:

[0137] NC-siRNA-F (SEQ ID NO.20): UUCUCCGAACGUGUCACGUUU。

[0138] NC-siRNA-R (SEQ ID NO.21): ACGUGACACGUUCGGAGAAUU。

[0139] Example 7: Detection of the interference of the cyclic peptide inhibitor of CypA with the interaction between CypA and P72

[0140] Cyclosporin A (CsA) is the most classical inhibitor of CypA and is also a drug widely used clinically to prevent organ transplant rejection. NIM811 and Alisporivir are derivatives of CsA, but these two derivatives do not have immunosuppressive effects. They are both cyclic small molecules with a size of approximately 1.2 kDa.

[0141] Structural data (1CWA, 1CWM, and 5HSV) in the PDB database show that CsA, NIM811, or Alisporivir can specifically bind to the CypA protein ( Figure 7 A). Moreover, the region where they bind to CypA is also the region where P72 binds ( Figure 7In Figures A and 7B, it was hypothesized that these three cyclic peptide inhibitors of CypA would interfere with the formation of the CypA-P72 complex.

[0142] In the present invention, in vitro binding experiments were conducted to test the effects of these three cyclic peptide inhibitors on the binding of CypA to P72. These three cyclic peptide inhibitors were separately dissolved in DMSO to prepare stock solutions with a concentration of 5 mM. An appropriate amount of strep-P72 recombinant protein was mixed with GST-CypA protein, and an equimolar amount of CypA cyclic peptide inhibitor (both at a concentration of 5 μM) was added simultaneously, and the mixture was incubated at 4 °C for 2 hours. Meanwhile, the DMSO-treated group was used as a control. Then, co-precipitation enrichment was carried out using Strep-Tactin beads, and finally, immunoblotting experiments were performed (for specific steps, refer to Examples 1 and 5).

[0143] The results are as Figure 7 shown in Figure C. The upper panel shows the results of co-precipitation, and the lower panel shows the results of non-co-precipitation. In the presence of DMSO, a high level of GST-CypA protein was detected in the enrichment product of strep-p72; however, the addition of the three cyclic peptide inhibitors significantly reduced the protein level of GST-CypA in the enrichment product. This indicates that the three cyclic peptide inhibitors significantly interfere with the recruitment of CypA protein by P72 protein in vitro. This result not only identified the region on CypA responsible for binding to P72 but also demonstrated that the CypA cyclic peptide inhibitor can block the formation of the P72-CypA complex.

[0144] Example 8: Detection of the degradation of P72 protein caused by the cyclic peptide inhibitor of CypA

[0145] HEK293T cells were seeded in a 12-well plate and cultured overnight. After the cells grew to 90% confluence, 1 μg of Strep-P72 expression plasmid and 1 μg of B602L-His plasmid were co-transfected. Twenty-four hours after transfection, different concentrations of the cyclic peptide inhibitor CsA were added, and the cells were cultured for another 24 hours. Then, the culture medium was removed, 200 μL of 1X SDS loading buffer was added for cell lysis, and the samples were collected and heated for denaturation. Finally, the samples were detected using immunoblotting experiments.

[0146] The results are as Figure 8 shown. As the concentration of the cyclic peptide inhibitor CsA increased, the protein level of P72 gradually decreased ( Figure 8 ), indicating that the addition of CsA significantly reduced the protein stability of P72, resulting in a decrease in P72 protein. This also suggests that the CypA cyclic peptide inhibitor is likely to affect the infection of ASFV.

[0147] Example 9: The cyclic peptide inhibitor of CypA can effectively inhibit the infection and replication of ASFV

[0148] PAMs cells were infected with the ASFV Pig / HLJ / 2018 strain (MOI = 0.2), and three cyclic peptide inhibitor stock solutions with 5 mM DMSO as the solvent were added to the infected cells at a final concentration of 10 μM. DMSO was used as the drug control, uninfected PAMs cells treated with DMSO were used as the negative control group, and infected PAMs cells treated with DMSO were used as the infection control group. After 48 hours of infection, the infected supernatant was collected for nucleic acid extraction, and qPCR quantitative analysis of the B646L gene fragment was performed (the same method as in Example 6), and the relative level of the ASFV genome was calculated. In the same method as in Example 5, WB detection was performed on the African swine fever virus P30 protein in the infected supernatant.

[0149] The results are as Figure 9 shown in A. The infected group treated with DMSO showed a high level of ASFV genome content, indicating that ASFV can proliferate normally in the presence of DMSO, but the three cyclic peptide inhibitors of CypA can significantly inhibit the proliferation of ASFV virus.

[0150] PAMs cells were infected with the ASFV HLJ / 18-7GD strain (MOI = 0.2), and different concentrations (0 μM, 1 μM, 5 μM, 10 μM, and 20 μM) of CsA and NIM811 were added simultaneously. After 48 hours of infection, the infected supernatants of each group were collected to detect the copy number of the virus genome B646L gene fragment (the same method as in Example 6), and at the same time, cell lysates were lysed with 1X SDS loading buffer to prepare samples for immunoblot analysis. The results showed that as the concentration of the cyclic peptide inhibitor gradually increased, the synthesis of viral protein (P30) gradually decreased ( Figure 9 B). Especially when the inhibitor concentration was greater than 10 μM, the synthesis of viral protein basically stopped. In addition, the genomic content of virus particles in the infected supernatant also decreased significantly with the increase in the inhibitor concentration ( Figure 9 C). These results indicate that these three cyclic peptide inhibitors can significantly inhibit the replication and assembly of ASFV virus particles.

[0151] Example 10: Test on the combined use of cyclic peptide inhibitors of CypA

[0152] PAMs cells were seeded into 6-well plates. After the cells adhered, they were infected with the ASFV HLJ / 18-7GD strain (MOI = 0.2). Simultaneously with the infection, a cyclic peptide inhibitor of CypA (total final concentration of 10 μM) was added for treatment. The cyclic peptide inhibitor of CypA was added alone (10 μM CsA, 10 μM NIM811, or 10 μM Alisporivir), or added after different combinations: 5 μM CsA + 5 μM NIM811, 5 μM CsA + 5 μM Alisporivir, 5 μM NIM811 + 5 μM Alisporivir, 3.3 μM CsA + 3.3 μM NIM811 + 3.3 μM Alisporivir. After 48 hours of infection, the infected supernatant was collected for nucleic acid extraction, and qPCR was used to quantitatively analyze the B646L gene fragment in the virus particles in the supernatant (the method was the same as in Example 6).

[0153] The results are shown in Figure 10 . It can be seen that the inhibitory effect of ASFV when these three cyclic peptide inhibitors are paired pairwise or used together is between that of single inhibitors. The three cyclic peptide inhibitors do not show obvious mutual synergy or antagonism in resisting African swine fever virus replication and can be used in combination.

[0154] Example 11: Test on the effect of the cyclic peptide inhibitor of CypA on the expression of the ASFV transcriptome and on host inflammation

[0155] PAMs were seeded into 6-cm culture dishes. After the cells adhered, infection and drug addition (simultaneous treatment) were carried out. A total of 4 large groups were set up: non-infected group (Mock) + DMSO; infected group (ASFV, MOI = 0.2) + DMSO; Mock + NIM811 (10 μM); and ASFV (MOI = 0.2) + NIM811 (10 μM). The infected strain was the ASFV HLJ / 18-7GD strain. After 48 hours of infection, the supernatant was discarded, and Trizol lysis buffer was added to lyse the cells. The cells were snap-frozen in liquid nitrogen for 10 minutes, then stored in a -80 °C refrigerator, and then RNA-seq sequencing was performed.

[0156] After the sequencing was completed, the sequencing data were pre-processed, including quality control (FastQC), filtering of low-quality data (SOAPnuke), alignment to the reference genome (Bowtie2), etc. Then, differential analysis and visualization analysis of gene expression between different groups were performed.

[0157] The results are shown in Figure 11 A and Figure 11 B. Figure 11 A and Figure 11B is the differential analysis data of the ASFV transcriptome. The results show that the transcriptome of ASFV is significantly expressed after ASFV infection ( Figure 11 A, |Log2FC| > 2 and p-adjust < 0.01 are considered significantly different), but in the presence of NIM811, the expression of the ASFV transcriptome is significantly inhibited ( Figure 11 B). This indicates that NIM811 can effectively directly or indirectly interfere with the overall expression of the viral genome under ASFV infection conditions.

[0158] Figure 11 C and Figure 11 D are the differential analysis data of the host (PAMs cells) transcriptome. The red dots are the host genes significantly upregulated after virus infection. The results show that when ASFV infects, a large number of genes in the host cells are upregulated ( Figure 11 C, |Log2FC| > 2 and p-adjust < 0.01 are considered significantly different). The top ten genes with the most significant changes are: CYP19A3, BMP8A, IL13, DMRT1, OPALIN, SLC16A12, PRDM5, FAP, GMNC, and GCG. However, in the presence of NIM811, the host genes upregulated after ASFV infection are significantly reduced ( Figure 11 D), indicating that various stress responses (including inflammatory responses) of the host under infection conditions are effectively alleviated. These data prove that the cyclic peptide inhibitor of CypA can significantly inhibit or alleviate virus replication and the overall host response caused by it.

[0159] In summary, this type of cyclic peptide small molecule can be used as a drug to resist ASFV infection.

[0160] As is known by common technical knowledge, the present invention can be implemented by other embodiments that do not depart from its spiritual essence or essential features. Therefore, the above-disclosed embodiments are illustrative in all aspects and not exclusive. All changes within the scope of the present invention or equivalent to the present invention are encompassed by the present invention.

Claims

1. A composition, which is composition 1, composition 2, composition 3 or composition 4; The active ingredients of the composition 1 include a first active substance and a second active substance; The active ingredients of the composition 2 include a second active substance and a third active substance; The active ingredients of the composition 3 include a first active substance and a third active substance; The active ingredients of the composition 4 include a first active substance, a second active substance and a third active substance; The first active substance is cyclosporine A, a pharmaceutically acceptable salt of cyclosporine A, or a combination thereof; The second active substance is alisporivir, a pharmaceutically acceptable salt of alisporivir, or a combination thereof; The third active substance is [MeIle]4-cyclosporine, a pharmaceutically acceptable salt of [MeIle]4-cyclosporine, or a combination thereof; The structural formula of cyclosporine A is as follows: The structural formula of alisporivir is as follows: [MeIle]4-cyclosporine has the following structural formula:

2. The composition according to claim 1, characterized in that The molar ratio of any two active substances in the composition is 1:0.1-10.

3. The composition according to claim 1, characterized in that The pharmaceutically acceptable salt of cyclosporine A includes: hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurine, gluconate, fructose, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, citrate, fumarate and ascorbate of cyclosporine A; The pharmaceutically acceptable salts of alisporivir include: hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurine, gluconate, fructose, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, citrate, fumarate and ascorbate of alisporivir; The pharmaceutically acceptable salts of [MeIle]4-cyclosporine include: hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurine, gluconate, fructose, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, citrate, fumarate and ascorbate of [MeIle]4-cyclosporine.

4. The composition according to claim 1, characterized in that The composition also contains pharmaceutically acceptable excipients.

5. An siRNA, wherein the siRNA is any one of the first siRNA, the second siRNA and the third siRNA, a combination of any two of them, or a combination of the three; The first siRNA contains RNAs with sequences as shown in SEQ ID NO.14 and SEQ ID NO.15 respectively; The second siRNA contains RNAs whose sequences are shown in SEQ ID NO.16 and SEQ ID NO.17 respectively; The first and third siRNAs contain RNAs with sequences as shown in SEQ ID NO.18 and SEQ ID NO.19 respectively.

6. Use of a cyclophilin A inhibitor in the preparation of a preparation for preventing African swine fever, treating African swine fever, slowing down African swine fever or inhibiting the proliferation of African swine fever virus.

7. The use according to claim 6, characterized in that The cyclophilin A inhibitor is selected from the group consisting of the composition of any one of claims 1 to 4, the first active substance in the composition of any one of claims 1 to 4, the second active substance in the composition of any one of claims 1 to 4, the third active substance in the composition of any one of claims 1 to 4, siRNA targeting the cyclophilin A, shRNA targeting the cyclophilin A, sgRNA targeting the cyclophilin, antisense RNA targeting the cyclophilin, a competitive inhibitor of African swine fever virus P72 protein binding to the cyclophilin A, and an antibody or a derivative thereof that specifically binds to the cyclophilin A; Wherein, the African swine fever virus P72 protein binding competitive inhibitor of the cyclophilin A can specifically bind to the African swine fever virus P72 protein and can block the 139-142 amino acid residues of the African swine fever virus P72 protein; the African swine fever virus P72 protein binding competitive inhibitor of the cyclophilin is selected from an antibody that specifically binds to the African swine fever virus P72 protein, an antigen-binding fragment of an antibody that specifically binds to the African swine fever virus P72 protein, and a fusion protein containing an antigen-binding fragment of an antibody that specifically binds to the African swine fever virus P72 protein; The antibody or its derivative that specifically binds to the cyclophilin A can specifically bind to the cyclophilin A and can block amino acid residues 55, 60, 61, 63, 101, 113, 121, 122 and 126 of the cyclophilin A. The antibody derivative that specifically binds to the cyclophilin A is selected from the antigen-binding fragment of the antibody that specifically binds to the cyclophilin A and a fusion protein containing the antigen-binding fragment of the antibody that specifically binds to the cyclophilin A.

8. The use according to claim 6 or 7, characterized in that The preparation is a medicine or a feed additive; and / or The siRNA targeting the cyclophilin A is the siRNA according to claim 5.

9. The use according to any one of claims 6 to 8, characterized in that: The mechanism for preventing African swine fever, treating African swine fever, slowing down African swine fever or inhibiting the proliferation of African swine fever virus is selected from: The cyclophilin A inhibitor hinders or weakens the binding of cyclophilin A to the African swine fever virus P72 protein; The cyclophilin A inhibitor reduces the stability of the African swine fever virus P72 protein; The cyclophilin A inhibitor hinders the correct folding of the African swine fever virus P72 protein; The cyclophilin A inhibitor promotes the degradation of African swine fever virus P72 protein; The cyclophilin A inhibitor reduces the expression level of African swine fever virus P72 protein; The cyclophilin A inhibitor interferes with the assembly of African swine fever virus particles.

10. The use according to claim 6, characterized in that Selected from A1, A2 and A3 as follows; A1: The African swine fever strain selected from GenBank is MK333180.1; A2: The amino acid sequence of the African swine fever virus P72 protein is shown in GenBank No. QBH90570.1; A3: The protein sequence of the cyclophilin A is shown in SEQ ID NO.1.

Images