Application of a GSDMD peptide in the prevention and / or treatment of RNA virus infections

Abstract

This invention belongs to the fields of biotechnology and medicine, specifically relating to the application of a GSDMD polypeptide in the preparation of drugs for the prevention and / or treatment of RNA virus infectious diseases. Through animal model experiments, this invention has found that in infectious diseases caused by VSV infection, knocking out… Gsdmd Inhibition of type I interferon secretion, promotion of viral replication, and reduction of survival time in infected mice indicate that GSDMD positively regulates the antiviral immune response. Furthermore, the GSDMD protein SSSRFWKPRYSCVNL motif was found to effectively inhibit VSV replication.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, specifically relating to the application of the GSDMD protein SSSRFWKPRYSCVNL motif in the preparation of drugs for the prevention and / or treatment of RNA virus infectious diseases. Background Technology

[0002] Because RNA viruses (such as influenza virus, Ebola virus, Zika virus, and novel coronavirus) have a wide range of transmission and extremely high mutation rate, the development of broad-spectrum and highly effective antiviral drugs to deal with 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. The innate immune system, as the first line of defense against the invasion of external pathogens, 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 in 2004 as an intracellular PRR that can bind double-stranded RNA (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 tissues and cells, including immune cells. It recognizes short-chain dsRNA and 5'-blunt-terminated triphosphate dsRNA, and is mainly located in the cytoplasm. It can also translocate to the nucleus to exert its antiviral immune response (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 a self-inhibited state. However, when it recognizes viral RNA, RIG-I undergoes a conformational change, followed by K63 ubiquitination activation, which in turn activates proteins such as TRAF3. This leads to the phosphorylation of downstream molecules such as TBK1 and IRF3. Phosphorylated IRF3 enters the nucleus and 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] Pyroptosis, a novel inflammatory cell death mechanism, originated from research on macrophages infected with Salmonella. It is one of the body's immune system's mechanisms for combating invading pathogens and is also a new hot topic in the field of tumor immunotherapy. The N-terminal p30 protein, generated by the cleavage of GSDMD by its upstream protein, not only mediates pyroptosis but also regulates the non-canonical release of IFN-I, activates the cGAS-STING signaling pathway, and affects mitochondrial membrane permeability. After intracellular DNA is sensed by the AIM2 inflammasome, caspase-1 is activated. Subsequently, GSDMD is cleaved and activated, forming a pore on the cell membrane to mediate pyroptosis. Intracellular IL-1β, IL-18 and potassium ions can be released into the extracellular space through this pore. 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] Under 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 function. Unlike the N-terminal domain in macrophages, GSDMD in neutrophils also generates an N-terminal domain after cleavage by elastase. This domain does not polymerize on the cell membrane to form pores, but rather communicates through LC3. + Autophagosomes mediate IL-1β release (Karmakar, M., et al., N-GSDMD trafficking to neutrophil organelles facilitates IL-1β release independently of plasma membrane pores and pyroptosis. Nature Communications, 2020.11(1):p.2212.). GSDMD in intestinal epithelial cells, under physiological conditions, via Ca2+... 2+-Scinderin-dependent cytoskeleton remodeling affects mucus vesicle efflux, mediating intestinal goblet cells to secrete mucin in a non-pyroptotic manner to form the intestinal mucus layer, thereby maintaining intestinal immune homeostasis. This study reveals the important physiological functions of GSDMD in non-immune cells that are pyroptotic and inflammation-independent (Jian Zhang, 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 cleaved at the N-terminus by food antigens to generate a pyroptosis-independent p13 fragment of 88 amino acids. After entering the nucleus, the p13 fragment enhances the regulation of Ciita transcription by STAT1, enhances the expression of MHCII molecules, and then induces the upregulation of Tr1 cells, 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 the virus and the host. In addition to the host cell restricting viral replication, the virus has evolved a variety of strategies to escape the host's antiviral response. Studies have shown that viral proteins can escape the host's inhibition of viral infection by non-classical cleaving of the host protein GSDMD. African swine fever, which causes huge economic losses to the livestock industry, is caused by pigs infected with African swine fever virus (ASFV). The S273R viral protein encoded by ASFV cleaves the G107-A108 position of pig GSDMD, producing the GSDMD-N1-107 splice that does not cause pyroptosis. In addition, the enterovirus EV71, which causes frequent outbreaks of hand-foot-mouth disease in children, encodes viral protease 3C, which evades the body's antiviral response by cleaving GSDMD at the Q193-G194 position to inhibit host cell pyroptosis (Zhao, G., et al., African swine fever virus cysteineprotease pS273R inhibits pyroptosis by noncanonically cleaving gasdermin D. Journal of Biological Chemistry, 2022, 298(1): p.101480.; Lei, X., et al., Enterovirus 71 Inhibits Pyroptosis through Cleavage of Gasdermin D. Journal of Virology, 2017, 91(18). Therefore, studies on different physiological conditions, different cell types, and different GSDMD cleavage products have broadened and enriched the regulatory network of GSDMD.

[0007] Chinese invention patent application CN 114249797A discloses the preparation and application of small molecule peptide drugs based on the RFWK motif of the GSDMD protein. It was found that the small molecule peptides can inhibit inflammasome activation and suppress the occurrence of sepsis in mice. The patent protects the related peptides in improving the pathogenesis of sepsis; however, the function of the RFWK motif in protecting the host from viral infection was not observed. Summary of the Invention

[0008] This invention proposes the application of the GSDMD protein SSSRFWKPRYSCVNL motif in the preparation of drugs for the prevention and / or treatment of RNA virus infectious diseases.

[0009] This invention, through animal model experiments, found that in VSV-induced infectious diseases, knockout of Gsdmd inhibits type I interferon secretion, promotes viral replication, and reduces the survival time of infected mice, indicating that Gsdmd positively regulates the antiviral immune response. Furthermore, it was found that the Gsdmd protein SSSRFWKPRYSCVNL motif effectively inhibits VSV replication. The inventors' research revealed that the absence of Gsdmd significantly inhibits the antiviral immune response. In VSV-infected mice, compared to wild-type mice, Gsdmd... - / - Mice exhibited downregulation of interferon and interferon-induced gene mRNA, increased viral titer, and decreased survival rate. Compared with bone marrow-derived macrophages from mice pretreated with the GSDMD protein SSSRFWKPRYSCVNL peptide, VSV replication was significantly inhibited, while RIG-I protein was significantly increased. This suggests that the GSDMD protein SSSRFWKPRYSCVNL peptide has potential applications in the prevention and treatment of viral infectious diseases.

[0010] Therefore, in a first aspect, the present invention provides the application of the GSDMD peptide in the prevention and / or treatment of RNA viral infectious diseases. Additionally, the DNA encoding the GSDMD peptide and the RNA expressing the GSDMD peptide can also be used in the prevention and / or treatment of RNA viral infectious diseases. The GSDMD peptide contains the SSSRFWKPRYSCVNL motif.

[0011] This invention investigates the role of the GSDMD protein SSSRFWKPRYSCVNL peptide in the infection process of RNA viruses such as VSV at both in vivo and in vitro levels.

[0012] First, the effect of VSV infection on GSDMD cleavage was examined. Cell death and lactate dehydrogenase (LDH) release in the culture supernatant were also investigated. The results showed that VSV infection did not induce GSDMD cleavage or pyroptosis in cells, and that VSV infection upregulated GSDMD transcription and translation in cells.

[0013] Next, wild-type mice (WT) and Gsdmd knockout mice (Gsdmd) were separated. - / - Primary macrophages were then infected with VSV. Viral load, changes in type I interferon (IFN-I) and interferon-stimulated genes, and activation of IFN-I upstream and downstream signaling pathways were measured. GSDMD was found to upregulate RIG-I protein levels in cells, enhance IFN-I secretion, and inhibit viral replication. In vivo experiments showed that GSDMD... - / - Mice are more susceptible to VSV infection in Gsdmd - / -Increased viral load was detected in mouse lung, liver, spleen, and serum. These experimental results indicate that GSDMD positively regulates the antiviral immune response through a non-pyroptotic mechanism.

[0014] Finally, the SSSRFWKPRYSCVNL peptide, which exhibits antiviral function in GSDMD, was screened. At a safe dosage, the SSSRFWKPRYSCVNL peptide demonstrated a strong antiviral effect, proving that the GSDMD protein SSSRFWKPRYSCVNL peptide has the potential to prevent and treat RNA virus infections.

[0015] In a second aspect, the present invention provides a medicament for the prevention and / or treatment of RNA virus infectious diseases, wherein the active ingredient in the medicament is a GSDMD polypeptide containing the SSSRFWKPRYSCVNL motif.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention is the first to demonstrate in vivo and in vitro that GSDMD can promote IFN-I-mediated antiviral innate immune responses in a non-pyroptosis-dependent manner, thereby inhibiting infections by RNA viruses such as VSV.

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

[0019] This invention is the first to demonstrate that the GSDMD protein SSSRFWKPRYSCVNL polypeptide can effectively inhibit VSV replication. Attached Figure Description

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

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

[0022] Figure 3 Knocking out Gsdmd increases viral load in VSV-infected mouse primary macrophages. VSV (MOI=1) infection of WT and Gsdmd cells... - / - Mouse peritoneal macrophages; Figure A shows the transcriptional level of VSVG detected by RT-qPCR; Figure B shows TCID. 50 The load of VSV in the cell supernatant was detected.

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

[0024] Figure 5 Knocking out Gsdmd inhibited the transcription of Ifnb1 and other genes induced by poly(I:C) stimulation in primary mouse macrophages. Lipo2000 was used to transfect poly(I:C) cells, and RT-qPCR was used to detect the mRNA levels of Ifnb1 (A), Il6 (B), Ifit2 (C), and Ip10 (D).

[0025] Figure 6 VSV infects WT and Gsdmd - / - After mouse treatment, the content of VSVG in the lung, liver and spleen tissues of mice was measured.

[0026] Figure 7 VSV infects WT and Gsdmd - / - After mouse treatment, the VSV titers in the lung, liver, spleen, and serum of mice were analyzed.

[0027] Figure 8 VSV infects WT and Gsdmd - / - The mortality curve of mice after treatment.

[0028] Figure 9 The 1-69 amino acid truncated form of GSDMD can stabilize RIG-I protein expression. Figure A shows a schematic diagram of different GSDMD truncated forms; Figure B shows the stability of RIG-I protein by GSDMD-D276A mutant and GSDMD-C truncated form as detected by Western blot; Figure C shows the stability of RIG-I protein by GSDMD truncated forms 1-138 and 139-276; Figure D shows the stability of RIG-I protein by GSDMD truncated forms 1-69 and 70-138.

[0029] Figure 10 Biotin-labeled GSDMD peptides interact with the STUB1 protein. A shows a schematic diagram of the 12 peptides derived from GSDMD, where peptide10 has the sequence SSSRFWKPRYSCVNL; B shows the pull-down assay used to detect the binding of GSDMD peptides to STUB1; C shows the ELISA assay used to detect the binding of GSDMD peptides to STUB1.

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

[0031] Figure 12Peptide 10 performed optimally in stabilizing RIG-I expression and resisting VSV infection in the iBMM cell line. iBMM cells were co-incubated with 5 μM Biotin-CPP-conjugated GSDMD peptides 2, 6, 7, and 10, as well as Biotin-CPP-conjugated 2×Flag peptide, for 2 h, followed by VSV infection (MOI=1) for 8 h. A shows the VSVG load in cells as detected by RT-qPCR; B shows the RIG-I protein expression in cells as detected by Western blot. Mock samples represent the VSV-uninfected and peptide-untreated groups.

[0032] Figure 13 Peptide 10 exhibited optimal function in stabilizing RIG-I expression and resisting VSV infection in mouse bone marrow-derived macrophages. Mouse bone marrow-derived macrophages were co-incubated with 5 μM Biotin-CPP-conjugated GSDMD peptides 2, 6, 7, and 10 (peptides 2, 6, 7, and 10) and Biotin-CPP-conjugated 2×Flag peptide for 2 h, followed by VSV infection (MOI=1) for 8 h. A shows the VSVG load in cells as detected by RT-qPCR; B shows the RIG-I protein expression in cells as detected by Western blot. Mock samples represent the VSV-uninfected and peptide-untreated groups. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

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

[0035] As used in this invention, "containing", "having" or "including" includes "comprising", "mainly composed of", "substantially composed of", and "composed of"; "mainly composed of", "substantially composed of", and "composed of" are subordinate concepts of "containing", "having" or "including".

[0036] Throughout the application, the term “about” means: a value includes the standard deviation of the error of the apparatus or method used to determine that value.

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

[0038] As used in this invention, the term "GSDMD" has its broad meaning and includes the GSDMD gene, GSDMD mRNA, and GSDMD protein.

[0039] GSDMD can be derived from humans, non-human primates (such as chimpanzees and apes), rodents (such as rats, mice, and guinea pigs), pets (such as cats and dogs), and livestock (such as horses, cattle, sheep, pigs, and rabbits).

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

[0041] As used in this invention, the term "viral infection" refers to the process by which a virus invades the body through various pathways and proliferates in susceptible host cells. Viral pathogenesis begins with invasion of the host and infection of cells, and its pathogenic effects manifest at both the systemic and cellular levels. The virus infecting the body in this invention is an RNA virus. In some specific embodiments, the virus in this invention is VSV.

[0042] As used herein, the term "treatment" means, after the occurrence of a disease, exposing (e.g., administering medication) a subject to a polypeptide of the GSDMD protein SSSRFWKPRYSCVNL motif of the present invention, thereby partially or completely reducing, improving, alleviating, suppressing, delaying the onset of, reducing the severity of, and / or reducing the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition compared to non-exposure, without implying the necessity of completely suppressing the symptoms of the disease. The occurrence of a disease means the appearance of symptoms of a disease in the body.

[0043] As used in this invention, the term "prevention" means: before the onset of disease, by exposing the subject to (e.g., administering medication) the polypeptide of the GSDMD protein SSSRFWKPRYSCVNL motif of this invention, thereby reducing the symptoms of the disease compared to when there is no exposure, and does not imply that the disease must be completely suppressed.

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

[0045] As used in this invention, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse reactions (such as toxicity, irritation, and allergic reactions), i.e., 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 embodiments (although illustrating specific implementations of this disclosure) are given for illustrative purposes only, as various changes and modifications that can be made within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description.

[0047] The following description is based on specific embodiments. The reagents and samples are commercially available or otherwise readily available to the public; they are merely examples and not the only ones applicable to this invention. Other suitable tools and biological materials can be substituted. The experimental procedures involved can be performed according to the conditions and methods described in *Molecular Cloning: A Laboratory Manual (Third Edition)* (Science Press, 2002), and according to the manufacturer's instructions for commercially available enzymes and reagent kits. Other experimental methods not described in detail are conventional methods well-known to those skilled in the art unless otherwise specified.

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

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

[0050] 1.1 Extraction and culture of primary mouse cells

[0051] 1.1.1 Extraction and culture of mouse peritoneal macrophages

[0052] (1) Select mice that are about eight weeks old and inject them intraperitoneally with 3% mercaptoacetate, 2 mL / mouse, three days in advance.

[0053] (2) Take out RPMI-1640 complete culture medium, PBS, and the required six-well plates, twelve-well plates, scissors, forceps, syringes, pipettes, etc. in advance and place them in a clean bench for UV irradiation for 30 minutes. Anesthetize mice with isoflurane and euthanize them by dislocation. Place them in a large beaker containing 75% alcohol and disinfect for 3 minutes. Hold the mouse's abdominal fur with forceps, cut a small incision with scissors, and then slowly tear open the skin to fully expose its peritoneum. Draw 5 mL of PBS into the mouse's peritoneal cavity with a 5 mL syringe and inject it (reserve 1 mL of PBS for blowing into the peritoneal cavity). Remove the syringe needle, grasp the mouse's limbs and shake it to mix the PBS thoroughly in the mouse's peritoneal cavity. Then, aspirate the PBS and transfer it to a 15 mL centrifuge tube. Repeat this process 2-3 times.

[0054] (3) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and gently tap the bottom of a 15 mL centrifuge tube. Add 2 mL of RPMI-1640 complete culture medium and mix thoroughly. Dilute the mixed cell suspension a certain factor and count the cells using a cell counter. Place 1 × 10⁻⁶ cells per well in a 12-well plate. 6 10 cells, 2 × 10⁶ cells per well in a six-well plate 6 Collect the cells, shake well, and incubate at 37°C. After 1–2 hours of incubation, replace with fresh culture medium and continue culturing for subsequent experiments.

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

[0056] (1) Take out the DMEM complete culture medium in advance, and place PBS, 75% alcohol, six-well plate, twelve-well plate, scissors, tweezers, syringe, pipette and other items in the ultra-clean workbench and irradiate them with ultraviolet light for 30 minutes.

[0057] (2) Select mice approximately eight weeks old. After anesthetizing with isoflurane, euthanize the mice by cervical dislocation and place them in a large beaker containing 75% alcohol for 3 minutes for disinfection. Hold the femur with forceps and cut off the paw at the ankle. Carefully separate the muscles on the femur with scissors and separate the femur from the tibia at the joint with forceps. Rinse the femur (with muscles removed) in 75% alcohol and PBS in sequence. Hold the femur with forceps and cut a small piece from each end of the femur with scissors. Use a 1mL syringe to draw PBS and rinse the bone marrow repeatedly from top to bottom of a 15mL centrifuge tube until the bone marrow turns white. Centrifuge at 1000rpm for 5 minutes.

[0058] (3) Discard the supernatant, add 2 mL of red blood cell lysis solution, let stand for 2 min, then add an equal volume of DMEM complete medium, centrifuge at 1000 rpm for 5 min. Discard the supernatant, gently tap the bottom of the centrifuge tube, add 2 mL of DMEM complete medium (containing 10% FBS + 1% PS + 1% GMG1412) and mix thoroughly. Dilute the mixed cell suspension a certain factor and count the cells using a cell counter. 1 × 10⁻⁶ cells per well in a 12-well plate. 6 10 cells, 2 × 10⁶ cells per well in a six-well plate 6 After shaking the cells thoroughly, they were placed in a 37°C incubator. On the third day of culture, an equal volume of DMEM complete medium was added. On the fifth day, the DMEM complete medium was replaced, and the cells were cultured for subsequent experiments on the seventh day.

[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 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 stand for 5 min until the layers separate.

[0061] (2) After settling, centrifuge at 12000 rpm for 15 min in a centrifuge pre-cooled to 4°C. After centrifugation, the sample separated into three layers: a pink organic phase at the bottom, a white protein layer in the middle, and a colorless aqueous phase at the top. Transfer the upper aqueous phase to a new 1.5 mL EP tube and add an equal volume of isopropanol, inverting the tube to mix thoroughly. Incubate at room temperature for 20 min.

[0062] (3) After standing, centrifuge at 12000 rpm for 15 min at 4℃. After centrifugation, discard the supernatant, add 500 μL of pre-cooled 75% alcohol (prepared with DEPC H2O) to wash the precipitate, centrifuge at 12000 rpm for 5 min, discard the supernatant after centrifugation, repeat this process three times, then centrifuge at 12000 rpm for 2 min, and use a pipette to remove the residual liquid.

[0063] (4) Place the 1.5 mL EP tube containing the precipitate in a fume hood to dry, add an appropriate amount of DEPC H2O, and heat 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 at -80℃.

[0065] 1.3 Reverse transcription

[0066] (1) Place the measured concentrations of RNA, DEPC H2O, 4×gDNA wiper Mix and 5×PrimeScript RTMaster Mix in a foam box containing crushed ice.

[0067] (2) Add 1 μg RNA sample and 4 μL 4×g DNA wiper Mix to a 0.5 mL microcentrifuge tube, and finally add DEPC H2O to make up to 16 μL.

[0068] (3) Mix the above components thoroughly and centrifuge. Place the mixture in a PCR instrument to perform the first step of reverse transcription. The reverse transcription conditions are: 42℃, 2 min.

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

[0070] 1.4 Quantitative Real-Time PCR Reaction

[0071] (1) Place the reverse transcribed cDNA, 2×TB Green Premix Ex Taq (Tli RNaseH Plus), PCR upstream primer, PCR downstream primer, and DEPC H2O on ice. Prepare the PCR reaction system according to the following system.

[0072] Real-time 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 H2O]]> 4μL

[0074] (2) After the above reagents are mixed, the test is performed according to the following procedure.

[0075] RT-qPCR amplification system

[0076]

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

[0078] 1.5 Protein Immunoblotting (Western blot)

[0079] (1) Extraction of cell proteins

[0080] ① Remove the cells to be extracted from 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 off with a cell scraper, transfer them to a 1.5 mL EP tube, and centrifuge at 4°C and 5000 rpm for 5 min.

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

[0083] ④ After lysis, centrifuge at 12,000 rpm for 20 min in a centrifuge at 4℃, and transfer the supernatant to a new 1.5 mL EP tube.

[0084] ⑤ After centrifugation, BCA protein quantification was performed. BCA working solution was prepared at a ratio of solution A:solution B = 50:1. 18 μL of PBS, 2 μL of protein solution, and 200 μL of BCA working solution were added to each well of a 96-well plate, and the plate was incubated at 37°C in the dark for 30 minutes.

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

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

[0087] (2) SDS-PAGE gel preparation

[0088] ① Rinse the glass bonding plate with clean water first, then scrub it with dish soap, and finally rinse off the foam with distilled water before placing it in a 37℃ constant temperature oven to dry.

[0089] ② Prepare the appropriate concentration of separating gel according to the size of the strip, pour it into the clamped glass gel preparation plate and add isopropanol to remove air bubbles, and wait for 20-30 minutes.

[0090] ③ After the separating gel has completely solidified, discard the upper layer of isopropanol and invert it onto filter paper to absorb the isopropanol.

[0091] ④ Prepare the upper layer of concentrated adhesive. After adding it to the glass adhesive mixing plate, quickly insert the washed comb and let it stand for 20-30 minutes. Once it has solidified, it is ready for use.

[0092] (3) Protein electrophoresis

[0093] ① Prepare 1× electrophoresis solution by distilling 10× electrophoresis solution with single-distilled water.

[0094] ② Place the solidified gel in the electrophoresis tank, pour in the electrophoresis buffer, and remove the comb. After ensuring that no electrophoresis buffer is missed, load the protein sample. The protein amount is 30 μg.

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

[0096] (4) Transfer membrane

[0097] ① Prepare a 1× transfer solution from 10× transfer solution, and completely saturate the filter paper with the transfer solution.

[0098] ② Take a pre-cut PVDF membrane, place it in methanol and 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 bar, cut off the upper layer of gel, and place the lower layer of gel into the transfer solution.

[0100] ④ Place the filter paper, PVDF membrane, gel, and filter paper in the semi-dry transfer apparatus from bottom to top, and use rollers to remove air bubbles. Then close the lid of the transfer apparatus. Maintain a constant voltage of 18V and transfer the membrane for 2 hours. After that, collect the membrane and seal it in 5% skim milk powder.

[0101] (5) Antibody incubation and exposure

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

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

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

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

[0106] ⑤ Discard the secondary antibody after blocking, and wash three times with PBST for 10 minutes each time.

[0107] ⑥ Place the PVDF membrane in a chemiluminescence imaging instrument, drop the prepared chemiluminescence liquid onto the PVDF membrane, shake it evenly, expose it for color development, and save the results.

[0108] 1.6TCID 50 experiment

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

[0110] (2) Dilute the virus solution serially 10-fold with DMEM complete medium, and inoculate one row of 8 replicates for each dilution. 100 μL / well and incubate at 37°C.

[0111] (3) 36–48 hours after cell infection, observe the number of cytopathic effects under a microscope and calculate the TCID of the virus 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 allow it to return to room temperature.

[0114] (2) After it returns to room temperature, take out the pre-coated strips required for the experiment from the sealed bag.

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

[0116] (4) 20 minutes before the next step, dilute the 30× biotinylated antibody with diluent to make a 1× working solution.

[0117] (5) Take the microplate out of the 37°C constant temperature incubator, discard the liquid, tap it on absorbent paper 5-6 times, add 200μL of diluted washing solution per well, let it stand at room temperature for 1 minute, discard the liquid, continue to tap it on absorbent paper 5-6 times, and repeat five times.

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

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

[0120] (8) Take the microplate out of the 37°C constant temperature incubator, discard the liquid, tap it on the absorbent paper 5-6 times, add 200μL of diluted washing solution per well, let it stand at room temperature for 1 minute, discard the liquid, continue to tap it on the absorbent paper 5-6 times, and repeat five times.

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

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

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

[0124] (12) After the difference is obvious, add 100 μL of reaction stop solution to each well, mix well, and measure the value at OD 450 using an ELISA reader within 3 min.

[0125] (13) Based on the measured OD values, draw a standard curve according to the instructions and calculate the sample concentration.

[0126] 1.8PI staining

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

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

[0129] (3) Dilute PI (1000×) 1000 times with detection buffer, i.e., add PI (1000×) to 1 mL of detection buffer (protect 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 1mL of detection working solution to each well, and incubate at 37℃ in the dark for 30min.

[0131] (5) After the incubation in the dark, observe the staining effect under a fluorescence microscope and take pictures to record it. The entire experimental process requires operation in the dark.

[0132] 1.9LDH determination

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

[0134] (2) Discard the cell culture medium and infect the cells with VSV (MOI=1). Incubate in an incubator for 8 hours and collect the supernatant.

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

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

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

[0138] Example 2: VSV infection does not induce pyroptosis in mouse peritoneal macrophages.

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

[0140] Western blot analysis was performed on GSDMD cleavage in mouse peritoneal macrophages at 0, 4, 8 and 12 h after VSV infection.

[0141] VSV infection of mouse peritoneal macrophages at 0 and 8 h was followed by PI staining to detect cell death.

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

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

[0144] Analysis results as follows Figure 1 As shown, VSV infection does not cause GSDMD to cleave and generate P30 fragments; VSV infection does not increase PI. + Cellular 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 indicate that VSV infection did not induce pyroptosis in mouse peritoneal macrophages.

[0146] Example 3: Knocking out Gsdmd to inhibit the 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. - / - Mouse peritoneal macrophages were harvested at 0, 4, and 8 h after VSV infection, and RNA was extracted. The mRNA levels of Ifnb1 and IL-6 were detected by RT-qPCR. At 0 and 8 h after VSV infection, the culture supernatant was harvested, and the protein levels of IFN-β and IL-6 were detected by ELISA. At 0, 4, and 8 h after VSV infection, cells were harvested, proteins were extracted, and the overall and phosphorylation levels of RIG-I protein, TBK1, IRF3, p65, and IκBα were detected by Western blot.

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

[0149] The results of this embodiment indicate that knocking out Gsdmd inhibits the activation of RIG-I-mediated antiviral immune responses 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 from mice were harvested at 0, 4, and 8 hours after VSV infection. RNA was extracted, and the mRNA level of VSVG, the gene encoding the VSV viral membrane protein, was detected by RT-qPCR. Wild-type and Gsdmd mice were isolated and cultured. - / - Mouse peritoneal macrophages were infected with VSV 8 hours prior, and the cell culture supernatant was harvested. TCID10 was then added. 50 The VSV titer was tested experimentally.

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

[0153] The results of this embodiment indicate that Gsdmd knockout promotes VSV replication in mouse peritoneal macrophages.

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

[0155] VSV infection was induced in domesticated WT mice with a genetic background of C57BL / 6J and Gsdmd. - / - At 0, 4, and 8 hours after inoculation with mouse iBMM cell lines, 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 were isolated and cultured. - / - Mouse iBMM cells were harvested 8 hours after VSV infection, and the cell culture supernatant was collected. TCID10 was then added. 50Experimental detection of VSV titer. iBMM cells were infected with VSV-GFP for 8 hours. GFP was observed in the cells using an inverted fluorescence microscope, and GFP was detected by flow cytometry. + Cell content.

[0156] Analysis results as follows Figure 4 As shown, compared with the WT group, VSV infection with Gsdmd - / - The transcriptional level of VSVG in the VSV group was significantly increased; compared with the WT group, VSV infection of Gsdmd... - / - VSV load was significantly increased in the group; compared with the WT group, after VSV-GFP infection of cells, Gsdmd - / - The group had a higher GFP content.

[0157] The results of this embodiment indicate that Gsdmd knockout promotes VSV replication in mouse iBMM cells.

[0158] Example 6: Knocking out Gsdmd to inhibit the transcription of Ifnb1 and other substances induced by poly(I:C) stimulation of mouse primary macrophages.

[0159] Isolate and culture WT mice and Gsdmd mice with a genetic background of C57BL / 6J. - / - Mouse peritoneal macrophages were transfected with poly(I:C) using Lipo2000. Cells were harvested at 0, 4, and 8 h post-transfection, and RNA was extracted. The mRNA levels of Ifnb1, Il6, Ifit2, and Ip10 were detected by RT-qPCR.

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

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

[0162] Example 7: Knockout of Gsdmd in mice exacerbates VSV load in various organs.

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

[0164] VSVG levels in lung, liver, and spleen tissues, as follows Figure 6 As shown, the viral load in lung tissue, liver tissue, spleen tissue, and serum is as follows: Figure 7 As shown.

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

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

[0167] Example 8: Knocking out Gsdmd in mice increases the mortality rate caused by VSV infection.

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

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

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

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

[0172] The following plasmids were constructed: GSDMD-D276A mutant plasmid, GSDMD-C truncated plasmid, GSDMD truncated plasmids (amino acids 1-138, 139-276, 1-69, and 70-138). The RIG-I plasmid was co-transfected into HEK293T cells with the above plasmids. After 24 h of transfection, the protein was extracted, and the expression of RIG-I was detected by Western blot.

[0173] The relevant plasmid construction methods are as follows:

[0174] Construction of the full-length GSDMD plasmid (pcDNA3.1-GSDMD-Flag plasmid): RNA was extracted from mouse peritoneal macrophages and reverse transcribed into cDNA. Using this cDNA as a template, it was constructed into the eukaryotic expression plasmid pcDNA3.1-Flag through two restriction enzyme sites, EcoRI and BamHI.

[0175] Construction of the GSDMD-D276A mutant plasmid (pcDNA3.1-GSDMD-D276A-Flag plasmid): The plasmid was constructed using a point mutation kit with pcDNA3.1-GSDMD-Flag plasmid as a template.

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

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

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

[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 Figure 9 As shown, the GSDMD-D276A mutant is stable for RIG-I, while the GSDMD-C truncated mutant is not. The 1-138 amino acid truncated mutant of GSDMD is stable for RIG-I, while the 139-276 amino acid truncated mutant of GSDMD is not. The 1-69 amino acid truncated mutant of GSDMD is stable for RIG-I, while the 70-138 amino acid truncated mutant of GSDMD is not.

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

[0182] Example 10: GSDMD's peptide 10 interacts strongly with STUB1.

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

[0184] The results are as follows Figure 10 As shown, among the 12 peptides of GSDMD, pull-down assays revealed that peptides 6, 7, and 10 interact strongly with STUB1 protein; ELISA assays showed that peptide 10 binds most strongly to STUB1 among peptides 6, 7, and 10.

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

[0186] Example 11: Peptide 10 of the GSDMD protein is most effective in stabilizing RIG-I and inhibiting VSV replication.

[0187] To facilitate the intracellular entry of GSDMD peptides, we conjugated screened peptides 6, 7, and 10 with a transmembrane peptide (CPP) to resynthesize biotin-labeled CPP-conjugated GSDMD peptides 6, 7, and 10. Simultaneously, we synthesized biotin-labeled GSDMD peptide 2 and a 2×Flag (DYKDDDDKDYKDDDDK) peptide as negative controls. First, we assessed the effects of the relevant peptides on cell viability. After setting a series of dose ranges (0 μM, 2.5 μM, 5 μM, 10 μM) of biotin-labeled CPP-conjugated GSDMD, we evaluated the safety of these dose ranges in mouse peritoneal macrophages using PI staining. Immunofluorescence assays were then used to assess whether safe doses of the peptides could enter mouse peritoneal macrophages.

[0188] The results are as follows Figure 11 As shown, the relevant peptide had no effect on cell viability after treatment with 5 μM for 12 h. Furthermore, the 5 μM peptide was able to enter the cells.

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

[0190] The results are as follows Figure 12 As shown, RT-qPCR results showed that the VSVG content was lowest in the peptide 10 treatment group; 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 and 2×Flag groups, with the peptide 10 group showing the highest expression.

[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 h, followed by VSV (MOI=1) infection for 8 h. The mRNA level of VSVG was detected by RT-qPCR, and the expression of RIG-I protein in cells was detected by Western blot.

[0192] The results are as follows Figure 13 As shown, RT-qPCR results indicated that the VSVG content was lowest in the peptide 10 treatment group; 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 and 2×Flag groups, with the peptide 10 group showing the highest expression.

[0193] The results of this embodiment indicate that peptide 10 of the GSDMD protein is the most effective 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, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of GSDMD peptide, DNA encoding GSDMD peptide, and RNA expressing GSDMD peptide in the preparation of medicaments for the prevention and / or treatment of RNA virus infectious diseases, wherein the sequence of GSDMD peptide is SSSRFWKPRYSCVNL, and the RNA virus is VSV.

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

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