Application of natural immune signal linker protein MAVS mutant in resisting African swine fever virus

Abstract

The invention discloses an application of a protease cleavage site mutant of a natural immune signal linker protein MAVS in resisting African swine fever viruses, aiming at three sites of African swine fever protease pS273R cleavage MAVS, the research finds that the site mutant can efficiently inhibit the replication of the African swine fever viruses; and breeding the variety pigs capable of resisting African swine fever by utilizing a gene editing technology.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to the application of a MAVS protein mutant in resisting African swine fever virus. Specifically, it relates to the application of a mutant in which the African swine fever virus protease pS273R cuts the precise site of the host innate immune signaling adaptor protein MAVS in resisting African swine fever virus. Background Art

[0002] African swine fever (ASF) is a highly contagious and devastating disease affecting domestic and wild pigs, typically causing an acute, hemorrhagic illness with a mortality rate as high as 100%. African swine fever virus (ASFV) is a large, double-stranded DNA nucleocytoplasmic virus belonging to the Assurviridae family, the only member of this family and the only DNA virus transmitted by arthropod ticks (soft ticks). ASFV is an enveloped virus with an icosahedral internal structure approximately 200 nm in diameter. The ASFV virion consists of five components: the outer viral membrane, the capsid, the inner membrane, the core capsid, and the nuclear genome. The genome size varies slightly between strains, ranging from approximately 170 to 190 kb, encoding 151 to 167 open reading frames, including 54 structural proteins and over 100 nonstructural proteins.

[0003] The ASFV protease pS273R, encoded by the viral S273R gene, is a 31 kDa protein consisting of two domains: the N-terminal "Arm Domain" (AD) and the C-terminal "Core Domain" (CD). pS273R belongs to the SUMO-1 protease family and produces cysteine ​​proteases. It plays a key role in catalyzing the maturation of the ASFV polyproteins pp220 and pp62 into core capsid proteins. pS273R precisely cleaves the polyproteins pp220 and pp62 to produce the six main structural components of the virion core capsid. p37, p34, p40, and p150 are derived from the polyprotein pp220, while p15 and p35 are derived from the polyprotein pp62. Protease pS273R not only participates in virion assembly but also targets and cleaves host immune proteins, enabling immune evasion and effective viral replication. Protease pS273R specifically cleaves the host protein Gasdermin D (GSDMD) between glycine 107 and alanine 108 (G107-A108), generating two cleavage fragments: GSDMD-N1-107 and GSDMD-C108-279. These fragments fail to trigger pyroptosis and instead inhibit it, thereby facilitating viral evasion of the host's antiviral immune response. Similarly, protease pS273R specifically cleaves the stress granule protein G3BP1 between glycine 140 and phenylalanine 141 (G140-F141), generating two nonfunctional fragments: G3BP1-N1-140 and G3BP1-C141-456. These fragments inhibit stress granule formation and promote viral replication.

[0004] The innate immune response, as the first line of defense against viral infection, utilizes pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) to detect invading pathogens. Among various PRRs, nucleic acids are the primary PRRs during viral infection. Therefore, nucleic acid-sensing RIG-I-like receptors (RLRs) and cytoplasmic DNA receptors (CDRs) play a key role in the cellular antiviral response. ASFV is a nucleocytoplasmic giant DNA virus that primarily activates the DNA-sensing cGAS-STING signaling pathway. cGAS, a member of the CDR and a nucleotidyltransferase, uses GTP and ATP as substrates. In response to the activator DNA, cGAS synthesizes the second messenger 2′3′-cGAMP, which binds to and activates the signaling protein STING in the endoplasmic reticulum (ER). STING then oligomerizes and translocates from the ER to the Golgi apparatus via the ER-Golgi intermediate compartment (ERGIC). Upon activation, the [(D / E)xPxPLR(S / T)D] motif within the STING C-terminal tail (CTT) recruits the TBK1 kinase. Recruited TBK1 then self-activates and phosphorylates a conserved serine (S) within the pLxIS motif within the STING CTT, further recruiting the transcription factor IRF3, which is then phosphorylated by the nearby TBK1. Phosphorylated IRF3 forms a dimer and translocates to the nucleus, inducing the expression of type I interferons (IFN-I). STING also activates NF-κB, to a lesser extent, through the IKKα / β / γ complex. This NF-κB, in concert with IRF3, induces IFN expression and the production of proinflammatory cytokines.

[0005] RIG-I and MDA5, members of the RLR family, are the primary viral RNA receptors in the cytoplasm. They share similar structures and the same downstream signaling adaptor protein, MAVS. RIG-I recognizes short double-stranded RNA (dsRNA) with a 5'-PPP cap, while MDA5 recognizes longer dsRNA. When viral dsRNA binds to the RD region of RIG-I / MDA5, the conformation of RIG-I / MDA5 changes. This conformational shift recruits the adaptor protein MAVS, which in turn binds to and phosphorylates TBK1 and IKKε. Finally, phosphorylated TBK1 and IKKε activate IRF3 or NF-κB nuclear translocation, initiating type I IFN transcription.

[0006] ASFV, a nucleocytoplasmic giant double-stranded DNA virus, activates the DNA receptor cGAS-STING signaling pathway to induce type I IFN production. However, recent studies have shown that early in ASFV infection, RIG-I and MDA5 transcription levels increase significantly but then decrease with increasing viral replication. The ASFV I267L protein interacts with the E3 ubiquitin ligase Riplet, disrupting the Riplet-RIG-I interaction. This impairs Riplet-mediated K63 polyubiquitination of RIG-I and its activation-induced IFN transcription, interfering with the RLR-IFN signaling pathway and thereby inhibiting the RNA polymerase III-RIG-I-mediated antiviral response. These results suggest that ASFV infection activates not only the innate immune DNA receptor cGAS-STING signaling pathway and its mediation of antiviral responses, but also the innate immune RNA receptor RIG-I / MDA5-MAVS signaling pathway and its mediation of antiviral responses. ASFV has a large genome, encoding over 150 proteins, but the mechanisms by which many of these proteins evade the host antiviral response remain unclear. Our screening results above indicate that ASFV protease pS273R can cleave the adaptor protein porcine (p)MAVS in the RNA receptor signaling pathway. Therefore, further investigation is needed to explore the inhibitory effect of protease pS273R on the porcine (p)RIG-I / MDA5-MAVS signaling pathway.

[0007] MAVS is an adaptor protein in the innate immune RNA receptor (RLR) signaling pathway and a key antiviral protein whose expression level directly influences the host's antiviral immune response. However, limited research has been conducted on the immune escape and innate antiviral effects of the African swine fever virus protease pS273R. Currently, the sites and target genes for the precise cleavage of the innate immune antiviral protein MAVS by the African swine fever virus protease pS273R have been identified, creating a bottleneck that urgently needs to be addressed in the development and production of African swine fever-resistant pigs. Summary of the Invention

[0008] The present invention aims to solve the technical problem to be solved, overcomes the shortcomings of the existing technology and provides an application of a natural immune signaling adaptor protein MAVS mutant in resistance to African swine fever virus, provides a new target for the breeding or preparation of African swine fever virus-resistant pigs, and is of great significance to improving the level of disease-resistant breeding research.

[0009] One of the objects of the present invention is to provide a MAVS protein mutant, wherein the MAVS protein mutant is mutated at amino acid residue 192 and / or amino acid residue 270 and / or amino acid residue 388 of the sequence shown in SEQ ID NO.1; and the amino acid residue site 192 is mutated to Ala, the amino acid residue site 270 is mutated to Ala, and the amino acid residue site 388 is mutated to Ala.

[0010] This study found that the ASFV protease pS273R cleaves three precise sites on the host innate immune RNA receptor signaling pathway adaptor protein porcine (p)MAVS: G192-H193, G270-A271, and G388-T389, disrupting the porcine MAVS molecular structure and causing it to lose its antiviral signaling function. By targeting these three sites on MAVS cleaved by the ASFV protease pS273R, gene editing technology was used to collaboratively edit porcine MAVS-related genes, enabling the breeding of African swine fever-resistant sows. Therefore, the discovery of multiple protease cleavage sites provides important key targets for genetic breeding of ASFV-resistant pigs and offers new insights into ASFV prevention and control.

[0011] The technical solution further optimized by the present invention is as follows: The MAVS protein mutant undergoes mutations at amino acid residues 192, 270, and 388 in the sequence shown in SEQ ID NO. 1; the amino acid residues 192, 270, and 388 are mutated to Ala.

[0012] A second object of the present invention is to provide a polynucleotide molecule encoding the above-mentioned MAVS protein mutant.

[0013] The third object of the present invention is to provide a vector containing the above-mentioned polynucleotide molecule.

[0014] A fourth object of the present invention is to provide a host cell, wherein the host cell contains the above-mentioned vector or the chromosome has the above-mentioned polynucleotide molecule integrated therein.

[0015] The fifth object of the present invention is to provide the use of the MAVS protein mutant in resisting African swine fever virus.

[0016] In the above application, by site-directed mutation of the amino acids at positions 192 and / or 270 and / or 388 of the MAVS protein, changing them from glycine to alanine, and then complementing them into monkey kidney epithelial cells in which the MAVS gene was knocked out, the replication of African swine fever virus in cells infected with African swine fever virus was inhibited.

[0017] In the above application, the MAVS protein mutant is used to resist cleavage by ASFV protease.

[0018] Furthermore, the MAVS protein mutant is used in the genetic breeding of pigs resistant to African swine fever.

[0019] The present invention systematically verified the molecular mechanism of ASFV's immune escape from the innate immune antiviral effect of its protease pS273R at the cellular level. The ASFV protease pS273R cleaves three precise sites, G192-H193, G270-A271, and G388-T389, on the host innate immune RNA signaling pathway adaptor protein MAVS. Through gene editing technology, the relevant genes on pig MAVS were edited to cultivate gene-edited pigs resistant to African swine fever virus. Therefore, the ASFV protease pS273R precisely cleaves multiple sites of the innate immune antiviral protein MAVS and its mutants, providing an important key target for the genetic breeding of ASFV-resistant pigs and a new approach to the prevention and control of ASFV. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the result of screening the cleavage effect of ASFV protease pS273R on porcine innate immune signaling adaptor protein in HEK293T cells.

[0021] Figure 2 Figure 2 shows the results of Western blotting analysis of the dose-dependent cleavage of pMAVS by protease pS273R. Figure A shows the Western blotting results of HEK293T cells co-transfected with mCherry-pS273R plasmid and GFP-pMAVS, and Figure B shows the Western blotting results of HEK293T cells co-transfected with pS273R-HA plasmid and GFP-pMAVS-mCherry.

[0022] Figure 3 Figure 2 is the result of the interaction between protease pS273R and pMAVS and their influence. Figure A is the Western blotting detection result of immunoprecipitation after HEK293T cells were co-transfected with mCherry-pS273R plasmid and GFP-pMAVS, B is the Western blotting detection result of immunoprecipitation after MA104 cells were infected with or without ASFV-GFP virus, C is the immunofluorescence detection result after primary porcine alveolar macrophages (PAM) were infected with or without YZ-1ASFV strain, D is the Western blotting detection result of immunoprecipitation after HEK293T cells were co-transfected with increasing amounts of mCherry-pS273R plasmid, GFP-pMAVS and pRIG-I-HA, and E is the Western blotting detection result of immunoprecipitation after HEK293T cells were co-transfected with increasing amounts of mCherry-pS273R plasmid, GFP-pMAVS and pMAD5-HA.

[0023] Figure 4Figure 3 is the result of Western blotting detection of the effect of pS273R enzyme activity on its cleavage of pMAVS. A is a schematic diagram of pS273R point mutants and truncation mutants. B is the result of Western blotting detection after HEK293T cells were co-transfected with GFP-pMAVS plasmid and pS273R-WT or 5-point pS273R mutants.

[0024] Figure 5 Figure 1 shows the identification results of the sites on the pMAVS protein cleaved by the protease pS273R. A is a schematic diagram of the pMAVS Gly-Gly site, B is a schematic diagram of the pMAVS point mutants, and C is the Western blotting detection results after HEK293T cells were co-transfected with the mCherry-pS273R plasmid and pMAVS-WT or five point mutants.

[0025] Figure 6 Schematic diagram of the sites where protease pS273R cleaves pMAVS based on multiple point mutations. A is a schematic diagram of the double and triple mutants of pMAVS. B is the Western blotting results after HEK293T cells were co-transfected with the mCherry-pS273R plasmid and pMAVS-WT or four multiple-point mutants.

[0026] Figure 7 Figure 3 is a schematic diagram of the signaling activity of the pMAVS fragment cleaved by protease pS273R. A is the sequence alignment of porcine and human MAVS. B is a schematic diagram of the pMAVS fragment cleaved by protease pS273R. C is a diagram of the promoter activity after HEK293T cells were transfected with pS273R or vector, GFP-pMAVS, or 9 cleaved pMAVS fragments, as well as IFNβ-luc and RL-TK plasmids.

[0027] Figure 8 The immunoprecipitation results of the pMAVS fragment cleaved by pS273R protease and its interaction with pS273R are shown.

[0028] Figure 9 The CD domain of pS273R is the key region for interaction with pMAVS.

[0029] Figure 10Figure 3 shows the effect of pS273R on the signaling activity mediated by exogenously expressed RNA receptor pathways pRIG-I, pMDA5, and pMAVS. Figure A shows the promoter activity assay results after HEK293T cells were transfected with increasing amounts of pS273R and 20 ng of pRIG-I-HA. Figure B shows the promoter activity assay results after HEK293T cells were transfected with increasing amounts of pS273R and pMDA5-HA. Figure C shows the promoter activity assay results after HEK293T cells were transfected with increasing amounts of pS273R and GFP-pMAVS.

[0030] Figure 11 Figure 3 shows the effect of pS273R on the signaling activity of the endogenous RNA receptor porcine RIG-I / pMDA5-pMAVS pathway. Figure A shows the RT-qPCR detection results of downstream IFNβ gene transcription induced by 3D4 / 21 cells transfected with increasing amounts of mCherry-pS273R plasmid and stimulated by poly(I:C) transfection. Figure B shows the RT-qPCR detection results of downstream ISG56 gene transcription induced by 3D4 / 21 cells transfected with increasing amounts of mCherry-pS273R plasmid and stimulated by poly(I:C) transfection. Figure C shows the RT-qPCR detection results of downstream TNF-a gene transcription induced by 3D4 / 21 cells transfected with increasing amounts of mCherry-pS273R plasmid and stimulated by poly(I:C) transfection. Figure D shows Western blotting of 3D4 / 21 cells transfected with increasing amounts of mCherry-pS273R plasmid and stimulated by poly(I:C) transfection. Blotting detection results, E is the Western blotting detection results after 3D4 / 21 cells were transfected with incremental mCherry-pS273R plasmid and stimulated by VSV-GFP virus infection.

[0031] Figure 12 These are the results of the effect of protease pS273R on the anti-ASFV effect of pRIG-I / pMDA5 signal. In the figure, A is the fluorescence microscopy observation result of protease pS273R interfering with the anti-ASFV effect of pRIG-I / pMDA5 signal, B is the flow cytometry detection result of protease pS273R interfering with the anti-ASFV effect of pRIG-I / pMDA5 signal, C is the RT-qPCR detection result of protease pS273R interfering with the anti-ASFV effect of pRIG-I / pMDA5 signal, and D is the Western blotting detection result of protease pS273R interfering with the anti-ASFV effect of pRIG-I / pMDA5 signal.

[0032] Figure 13These are the results of the effect of silencing pS273R expression on the anti-ASFV effect of RNA receptor signaling. In the figure, A is the fluorescence microscopy observation result of the anti-ASFV effect of pS273 expression silencing on RNA receptor signaling, B is the flow cytometry detection result of the anti-ASFV effect of pS273 expression silencing on RNA receptor signaling, C is the RT-qPCR detection result of the anti-ASFV effect of pS273 expression silencing on RNA receptor signaling, and D is the Western blotting detection result of the anti-ASFV effect of pS273 expression silencing on RNA receptor signaling.

[0033] Figure 14 This is the sequencing result of MAVS knockout MA104 cell clone.

[0034] Figure 15 These are the analysis results of ASFV-GFP infected MAVS knockout MA104 cells. Figure A shows the fluorescence microscopy observation results of ASFV-GFP infected MAVS knockout MA104 cells, B shows the flow cytometry detection results of ASFV-GFP infected MAVS knockout MA104 cells, C shows the qPCR detection results of ASFV-GFP infected MAVS knockout MA104 cells, and D shows the Western blotting detection results of ASFV-GFP infected MAVS knockout MA104 cells.

[0035] Figure 16 The figure shows the effect of silencing endogenous MAVS in primary porcine alveolar macrophages (PAM) on ASFV replication. Figure A shows the RT-qPCR result of silencing endogenous MAVS in PAM on ASFV replication, and Figure B shows the Western blotting result of silencing endogenous MAVS in PAM on ASFV replication. Figure 17 The figure shows the results of promoter activity verification of MAVS and its point mutants.

[0036] Figure 18 Replenishing MAVS for MAVS and point mutants - / - The results of ASFV infection in MA104 cells. A in the figure is MAVS and point mutant complemented MAVS - / - Fluorescence microscopy observation results of MA104 cells infected with ASFV, B is MAVS and point mutant complemented MAVS - / - Flow cytometry results of MA104 cells infected with ASFV. C is MAVS and point mutant complemented MAVS - / - qPCR results of MA104 cells infected with ASFV, D is MAVS and point mutant complemented MAVS - / - Western blotting results of MA104 cells infected with ASFV. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described in detail below in conjunction with the embodiments: This embodiment is implemented on the premise of the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection authority of the present invention is not limited to the following embodiments.

[0038] The materials and reagents mentioned in the present invention can be purchased by the public from domestic and foreign commercial channels and will not be described one by one here.

[0039] DNA sequences were amplified by PCR from porcine macrophage cell line 3D4 / 21 cells and cloned into pEGFP vector to obtain nine porcine (p) signal adaptor protein recombinant expression vectors pRIPK2, pSTING, pASC, pMAVS, pMyD88, pCARD9, pBCL10, pMALT1 and pTRIF with N-terminal GFP tags; pS273R protease was amplified by PCR from ASFV and cloned into pmCherry vector to obtain the recombinant expression vector mCherry-p containing pS273R protease. S273R; pMAVS was PCR amplified from ASFV, and the sequence was cloned into the pEGFP expression vector to obtain the recombinant expression vector GFP-pMAVS containing the pMAVS protein; HEK293T cells were 293T-CRL-3216 from ATCC; MA104 cells were CRL-2378.1 from ATCC; IgG was FLAG mouse antibody purchased from Quanshijin; the preparation method of anti-pS273R is shown in CN118184749A; porcine macrophages 3D4 / 21 were CRL-2843 from ATCC.

[0040] Log in to NCBI, search and download the nucleotide sequence of the porcine MAVS (pMAVS) gene (GenBank: NM_001097429), import the pMAVS sequence file into Benchling, and design cloning primers. The primer sequences are shown in Table 1. Using the pEGFP-C1-pMAVS plasmid as a template, PCR amplify the target gene using Gold Mix (green) enzyme. Purify the amplified product. Use two restriction endonucleases. Bgl II and Sal I. The dual-tag vector pEGFP-mCherry was double-enzyme digested, and the digested product was purified. The recovered pMAVS target fragment and the double-enzyme digested linear pEGFP-mCherry were seamlessly cloned using 2×MultiF Seamless Assembly Mix to obtain the pMAVS plasmid with N-terminal GFP and C-terminal mCherry dual tags.

[0041] The pS273R nucleotide sequence of ASFV China 2018 / 1 (GenBank: MH_766894) was introduced into Benchling, and cloning primers were designed. The primer sequences are shown in Table 1. PCR amplification was performed from ASFV-positive samples using Gold MIX (green) enzyme, and the amplified products were purified. EcoR I / EcoR The eukaryotic expression vector pCAGGS-HA was double-digested with enzymes V, and the digested product was purified. The recovered pS273R target fragment and the double-digested linearized pCAGGS-HA were seamlessly cloned using 2× MultiF Seamless Assembly Mix to obtain the pS273R-HA plasmid.

[0042] The porcine RIG-I and MDA5 sequences were cloned into pcDNA vectors with C-terminal HA tags to obtain recombinant expression vectors pRIG-I-HA and pMAD5-HA.

[0043] To prepare three point mutants at the catalytic triad site, H168R, N187A, and C232S, the S273R nucleotide sequence of ASFV China 2018 / 1 (GenBank: MH_766894) was introduced into the QuickChange Primer Design method for point mutant primer design. The sequences are shown in Primer Table 1. PCR amplification was performed using pmCherry-C1-pS273R as a template using KOD-Plus-Neo high-fidelity DNA polymerase, and the amplified products were cleaved with restriction endonucleases. Dpn I digestion was performed, and a sample was transformed into 50 μL of DH5α competent cells. After transformation, the transformation product was spread onto a kanamycin-resistant LB plate and incubated in an inverted position overnight at 37°C in a bacterial incubator. Plasmid DNA was extracted and sequenced to verify the corresponding mutation.

[0044] To prepare the truncated mutants, the N-terminal "Arm Domain" (AD, 1-83 aa) and the C-terminal "Core Domain" (CD, 84-273 aa) were introduced into Benchling by introducing the pS273R nucleotide sequence of ASFV China 2018 / 1 (GenBank: MH_766894), and the truncated mutant primers were designed. The primer sequences are shown in Table 1. PCR amplification was performed with mCherry-C1-pS273R using Gold MIX (green) enzyme, and gel recovery was performed after amplification. Bgl II / KpnI. The eukaryotic expression vector pmCherry-C1 was double-digested, and the digested products were recovered on gel. The recovered target fragment and the double-digested linear pmCherry-C1 were seamlessly cloned using 2×MultiF Seamless Assembly Mix.

[0045] To construct the pMAVS point mutants: G175A, G192A, G257A, G270A, and G388A, we first searched and downloaded the nucleotide sequence of the porcine MAVS (pMAVS) gene (GenBank: NM_001097429) from NCBI. The QuickChange PrimerDesign method was then used to design primers for the mutants. The primer sequences are shown in Table 1. PCR amplification was performed using pEGFP-C1-pMAVS as a template and KOD-Plus-Neo high-fidelity DNA polymerase. The products were cleaved with restriction endonucleases. Dpn I digestion was performed, and a sample was transformed into 50 μL of DH5α competent cells. After transformation, the transformation product was plated onto a kanamycin-resistant LB plate and incubated in an inverted position overnight at 37°C. The bacterial plasmid was then extracted and DNA sequencing was performed to verify the corresponding mutation.

[0046] Double-point mutants DM1 (G270A / G388A), DM2 (G192A / G388A), DM3 (G192A / G270A), and a triple-point mutant TM (G192A / G270A / G388A) were constructed. The DM1, DM2, and DM3 mutants were constructed as follows: First, the nucleotide sequence of the porcine MAVS (pMAVS) gene (GenBank: NM_001097429) was downloaded from NCBI. Primers for the mutants were designed using the QuickChange Primer Design method. Primer sequences are shown in Table 1 (primers for G192A, G270A, and G388A were used). PCR amplification was performed using KOD-Plus-Neo high-fidelity DNA polymerase, using the single mutants G192A, G270A, and G388A as templates. The products were cleaved with restriction enzymes. DpnI digestion was completed, and a sample was transformed into 50 μL of DH5α competent cells. After transformation, the transformation product was plated onto kanamycin-resistant LB plates and incubated upside down overnight in a 37°C incubator. Plasmid DNA was extracted and sequenced to verify the corresponding mutations. The TM mutants were constructed as follows: PCR amplification was performed using DM1 (G270A / G388A) as a template, primers targeting G192A, and KOD-Plus-Neo high-fidelity DNA polymerase. The product was digested with the restriction endonuclease DpnI, and a sample was transformed into 50 μL of DH5α competent cells. After transformation, the transformation product was plated onto kanamycin-resistant LB plates and incubated upside down overnight in a 37°C incubator. Plasmid DNA was extracted and sequenced to verify the corresponding mutations. In general, double-point mutants were constructed based on single-point mutants, and triple-point mutants were constructed based on double-point mutants. For example, after the G192A mutation was successful, the G270A primer was used to prepare the DM3 mutant. Similarly, DM1, DM2, and TM were all prepared using the above method.

[0047] The nine cleavage fragments of pMAVS (1-192, 193-270, 193-388, 193-524, 1-270, 271-388, 271-524, 1-388, and 389-524) were cloned and expressed as follows: First, the nucleotide sequence of the porcine MAVS (pMAVS) gene (GenBank: NM_001097429) was downloaded from NCBI and imported into the Benchling website. Primers for truncation mutants were designed as shown in Table 1. Using the pEGFP-C1-pMAVS plasmid as a template, the target gene was amplified using Gold Mix (green) enzyme. PCR products were recovered from gels. Simultaneously, two restriction endonucleases were used to amplify the target gene. Bgl II and Kpn I. Double-enzyme digestion of the vector pEGFP-C1 was performed, followed by gel recovery. The recovered pMAVS truncated mutant fragment was seamlessly cloned with the double-enzyme digested linear pEGFP-C1 using 2×MultiFSeamless Assembly Mix. Example 1

[0048] (1) Screening of the cleavage effect of ASFV protease pS273R on the adaptor protein of the innate immune signaling pathway Nine porcine (p) signaling adaptor proteins in the innate immune signaling pathway (i.e., pRIPK2, pSTING, pASC, pMAVS, pMyD88, pCARD9, pBCL10, pMALT1, and pTRIF) with N-terminal GFP tags were co-transfected into HEK293T cells with pS273R.

[0049] HEK293T cells were co-transfected with 500ng of pRIPK2, pSTING, pASC, pMAVS, pMyD88, pCARD9, pBCL10, pMALT1, and pTRIF, respectively, and 500ng of pS273R protein expression vector pmCherry or pS273R. After 24 hours, cells were collected and Western blotting was performed using GFP antibody (GFP antibody is an anti-GFP tag mouse monoclonal antibody (HT801-01), purchased from Beijing Quanshijin Biotechnology Co., Ltd.). The results showed that pS273R could specifically cleave pMyD88 and pMAVS (see Figure 1 ), among which pS273R had a very obvious effect of cutting pMAVS. In Western blotting, GFP mouse antibody incubation produced three cleavage bands: N1 (about 51 kDa), N2 (about 60 kDa), and N3 (about 85 kDa). Therefore, ASFV protease pS273R can specifically cut pMAVS, and pMAVS was selected as the host protein cut by ASFV protease pS273R.

[0050] (2) Protease pS273R interacts with porcine MAVS and cleaves MAVS To further determine the cleavage effect of protease pS273R on pMAVS, HEK293T cells were co-transfected with increasing concentrations of mCherry-pS273R plasmid and GFP-pMAVS plasmid. GFP mouse anti-Western blotting was used to detect the protein expression of pMAVS. HEK293T cells were co-transfected with mCherry-pS273R plasmid (250ng, 500ng and 750ng) and 500ng GFP-pMAVS. After 24h, the cells were collected and subjected to Western blotting. The results showed that with the increase in the dose of pS273R, the expression of the full-length protein of pMAVS gradually decreased, and three gradually obvious cleavage bands were observed: pMAVS-N1 (about 51 kDa), pMAVS-N2 (about 60 kDa), pMAVS-N3 (about 85 kDa) (see Figure 2 A).

[0051] Next, a pMAVS plasmid with an N-terminal GFP and C-terminal mCherry dual tag was constructed (primers are shown in Table 1, where F is the upstream primer and R is the downstream primer; restriction enzyme sites are underlined). This plasmid was then co-transfected with the pS273R plasmid into HEK293T cells to further investigate the cleavage of pS273R on pMAVS. HEK293T cells were co-transfected with 250 ng, 500 ng, and 750 ng of the pS273R-HA plasmid and 500 ng of GFP-pMAVS-mCherry. After 24 hours, cells were harvested and analyzed by Western blotting. The results showed that as the dose of pS273R increased, the expression of the full-length GFP-pMAVS-mCherry protein gradually decreased, while the expression of the three cleavage fragments tagged with N-terminal GFP (pMAVS-N1, pMAVS-N2, and pMAVS-N3) gradually increased. Furthermore, the expression of the three cleavage fragments tagged with C-terminal mCherry (pMAVS-C1, pMAVS-C2, and pMAVS-C3) also gradually increased (see Figure 2B). This indicates that the protease pS273R specifically cleaves pMAVS in a dose-dependent manner, producing multiple cleavage bands.

[0052] To date, no studies have shown that pS273R can interact with pMAVS. The present invention uses a co-immunoprecipitation (Co-IP) assay to verify that pS273R can interact with pMAVS. HEK293T cells were co-transfected with mCherry-pS273R plasmid (1 mg) and 1 mg GFP-pMAVS. After 24 h, the cells were collected and immunoprecipitated with HA antibody (HA antibody is anti-HA tag mouse monoclonal antibody (HT301-01), purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and Western blotting was performed. MA104 cells were infected or not with ASFV-GFP virus stored in the Animal Biosafety Level 3 Laboratory (ABSL-3) of Yangzhou University. After 72 h, the cells were collected and immunoprecipitated with IgG and anti-pS273R, and then Western blotting was performed. Primary porcine alveolar macrophages PAM were infected or not with YZ-1 ASFV strain stored in the Animal Biosafety Level 3 Laboratory (ABSL-3) of Yangzhou University, and immunofluorescence was performed after 72 h. 1 μg GFP-pMAVS and increasing amounts of mCherry-pS273R were added. Plasmids (0.25 μg, 0.5 μg, 0.75 μg) were co-transfected with 1 μg of pRIG-I-HA or 1 μg of pMAD5-HA into HEK293T cells. After 24 hours, cells were collected and immunoprecipitated with HA antibody and analyzed by Western blotting. Co-IP results showed that there was a significant interaction between exogenously expressed pS273R and pMAVS (see Figure 3 Next, ASFV-GFP virus was used to infect MA104 cells, and the interaction between virally expressed pS273R and endogenous MAVS was studied by Co-IP assay. The results showed that under ASFV infection, viral pS273R was able to interact with endogenous MAVS (see Figure 3 B). It was also observed that viral protease pS273R could not only cleave exogenously expressed pMAVS (see Figure 3 A), and is able to cleave endogenous MAVS (see Figure 3 In the endogenous MAVS cleavage, only three cleavage fragments (pMAVS-N1, pMAVS-N2, and pMAVS-N3) were detected due to the use of rabbit antibodies against the MAVS-N terminus (see Figure 3 Confocal immunofluorescence assay showed the same results as Co-IP, i.e., viral pS273R could co-localize with endogenous MAVS under ASFV infection (see Figure 3 C).

[0053] In addition, Co-IP experiments showed that pS273R not only inhibited the interaction between RIG-I and MAVS in a dose-dependent manner, but also inhibited the interaction between RIG-I / MDA5 and MAVS in a dose-dependent manner (see Figure 3 In conclusion, ASFV protease pS273R can interact with MAVS and cleave MAVS, thereby antagonizing the interaction between MAVS and RIG-I / MDA5.

[0054] (3) pS273R relies on its enzymatic activity to cleave the Gly192, Gly270, and Gly388 sites on pMAVS The crystal structure analysis of the pS273R protein showed that the catalytic triad Cys-His-Asn and the complete protein structure of the C-terminal core domain ("Core Domain", CD) play a crucial role in maintaining the activity of the protease pS273R. In order to clarify whether the cleavage of pMAVS by the protease pS273R depends on its enzymatic activity, three point mutants at the catalytic triad site, H168R, N187A and C232S, and two truncation mutants, the N-terminal "Arm Domain" (AD, 1-83 aa) and the C-terminal "CoreDomain" (CD, 84-273 aa) were prepared (primers are shown in Table 1). The pS273R point mutants and truncation mutants are shown in Table 1. Figure 4 A. pS273R and five mutants were co-transfected with pMAVS into HEK293T cells. GFP-pMAVS plasmid (500 ng) was co-transfected with 500 ng of the parental pS273R plasmid (WT, mCherry-C1-pS273R) or five pS273R mutants. After 24 hours, cells were harvested and analyzed by Western blotting. The results showed that, with the exception of pS273R-WT, none of the five pS273R mutants could cleave pMAVS, indicating that the pS273R protease cleaves pMAVS via its enzymatic activity (see ). Figure 4 B).

[0055] Since protease pS273R can specifically cleave the Gly-Gly (GG) amino acid sites in ASFV polyproteins pp220 and pp62. In order to determine the protease pS273R cleavage site in the pMAVS sequence, the amino acid sequences of pMAVS were aligned with those of pp220 and pp62. The results showed that the pMAVS sequence contained five regions with Gly-Gly (GG) amino acid sites, namely GG175, GG192, GG257, GG270 and GG388 sites. The pMAVS Gly-Gly site is shown in Figure 2. Figure 5Therefore, five pMAVS point mutants were constructed by replacing glycine with alanine: G175A, G192A, G257A, G270A, and G388A (primers are shown in Table 1). Figure 5 B. pMAVS and the five mutants were transfected with pS273R into HEK293T cells. HEK293T cells were co-transfected with 500 ng of the parental pMAVS (WT) or the five point mutants. After 24 hours, cells were harvested and analyzed by Western blotting. The results showed that, like the parental pMAVS protein, the G175A and G257A point mutants were cleaved by the pS273R protease to produce three fragments (pMAVS-N1, pMAVS-N2, and pMAVS-N3), indicating that the G175-P176 and G257-A258 sites on pMAVS are not cleavage sites for the pS273R protease. However, the G192A point mutant was cleaved by pS273R to produce pMAVS-N2 and pMAVS-N3 fragments; the G270A point mutant was cleaved by pS273R to produce pMAVS-N1 and pMAVS-N3 fragments; the G388A point mutant was cleaved by pS273R to produce pMAVS-N1 and pMAVS-N2 fragments. pS273R specifically cleaves the Gly192, Gly270, and Gly388 sites on pMAVS (see Figure 5 C). In summary, protease pS273R cleaves pMAVS at sites G192-H193, G270-A271, and G388-T389, generating pMAVS-N 1-192 、pMAVS-N 1-270 and pMAVS-N 1-388 , namely pMAVS-N1, pMAVS-N2 and pMAVS-N3.

[0056] To further confirm that the three sites Gly192, Gly270, and Gly388 are required for the cleavage of pMAVS by the protease pS273R, three double mutants and one triple mutant were constructed: DM1 (G270A / G388A), DM2 (G192A / G388A), DM3 (G192A / G270A), and TM (G192A / G270A / G388A) (primers are shown in Table 1 ). The pMAVS double and triple mutants are shown in Figure 6A. pMAVS and the four mutants were transfected with pS273R into HEK293T cells. 500ng of mCherry-pS273R plasmid was co-transfected with 500ng of pMAVS-WT or the four multiple mutants into HEK293T cells. After 24h, the cells were collected and analyzed by Western blotting. The results showed that the protease pS273R cleaved DM1 to produce only pMAVS-N1, cleaved DM2 to produce only pMAVS-N2, and cleaved DM3 to produce only pMAVS-N3, indicating that the Gly192, Gly270 and Gly388 sites are indeed the pS273R cleavage sites. In addition, it was found that the protease pS273R could not cleave TM, indicating that the three sites of Gly192, Gly270 and Gly388 are all the sites where the protease pS273R cleaves pMAVS (see Figure 6 B). The above results indicate that the protease pS273R specifically cleaves pMAVS at Gly192, Gly270, and Gly388, depending on its enzymatic activity.

[0057] (4) Signaling activity of the pMAVS fragment cleaved by protease pS273R Based on the results of pMAVS cleavage by protease pS273R, three cleavage fragments can be detected in Western blotting with N-terminal and C-terminal antibodies, indicating that this cleavage can be either complete or incomplete, and the two cleavage modes coexist. Therefore, it can be inferred that after complete and incomplete cleavage of the three sites on pMAVS by pS273R, a total of 9 cleavage fragments can be produced. In order to explore the signaling activity of the fragments produced by pS273R protease cleavage of pMAVS. First, the amino acid sequences of porcine and human MAVS were compared, the functional domains and motifs on the protein were annotated, and then the 9 cleavage fragments of pMAVS (1-192, 193-270, 193-388, 193-524, 1-270, 271-388, 271-524, 1-388 and 389-524) were cloned and expressed (primers are shown in Table 1). The sequence alignment results of porcine and human MAVS are shown in Table 1. Figure 7 A, protease pS273R cleavage of pMAVS fragment see Figure 7B. pMAVS and 9 fragment plasmids were transfected into HEK293T cells for promoter assay. HEK293T cells were transfected with pS273R (or mCherry-C1-pS273R vector) together with 20 ng GFP-pMAVS (or cut pMAVS fragment), 10 ng IFNβ-luc (purchased from Shanghai Qincheng Biotechnology Co., Ltd.) and 0.4 ng RL-TK plasmid (purchased from Zhili Zhongte (Wuhan) Biotechnology Co., Ltd.). The total amount of transfected plasmid per well was 50 ng. After 24 hours, the luciferase activity was detected using a dual-luciferase reporter kit. The results showed that overexpression of pMAVS could induce activation of the IFN-β promoter, while overexpression of the 9 cut fragments could not induce promoter activation. At the same time, pS273R could indeed inhibit promoter activation induced by pMAVS. The promoter activity results of the 9 cut fragments produced by pS273R cutting pMAVS were inactive. Figure 7 C. Studies have shown that the CARD and TM functional domains of MAVS play a crucial role in inducing IFN-β production, and neither is indispensable. Protease pS273R cleaves pMAVS, destroying its structural integrity. All resulting cleavage fragments do not contain both the CARD and TM domains and therefore cannot induce IFN-β production, thereby hindering MAVS signaling activity and antiviral responses, and promoting ASFV replication.

[0058] (5) Key domains of interaction between protease pS273R and pMAVS pMAVS and 9 fragment plasmids were co-transfected with pS273R in HEK293T cells, and co-immunoprecipitation (Co-IP) assay was performed to detect protein interaction. 1ug mCherry-pS273R plasmid was co-transfected with 1ug pMAVS-WT or 1ug 9 cut pMAVS fragments into HEK293T cells. After 24h, the cells were collected and immunoprecipitated with mCherry antibody, followed by Western blotting. The results showed that pMAVS and 1-192 aa, 1-270 aa, and 1-388 aa could interact with pS273R, while 193-270 aa, 193-388 aa, 193-524 aa, and 271-388 aa had no interaction with pS273R. The CARD region of pMAVS is the region that interacts with pS273R (see Figure 8 However, no protein expression was detected in the cell lysate for the fragments 271-524 aa and 389-524 aa. However, since the fragments 193-524 aa did not interact with pS273R, the possibility that the fragments 271-524 aa and 389-524 aa interacted with pS273R was also ruled out.

[0059] The CARD domain of MAVS is the structural basis for its interaction with other proteins. The three cleavage fragments of pMAVS (1-192 aa, 1-270 aa, and 1-388 aa) all contain the CARD domain and therefore retain the ability to interact with pS273R. However, due to the loss of the TM domain, they cannot induce IFN-β production. These results indicate that the ASFV protease pS273R destroys the structural integrity of pMAVS by cleaving it, disrupting its structure and signaling, inhibiting type I IFN production and antiviral responses, and thus promoting viral replication.

[0060] To determine which domain of pS273R is the key region for its interaction with pMAVS, pS273R and two truncation mutants AD (1-83 aa) and CD (84-273 aa) were co-transfected with pMAVS into HEK293T cells, and Co-IP assay was performed. 1 μg of mCherry-pS273R or AD or CD plasmid was co-transfected with 1 μg of pMAVS-WT into HEK293T cells. After 24 hours, the cells were collected and immunoprecipitated with GFP antibody, followed by Western blotting. The results showed that the CD domain of pS273R is the key region for its interaction with pMAVS (see Figure 9 ).

[0061] (6) pS273R can inhibit the activity of the porcine RIG-I / MDA5-MAVS signaling pathway pS273R was co-transfected with RNA receptor signaling pathway proteins pRIG-I, pMDA5, and pMAVS, respectively, into HEK293T cells for ISRE, IFN-β, and NF-κB promoter activity assays. Increasing amounts of pS273R (10 ng, 20 ng, and 30 ng) were co-transfected with 20 ng of pRIG-I-HA, pMDA5-HA, or GFP-pMAVS, along with 10 ng of ISRE-luc (purchased from Nanjing Kebai Biotechnology Co., Ltd.), IFNβ-luc, or NF-κB-luc (purchased from BioVector NTCC Type Culture Collection), and 0.4 ng of RL-TK plasmid, resulting in a total of 50 ng of plasmid per well. Luciferase activity was measured 24 hours later using a dual-luciferase reporter assay. The results showed that pS273R significantly inhibited the activities of ISRE, IFN-β and NF-κB promoters induced by exogenous expression of pRIG-I, pMDA5 and pMAVS in a dose-dependent manner, and pS273R could inhibit the activity of porcine RIG-I / MDA5-MAVS signaling pathway (see Figure 10 AC).

[0062] To further investigate the effect of the protease pS273R on endogenous porcine RNA receptor signaling, we stimulated the RNA receptor RIG-I / MDA5-MAVS signaling pathway in porcine macrophage 3D4 / 21 cells using RNA stimulator poly(I:C) (purchased from InvivoGen) transfection and GFP-tagged vesicular stomatitis virus (VSV-GFP, an RNA virus). 3D4 / 21 cells were transfected with increasing amounts of mCherry-pS273R plasmid (250 ng, 500 ng, and 750 ng) and stimulated with 1 μg of poly(I:C) 24 hours later. Six hours later, the cells were harvested and analyzed by RT-qPCR and Western blotting. Furthermore, 3D4 / 21 cells were transfected with increasing amounts of mCherry-pS273R plasmid (250 ng, 500 ng, and 750 ng) and stimulated with VSV-GFP virus 24 hours later for 12 hours. The cells were then harvested and analyzed by Western blotting. Reverse transcription quantitative PCR (RT-qPCR) results showed that pS273R could significantly inhibit the transcriptional expression of downstream IFNβ, ISG56 and IL-8 genes induced by poly(I:C) transfection in a dose-dependent manner (see Figure 11 In addition, Western blotting results showed that exogenous expression of pS273R in 3D4 / 21 cells could inhibit the phosphorylation of TBK1 and IRF3 induced by poly(I:C) transfection in a dose-dependent manner (see Figure 11 Similarly, after 12 h of VSV infection and stimulation of 3D4 / 21 cells, exogenous expression of pS273R could inhibit the phosphorylation of TBK1 and IRF3 in a dose-dependent manner and promote the replication of VSV (see Figure 11 These results indicate that the protease pS273R can inhibit the antiviral signaling activity mediated by the RNA receptor porcine RIG-I / MDA5-MAVS pathway.

[0063] (7) Protease pS273R can inhibit the anti-ASFV immune response mediated by pRIG-I / pMDA5 To determine whether the protease pS273R interferes with the anti-ASFV effect mediated by the RNA receptor signaling pathway, MA104 cells were transfected with increasing amounts of mCherry-pS273R plasmid (250 ng, 500 ng) and stimulated with 1 μg poly(I:C) for 6 h to activate the endogenous RNA receptor RIG-I and MDA5 signaling pathways. MA104 cells were infected with ASFV-GFP virus in the presence and absence of pS273R for 48 h, and viral replication was analyzed by fluorescence microscopy, RT-qPCR, Western blotting, and flow cytometry (see Figure 12 ). Fluorescence microscopy observation and flow cytometry analysis showed that ASFV replication was inhibited after poly(I:C) transfection stimulation compared with the control group, indicating that the innate immune RNA receptor signaling pathway played an anti-ASFV role. Exogenous expression of pS273R can interfere with the inhibitory effect of poly(I:C) on ASFV, and the interference effect is pS273R dose-dependent. The results are shown in Figure 12 Similarly, RT-qPCR assays also showed that poly(I:C) transfection stimulated and inhibited ASFV replication, while exogenous expression of pS273R upregulated ASFV replication in a dose-dependent manner (see Figure 12 In addition, Western blotting results showed that poly(I:C) transfection stimulated the inhibition of ASFV protein p30 and p72 expression, while exogenous expression of pS273R interfered with the inhibitory effect of poly(I:C) and increased the expression levels of ASFV infection proteins p30 and p72 in a dose-dependent manner (see Figure 12 To further determine the effect of reduced pS273R expression on RNA receptor signaling against ASFV, two pairs of pS273R siRNAs were synthesized to silence pS273R expression in ASFV-infected cells, and two pairs of control siRNAs were also synthesized (see Table 1 for synthetic primers). MA104 cells were transfected with the two pairs of pS273R siRNAs and the control siRNA for 24 hours, then stimulated with poly(I:C) for 6 hours, and then infected with ASFV-GFP for 48 hours. Viral replication was assessed by fluorescence microscopy, RT-qPCR, Western blotting, and flow cytometry (see Table 1 for synthetic primers). Figure 13 Fluorescence microscopy, flow cytometry and RT-qPCR analysis showed that compared with the control group, poly(I:C) transfection stimulated the activation of RNA receptor signaling to inhibit ASFV replication, while silencing ASFVpS273R expression could enhance the inhibitory effect of poly(I:C) on ASFV replication (see Figure 13Similarly, Western blotting results showed that poly(I:C) transfection stimulation inhibited the expression of ASFV proteins p30 and p72, while silencing pS273R expression further inhibited the expression of ASFV proteins p30 and p72 (see Figure 13 These results suggest that protease pS273R promotes viral replication by antagonizing the anti-ASFV immune response mediated by the RNA receptor signaling pathway.

[0064] (8) MAVS knockout promotes ASFV replication To study the role of MAVS in anti-ASFV, CRISPR-Cas9 gene editing technology was used to prepare MA104 cells with MAVS knockout, and a heterozygous MAVS knockout cell was obtained. + / - Cell clones and a homozygous knockout of MAVS - / - Cell clones, genome sequencing results are as follows Figure 14 As shown in the figure, the number of bases deleted (-) or inserted (+) for each allele is marked above the sequencing results. PCR detection primers for detecting MAVS gene editing and gene knockout are shown in Table 1. The specific method is: extract the whole genome DNA sample of the knockout cell clone for PCR amplification of the target fragment, clone the PCR fragment into the T vector, transform the bacteria and amplify it, then extract the plasmid for sequencing, and infer the gene editing status of the cell clone genomic DNA based on the sequencing results, thereby preparing MAVS gene heterozygous knockout and homozygous knockout cells. Next, MAVS was infected with ASFV-GFP. + / - MA104 cells and MAVS - / - At 36 h and 72 h, the viral replication in MA104 cells was analyzed by fluorescence microscopy, RT-qPCR, Western blotting, and flow cytometry (see Figure 15 Fluorescence microscopy, flow cytometry, and RT-qPCR analysis showed that MAVS + / - ASFV replication levels increased in MA104 cells, while MAVS - / - The increase in ASFV replication levels in MA104 cells was more significant (see Figure 15 Similarly, Western blotting results showed that MAVS + / - The expression of ASFV proteins p30, p72, and pS273R increased in MA104 cells, while MAVS - / - The expression of ASFV proteins p30, p72, and pS273R in MA104 cells was further increased (see Figure 15D). These results indicate that the signaling adaptor protein MAVS can mediate anti-ASFV immune responses and plays an important role in anti-ASFV immunity. They also clearly demonstrate that the innate immune RNA receptor RIG-I / MDA5-MAVS signaling pathway plays a crucial role in anti-ASFV immunity and that MAVS knockout promotes ASFV replication.

[0065] To further validate the above experimental results, two pairs of pMAVS siRNA were synthesized to silence MAVS in primary porcine alveolar macrophages (PAMs) (synthesized primers are shown in Table 1). PAMs were transfected with the two pairs of pMAVS siRNA and control siRNA, and then infected with isolated wild-type ASFV YZ-1. Viral replication was analyzed by RT-qPCR and Western blotting 36 and 72 hours after infection. MAVS knockdown promoted ASFV replication (see Table 1). Figure 16 ). RT-qPCR results showed that both pairs of pMAVS siRNAs promoted ASFV replication compared with the control siRNA (see Figure 16 Similarly, Western blotting results showed that both pairs of pMAVS siRNAs could promote the expression of viral p30 and p72 proteins compared with the control siRNA (see Figure 16 B). Therefore, the innate immune RNA receptor RIG-I / MDA5-MAVS signaling pathway plays an important role in anti-ASFV immunity.

[0066] (9) Effects of complementation of pMAVS and its mutants on ASFV replication Based on these findings, pMAVS and all of its pS273R cleavage-related point mutants (G175A, G192A, G257A, G270A, G388A, DM1, DM2, DM3, and TM) were transfected with pS273R in HEK293T cells for IFN-β promoter assays. HEK293T cells were co-transfected with pS273R (or vector) and 20 ng of GFP-pMAVS (or any of the nine mutants), along with 10 ng of IFNβ-luc and 0.4 ng of RL-TK plasmid, for a total of 50 ng of plasmid per well. Luciferase activity was measured 24 hours later using a dual-luciferase reporter assay. The results revealed that the cleavage site on MAVS significantly impacted its signaling in the presence of ASFV protease. On the one hand, like the parental pMAVS, the expression of all nine mutant pMAVS can induce the same degree of activation of the IFN-β promoter; on the other hand, pS273R can significantly inhibit the activation of the IFN-β promoter induced by the expression of pMAVS, G175A and G257A. Although pS273R can still inhibit the IFN-β promoter activity of the other six mutants of pMAVS (G192A, G270A, G388A, DM1, DM2, DM3), the inhibitory effect is weaker than that of the parental pMAVS, especially the inhibitory effect on the pMAVS double-point mutants (DM1, DM2, DM3) is further weakened. It is worth noting that pS273R cannot inhibit the IFN-β promoter activity induced by the expression of the pMAVS triple-point mutant (TM) (see Figure 17 These results indicate that the cleavage site on MAVS has an important influence on its signaling in the presence of ASFV protease.

[0067] Subsequently, 1 μg of pMAVS and all point mutants were transfected back into MAVS - / - MA104 cells were then infected with ASFV-GFP, and viral replication was detected by fluorescence microscopy, RT-qPCR, Western blotting, and flow cytometry. Point mutations at the cleavage site on pMAVS can resist cleavage by ASFV protease (see Figure 18 ). Fluorescence microscopy, flow cytometry, RT-qPCR, and Western blotting analysis showed that compared with the control group, both the complemented pMAVS and mutants could inhibit ASFV replication, and the inhibitory effect of the cleavage site mutant was more obvious. Among them, the ASFV replication levels of the complemented G192A, G270A, and G388A were lower than those of the complemented parental pMAVS, G175A, and G257A; the ASFV replication levels of the complemented double-point mutants DM1, DM2, and DM3 were even lower; and the ASFV replication level of the complemented triple-point mutant TM was the lowest (see Figure 18These results suggest that point mutations in the cleavage sites of pMAVS can resist cleavage by ASFV protease and thus play a unique role in protecting against ASFV infection.

[0068] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person familiar with the technology can understand and think of any changes or replacements within the technical scope disclosed by the present invention, which should be included in the scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

[0069] Table 1. Amplification primer sequences and PCR amplification primer sequences for mutant genes

Claims

1. A MAVS protein mutant, characterized in that: The MAVS protein mutant is mutated at amino acid residue 192 and / or amino acid residue 270 and / or amino acid residue 388 of the sequence shown in SEQ ID NO.1; and the amino acid residue site 192 is mutated to Ala, the amino acid residue site 270 is mutated to Ala, and the amino acid residue site 388 is mutated to Ala.

2. The MAVS protein mutant according to claim 1, characterized in that: The MAVS protein mutant undergoes mutations at amino acid residues 192, 270, and 388 in the sequence shown in SEQ ID NO. 1; the amino acid residues 192, 270, and 388 are mutated to Ala.

3. A polynucleotide molecule, characterized in that The polynucleotide molecule encodes the MAVS protein mutant according to claim 1 or 2.

4. A carrier, characterized in that The vector contains the polynucleotide molecule according to claim 3.

5. A host cell, characterized in that The host cell contains the vector according to claim 3 or the chromosome is integrated with the polynucleotide molecule according to claim 3.

6. Use of the MAVS protein mutant according to claim 1 or 2 in resisting African swine fever virus.

7. The use according to claim 6, characterized in that By site-directed mutation of the amino acids at positions 192 and / or 270 and / or 388 of the MAVS protein, changing them from glycine to alanine, and then complementing them into monkey kidney epithelial cells in which the MAVS gene was knocked out, the replication of African swine fever virus in cells infected with the African swine fever virus was inhibited.

8. The use according to claim 6, characterized in that The MAVS protein mutant is used to resist cleavage by ASFV protease.

9. The use according to claim 6, characterized in that Application of the MAVS protein mutant in genetic breeding of African swine fever-resistant pigs.

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