Degradant motif in natural immune signal channel and application thereof

Abstract

The invention relates to a degradant motif in a natural immune signal channel and application of the degradant motif. According to the invention, through comparative analysis of key molecules RIG-I and MDA5 in an RLR signal channel and protein sequences, conservative KENW or KEHW or KEDW motifs exist in CARD structural domains of the key molecules RIG-I and MDA5; and a gene engineering means is further utilized to carry out deletion and point mutation on the "KENW" or "KEHW" or "KEDW" motif, which verifies that the motif is necessary for "activation" and "closing" of RIG-I and MDA5 signal pathways, and proves that the motif is a degradation submotif in a natural immune signal pathway. The invention also proves that the E3 ubiquitin ligase RNF20 can specifically recognize the motif and is used for regulating and controlling the immune homeostasis mediated by the RLR signal channel, so that the E3 ubiquitin ligase RNF20 can be used for treating immune-related diseases caused by the RLR signal channel.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular, relates to a degradation sub-motif in a natural immune signaling pathway and an application thereof. Background Art

[0002] The innate immune system is the first line of defense for organisms. It not only plays an important role in resisting infection by pathogenic microorganisms, but is also essential for maintaining the health of the body. Among them, the RLR signaling pathway plays a key role in the innate immune response. There are two important members in this signaling pathway: RIG-I and MDA5. It is mainly responsible for recognizing RNA virus infection and triggering innate immune response. The normal function of RIG-I and MDA5 proteins is the key prerequisite to ensure that the body can quickly activate the immune response and effectively control viral infection. However, abnormal activation of RIG-I and MDA5 proteins can lead to autoimmune diseases such as systemic lupus erythematosus (SLE) and Sjögren's syndrome; inflammatory diseases such as rheumatoid arthritis and Crohn's disease; and neurological diseases such as Alzheimer's disease. Therefore, regulating the normal activation of the innate immune signaling pathway mediated by RIG-I and MDA5 is the key to maintaining host health and an important scientific issue that needs to be solved urgently at home and abroad.

[0003] In the innate immune signaling pathway, specific protein sequences or structural motifs (called "degradons") can be recognized and trigger signal transduction or protein degradation processes, thereby mediating the "activation" or "shutdown" of immune signals. "Degradons" were originally defined as protein motifs that cause metabolic instability of some or all peptide bonds in proteins, which can be specifically recognized by certain E3 ubiquitin ligases, ultimately leading to protein degradation or activation. Human cells encode more than 600 E3 ubiquitin ligases, but only a few E3 ubiquitin ligases have been identified with specific degradon motifs. However, the current understanding of these "degradon" motifs is still insufficient, which limits the development of treatment strategies for related diseases. Therefore, it is crucial to screen and identify the "degradon" motifs that regulate the innate immune signaling pathway in the RLR signaling pathway as drug targets for regulating the "activation" or "shutdown" of innate immunity and treating immune-related diseases induced by RIG-I and MDA5.

[0004] Sequence analysis of proteins can reveal the evolution and changes of target proteins during species evolution. By comparing the amino acid sequences of corresponding proteins in different species, the evolutionary relationships and functional conservation of these proteins can be inferred. This analysis helps to understand the roles of proteins in different organisms and their adaptive changes during evolution. "Degron" is exactly a type of protein motif that is highly conserved during evolution. Compared with traditional screening methods such as genome-wide library knockout, mass spectrometry identification, gene interference, etc., protein sequence analysis can screen out potential degron motifs in large quantities and quickly. Therefore, this method provides an efficient and rapid research scheme for degron screening.

[0005] Reverse genetic techniques of genes can be used for the study of deletion or point mutation of functional domains of target genes. This method is crucial for verifying the functional domains of target genes. By constructing mutants of degron motifs, the effects of the deletion or point mutation of this motif on the phenotypes of organisms can be observed, thereby inferring the role of this motif. In addition, reverse genetic techniques can also be used to study gene regulatory networks. By knocking out or knocking in specific genes, researchers can reveal the interactions and regulatory relationships between genes. This method provides a powerful tool for gene function research and has been widely applied especially in model organisms. Summary of the Invention

[0006] Aiming at the deficiencies existing in the prior art, the present invention provides a degron motif in the innate immune signaling pathway and its application. The present invention discovers and verifies the degron motif in the RLR signaling pathway, provides a drug target "KENW", "KEHW" or "KEDW" for preventing and / or treating immune-related diseases, and further uses the "KENW", "KEHW" or "KEDW" motif for the application in the treatment of diseases such as immune overreaction.

[0007] Specifically, the present invention determines the existence of a conserved "degron" motif in the RLR signaling pathway by analyzing the protein sequences of the key molecules RIG-I and MDA5 in the RLR signaling pathway, constructs deletion and point mutation plasmids of the degron motif in the RLR signaling pathway by means of reverse genetics, and verifies their regulatory effects on innate immune responses. Through E3 ubiquitin ligase screening, it is verified that the E3 ubiquitin ligase RNF20 specifically recognizes this motif and mediates the degradation of RLR.

[0008] To achieve the above invention objectives, the technical solutions adopted by the present invention include:

[0009] In a first aspect, the present invention provides a degron motif in the innate immune signaling pathway, and the amino acid sequence of the degron motif is any one of the following sequences: KENW (SEQ ID NO.1), KEHW (SEQ ID NO.2), KEDW (SEQ ID NO.3).

[0010] As a preferred embodiment, the DNA sequence encoding the degron motif is any one of SEQ ID NOs. 4 to 6.

[0011] As a preferred embodiment, the degron motif can activate or shut down the RLR signaling pathway, thereby regulating the innate immune response.

[0012] As a preferred embodiment, the degron motif is derived from MDA5 and / or RIG-I genes of species including humans, chickens, pigs, ducks, bats, etc., but is not limited thereto.

[0013] As a preferred embodiment, when the degron motif is knocked out, the RLR signaling pathway cannot be normally activated, and the IFN-β promoter activity can be reduced, thereby leading to an immunosuppressive phenomenon;

[0014] When the degron motif is mutated, the RLR signaling pathway cannot be normally shut down after activation, and the IFN-β promoter activity can be increased, thereby leading to an immune overreaction.

[0015] As a preferred embodiment, the mutation is to mutate the "K" in the amino acid sequence of the degron motif to "R". The amino acid sequences of the mutated degron motifs are shown in SEQ ID NOs. 15 to 17, and the DNA sequences encoding the mutated degron motifs are shown in SEQ ID NOs. 18 to 20.

[0016] The present invention also provides a degron motif deletion plasmid, which includes the RIG-I or MDA5 protein sequence with the degron motif knocked out; the degron motif is any one of the following sequences: KENW, KEHW, KEDW.

[0017] The present invention also provides a degron motif mutant plasmid, which includes the RIG-I or MDA5 protein sequence with the degron motif mutated to RENW, REHW, REDW; the degron motif is any one of the following sequences: KENW, KEHW, KEDW.

[0018] In a second aspect, the present invention provides the use of the degron motif in the innate immune signaling pathway according to claim 1 in the preparation of a preparation for preventing and / or treating innate immune-related diseases.

[0019] As a preferred embodiment, the preparation can specifically recognize and bind to the degron motif or its functional fragment.

[0020] As a further preferred embodiment, the preparation capable of specifically recognizing the degron motif or its functional fragment includes an E3 ubiquitin ligase, such as RNF20. However, it is not limited thereto.

[0021] As a preferred embodiment, the preparation can regulate the degron motif, thereby regulating the immune response mediated by the RLR signaling pathway.

[0022] As a further preferred embodiment, the preparation for regulating the degron motif includes recombinant proteins, plasmids, drugs, genes, etc. The recombinant proteins and plasmids are obtained by conventional methods, and the present invention does not make special limitations.

[0023] As a preferred embodiment, the innate immunity-related diseases include viral infections, autoimmune diseases, and inflammatory diseases.

[0024] As a preferred embodiment, the innate immunity-related diseases include autoimmune diseases caused by abnormal activation of the RLR signaling pathway.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention has first discovered a degron motif existing in the innate immune signaling pathway through sequence alignment and verification. The sequence alignment method adopted can provide an efficient and rapid way for the screening of degrons.

[0027] 2. By deeply studying the role of the degron motif (KENW, KEHW or KEDW) in the RLR signaling pathway, the present invention reveals its key regulatory role in the innate immune response. The present invention not only provides a new perspective for understanding the innate immune signaling pathway, but also provides a new target for developing therapeutic strategies for immune-related diseases.

[0028] 3. The present invention also provides an effective method to verify and utilize these motifs. By constructing specific deletion or point mutation plasmids, researchers can further explore the interaction and regulatory relationship between genes, thereby providing a scientific basis for disease treatment and the construction of biosensors.

[0029] 4. The implementation of the present invention is not limited to laboratory research, but also has broad application prospects, including but not limited to drug development, disease diagnosis, and the biotechnology industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] By reading the following detailed description of the non-limiting embodiments with reference to the accompanying drawings, other features, objects, and advantages of the present invention will become more apparent:

[0031] Figure 1Results of the amino acid sequence analysis of RIG-I and MDA5 proteins in Example 1; as shown in the figure, conserved "KENW" or "KEHW" or "KEDW" motifs exist in RIG-I and MDA5 of different species;

[0032] Figure 2 Results of the construction and functional analysis of plasmids lacking the "KENW" or "KEHW" or "KEDW" motifs of RIG-I and MDA5 proteins in Example 2; among them, Figure 2 A shows the results of the dual-luciferase assay of the plasmid lacking the "KENW" motif in human RIG-I; Figure 2 B shows the results of the dual-luciferase assay of the plasmid lacking the "KENW" motif in MDA5; Figure 2 C shows the results of the dual-luciferase assay of the plasmid lacking the "KEHW" motif in duck RIG-I; Figure 2 D shows the results of the dual-luciferase assay of the plasmid lacking the "KEDW" motif in chicken MDA5; as shown in the figure, after the deletion of the "KENW" or "KEHW" or "KEDW" motif of RIG-I and MDA5, the function of activating the IFN-β promoter was significantly inhibited; the results are expressed as mean ± standard error, **p<0.01, ***p<0.001 indicating extremely significant differences;

[0033] Figure 3 Results of the construction and functional analysis of plasmids with mutated "KENW" or "KEHW" or "KEDW" motifs of RIG-I and MDA5 proteins in Example 3; among them, Figure 3 A shows the results of the dual-luciferase assay of the plasmid with mutated "KENW" in human RIG-I; Figure 3 B shows the results of the dual-luciferase assay of the plasmid with mutated "KENW" motif in human MDA5; Figure 3 C shows the results of the dual-luciferase assay of the plasmid with mutated "KEHW" motif in duck RIG-I; Figure 3 D shows the results of the dual-luciferase assay of the plasmid with mutated "KEDW" motif in chicken MDA5; as shown in the figure, after the "KENW" or "KEHW" or "KEDW" motif of RIG-I and MDA5 was mutated to "RENW", or "REHW" or "REDW", the function of activating the IFN-β promoter was significantly enhanced; the results are expressed as mean ± standard error, **p<0.01, ***p<0.001 indicating extremely significant differences;

[0034] Figure 4 Results of the regulation of RIG-I and MDA5 ubiquitination by E3 ubiquitin ligase RNF20 in Example 4; among them, Figure 4 A shows the Western blot imaging results of the interaction between RNF20 and RIG-I; Figure 4B shows the Western blot development results of the interaction between RNF20 and MDA5; Figure 4 C shows the Western blot development results of RNF20 catalyzing the ubiquitination modification of MDA5; Figure 4 D shows the Western blot development results of RNF20 catalyzing the ubiquitination modification of RIG-I; as shown in the figure, there is a physical interaction between RNF20 and RIG-I or MDA5, and RNF20 can promote the ubiquitination of RIG-I or MDA5;

[0035] Figure 5 It is the ubiquitination analysis of RIG-I or MDA5 mediated by RNF20 after the "KENW" motif of RIG-I or MDA5 protein in Example 4 is mutated to "RENW"; among them, Figure 5 A shows the Western blot development results of the ubiquitination of RIG-I or MDA5 mediated by RNF20 after the "KENW" motif of RIG-I protein is mutated; Figure 5 B shows the Western blot development results of the ubiquitination of RIG-I or MDA5 mediated by RNF20 after the "KENW" motif of MDA5 protein is mutated; as shown in the figure, after the "KENW" motif in RIG-I or MDA5 protein is mutated to "RENW", the level of ubiquitination catalyzed by RNF20 on them is weakened. Detailed implementation mode

[0036] The present invention will be described in more detail below through specific implementation modes, so as to facilitate the understanding of the technical solution of the present invention, but it is not used to limit the protection scope of the present invention.

[0037] In order to better understand the present invention rather than limit the scope of the present invention, all numbers representing dosages, percentages, and other numerical values used in this application should be understood as being modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to different desired properties. Each numerical parameter should be regarded as being obtained at least according to the reported significant figures and by the conventional rounding method.

[0038] In order to screen and identify the "degron" motif that regulates the innate immune signaling pathway in the RLR signaling pathway, the following examples provide a method for discovering and identifying the "degron" motif in the innate immune signaling pathway, including the following steps:

[0039] a) Collect the protein sequence data of RIG-I and MDA5 from different species;

[0040] b) Analyze the collected protein sequences using bioinformatics tools to identify potential "degron" motifs;

[0041] Among them, the bioinformatics tools used in step b) include but are not limited to sequence alignment, secondary structure prediction, evolutionary conservation analysis, etc.

[0042] The following examples also provide a method for verifying the function of "degron" motifs, constructed based on reverse genetics technology, including the following steps:

[0043] a) Design primers for deletion or point mutation of the "degron" motif in the RIG-I / MDA5 plasmid;

[0044] b) Amplify the DNA fragment with deletion or point mutation of the degron motif by PCR;

[0045] c) Connect the DNA fragments into circular DNA by homologous recombination, transform the obtained ligation product into competent cells, streak and pick monoclonal strains containing the target sequence, and extract plasmids after amplification;

[0046] d) Transfect the plasmid with deletion or point mutation of the degron motif into cells and analyze the innate immune response;

[0047] e) Screen for E3 ubiquitin ligases that mediate the degradation of MDA5 and / or RIG-I;

[0048] f) Co-transfect the degron mutant plasmid and the screened E3 ubiquitin ligase into 293T cells and analyze the ubiquitination levels of MDA5 and / or RIG-I.

[0049] Among them, the experimental verification techniques used in step f) also include protein interaction analysis, signal transduction activity detection, etc.

[0050] The "degron" motif screened and identified by the foregoing method has the following applications:

[0051] 1. As a drug target for developing drugs for treating innate immunity-related diseases;

[0052] 2. For constructing or optimizing biosensors related to the innate immune signaling pathway;

[0053] 3. For studying the mechanism of the innate immune signaling pathway and the molecular basis of disease occurrence and development.

[0054] Sources of materials used in the following examples

[0055] Cells: The chicken fibroblast cell line (DF-1) and human embryonic kidney cells (293T cells) are from ATCC. DH5α competent cells are from Shanghai Qingke Biotechnology Co., Ltd.

[0056] Reagents and antibodies: The mouse monoclonal antibody against β-tubulin was purchased from Abmart; the mouse monoclonal antibodies against EGFP, Flag, and HA were purchased from Shanghai Sangon Biotech Co., Ltd.; the rabbit and mouse secondary antibodies were purchased from Norland Biolabs; the DMEM medium and 0.25% EDTA phenol red-containing trypsin were purchased from Gibco; fetal bovine serum (FBS) and liposomes were purchased from Shanghai Norland Co., Ltd.; the reverse transcription kit, AceQ Universal SYBR qPCR Master Mix, 2×Phanta Max Master Mix (Dye Plus) PCR enzyme, genomic DNA extraction kit, and gel extraction kit were purchased from Novoprotein; the endotoxin-free plasmid extraction kit was purchased from Omega; MG132 was purchased from MCE; the dual-luciferase reporter reagent used in the dual-luciferase reporter system was purchased from Promega; the HA-Ub plasmid, pcDNA3.1 plasmid, pGL3.0-basic plasmid, and pR-TK plasmid were purchased from Addgene; pcDNA3.1-huRIG-I, pcDNA3.1-huMDA5, pcDNA3.1-duRIG-I, pcDNA3.1-chMDA5, pcDNA4.0-RNF20-EGFP, pcDNA3.1-RNF20-Flag, pcDNA3.1-huRIG-I-Myc, pcDNA3.1-huMDA5-Myc, pcDNA3.1-huMDA5-K174R-Myc, pcDNA3.1-huRIG-I-K164R-Myc, pGL3.0-basic-huIFN-β, key molecules of the RLR signaling pathway, and ubiquitin Ub plasmid were all stored in the Key Laboratory of Veterinary Microbiology, Shanghai Jiao Tong University and obtained by conventional biological methods.

[0057] The following provides a detailed description of the screening of the "degron" motif in the RLR signaling pathway, the deletion of the "degron" motif, the mutation of lysine sites, functional verification, the screening of E3 ubiquitin ligases interacting with the "degron", and the application of ubiquitination analysis. The above is an explanation of the present invention rather than a limitation.

[0058] Example 1: Obtaining the "degron" motif by analyzing the amino acid sequences of key proteins RIG-I and MDA5 in the RLR signaling pathway

[0059] This example provides a method for discovering the "degron" motif in the innate immune signaling pathway. The specific steps are as follows:

[0060] a) Collect protein sequence data of the RLR signaling pathway (data source: https: / / www.uniprot.org / ), including the sequences of RIG-I and MDA5 from different species.

[0061] b) Use bioinformatics tools such as BLAST, Mega11, etc. for sequence alignment (in this example, Mega11 is specifically used to analyze the protein sequence), use SMART for secondary structure prediction, and then conduct evolutionary conservation analysis through ConSurf to identify potential "degron" motifs.

[0062] As Figure 1 shown. It can be Figure 1 seen that both the CARD domains of RIG-I and MDA5 have conserved "KENW" or "KEHW" or "KEDW" motifs, and these motifs are highly conserved among species.

[0063] Example 2: Deletion and functional verification of the "degron" motif in the RLR signaling pathway

[0064] In this example, plasmids with deletions of the "degron" motifs of RIG-I and MDA5 proteins in the RLR signaling pathway were constructed to verify the effects of "degron" deletion on their functions. The specific steps are as follows:

[0065] 1. Construction of plasmids with deletions of the "degron" motifs of RIG-I or MDA5 proteins

[0066] 1.1 The amino acid sequences of the "degron" motifs of the RIG-I or MDA5 proteins are: KENW (SEQ ID NO.1), KEHW (SEQ ID NO.2), KEDW (SEQ ID NO.3), and the DNA sequences are as follows:

[0067] 5’-aaagaaaactgg-3’ (SEQ ID NO.4);

[0068] 5’-aaggagcactgg-3’ (SEQ ID NO.5);

[0069] 5’-aaggaggactgg-3’ (SEQ ID NO.6);

[0070] By the reverse PCR amplification method, using the plasmids pcDNA3.1-huRIG-I / pcDNA3.1-huMDA5 / pcDNA3.1-duRIG-I / pcDNA3.1-chMDA5 as templates, linearly amplify the fragments with the "degron" motif removed. The sequences of the reverse PCR primers are shown in Table 2. The reverse PCR amplification system includes: 2×Phanta Max Master Mix (Dye Plus), ddH2O, upstream and downstream primers, and templates; the amplification reaction conditions are: pre-denaturation: 95°C, 3 min; denaturation: 95°C, 15 sec; annealing: 56°C, 15 sec; extension: 72°C, 90 sec; final extension: 72°C, 5 min; 30 cycles from pre-denaturation to extension steps.

[0071] Table 2 Primer sequences for KENW deletion amplification

[0072]

[0073]

[0074] 1.2 After recovering the above PCR products using a gel extraction kit, perform homologous recombination, and transform the homologous recombination products into DH5α competent cells. After streaking and picking monoclonal colonies, verify by sequencing.

[0075] 1.3 Expand the culture of the monoclonal strains with correct sequencing, and extract the plasmids with "KENW" or "KEHW" or "KEDW" deletion in the RIG-I / MDA5 plasmids (pcDNA3.1-huRIG-I-dKENW, pcDNA3.1-huMDA5-dKENW, pcDNA3.1-duRIG-I-dKENW, pcDNA3.1-chMDA5-dKEDW).

[0076] 2. Use the dual-luciferase reporter system to verify the effects of overexpressing the plasmids pcDNA3.1-huRIG-I, pcDNA3.1-huRIG-I-dKENW, pcDNA3.1-huMDA5, pcDNA3.1-huMDA5-dKENW in the 293T cell line, or overexpressing the plasmids pcDNA3.1-duRIG-I, pcDNA3.1-duRIG-I-dKEHW, pcDNA3.1-chMDA5, pcDNA3.1-chMDA5-dKEDW in the chicken DF-1 cell line on the expression of IFN-β. The specific steps are as follows:

[0077] The 293T cell line was seeded in a 24-well cell culture plate. After the cell confluence reached over 90%, 150 ng / well of pcDNA3.1-huRIG-I or pcDNA3.1-huRIG-I-dKENW, pcDNA3.1-huMDA5 or pcDNA3.1-huMDA5-dKENW plasmids were co-transfected with 120 ng / well of pGL3.0-basic-huIFN-β and 60 ng / well of pR-TK into 293T cells using liposomes, and cell lines overexpressing pcDNA3.1-huRIG-I, pcDNA3.1-huRIG-I-dKENW, pcDNA3.1-huMDA5, and pcDNA3.1-huMDA5-dKENW were obtained respectively for detecting the IFN-β promoter activity. The dual-luciferase level was detected 24 h after transfection, and their ratio could represent the IFN-β promoter activity and was used to reflect the IFN-β expression level in cells. The test results of the dual-luciferase are as Figure 2 A and Figure 2 shown in B. The value of the IFN-β promoter activity in the 293T cell line transfected with the overexpression plasmid of pcDNA3.1-huRIG-I-dKENW or pcDNA3.1-huMDA5-dKENW was significantly lower than that in the cell line transfected with pcDNA3.1-huRIG-I or pcDNA3.1-huMDA5.

[0078] The chicken DF-1 cell line was seeded in a 24-well cell culture plate. After the cell confluence reached over 90%, 150 ng / well of pcDNA3.1, pcDNA3.1-chMDA5 or pcDNA3.1-chMDA5-dKENW, pcDNA3.1-duRIG-I or pcDNA3.1-duRIG-I-dKEHW plasmids were co-transfected with 120 ng / well of pGL3.0-basic-huIFN-β and 60 ng / well of pR-TK into chicken DF-1 cells using liposomes, and cell lines overexpressing pcDNA3.1, pcDNA3.1-duRIG-I, pcDNA3.1-duRIG-I-dKENW, pcDNA3.1-chMDA5, and pcDNA3.1-chMDA5-dKENW were obtained respectively for detecting the IFN-β promoter activity. The dual-luciferase level was detected 24 h after transfection, and their ratio could represent the IFN-β promoter activity and was used to reflect the IFN-β expression level in cells. The test results of the dual-luciferase are as Figure 2 C and Figure 2As shown in D. Similarly, it was found in chicken DF-1 cells that the value of IFN-β promoter activity in the chicken DF-1 cell line transfected with the overexpression plasmid pcDNA3.1-duRIG-I-dKEHW or pcDNA3.1-chMDA5-Dkedw was significantly lower than that in the cell line transfected with pcDNA3.1-duRIG-I or pcDNA3.1-chMDA5.

[0079] The results from Figure 2 showed that the "KENW" or "KEHW" or "KEDW" motif in the RIG-I or MDA5 protein sequence is crucial for activating the innate immune response. After knocking out the degron motif, the RLR signaling pathway cannot be normally activated, and the IFN-β promoter activity decreases, resulting in an immunosuppressive phenomenon.

[0080] Example 3. Mutation and functional verification of the "degron" motif in the RLR signaling pathway

[0081] In this example, mutant plasmids were constructed by mutating the "KENW" motif of the RIG-I and MDA5 proteins in the RLR signaling pathway to "RENW" (SEQ ID NO.15), the "KEHW" motif to "REHW" (SEQ ID NO.16), and the "KEDW" motif to "REDW" (SEQ ID NO.17) to verify the effects of "RENW" or "REHW" or "REDW" on their functions. The specific steps are as follows:

[0082] 1. Construction of mutant plasmids of the RIG-I or MDA5 protein with "RENW" or "REHW" or "REDW"

[0083] 1.1 The DNA sequences in which the "KENW" motif of the RIG-I or MDA5 protein is mutated to the "RENW" motif, the "KEHW" motif is mutated to the "REHW" motif, and the "KEDW" motif is mutated to the "REDW" motif are as follows:

[0084] 5’-agagaaaactgg-3’ (SEQ ID NO.18);

[0085] 5’-agggagcactgg-3’ (SEQ ID NO.19);

[0086] 5’-agggaggactgg-3’ (SEQ ID NO.20).

[0087] By the reverse PCR amplification method, using the pcDNA3.1-huRIG-I / pcDNA3.1-huMDA5 / pcDNA3.1-duRIG-I / pcDNA3.1-chMDA5 plasmids as templates, linearly amplify the fragments with mutations in the "KENW" motif. The sequences of the reverse PCR primers are shown in Table 3. The reverse PCR amplification system includes: 2×Phanta Max Master Mix (Dye Plus), ddH2O, upstream and downstream primers, and templates; the amplification reaction conditions are: pre-denaturation: 95°C, 3 min; denaturation: 95°C, 15 sec; annealing: 56°C, 15 sec; extension: 72°C, 90 sec; final extension: 72°C, 5 min; 30 cycles from pre-denaturation to extension step.

[0088] Table 3 Primer sequences for amplification of KENW point mutations

[0089]

[0090] 1.2 After recovering the above PCR products by using a gel extraction kit, perform homologous recombination, and transform the homologous recombination products into DH5α competent cells. After streaking and picking monoclonal colonies, verify by sequencing.

[0091] 1.3 Expand the culture of the monoclonal strains with correct sequencing, and extract the plasmids (pcDNA3.1-huRIG-I-K164R, pcDNA3.1-huMDA5-K174R, pcDNA3.1-duRIG-I-K164R, pcDNA3.1-chMDA5-K174R) in which "KENW" in the RIG-I / MDA5 plasmids is mutated to "RENW", "KEHW" is mutated to "REHW", and "KEDW" is mutated to "REDW".

[0092] 2. Use the dual-luciferase reporter system to verify the overexpression of pcDNA3.1-huRIG-I, pcDNA3.1-huRIG-I-K164R, pcDNA3.1-huMDA5, pcDNA3.1-huMDA5-K174R plasmids in the 293T cell line, or the overexpression of pcDNA3.1-duRIG-I, pcDNA3.1-duRIG-I-K162R, pcDNA3.1-chMDA5, pcDNA3.1-chMDA5-K172R plasmids in the DF-1 cell line, and analyze their effects on IFN-β expression. The specific steps are as follows:

[0093] The 293T cell line was seeded in a 24-well cell culture plate. After the cell confluence reached over 90%, 150 ng / well of pcDNA3.1, pcDNA3.1-huRIG-I or pcDNA3.1-huRIG-I-K164R, pcDNA3.1-MDA5 or pcDNA3.1-MDA5-174R plasmids were co-transfected into 293T cells with 120 ng / well of pGL3.0-basic-huIFN-β and 60 ng / well of pR-TK by liposomes, respectively obtaining cell lines overexpressing pcDNA3.1, pcDNA3.1-huRIG-I, pcDNA3.1-huRIG-I-K164R, pcDNA3.1-huMDA5, pcDNA3.1-huMDA5-K174R for detecting the IFN-β promoter activity. The dual-luciferase level was detected 24 h after transfection, and their ratio could represent the IFN-β promoter activity for reflecting the IFN-β expression level in cells. The experimental results of the dual-luciferase are shown as Figure 3 A and Figure 3 shown in B. The value of the IFN-β promoter activity in the 293T cell line transfected with the pcDNA3.1-huRIG-I-K164R or pcDNA3.1-huMDA5-K174R overexpression plasmid was significantly higher than that in the cell line transfected with pcDNA3.1-huRIG-I or pcDNA3.1-huMDA5.

[0094] The chicken DF-1 cell line was seeded in a 24-well cell culture plate. After the cell confluence reached over 90%, 150 ng / well of pcDNA3.1, pcDNA3.1-duRIG-I or pcDNA3.1-duRIG-I-K162R, pcDNA3.1-chMDA5 or pcDNA3.1-chMDA5-172R plasmids were co-transfected into 293T cells or DF-1 cells with 120 ng / well of pGL3.0-basic-huIFN-β and 60 ng / well of pR-TK by liposomes, respectively obtaining cell lines overexpressing pcDNA3.1, pcDNA3.1-duRIG-I, pcDNA3.1-duRIG-I-K162R, pcDNA3.1-chMDA5, pcDNA3.1-chMDA5-K172R for detecting the IFN-β promoter activity. The dual-luciferase level was detected 24 h after transfection, and their ratio could represent the IFN-β promoter activity for reflecting the IFN-β expression level in cells. The experimental results of the dual-luciferase are shown as Figure 3 A and Figure 3As shown in Figure B. In chicken DF-1 cells, the IFN-β promoter activity in the chicken DF-1 cell line transfected with the pcDNA3.1-duRIG-I-K162R or pcDNA3.1-chMDA5-K172R overexpression plasmid was also significantly higher than that in the cell line transfected with pcDNA3.1-duRIG-I or pcDNA3.1-chMDA5.

[0095] The results from Figure 3 showed that the "KENW" motif in the RIG-I or MDA5 protein sequence is crucial for maintaining the immune homeostasis mediated by the RLR signaling pathway. After the degron motif mutation, the activation of the RLR signaling pathway cannot be normally turned off, and the IFN-β promoter activity is significantly increased, leading to immune overactivation.

[0096] Example 4. Application of the "degron" motif of the RLR signaling pathway

[0097] This example provides an application based on the "degron" motif of the RLR signaling pathway. Since the degron motif is mainly recognized by the E3 ubiquitin ligase and mediates its ubiquitination and degradation to maintain its function. Therefore, this example mainly verified an E3 ubiquitin ligase RNF20 that specifically recognizes the "KENW" motif of the RLR signaling pathway and explored its regulatory effect on the RLR degron. The specific steps are as follows:

[0098] 1. Protein immunoprecipitation analysis of the interaction between E3 ubiquitin ligase RNF20 and RIG-I or MDA5

[0099] 1.1 Seed 293T cells in a 6-well cell culture plate. After the cell confluence reaches more than 90%, transiently transfect the pcDNA3.1-RNF20-Flag, pcDNA3.1-huMDA5-Myc or pcDNA3.1-huRIG-I-Myc expression plasmid into 293T cells by liposome. After 36 h of transfection, add 200 μL of RIPA lysis buffer containing protease inhibitors (obtained by adding 200 mM AEBSF, 30 μM Aprotinin, 13 mM Bestatin, 1.4 mM E64 and 1 mM Leupeptin to DMSO) to lyse the cells sufficiently, and then centrifuge to collect the supernatant.

[0100] 1.2 Take 150 μL of the supernatant and incubate it with magnetic beads conjugated with anti-Flag antibody for 4 h. After the incubation, separate by a magnetic stand, add 50 μL of loading buffer, and boil for 10 min. After separation by the magnetic stand, transfer the supernatant to a new EP tube. Separate the proteins using a precast gel (SDS-PAGE) with the program: 80 V for 25 minutes; 120 V for 40 - 50 minutes. Transfer the membrane using a rapid membrane transfer instrument from ACE Company with the program: 26 V, 1.5 A, for 10 minutes. Then block with 5% non-fat milk for 1 h and wash with TBST.

[0101] 1.3 Dilute mouse anti-Myc and anti-Flag antibodies in the primary antibody diluent at a dilution ratio of 1:1000. After diluting the mouse anti-β-tubulin primary antibody with the diluent at a dilution ratio of 1:5000, incubate with the membrane washed above at room temperature for 4 h, wash 3 times with TBST, 5 minutes each time. Then add HRP-labeled mouse secondary antibody (diluted 1:5000) and incubate at room temperature for 1 h, followed by washing with TBST. Finally, develop the image using an ECL chemiluminescence developer.

[0102] The Western blot development results are as Figure 4 A and Figure 4 B show that Flag magnetic beads can conjugate with RNF20-Flag protein and RIG-I-Myc or MDA5-Myc protein, but cannot conjugate with pcDNA3.1-Flag protein and RIG-I-Myc or MDA5-Myc protein. This indicates that there is an interaction between RNF20 and RIG-I or MDA5.

[0103] 1.5 Protein immunoprecipitation analysis shows that the E3 ubiquitin ligase RNF20 catalyzes the ubiquitination modification of RIG-I or MDA5. The specific steps are as follows:

[0104] Seed 293T cells in a 6-well cell culture plate. After the cell confluence reaches over 90%, transiently transfect pcDNA4.0-RNF20-EGFP, pcDNA3.1-huMDA5-Myc or pcDNA3.1-huRIG-I-Myc, Ub-HA expression plasmids into 293T cells using liposomes. 30 h after transfection, add MG132 (10 μM). After 6 h, harvest the cells, add 200 μL of RIPA lysis buffer containing protease inhibitors (the same as above), fully lyse the cells, and centrifuge to obtain the supernatant. The subsequent steps are the same as steps 1.2 - 1.3 above.

[0105] The Western blot development results are as Figure 4 C and Figure 4As shown in Figure D, RNF20 can significantly increase the ubiquitination modification of RIG-I / MDA5. This indicates that RNF20 can catalyze the ubiquitination of RIG-I / MDA5, thereby regulating the innate immune response mediated by RLR.

[0106] 2. Protein co-immunoprecipitation analysis shows that the E3 ubiquitin ligase RNF20 catalyzes the ubiquitination modification of the "KENW" motif of RIG-I or MDA5 protein

[0107] Seed 293T cells in a 6-well cell culture plate. After the cell confluence reaches over 90%, transiently transfect the pcDNA3.1-RNF20-Flag, pcDNA3.1-huMDA5-Myc / pcDNA3.1-huMDA5-K174R-Myc or pcDNA3.1-huRIG-I-Myc / pcDNA3.1-huRIG-I-K164R-Myc, Ub-HA expression plasmids into 293T cells using liposomes. Harvest the cells 30 h after transfection and then 6 h later, add 200 μL of RIPA lysis buffer containing protease inhibitors to fully lyse the cells, and centrifuge to collect the supernatant. The subsequent steps are the same as steps 1.2 - 1.3 above.

[0108] The Western blot development results are as Figure 5 shown in Figure 5 Figures A and

[0109] B. After point mutation of the "KENW" motif of RIG-I or MDA5 protein (mutation of the ubiquitination site K to R), the ubiquitination bands of RIG-I and MDA5 catalyzed by RNF20 are significantly weakened. This indicates that RNF20 can catalyze the ubiquitination of the "KENW" motif of RIG-I / MDA5 protein, thereby regulating the innate immune response mediated by RLR.

[0110] Specific applications include: Based on the degron motif of the present invention, it can be used to prepare preparations for preventing and / or treating innate immunity-related diseases, and the preparations can regulate the degron motif, thereby regulating the immune response mediated by the RLR signaling pathway. The innate immunity-related diseases include viral infections, autoimmune diseases, and inflammatory diseases; in particular, autoimmune diseases caused by abnormal activation of the RLR signaling pathway are included.

[0111] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A degron motif in the innate immune signaling pathway, characterized in that, The amino acid sequence of the degron motif is any one of the following sequences: KENW, KEHW, KEDW.

2. The degron motif in the innate immune signaling pathway according to claim 1, wherein, The DNA sequence encoding the degron motif is any one of SEQ ID NOs. 4 to 6.

3. The degron motif in the innate immune signaling pathway according to claim 1 or 2, characterized in that, The degron motif can activate or shut down the RLR signaling pathway, thereby regulating the innate immune response.

4. The degron motif in the innate immune signaling pathway according to claim 3, characterized in that, Knocking out the degron motif results in the failure of the RLR signaling pathway to be normally activated and a decrease in the IFN-β promoter activity, thereby leading to an immunosuppressive phenomenon; Mutating the degron motif results in the failure of the activation of the RLR signaling pathway to be normally shut down and a significant increase in the IFN-β promoter activity, thereby leading to immune hyperactivity.

5. The degron motif in the innate immune signaling pathway according to claim 4, wherein The mutation is the mutation of "K" in the amino acid sequence of the degron motif to "R".

6. Use of the degron motif in the natural immune signaling pathway according to claim 1 in the preparation of a preparation for preventing and / or treating natural immunity-related diseases.

7. The use according to claim 6, characterized in that, The preparation can specifically recognize and bind to the degron motif or its functional fragment.

8. The use according to claim 6, characterized in that, The preparation can regulate the degron motif, thereby regulating the immune response mediated by the RLR signaling pathway.

9. The use according to claim 6, wherein The natural immunity-related diseases include viral infections, autoimmune diseases, and inflammatory diseases.

10. The use according to claim 9, wherein The natural immunity-related diseases include autoimmune diseases caused by abnormal activation of the RLR signaling pathway.

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