Application of GSDMD polypeptide in prevention and / or treatment of RNA virus infection

Abstract

The invention belongs to the field of biotechnology and medicine, and particularly relates to application of GSDMD polypeptide in preparation of medicine for preventing and / or treating RNA virus infectious diseases. Animal model tests show that in infectious diseases caused by VSV infection, knockout of Gsdmd inhibits I-type interferon secretion, promotes virus replication and reduces the lifetime of infected mice, which indicates that GSDMD positively regulates and controls antiviral immune response. Furthermore, it is found that the SSSRFWKPRYSCVNL motif of the GSDMD protein can effectively inhibit VSV replication.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and medicine, and specifically relates to an application of a GSDMD protein SSSRFWKPRYSCVNL motif in preparing a medicine for preventing and / or treating RNA virus infectious diseases. Background Art

[0002] Since RNA viruses (such as influenza virus, Ebola virus, Zika virus and new coronavirus) have a wide range of transmission and extremely high mutation rate, the development of broad-spectrum and highly effective antiviral drugs to respond to sudden and recurrent viral infections has always been a research hotspot in the field.

[0003] There is a long-term game between the host and the virus. As the first line of defense for the host to defend against the invasion of external pathogens, the innate immune system plays an important role in the body's resistance to viral infection. Host cells express a variety of pattern recognition receptors (PRRs) to recognize invading pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) in the body. After PRRs recognize viral nucleic acids, they trigger an antiviral cascade reaction with type I interferon (IFN-I) as the core. RIG-I was identified as an intracellular PRR that can bind to double-stranded RNA in 2004 (Yoneyama, M., et al., The RNA helicase RIG-I has anessential function in double-stranded RNA-induced innate antiviral responses. Nature Immunology, 2004. 5 (7): p. 730-737.). This protein is widely expressed in various tissue cells including immune cells. It recognizes short-chain dsRNA and 5'-flat-end triphosphate dsRNA. It is mainly located in the cytoplasm and can also be translocated to the nucleus to play an antiviral immune response role (Schmidt, A., 5 ,-triphosphate RNArequires base-paired structures to activate antiviral signaling via RIG-I. al., Nuclear-resident RIG-I senses viral replication inducing antiviral immunity. Nature Communications, 2018.9(1):p.3199.). In the resting state, RIG-I is in an autoinhibitory state. When it recognizes viral RNA, the conformation of RIG-I changes, followed by K63 ubiquitination activation, followed by activation of TRAF3 and other proteins, which in turn phosphorylate downstream molecules such as TBK1 and IRF3. After phosphorylated IRF3 enters the nucleus, it promotes the transcription of IFN-I and its downstream interferon-stimulated genes (ISGs). At the same time, IκBα is degraded by IKKα / β, and the released free NF-κB enters the nucleus to induce the transcription of inflammatory cytokines such as IL-6, further triggering an antiviral inflammatory response (Thoresen, D., et al., The molecular mechanism of RIG-I activation and signaling. Immunological Reviews, 2021. 304(1): p. 154-168.).

[0004] As a new type of inflammatory cell death, pyroptosis originated from the study of macrophages infected with Salmonella. It is one of the means by which the body's immune system fights against the invasion of external pathogens, and it is also one of the new hot spots in the field of tumor immunotherapy. GSDMD is cleaved by its upstream protein to generate the N-terminal p30 protein, which not only mediates the occurrence of pyroptosis, but also regulates the non-classical release of IFN-Ⅰ, the activation of the cGAS-STING signaling pathway, and the permeability of mitochondrial membranes. After intracellular DNA is sensed by AIM2 inflammasome, caspase-1 is activated, and GSDMD is subsequently cleaved and activated to form pores on the cell membrane to mediate cell pyroptosis. Intracellular IL-1β, IL-18 and potassium ions can be released to the extracellular space through the pores. At the same time, the intracellular potassium ion content is essential for the activation of the cGAS-STING signaling pathway. Therefore, GSDMD inhibits the activation of the cGAS-STING signaling pathway and the secretion of downstream IFN-Ⅰ by inhibiting the oligomerization activation of cGAS (Banerjee, I., et al., Gasdermin D restrains type I interferon response to cytosolic DNA by disrupting ionic homeostasis. Immunity, 2018. 49(3): p. 413-426.).

[0005] In physiological conditions, in addition to being highly expressed in immune cells and mediating inflammatory responses, GSDMD is also highly expressed in other tissues, playing a pyroptosis-independent role. Unlike the N-terminal domain in macrophages, GSDMD in neutrophils also generates an N-terminal domain after being cleaved by elastase. This domain does not polymerize on the cell membrane to form holes, but rather is expressed through LC3 + Autophagosomes mediate IL-1β release (Karmakar, M., et al., N-GSDMD trafficking to neutrophil organelles facilitates IL-1beta release independently of plasmamembrane pores and pyroptosis. Nature Communications, 2020.11(1): p.2212.). GSDMD in intestinal epithelial cells is activated by Ca under physiological conditions. 2+-Scinderin-dependent cytoskeleton remodeling affects the excretion of mucus vesicles, mediates the secretion of mucin by intestinal goblet cells in a non-pyroptotic manner to form the intestinal mucus layer, and thus maintains the homeostasis of intestinal immunity. This study reveals the important physiological functions of GSDMD in non-immune cells that are independent of pyroptosis and inflammation (JianZhang, QY, et al, Epithelial Gasdermin D shapes the host-microbial interface by driving mucus layer formation. Science Immunology, 2022. 7(68): p.eabk2092.). Another research group found that GSDMD in mouse duodenal epithelial cells can be induced by food antigens to cleave the N-terminus to generate a pyroptosis-independent 88-amino acid p13 fragment. After entering the nucleus, the p13 fragment enhances STAT1 regulation of Ciita transcription and enhances MHCⅡ molecule expression, thereby inducing Tr1 cell upregulation, ultimately promoting the formation of intestinal food tolerance and playing an important role in maintaining host intestinal homeostasis (He, K., et al., Gasdermin D licenses MHCII induction to maintain food tolerance in small intestine. Cell, 2023.186(14): p.3033-3048.).

[0006] There is a long-term game between viruses and hosts. In addition to host cells restricting viral replication, viruses have evolved a variety of strategies to escape the body's antiviral response. Studies have shown that viral proteins can escape the body's inhibition of viral infection through non-classical cleavage of the host protein GSDMD. African swine fever, which causes huge economic losses to the livestock industry, is caused by African swine fever virus (ASFV) infecting pigs. The S273R viral protein encoded by ASFV cuts the G107-A108 position of pig GSDMD to produce GSDMD-N1-107 splice that does not cause cell pyroptosis. In addition, the viral protease 3C encoded by enterovirus EV71, which causes frequent hand, foot and mouth disease in children, escapes the body's antiviral response by cleaving GSDMD at the Q193-G194 site to inhibit host cell pyroptosis (Zhao, G., et al., African swine fever virus cysteineprotease pS273R inhibits pyroptosis by noncanonically cleaving gasderminD. Journal of Biological Chemisty, 2022. 298(1): p.101480.; Lei, X., et al., Enterovirus 71Inhibits Pyroptosis through Cleavage of Gasdermin D. Journal of Virology, 2017. 91(18).). Therefore, the study of different physiological conditions, different cell types and different GSDMD cleavage products broadens and enriches the regulatory network of GSDMD.

[0007] The Chinese invention patent application with application publication number CN 114249797A discloses the preparation and application of small molecule peptide drugs based on the RFWK motif of GSDMD protein, and finds that small molecule peptides can inhibit the activation of inflammasomes and inhibit the occurrence of sepsis in mice. It protects related peptides in improving the onset of sepsis, but does not show the function of RFWK motif in protecting the host from viral infection. Summary of the invention

[0008] The present invention proposes an application of a GSDMD protein SSSRFWKPRYSCVNL motif in preparing a medicine for preventing and / or treating RNA virus infectious diseases.

[0009] Through animal model experiments, the present invention found that in infectious diseases caused by VSV infection, knocking out Gsdmd inhibited type I interferon secretion, promoted viral replication, and reduced the survival of infected mice, indicating that GSDMD positively regulates antiviral immune response. Further, it was found that the SSSRFWKPRYSCVNL motif of the GSDMD protein can effectively inhibit VSV replication. The inventors found through research that the loss of Gsdmd significantly inhibited the antiviral immune response. It was observed in VSV-infected mice that Gsdmd was significantly downregulated compared to wild-type mice. - / - The mice showed downregulation of interferon and interferon-induced gene mRNA, increased virus titer and decreased mouse survival rate. Compared with the bone marrow-derived macrophages of mice pretreated with the negative control peptide infected with VSV, the VSV replication was significantly inhibited in the cells pretreated with the GSDMD protein SSSRFWKPRYSCVNL peptide, while the RIG-I protein was significantly increased. This suggests that the GSDMD protein SSSRFWKPRYSCVNL peptide has application prospects in preventing and treating viral infectious diseases.

[0010] Thus, in a first aspect, the present invention provides the use of a GSDMD polypeptide in the prevention and / or treatment of RNA viral infectious diseases. In addition, a DNA encoding a GSDMD polypeptide or an RNA expressing a GSDMD polypeptide can also be used in the prevention and / or treatment of RNA viral infectious diseases. The GSDMD polypeptide contains a SSSRFWKPRYSCVNL motif.

[0011] The present invention studies the effect of GSDMD protein SSSRFWKPRYSCVNL polypeptide in the infection process of RNA virus such as VSV at in vivo and in vitro levels.

[0012] First, the effect of VSV infection on GSDMD shearing in mouse peritoneal macrophages (PMs) was detected. The death of infected cells and the release of lactate dehydrogenase (LDH) in the culture supernatant were also detected. The experimental results showed that VSV infection did not cause GSDMD shearing or cell pyroptosis in cells, but VSV infection upregulated the transcription and translation of GSDMD in cells.

[0013] Next, we isolated wild-type mice (WT) and Gsdmd knockout mice (Gsdmd - / - ) primary macrophages were infected with VSV and the viral load, changes in type I interferon (IFN-I) and interferon-stimulated genes, and activation of IFN-I upstream and downstream signaling pathways were detected. It was found that GSDMD could upregulate the RIG-I protein content in cells, enhance IFN-I secretion, and inhibit viral replication. In vivo experiments found that Gsdmd - / - Mice are more susceptible to VSV infection in Gsdmd - / -More viral loads were detected in the lung tissue, liver tissue, spleen tissue and serum of mice. The above experimental results indicate that GSDMD positively regulates antiviral immune response through non-pyroptosis.

[0014] Finally, the SSSRFWKPRYSCVNL peptide of GSDMD was screened for its antiviral function. At a safe dose, the SSSRFWKPRYSCVNL peptide showed a strong antiviral effect, which proves that the GSDMD protein SSSRFWKPRYSCVNL peptide has the potential to prevent and treat RNA virus infection.

[0015] In a second aspect, the present invention provides a drug for preventing and / or treating RNA virus infectious diseases, wherein the active ingredient in the drug is a GSDMD polypeptide, and the GSDMD polypeptide contains a SSSRFWKPRYSCVNL motif.

[0016] The beneficial effects of the present invention are:

[0017] The present invention demonstrates for the first time in vivo and in vitro that GSDMD can promote IFN-I-mediated antiviral innate immune response in a non-pyroptosis-dependent manner, thereby inhibiting RNA virus infection such as VSV.

[0018] The present invention proves for the first time that the GSDMD protein SSSRFWKPRYSCVNL polypeptide enhances the antiviral immune response by stabilizing the RIG-I protein.

[0019] The present invention proves for the first time that the GSDMD protein SSSRFWKPRYSCVNL polypeptide can effectively inhibit VSV replication. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :VSV infection did not cause pyroptosis of mouse peritoneal macrophages. Figure A shows the shearing of GSDMD after VSV (MOI=1) infected mouse peritoneal macrophages; Figure B shows PI staining after VSV (MOI=1) infected mouse peritoneal macrophages to detect PI-positive cells, scale bar: 100μm. Figure C shows the content of LDH in the culture supernatant after VSV (MOI=1) infected mouse peritoneal macrophages. Figure D shows the mRNA level of Gsdmd detected by RT-qPCR after VSV (MOI=1) and SeV (MOI=0.1) infected mouse peritoneal macrophages. Figure E shows the expression of GSDMD detected by Western blot after VSV (MOI=1) and SeV (MOI=0.1) infected mouse peritoneal macrophages.

[0021] Figure 2: Knockout of Gsdmd inhibits the activation of RIG-I-mediated antiviral immune response induced by VSV infection in primary mouse macrophages. VSV (MOI = 1) infected WT and Gsdmd - / - Mouse peritoneal macrophages, Figure A is RT-qPCR detection of the transcription levels of Ifnb1 and Il6; Figure B is ELISA detection of the content of IFN-β and IL-6 in the cell supernatant; Figure C is Western blot detection of the expression of RIG-I, a key protein in the upstream and downstream signaling pathways of IFN-I, and the overall levels and phosphorylation levels of TBK1, IRF3, p65 and IκBα.

[0022] Figure 3 : Knockout of Gsdmd increases the viral load in primary macrophages of mice infected with VSV. VSV (MOI = 1) infected WT and Gsdmd - / - Mouse peritoneal macrophages. Figure A is RT-qPCR detection of VSVG transcription levels; Figure B is TCID 50 The VSV load in the cell supernatant was detected.

[0023] Figure 4 :Knockout of Gsdmd increases the viral load in VSV-infected iBMM cells. Figure A shows VSV (MOI = 0.1) infected WT and Gsdmd - / - iBMM cells, RT-qPCR detection of VSVG transcription level; Figure B shows VSV (MOI = 0.1) infected WT and Gsdmd - / - iBMM cells, TCID 50 Detection of VSV load in cell supernatant; Figure C shows VSV-GFP (MOI = 0.1) infected WT and Gsdmd - / - iBMM cells, GFP fluorescence was observed under an inverted fluorescence microscope, scale bar: 100 μm. Figure D shows VSV-GFP (MOI = 0.1) infected WT and Gsdmd - / - iBMM cells, flow cytometry detection of GFP + Cell content.

[0024] Figure 5 :Knockout of Gsdmd inhibits the transcription of Ifnb1 and other genes induced by poly(I:C) stimulation in primary mouse macrophages. Poly(I:C) was transfected into cells by Lipo2000, and the mRNA levels of Ifnb1(A), Il6(B), Ifit2(C) and Ip10(D) were detected by RT-qPCR.

[0025] Figure 6 :VSV infection of WT and Gsdmd - / - The content of VSVG in the lung, liver and spleen tissues of mice was evaluated after the inoculation of mice with VSVG.

[0026] Figure 7 :VSV infection of WT and Gsdmd - / - After the mice were infected, the VSV titers in the lung tissue, liver tissue, spleen tissue and serum of the mice were analyzed.

[0027] Figure 8 :VSV infection of WT and Gsdmd - / - The mortality curve of mice after the mice were implanted.

[0028] Fig. 9 :1-69 amino acid truncations of GSDMD can stabilize RIG-I protein expression. Figure A is a schematic diagram of different truncations of GSDMD; Figure B is a Western blot detection of the stabilization of RIG-I protein by GSDMD-D276A mutant and GSDMD-C truncations; Figure C is a Western blot detection of the stabilization of RIG-I protein by GSDMD 1-138 amino acid truncations and GSDMD 139-276 amino acid truncations; Figure D is a Western blot detection of the stabilization of RIG-I protein by GSDMD 1-69 amino acid truncations and GSDMD 70-138 amino acid truncations.

[0029] Fig.10 :The biotin-labeled GSDMD peptide interacts with the STUB1 protein. A is a schematic diagram of the 12 peptides divided by GSDMD, among which the peptide 10 sequence is SSSRFWKPRYSCVNL; B is a pull-down experiment to detect the binding of the GSDMD peptide to STUB1; C is an ELISA experiment to detect the binding of the GSDMD peptide to STUB1.

[0030] Fig.11 :5μM biotin-labeled CPP-coupled GSDMD peptide can enter mouse peritoneal macrophages. A is the effect of different concentrations of peptide on cytotoxicity detected by PI staining, scale bar: 100μm; B is the detection of GSDMD peptide entering cells by Cy5-labeled streptavidin, scale bar: 100μm; C is a statistical chart of multiple fields of view in Figure B (n=5).

[0031] Fig.12:Peptide 10 plays the best role in stabilizing RIG-I expression and resisting VSV infection in iBMM cell lines. 5μM Biotin-CPP-coupled GSDMD peptides 2, 6, 7, 10 (peptide 2, 6, 7, 10) and Biotin-CPP-coupled 2×Flag peptide were co-incubated with iBMM cells for 2h, and VSV (MOI=1) was infected for 8h. A is the amount of VSVG detected by RT-qPCR; B is the expression of RIG-I protein in cells detected by Western blot. Mock is a group not infected with VSV and not treated with peptides.

[0032] Fig.13 :Peptide 10 plays the best role in stabilizing RIG-I expression and resisting VSV infection in mouse bone marrow-derived macrophages. 5μM Biotin-CPP-coupled GSDMD peptides 2, 6, 7, 10 (peptide2, 6, 7, 10) and Biotin-CPP-coupled 2×Flag peptide were co-incubated with mouse bone marrow-derived macrophages for 2 hours and infected with VSV (MOI=1) for 8 hours. A is the RT-qPCR detection of VSVG loading in cells; B is the Western blot detection of RIG-I protein expression in cells. Mock is a group not infected with VSV and not treated with peptides. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] All numerical ranges provided in the present invention are intended to clearly include all values ​​falling between the range endpoints and the numerical ranges therebetween. Features mentioned in the present invention or features mentioned in the embodiments may be combined. All features disclosed in the present specification may be used in combination with any form of composition, and each feature disclosed in the specification may be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0035] As used in the present invention, "containing", "having" or "including" include "comprising", "mainly consisting of", "substantially consisting of", and "consisting of"; "mainly consisting of", "substantially consisting of" and "consisting of" are subordinate concepts of "containing", "having" or "including".

[0036] Throughout this application, the term "about" indicates that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0037] When used in the claims or specification, a selected / optional / preferred "numerical range" includes both the numerical endpoints at both ends of the range and all natural numbers covered between the numerical endpoints relative to the aforementioned numerical endpoints.

[0038] As used herein, the term "GSDMD" has its broad meaning, including the GSDMD gene, the mRNA of GSDMD, and the GSDMD protein.

[0039] GSDMD can be derived from humans, non-human primates (eg, gorillas, apes), rodents (eg, rats, mice, guinea pigs), pets (eg, cats, dogs), livestock (eg, horses, cows, sheep, pigs, rabbits), etc.

[0040] The sequence of GSDMD is: in homo sapiens (human) it corresponds to NCBI Gene ID: 79792; in mouse [Mus musculus (house mouse)] it corresponds to Gene ID: 69146.

[0041] As used in the present invention, the term "virus infection" refers to the process in which a virus invades the body through various pathways and proliferates in susceptible host cells. Virus pathogenicity begins by invading the host and infecting cells, and the pathogenic effect is manifested in both the body as a whole and the cells. The virus that infects the body in the present invention is an RNA virus. In some specific embodiments, the virus in the present invention is VSV.

[0042] As used in the present invention, the term "treatment" means: after suffering from a disease, the subject is contacted with (e.g., administered with) a polypeptide having a GSDMD protein SSSRFWKPRYSCVNL motif of the present invention, thereby partially or completely alleviating, improving, alleviating, inhibiting, delaying the onset of the disease, reducing the severity and / or reducing the incidence of one or more symptoms or features of a specific disease, disorder and / or condition compared to when not contacted, and does not mean that the symptoms of the disease must be completely suppressed. Suffering from a disease means that the body has symptoms of the disease.

[0043] As used in the present invention, the term "prevention" means that before a subject develops a disease, the subject is contacted with (e.g., administered) a polypeptide having the SSSRFWKPRYSCVNL motif of the GSDMD protein of the present invention, thereby reducing the symptoms after developing the disease compared to when the subject has not been contacted, but does not necessarily mean that the disease must be completely suppressed.

[0044] As used herein, the term "subject" may be suspected of being infected with a virus, or may be suspected of suffering from a disease caused by a viral infection, and "subject" includes mammals, including but not limited to domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys) and rodents (e.g., mice and rats).

[0045] As used herein, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and / or animals without excessive adverse reactions (such as toxicity, irritation and allergic response), ie, at a reasonable benefit / risk ratio.

[0046] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although representing specific embodiments of the present disclosure) are given for illustrative purposes only, because after reading the detailed description, various changes and modifications made within the spirit and scope of the present disclosure will become apparent to those skilled in the art.

[0047] The following is described in conjunction with specific embodiments, wherein reagents and samples are commercially available or available to the public in other ways. They are only examples and are not exclusive to the present invention. Other suitable tools and biological materials can be used to replace them. The experimental operations involved can be carried out according to the conditions and methods described in "Molecular Cloning Experiment Guide (3rd Edition)" (Science Press, 2002), and can be carried out according to the manufacturer's instructions of commercial enzymes and kits. Other experimental methods not described in detail are conventional methods known to those skilled in the art unless otherwise specified.

[0048] The wild-type mice (WT) with a genetic background of C57BL / 6J used in the examples were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. - / - Mice were kindly provided by Dr. Zhang Hailong from Henan University.

[0049] Example 1: Experimental methods involved in Examples 2 to 10

[0050] 1.1 Extraction and culture of mouse primary cells

[0051] 1.1.1 Extraction and culture of mouse peritoneal macrophages

[0052] (1) Mice of about eight weeks old were selected and intraperitoneally injected with 3% thioglycolate (2 mL / mice) three days in advance.

[0053] (2) Take out RPMI-1640 complete medium, PBS and the required six-well plates, twelve-well plates, scissors, tweezers, syringes, pipettes, etc. in advance and place them on the clean bench for ultraviolet irradiation for 30 minutes. After isoflurane anesthesia, the mouse was killed by dislocating the neck and placed in a large beaker containing 75% alcohol for disinfection for 3 minutes. Clamp the mouse's abdominal fur with tweezers, cut a small hole with scissors, and then slowly tear the skin open with your hands to fully expose its peritoneum. Use a 5mL syringe to draw 5mL of PBS and inject it into the mouse's abdominal cavity (keep 1mL PBS for blowing the abdominal cavity), pull out the syringe needle, grab the mouse's limbs and shake it to fully mix the PBS in the mouse's abdominal cavity, then aspirate the PBS and transfer it to a 15mL centrifuge tube, and repeat this 2 to 3 times.

[0054] (3) Centrifuge at 1000 rpm for 5 min, discard the supernatant, flick the bottom of the 15 mL centrifuge tube by hand, add 2 mL of RPMI-1640 complete medium and mix thoroughly. Dilute the mixed cell suspension a certain multiple and count with a counter. 1×10 cells per well of a 12-well plate 6 Cells, 2×10 per well of a six-well plate 6 After shaking evenly, place the cells in a 37°C incubator for culture. After 1 to 2 hours of culture, replace with fresh culture medium and continue to culture for subsequent experiments.

[0055] 1.1.2 Extraction and culture of mouse bone marrow-derived macrophages

[0056] (1) Take out DMEM complete medium, PBS, 75% alcohol, six-well plate, twelve-well plate, scissors, tweezers, syringe, pipette, etc. in advance and place them in the clean bench for ultraviolet irradiation for 30 minutes.

[0057] (2) Select mice about eight weeks old, anesthetize them with isoflurane, and kill them by dislocating their necks. Place them in a large beaker containing 75% alcohol and disinfect for 3 minutes. Clamp the mouse femur with tweezers, cut off the soles of the feet at the ankles, and then carefully separate the muscles on the femur with scissors, and separate the femur and tibia at the joint with tweezers. Rinse the femur without muscles in 75% alcohol and PBS in turn, clamp the femur with tweezers, and use scissors to cut a small piece at each end of the femur. Use a 1mL syringe to draw PBS directly above a 15mL centrifuge tube and repeatedly rinse the bone marrow alternately up and down until the bone marrow turns white. Centrifuge at 1000rpm for 5 minutes.

[0058] (3) Discard the supernatant, add 2 mL of red cell breaking solution, let stand for 2 minutes, then add an equal volume of DMEM complete medium, centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, flick the bottom of the centrifuge tube by hand, add 2 mL of DMEM complete medium (containing 10% FBS + 1% PS + 1% GMG1412) and mix thoroughly. Dilute the mixed cell suspension a certain multiple and count with a counter. 1×10 cells per well of a 12-well plate 6 Cells, 2×10 per well of a six-well plate 6 The cells were shaken evenly and placed in a 37°C incubator for culture. On the third day of culture, an equal volume of DMEM complete medium was added, and on the fifth day, DMEM complete medium was replaced. The cells cultured on the seventh day were used for subsequent experiments.

[0059] 1.2 RNA extraction

[0060] (1) Discard the cell supernatant in the 12-well plate, slowly add 1 mL of PBS along the wall of the plate with a pipette to rinse and then discard it. Then add 500 μL of Trizol to the 12-well plate, pipette repeatedly 5 to 10 times, transfer to a pre-labeled 1.5 mL EP tube, add 100 μL of chloroform and shake vigorously to mix thoroughly, and let it stand for 5 min until layers are separated.

[0061] (2) After standing, centrifuge at 12000 rpm for 15 min in a centrifuge precooled to 4°C. After centrifugation, the sample is divided into three layers: the bottom layer is a pink organic phase, the middle layer is a white protein layer, and the upper layer is a colorless aqueous phase. Transfer the upper aqueous phase to a new 1.5 mL EP tube and add an equal volume of isopropanol and mix thoroughly by turning it upside down. Leave it at room temperature for 20 min.

[0062] (3) After standing, centrifuge at 12000 rpm for 15 min at 4°C. After centrifugation, discard the supernatant and add 500 μL of pre-cooled 75% alcohol (DEPC H 2 The precipitate was washed by centrifugation at 12000 rpm for 5 min, and the supernatant was discarded. This was repeated three times, and then centrifuged at 12000 rpm for 2 min, and the residual liquid was aspirated with a pipette.

[0063] (4) Place the 1.5 mL EP tube containing the precipitate in a fume hood to dry, and add an appropriate amount of DEPC H 2 After that, it was heated in a metal bath at 60°C for 10 min.

[0064] (5) After the precipitate is dissolved, place it on ice and measure the RNA concentration and purity. Store it in a -80℃ refrigerator.

[0065] 1.3 Reverse transcription

[0066] (1) Place the RNA and DEPC H 2 O, 4× gDNA wiper Mix, and 5× PrimeScript RTMaster Mix were placed in a foam box containing crushed ice.

[0067] (2) Add 1 μg RNA sample and 4 μL 4×g DNA wiper Mix into a 0.5 mL microcentrifuge tube and finally add DEPC H 2 Add 3% HO to 16 μL.

[0068] (3) The above components were mixed and centrifuged, and placed in a PCR instrument for the first step of reverse transcription reaction. The reverse transcription conditions were: 42°C, 2 min.

[0069] (4) After adding 4 μL of 5× PrimeScript RT Master Mix to the 0.5 mL microcentrifuge tube where the first step of reverse transcription was completed, the second step of reverse transcription reaction was carried out under the following conditions: 50°C, 15 min; 85°C, 5 s; 4°C, ∞.

[0070] 1.4 Fluorescence quantitative PCR reaction

[0071] (1) Reverse transcribed cDNA, 2×TB Green Premix Ex Taq (Tli RNaseH Plus), PCR upstream primer, PCR downstream primer, DEPC H 2 O was placed on ice. The PCR reaction system was prepared according to the following system.

[0072] Fluorescence quantitative PCR reaction system

[0073] Reagents volume 2×TB Green Premix Ex Taq 5μL Upstream primer (10 μM) 0.25μL Downstream primer (10 μM) 0.25μL cDNA 0.5μL <![CDATA[DEPC H 2 The]]> 4μL

[0074] (2) After mixing the above reagents, perform the test according to the following procedure.

[0075] RT-qPCR amplification system

[0076]

[0077] (3) After the reaction is completed, the Ct value is derived and quantitative calculation is performed.

[0078] 1.5 Western blot

[0079] (1) Extraction of cell proteins

[0080] ① Take the cells to be extracted out of the incubator, discard the cell supernatant, and slowly add 2 mL of pre-cooled PBS along the wall of the culture dish to rinse the cells twice.

[0081] ② Place the cell culture dish on ice, add 1 mL of pre-cooled PBS, scrape the cells with a cell scraper, transfer them to a 1.5 mL EP tube, and centrifuge at 4°C, 5000 rpm for 5 min.

[0082] ③After centrifugation, discard the supernatant, add an appropriate amount of prepared cell lysis buffer according to the cell quantity, and lyse on ice for 30 minutes.

[0083] ④ After lysis, centrifuge at 12000 rpm for 20 min in a 4°C centrifuge, and transfer the supernatant to a new 1.5 mL EP tube.

[0084] ⑤ After centrifugation, perform BCA protein quantification. Prepare BCA working solution at a ratio of A solution: B solution = 50:1. Add 18μL PBS, 2μL protein solution and 200μL BCA working solution to the 96-well plate, and place it in a 37℃ incubator away from light for 30 minutes.

[0085] ⑥ Calculate the volume of protein supernatant, add 4× Loading Buffer and mix thoroughly, then boil in a 100℃ metal bath for 10 minutes. Store in a -80℃ refrigerator for later use.

[0086] ⑦After incubation, measure the protein OD value at 562nm using an enzyme reader and calculate the protein concentration.

[0087] (2) SDS-PAGE gel preparation

[0088] ① Rinse the glass plate with clean water first, then scrub it with detergent, and finally rinse off the foam with single distilled water, and place it in a 37℃ constant temperature box to dry.

[0089] ② Prepare separation gel of corresponding concentration according to the size of the band, pour it into the clamped glass gel plate and add isopropanol to remove bubbles, and wait for 20 to 30 minutes.

[0090] ③ After the separation gel is completely solidified, discard the upper layer of isopropanol and invert it on filter paper to absorb all the isopropanol.

[0091] ④ Prepare the upper layer of concentrated glue, add it to the glass glue plate, quickly insert the washed comb, and let it stand for 20 to 30 minutes. After it solidifies, use it for later use.

[0092] (3) Protein electrophoresis

[0093] ① Prepare 1× electrophoresis buffer by using single distilled water to prepare 1× electrophoresis buffer.

[0094] ② Place the solidified gel in the electrophoresis tank, pour in the electrophoresis solution and remove the comb to ensure that no electrophoresis solution is left out before loading the protein sample. The protein amount is 30μg.

[0095] ③ Perform electrophoresis at a constant voltage of 80V for 40 minutes, then change the voltage to 120V. When the blue indicator band reaches the bottom, stop electrophoresis and proceed to membrane transfer.

[0096] (4) Transfer

[0097] ① Prepare 10× transfer buffer into 1× transfer buffer and completely soak the filter paper with the transfer buffer.

[0098] ②Take a cut PVDF membrane and place it in methanol to activate it for about 30 seconds, then place it in the transfer solution for later use.

[0099] ③ Rinse the gel plate with tap water, carefully pry open the gel with a pry board, cut off the upper layer of gel, and place the lower layer of gel in the transfer solution.

[0100] ④ Place the filter paper, PVDF membrane, gel, and filter paper in the semi-dry transfer apparatus in the order of bottom to top, and use a roller to remove bubbles, then cover the transfer apparatus. Keep the voltage constant at 18V, transfer for 2 hours, and seal the membrane in 5% skim milk powder.

[0101] (5) Antibody incubation and exposure

[0102] ① After blocking the PVDF membrane in 5% skim milk at room temperature for 2 hours, cut the strips of appropriate size according to needs.

[0103] ② Pour the diluted antibody into the box containing the PVDF membrane and incubate on a shaker at 4°C overnight.

[0104] ③After recovering the primary antibody, rinse three times with PBST, each time for 10 minutes.

[0105] ④After rinsing, incubate the secondary antibody diluted in PBST at room temperature for 2 hours.

[0106] ⑤After the secondary antibody is blocked, discard it and rinse with PBST three times, 10 minutes each time.

[0107] ⑥ Place the PVDF membrane in a chemiluminescent imager, drop the prepared chemiluminescent liquid on the PVDF membrane, shake it evenly, expose it to color and save the results.

[0108] 1.6TCID 50 experiment

[0109] (1) Vero cells were evenly plated onto a 96-well plate one day in advance. The next day, when the cells grew to 80%, they could be infected with the virus.

[0110] (2) The virus solution was diluted 10 times in a series with DMEM complete medium, and each dilution gradient was inoculated into a row of 8 duplicate wells, 100 μL / well, and placed in a 37°C constant temperature incubator for culture.

[0111] (3) When cells are infected with the virus for 36 to 48 hours, the number of cytopathic effects is observed under a microscope and the TCID of the virus is calculated according to the Reed-Muench method. 50 .

[0112] 1.7 Enzyme-linked immunosorbent assay (ELISA)

[0113] (1) Take the ELISA kit out of the refrigerator 20 minutes before the experiment and return it to room temperature.

[0114] (2) After the strips have returned to room temperature, take out the coated strips required for the experiment from the sealed bag.

[0115] (3) Add the diluted standard of different concentrations into the wells in turn (100 μL / well), and add 100 μL of sample into the remaining corresponding wells. Seal the wells with sealing film and incubate in a 37°C constant temperature oven in the dark for 90 min.

[0116] (4) 20 min before the next step, dilute the 30× biotinylated antibody to 1× working solution.

[0117] (5) Take the ELISA plate out of the 37°C incubator, discard the liquid, tap it on absorbent paper 5 to 6 times, add 200 μL / well of the diluted washing solution, let it stand at room temperature for 1 min, discard the liquid, and continue tapping it on absorbent paper 5 to 6 times. Repeat five times.

[0118] (6) After discarding the washing solution, add 100 μL of biotinylated antibody working solution to each well. Seal the plate with a sealing film and place it in a 37°C constant temperature oven in the dark for 60 min.

[0119] (7) 20 min before the next step, dilute the 30× concentrated enzyme conjugate to a 1× working solution with diluent and place at room temperature away from light.

[0120] (8) Take the ELISA plate out of the 37°C incubator, discard the liquid, tap it on absorbent paper 5 to 6 times, add 200 μL / well of the diluted washing solution, let it stand at room temperature for 1 min, discard the liquid, and continue tapping it on absorbent paper 5 to 6 times. Repeat five times.

[0121] (9) Add 100 μL of the diluted enzyme conjugate working solution to each well. Seal the plate with a sealing film and incubate in a 37°C constant temperature oven in the dark for 30 min.

[0122] (10) Take the ELISA plate out of the 37°C incubator, discard the liquid, tap it on absorbent paper 5-6 times, add 200 μL / well of the diluted washing solution, let it stand at room temperature for 1 min, discard the liquid, tap it on absorbent paper 5-6 times, and repeat five times.

[0123] (11) Add 100 μL of chromogenic substrate to each well and incubate in a 37°C constant temperature oven away from light, and observe the degree of color change at any time.

[0124] (12) When the difference is obvious, add 100 μL of reaction stop solution to each well, mix well, and measure the value at OD450 using a microplate reader within 3 min.

[0125] (13) Draw a standard curve based on the measured OD value according to the instructions and calculate the sample concentration.

[0126] 1.8PI staining

[0127] (1) The isolated mouse peritoneal macrophages were inoculated into 12-well plates and cultured in an incubator overnight.

[0128] (2) Aspirate the cell culture medium, infect the cells with VSV (MOI=1), and culture in an incubator for 8 h.

[0129] (3) Dilute PI (1000×) 1000 times with detection buffer, that is, add PI (1000×) to 1 mL of detection buffer (keep away from light). Add 1 mL of detection working solution to each well of a 12-well plate.

[0130] (4) Aspirate the cell supernatant, rinse once with 1× PBS, thoroughly aspirate the PBS, add 1 mL of detection working solution to each well, and incubate at 37°C in the dark for 30 min.

[0131] (5) After the incubation in the dark, observe the staining effect under a fluorescence microscope and take photos. The entire experimental process needs to be kept away from light.

[0132] 1.9LDH determination

[0133] (1) The isolated mouse peritoneal macrophages were inoculated into 12-well plates and cultured in an incubator overnight.

[0134] (2) The cell culture medium was discarded, and the cells were infected with VSV (MOI=1). The cells were cultured in an incubator for 8 h, and the supernatant was collected.

[0135] (3) Prepare the color developing solution according to the instructions.

[0136] (4) Add 50 μL of the supernatant sample to each well of a 96-well plate, with three replicates for each treatment. Then add 50 μL of the prepared colorimetric solution to each well, mix for 15 seconds, and incubate at room temperature in the dark for 30 minutes.

[0137] (5) Determine OD using MμLtiskan GO microplate spectrophotometer 492 value.

[0138] Example 2: VSV infection does not cause pyroptosis of mouse peritoneal macrophages

[0139] Peritoneal macrophages from WT mice with a genetic background of C57BL / 6J were isolated and cultured.

[0140] Western blot was used to analyze the shearing of GSDMD in mouse peritoneal macrophages 0, 4, 8 and 12 h after VSV infection.

[0141] PI staining was used to detect cell death in mouse peritoneal macrophages 0 and 8 hours after VSV infection.

[0142] After 0 and 8 h of VSV infection of mouse peritoneal macrophages, the LDH release in the cell culture supernatant was detected by LDH kit.

[0143] 0, 4 and 8 h after VSV and SeV infection of mouse peritoneal macrophages, RT-qPCR and Western blot were used to analyze the transcription and translation of GSDMD in the cells.

[0144] The analysis results are as follows Figure 1 As shown, VSV infection does not induce GSDMD cleavage to generate P30 fragments; VSV infection does not increase PI + cell content; VSV infection did not significantly increase the LDH content in the culture supernatant; VSV and SeV infection significantly promoted the transcription and translation of GSDMD in cells.

[0145] The results of this example show that VSV infection does not cause pyroptosis of mouse peritoneal macrophages.

[0146] Example 3: Knockout of Gsdmd inhibits activation of RIG-I-mediated antiviral immune response induced by VSV infection

[0147] Isolate and culture WT mice and Gsdmd mice with a genetic background of C57BL / 6J - / - The peritoneal macrophages of mice were harvested at 0, 4 and 8 h after VSV infection, and RNA was extracted. RT-qPCR was used to detect the mRNA levels of Ifnb1 and Il6. At 0 and 8 h after VSV infection, the culture supernatant was harvested, and ELISA was used to detect the protein levels of IFN-β and IL-6. At 0, 4 and 8 h after VSV infection, the cells were harvested, protein was extracted, and Western blot was used to detect the overall levels and phosphorylation levels of RIG-I protein and TBK1, IRF3, p65 and IκBα.

[0148] The analysis results are as follows Figure 2 As shown, compared with the WT group, VSV infection of Gsdmd - / -The transcriptional activation levels of Ifnb1 and Il6 in the group were significantly inhibited; compared with the WT group, VSV infection of Gsdmd - / - The protein levels of IFN-β and IL-6 in the VSV-infected Gsdmd - / - The RIG-I protein content in the group was decreased, and the phosphorylation levels of TBK1, IRF3, p65 and IκBα were decreased.

[0149] The results of this example show that knocking out Gsdmd inhibits the activation of RIG-I-mediated antiviral immune response induced by VSV infection.

[0150] Example 4: Knockout of Gsdmd promotes VSV replication in mouse peritoneal macrophages

[0151] Isolate and culture WT mice and Gsdmd mice with a genetic background of C57BL / 6J - / - Peritoneal macrophages of mice were harvested at 0, 4, and 8 h after VSV infection, and RNA was extracted. RT-qPCR was used to detect the mRNA level of VSVG, the gene encoding the VSV viral membrane protein. Wild-type mice and Gsdmd - / - The peritoneal macrophages of mice were harvested 8 hours after VSV infection, and the cell culture supernatant was calculated. 50 The VSV titer was detected experimentally.

[0152] The analysis results are as follows Figure 3 As shown, compared with the WT group, VSV infection of Gsdmd - / - The transcription level of VSVG in the group was significantly increased compared with the WT group. - / - The VSV load in the group was significantly increased.

[0153] The results of this example show that Gsdmd knockout promotes the replication of VSV in mouse peritoneal macrophages.

[0154] Example 5: Knockout of Gsdmd promotes VSV replication in immortalized mouse bone marrow macrophages (iBMM cells)

[0155] VSV infection of acclimated WT mice and Gsdmd mice from a C57BL / 6J genetic background - / - At 0, 4 and 8 h after iBMM cell line was grown, cells were harvested, RNA was extracted, and VSVG mRNA levels were detected by RT-qPCR. Wild-type mice with a genetic background of C57BL / 6J and Gsdmd - / - Mouse iBMM cells, 8h after VSV infection, cell culture supernatant was harvested, TCID 50The experiment detected the VSV titer. VSV-GFP infected iBMM cells for 8 hours, and the GFP in the cells was observed by inverted fluorescence microscopy, and GFP was detected by flow cytometry. + Cell content.

[0156] The analysis results are as follows Figure 4 As shown, compared with the WT group, VSV infection of Gsdmd - / - The transcription level of VSVG in the group was significantly increased compared with the WT group. - / - Compared with the WT group, the VSV load in the Gsdmd - / - The GFP content in the group was higher.

[0157] The results of this example show that Gsdmd knockout promotes the replication of VSV in mouse iBMM cells.

[0158] Example 6: Knockout of Gsdmd inhibits transcription of Ifnb1 and other genes induced by poly(I:C) stimulation of primary mouse macrophages

[0159] Isolate and culture WT mice and Gsdmd mice with a genetic background of C57BL / 6J - / - Poly(I:C) was transfected into mouse peritoneal macrophages using Lipo2000. At 0, 4, and 8 h after transfection, the cells were harvested and RNA was extracted. RT-qPCR was used to detect the mRNA levels of Ifnb1, Il6, Ifit2, and Ip10.

[0160] The analysis results are as follows Figure 5 As shown, compared with the WT group, poly(I:C) stimulated Gsdmd - / - The transcriptional activation of Ifnb1, Il6, Ifit2, and Ip10 was significantly inhibited.

[0161] The results of this example show that Gsdmd knockout inhibits the activation of anti-RNA virus immune response.

[0162] Example 7: Knockout of Gsdmd in mice increases the burden of VSV in various organs

[0163] WT mice and Gsdmd mice with a genetic background of C57BL / 6J - / - Mice were injected via tail vein with 1 × 10 7 pfu / g of VSV, the mice were killed 12h later, and the levels of VSVG in lung tissue, liver tissue and spleen tissue were detected by RT-qPCR; TCID 50 The viral load was detected in lung tissue, liver tissue, spleen tissue and serum.

[0164] The levels of VSVG in lung, liver and spleen tissues were Figure 6 As shown, the viral loads in lung tissue, liver tissue, spleen tissue and serum were Figure 7 shown.

[0165] The analysis results showed that compared with the WT group of mice, Gsdmd - / - The levels of VSVG in the lung, liver and spleen tissues of the Gsdmd group mice were increased compared with those in the WT group. - / - The VSV load in the lung tissue, liver tissue, spleen tissue and serum of group A mice was increased.

[0166] The results of this example show that knocking out Gsdmd increases the VSV content in the lung tissue, liver tissue, spleen tissue and serum of mice.

[0167] Example 8: Knockout of Gsdmd in mice increases mortality caused by VSV infection

[0168] WT mice and Gsdmd mice with a genetic background of C57BL / 6J - / - Mice were injected via tail vein with 1 × 10 8 pfu / g of VSV, and observed the survival of mice every 12 hours, and calculated the mortality rate of mice.

[0169] The results are as follows Figure 8 As shown, compared with WT mice, Gsdmd - / - Mice are more susceptible to VSV and have a higher mortality rate.

[0170] The results of this example show that knocking out Gsdmd in mice increases the mortality rate caused by VSV infection.

[0171] Example 9: The 1-69 amino acid truncation of GSDMD can stabilize RIG-I protein expression

[0172] GSDMD-D276A mutant plasmid, GSDMD-C truncation plasmid, 1-138 amino acid truncation plasmid of GSDMD, 139-276 amino acid truncation plasmid of GSDMD, 1-69 amino acid truncation plasmid of GSDMD, and 70-138 amino acid truncation plasmid of GSDMD were constructed, and RIG-I plasmid was co-transfected into HEK293T cells with the above plasmids, respectively. After 24 hours of transfection, the protein was extracted and the expression of RIG-I was detected by Western blot.

[0173] The relevant plasmid construction method is as follows:

[0174] Construction of GSDMD full-length plasmid (pcDNA3.1-GSDMD-Flag plasmid): RNA was extracted from mouse peritoneal macrophages and reverse transcribed into cDNA. The cDNA was used as a template and constructed into the eukaryotic expression plasmid pcDNA3.1-Flag through two restriction sites, EcoR I and BamH I.

[0175] Construction of GSDMD-D276A mutant plasmid (pcDNA3.1-GSDMD-D276A-Flag plasmid): The pcDNA3.1-GSDMD-Flag plasmid was used as a template and constructed by a point mutagenesis kit;

[0176] Construction of GSDMD-C truncated plasmid (pcDNA3.1-GSDMD-C-Flag plasmid): PCR was performed using pcDNA3.1-GSDMD-Flag plasmid as a template, and the amplified nucleotides corresponding to the amino acids of GSDMD-C were constructed into the eukaryotic expression plasmid pcDNA3.1-Flag through the two restriction sites of EcoR I and BamH I;

[0177] Construction of the plasmids of the 1-138 amino acid truncations of GSDMD (pcDNA3.1-GSDMD-1-138-Flag plasmid) and the 139-276 amino acid truncations of GSDMD (pcDNA3.1-GSDMD-139-276-Flag plasmid): PCR was performed using the pcDNA3.1-GSDMD-Flag plasmid as a template, and the amplified nucleotides corresponding to the 1-138 amino acids of GSDMD and the 139-276 amino acids of GSDMD were respectively constructed into the eukaryotic expression plasmid pcDNA3.1-Flag through the two restriction sites of Nhe I and EcoR I;

[0178] Construction of the 1-69 amino acid truncation plasmid of GSDMD (pcDNA3.1-GSDMD-1-69-GFP plasmid) and the 70-138 amino acid truncation plasmid of GSDMD (pcDNA3.1-GSDMD-70-138-GFP plasmid): PCR was performed using the pcDNA3.1-GSDMD-Flag plasmid as a template, and the amplified nucleotides corresponding to the 1-69 amino acids of GSDMD and the 70-138 amino acids of GSDMD were constructed into the eukaryotic expression plasmid pcDNA3.1-GFP through the two restriction sites of Nhe I and EcoR I.

[0179] The nucleotide sequence of GSDMD is shown in SEQ ID NO:1, the nucleotide sequence of GSDMD-C is shown in SEQ ID NO:2, and the amino acid sequence of GSDMD is shown in SEQ ID NO:3.

[0180] The results are as follows Fig. 9 As shown, the GSDMD-D276A mutant can stabilize RIG-I, while the GSDMD-C truncate cannot stabilize RIG-I; the 1-138 amino acid truncate of GSDMD can stabilize RIG-I, while the 139-276 amino acid truncate of GSDMD cannot stabilize RIG-I; the 1-69 amino acid truncate of GSDMD can stabilize RIG-I; and the 70-138 amino acid truncate of GSDMD cannot stabilize RIG-I.

[0181] The results of this example show that the 1-69 amino acid truncation of GSDMD can stabilize the expression of RIG-I protein.

[0182] Example 10: Peptide 10 of GSDMD interacts strongly with STUB1

[0183] The 1-70 amino acids of GSDMD were divided into 12 peptides with a length of 15 amino acids and a step of 5 amino acids (peptide 1: 1-15 amino acids of GSDMD, peptide 2: 6-20 amino acids of GSDMD, peptide 3: 11-25 amino acids of GSDMD, peptide 4: 16-30 amino acids of GSDMD, peptide 5: 21-35 amino acids of GSDMD, peptide 6: 26-40 amino acids of GSDMD, peptide 7: 31-45 amino acids of GSDMD, peptide 8: 36-50 amino acids of GSDMD, peptide 9: 41-55 amino acids of GSDMD, peptide 10: 46-60 amino acids of GSDMD, peptide 11: 51-65 amino acids of GSDMD, peptide 12: 56-70 amino acids of GSDMD), biotin-labeled peptides were synthesized, and the peptides were expressed by pull-down. Down assay and ELISA assay were used to detect the interaction between the peptide and E3 ubiquitin ligase STUB1.

[0184] The results are as follows Fig.10 As shown in the figure, among the 12 peptides of GSDMD, the pull-down experiment found that peptide6, peptide 7, and peptide 10 had strong interactions with STUB1 protein; the ELISA experiment found that among the three peptides, peptide 6, peptide 7, and peptide 10, peptide 10 bound the most to STUB1.

[0185] The results of this example show that peptide 10 (SSSRFWKPRYSCVNL) of the GSDMD protein has a strong interaction with STUB1.

[0186] Example 11: Peptide 10 of GSDMD protein has the strongest function in stabilizing RIG-I and inhibiting VSV replication

[0187] In order to make GSDMD peptides more easily enter cells, we conjugated the screened peptide 6, peptide 7, and peptide 10 with a cell-penetrating peptide (CPP), resynthesized biotin-labeled cell-penetrating peptide (CPP)-coupled GSDMD peptide 6, peptide 7, and peptide 10, and synthesized biotin-labeled GSDMD peptide 2 and 2×Flag (DYKDDDDKDYKDDDDK) peptides as negative controls. First, the effects of related peptides on cell activity were evaluated. After setting a series of dose ranges (0μM, 2.5μM, 5μM, and 10μM) of biotin-labeled cell-penetrating peptide (CPP)-coupled GSDMD, the dose range was safely evaluated by PI staining in mouse peritoneal macrophages. Immunofluorescence experiments were used to evaluate whether safe doses of peptides could enter mouse peritoneal macrophages.

[0188] The results are as follows Fig.11 As shown, the related peptide had no effect on cell activity at 5 μM for 12 hours, and the 5 μM peptide was able to enter the cell.

[0189] After iBMM cells were pretreated with 5 μM 2×Flag peptide, peptide 2, peptide 6, peptide 7, and peptide 10 for 2 hours, they were infected with VSV (MOI=1) for 8 hours, and the mRNA level of VSVG was detected by RT-qPCR; the expression of RIG-I protein in the cells was detected by Western blot.

[0190] The results are as follows Fig.12 As shown, RT-qPCR results showed that the VSVG content in the peptide 10 treatment group was the lowest; Western blot results showed that compared with the Mock group, VSV infection upregulated RIG-I protein (Peptide 2, 2×Flag group), and the RIG-I protein expression in the peptide 6, peptide 7, and peptide 10 treatment groups was higher than that in the peptide 2, 2×Flag group, and the peptide 10 group was the highest.

[0191] Mouse bone marrow-derived macrophages were pretreated with 5 μM 2×Flag peptide, peptide 2, peptide 6, peptide 7, and peptide 10 for 2 hours and then infected with VSV (MOI=1) for 8 hours. RT-qPCR was used to detect the mRNA level of VSVG; Western blot was used to detect the expression of RIG-I protein in the cells.

[0192] The results are as follows Fig.13 As shown, RT-qPCR results showed that the VSVG content in the peptide 10 treatment group was the lowest; Western blot results showed that compared with the Mock group, VSV infection upregulated RIG-I protein (Peptide 2, 2×Flag group), and the RIG-I protein expression in the peptide 6, peptide 7, and peptide 10 treatment groups was higher than that in the peptide 2, 2×Flag group, and the peptide 10 group was the highest.

[0193] The results of this example show that peptide 10 of the GSDMD protein has the strongest function in stabilizing RIG-I and inhibiting VSV replication.

[0194] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a GSDMD polypeptide, a DNA encoding a GSDMD polypeptide, or an RNA expressing a GSDMD polypeptide in the preparation of a drug for preventing and / or treating RNA virus infectious diseases, wherein the GSDMD polypeptide contains a SSSRFWKPRYSCVNL motif.

2. The use according to claim 1, characterized in that: The RNA virus is VSV.

3. A drug for preventing and / or treating RNA virus infectious diseases, wherein the active ingredient of the drug is a GSDMD polypeptide, and the GSDMD polypeptide contains a SSSRFWKPRYSCVNL motif.

4. The drug according to claim 3, characterized in that The RNA virus is VSV.

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