Use of h1.2 as target in preparation of drug for enhancing antiviral immunity and antiviral infection

Abstract

The present invention relates to use of H1.2 as a target in the preparation of a drug for enhancing antiviral immunity and antiviral infection. It is proposed that H1.2 is an intranuclear negative regulatory element of cGAS, and the mechanism by which H1.2 regulates cGAS is clarified, providing new theoretical support for the in-depth research and development of cGAS, and also providing a new research target H1.2 for the development of antiviral drugs. The present invention also relates to use of a substance that inhibits the expression of H1.2 during infection in the preparation of a drug for enhancing antiviral immunity and antiviral infection. It was first found that during infection, the body regulates the expression of H1.2 by means of an Sp1-H1.2 pathway, thereby affecting the activity of downstream cGAS. Plicamycin, a small molecule inhibitor of Sp1, can inhibit Sp1, thereby inhibiting the expression of H1.2 during infection, and thus enhancing the antiviral immunity of the body.

Description

Application of H1.2 as a target in preparation of drugs for enhancing antiviral immunity and resisting antiviral infection TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, more specifically, it relates to the application of H1.2 as a target in preparation of drugs for enhancing antiviral immunity and resisting antiviral infection. BACKGROUND

[0002] Viruses have high environmental adaptability and high mutability, and the infectious diseases caused by viruses have brought great threat to human health and global property safety, causing high morbidity and mortality, and causing more than 14 million deaths each year. Viruses can cause various epidemics, and the high mutability and various immune escape mechanisms of viruses enable them to hide in the human body and escape immune surveillance. Existing antiviral drugs mostly focus on special viral protein subunit structures, and have insufficient broad-spectrum, so we focus on the host itself and resist viral infection by improving the antiviral immunity of the host.

[0003] cGAS is an important DNA sensor in cells, which can recognize and bind to free viruses, bacterial dsDNA in the cytoplasm, and the bound cGAS is activated to dimerize under the catalysis of ATP and GTP, forming 2', 3' cyclic GMP-AMP. Interferon activating protein STING is combined and activated in the endoplasmic reticulum. Activated STING dimerizes, recruits and activates molecules such as TBK1, IRF3 and NF-κB, and finally initiates the transcription of downstream cytokines such as IFN and IL6. The interferon response based on the cGAS-STING pathway is one of the important pathways for the body to resist viral infection.

[0004] H1.2 belongs to a subtype of the histone H1 family, and is also the most widely existing subtype in the H1 family, which is a housekeeping gene with strong species conservation. It is involved in DNA damage repair and tumor immunity. Our previous data show that H1.2 and cGAS have a binding effect, and the overexpression of H1.2 can significantly inhibit the interferon response of cells, so H1.2 may be an upstream regulatory element of intracellular cGAS, and has good development potential in the treatment of antiviral immunity. SUMMARY

[0005] In order to study the related mechanism of H1.2 regulating intracellular cGAS and provide a new idea for the treatment of antiviral immunity, the present application provides an application of H1.2 as a target in preparation of drugs for enhancing antiviral immunity and resisting antiviral infection.

[0006] In a first aspect, the present application discloses an application of H1.2 as a target in preparation of drugs for enhancing antiviral immunity and resisting antiviral infection.

[0007] Further, the H1.2 plays a role of enhancing anti-viral immune capacity and anti-viral infection by regulating cGAS.

[0008] In a second aspect, the application discloses an application of a substance inhibiting expression of H1.2 during infection in preparation of a medicine for enhancing anti-viral immune capacity and anti-viral infection.

[0009] Further, the substance inhibiting expression of H1.2 during infection is a Sp1 small molecule inhibitor.

[0010] Further, the substance inhibiting expression of H1.2 during infection is Plicamycin.

[0011] In summary, the application has the following beneficial effects:

[0012] 1. The application first proposes that H1.2 is a nuclear negative regulatory element of cGAS, clarifies the mechanism of H1.2 regulating cGAS, provides new theoretical support for in-depth research and development of cGAS, and provides a new research target H1.2 for development of anti-viral drugs.

[0013] 2. The application first discovers the role and spontaneous regulation mode of the Sp1-H1.2-cGAS axis in innate immunity, proposes that under the condition of viral infection, the body regulates the expression of H1.2 through the Sp1-H1.2 pathway, and then affects the activity of downstream cGAS.

[0014] 3. The application also first discovers that the small molecule inhibitor Plicamycin of Sp1 can inhibit the expression of H1.2 during infection by inhibiting Sp1, thereby enhancing the anti-viral immune capacity of the body. Therefore, Plicamycin can be used as a potential anti-viral drug in clinical treatment, especially in the case of co-infection of clinical tumor patients. BRIEF DESCRIPTION OF DRAWINGS

[0015] Fig. 1: cGAS and H1.2 have interaction;

[0016] (A: Immunoprecipitation results of expressing exogenous target proteins in HEK293T cells; B: GST-Pull down results of purifying two proteins in a prokaryotic system);

[0017] Fig. 2: Deletion of H1.2 can enhance the level of interferon in cells;

[0018] (A: Extract wild type mouse and H1.2 knockout mouse primary peritoneal macrophages, and infect them with HSV-1, detect the mRNA levels of intracellular IFN, isg15 and other cytokines; B: Extract wild type mouse and H1.2 knockout mouse primary peritoneal macrophages, infect them with HSV-1 for a specific time, detect the level of protein phosphorylation of STING, TBK1, IRF3, P65 and other proteins in the cells);

[0019] Figure 3: H1.2 deletion can enhance the body's anti-infection immune response;

[0020] (A: Mouse lung tissue HE staining section, confirming that the deletion of H1.2 can reduce the damage to the lung caused by viral infection; B: H1.2 deletion can reduce lung viral load; C: H1.2 deletion can enhance the expression of IFN in mouse peripheral blood) ;

[0021] Figure 4: Sp1-H1.2 axis is involved in immune regulation during infection;

[0022] (A: After HSV-1 infection, the cell spontaneously down-regulates the transcription level of H1.2; B: Sp1 as an upstream positive transcription factor of H1.2 can significantly promote the expression of H1.2 transcription level; C: Sp1 is involved in antiviral immunity, and Sp1 can significantly inhibit the activation level of STING and other proteins in the cell interferon pathway) ;

[0023] Figure 5: Plicamycin as an anti-infection drug for antiviral immunity;

[0024] (A: The activation of IFN signaling pathway in mouse peritoneal macrophages infected with HSV-1 under Plicamycin treatment; B: The effect of Plicamycin treatment on IFN-β transcription level under infection, and this effect is specific to H1.2; C: Plicamycin treatment can inhibit the replication ability of intracellular HSV-1; D: Plicamycin treatment can reduce the damage of virus to the tissue after infection). DETAILED DESCRIPTION

[0025] The technical solutions and effects of the present application are further described in detail below. It can be understood that the specific embodiments described herein are only used to explain the present application, and not to limit the present application.

[0026] Plicamycin, Flag-beads were purchased from MedChemExpress; WB chemiluminescent substrates were purchased from Vazyme; primary antibody H1.2 was purchased from Proteintech; primary antibody STING, TBK1, P65.IRF3 and their phosphorylation antibodies were purchased from Cell Signaling Technology; primary antibody HA, FLAG, GAPDH were purchased from Abclonal; primary antibody GFAP was purchased from Biolegend.

[0027] Statistical analysis was performed using GraphPad Prism software. The t-test was used to compare two groups, and one-way ANOVA was used to compare multiple groups. P<0.05 was considered statistically significant.

[0028] Example 1: cGAS directly interacts with H1.2

[0029] (1) cGAS interacts with H1.2 in cells (Figure 1-A).

[0030] Sample preparation:

[0031] 1) Transfect the designated plasmid into 70%-80% full 6cm dish HEK293T cells using cationic liposomes, change the liquid 4-6h, and collect the sample after 48h.

[0032] 2) Wash the cells twice with PBS, add 500μL IP lysis buffer, and lyse at 4℃ for 30min.

[0033] 3) 13200r, 4℃ centrifuge for 15min, 400μL supernatant, add Flag-beads for IP, 4℃ rotation overnight; 75μL supernatant as lysate, add 25μL 4x loading buffer, 100℃ deformation for 10min, standby.

[0034] 5) Wash beads: take out the EP tube, 1000r, centrifuge for 5min, IP tube is placed on the magnetic stand, aspirate the liquid, leave the beads, add 1mL 1xPBST, rotate for 5min, then place on the magnetic stand, aspirate the liquid, repeat 3 times, after the last time aspirate the supernatant, add 100μL lxloading(IP lysis buffer diluted 4x), 100℃, heat for 10min.

[0035] Electrophoresis:

[0036] 1) Prepare 10% separating gel (according to the instructions), and suck out the residual gel in the hole before loading.

[0037] 2) Load 3 μL marker protein, 10 μg of sample, and calculate the corresponding volume according to the protein concentration, and use loading buffer to make up the highest volume;

[0038] 3) Electrophoresis: concentrated gel 80 mV, 30 min; separating gel 110-120 mV, 80 min.

[0039] 4) Membrane transfer: cut 4 cm*8 cm PVDF membrane, activate in advance in methanol, install the membrane transfer clamp in the order of black clamp-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-white clamp, 100 V, 1 KD / min, and set the membrane transfer time.

[0040] 5) Blocking: 5% skim milk blocking for 1 h, and TBST (regular membrane washing solution) washing for three times, each for 5 min.

[0041] 6) Incubate the primary antibody: incubate the primary antibody at 4°C overnight, respectively.

[0042] 7) TBST washing for three times, each for 5 min, incubate the secondary antibody (the secondary antibody is the antibody combined with the primary antibody, and has an enzyme that can react with the substrate in the luminescence solution), 60 min, TBST washing for three times, each for 5 min.

[0043] 8) Development.

[0044] (2) cGAS has a binding with H1.2 outside the cell (Figure 1-B).

[0045] 1) The His-tagged and GST-tagged proteins are expanded in 100 mL LB to OD600 = 0.4-0.6, 1000x IPTG (final concentration 1 mM) is added, 30°C 180r induction for 4-6 h, or overnight induction;

[0046] 2) 8000r / 3min collect the bacteria, resuspend the bacteria with lysis buffer (100mL bacteria + 5mL lysis buffer mix well) and ultrasonic crushing (ultrasonic crushing needs to be carried out on ice water mixture); ultrasonic crushing: work for 3s, pause for 3s, total time 30min;

[0047] 3) 4°C, 13200r, 15min centrifugation to take the supernatant, 0.22 / 0.45um filter filtration, and add to new 1.5mL EP;

[0048] 4) His-tag protein: Take the Ni column, release 20% ethanol, rinse according to the order of ddH2O-20mM imidazole rinse-30mM imidazole rinse-filtered lysate-50mM imidazole rinse, add 300mM imidazole eluent to elute His-tag protein, recover 300mM eluent, a total of 5mL, add 1mL imidazole each time, discard the first 1mL and the last 1mL, add 150mM imidazole rinse after elution, and store in ddH2O-20% ethanol;

[0049] 5) GST-tag protein: Prepare 120μL lysate, take 1mL protein solution, add 20μL washed GST-beads, 4℃ small sun incubate for 3h, take out the EP tube and place it on the magnetic stand, aspirate the liquid, add 1mL PBST and rotate for 5min, aspirate the liquid, repeat 3 times. Add 1mL His-tag protein solution, 4℃ rotate overnight, PBST clean 3 times the next day, configure 100μL sample.

[0050] 6) Electrophoresis.

[0051] 7) Development.

[0052] Example 2: Deletion of H1.2 can enhance the level of cell interferon

[0053] (1) H1.2 deletion can enhance the mRNA level of IFN, ISG15 in cells after infection (Figure 2-A).

[0054] 1) Take the cells: ① Three days before the experiment, inject 1mL of 3% thioacetate broth into the abdominal cavity of the mouse to obtain more number of macrophages, sterilize the metal instruments such as ophthalmic scissors, ophthalmic forceps, etc. by autoclaving, irradiate the centrifuge tube for disinfection, and the mouse is fasted for 8h. Before the experiment, sterilize the ultraclean workbench with ultraviolet light for 30min. ② Kill the mouse by cutting off the neck, immerse the whole mouse in 70% alcohol for 3-5 seconds, lie on the stainless steel tray, inject normal saline or cell culture medium into the abdominal cavity, rub the mouse's abdomen several times, and let the liquid flow in the abdominal cavity for 3-5min. ③ Dissect and expose the peritoneum, use forceps to lift the lower abdominal skin and separate it from the peritoneum, cut a small opening in the forceps extraction site, then cut the entire abdominal skin, pinch the skin on both sides and slowly tear it apart, exposing the peritoneum. Extract the abdominal fluid, pinch the mouse's skin on one side and squeeze the internal organs to create a cavity in the abdominal cavity, insert the needle of the syringe into the gap to extract the abdominal fluid, and then inject the abdominal fluid into the centrifuge tube. ④ Centrifuge the collected abdominal lavage fluid at 1000r / min for 10min, discard the supernatant, resuspend with cell culture medium and count the cells. After adjusting the cell concentration, inoculate into cell 6-well plates, incubate in a 37℃ incubator for 4-6h, discard the supernatant after the macrophages are fully adherent, replace with fresh cell culture medium, and continue to culture in the incubator.

[0055] 2) Infection: Replace pure culture medium to starve cells before infection, add HSV-1 at MOI=10 for specific time.

[0056] 3) Collect sample: Add 1 mL TRIZOL (all EP tubes, washing, etc. are RNAase free), mix well by pipetting, and transfer to a new EP tube.

[0057] 4) Add 200 μL chloroform, cover the tube cap, shake vigorously for 15 seconds, and stand at room temperature for 5 minutes. Centrifuge at 12000 r for 15 minutes. The sample will be divided into three layers. Take 400 μL of the upper aqueous phase, add 400 μL of isopropanol, stand at room temperature for 10 minutes, centrifuge at 10000 r for 10 minutes, and discard the supernatant.

[0058] 5) Wash the RNA precipitate with 75% ethanol, centrifuge at 10000 r for 5 minutes, discard the supernatant, and repeat 3 times.

[0059] 6) Stand at room temperature to dry the RNA precipitate. Add an appropriate amount of RNase-free water, and dissolve the RNA by pipetting several times with a gun head. Detect the RNA concentration and purity.

[0060] 7) Reverse transcription: Use the HiScript IV RT SuperMix for qPCR (R423-01) kit from Vazyme Company, and follow the kit instructions to reverse transcribe into cDNA.

[0061] 8) qPCR: Follow the system of 0.2 μL upstream and downstream primers + 1 μL cDNA + 3.6 μL ddH2O + 5 μL 2X sybr, use ChamQ Blue Universal SYBR qPCR Master Mix from Vazyme Company, and use Roche 480 for data export.

[0062] (2) H1.2 deletion can enhance the activation of type I interferon pathway after infection (Figure 2-B).

[0063] 1) Take the cells: ①Three days before the experiment, inject 1 mL of 3% thioacetate broth into the mouse's abdominal cavity to obtain more macrophages. Sterilize metal instruments such as ophthalmic scissors, ophthalmic forceps, etc. with high-pressure sterilization. Centrifuge tubes are sterilized by irradiation. The mice are fasted for 8 hours. The ultraclean workbench is sterilized with ultraviolet light for 30 minutes before the experiment. ②Euthanize the mouse by cutting off the neck, immerse the whole mouse in 70% alcohol for 3-5 seconds, and place it on a stainless steel tray on its back. Inject normal saline or cell culture medium into the abdominal cavity, gently rub the mouse's abdomen several times, and let it stand for 3-5 minutes to allow the liquid to flow freely in the abdominal cavity. ③Dissect and expose the peritoneum, use forceps to lift the lower abdominal skin and separate it from the peritoneum. Cut a small opening in the skin at the point where the forceps are extracted, then cut the entire abdominal skin. Hold the skin on both sides and slowly tear it apart to expose the peritoneum. Extract the peritoneal fluid by pinching the mouse's skin on one side and squeezing the internal organs. Create a cavity in the abdominal cavity by pinching the mouse's skin on one side and squeezing the internal organs. Insert the needle of a syringe into the gap to extract the peritoneal fluid. Then inject the peritoneal fluid into a centrifuge tube. ④Centrifuge the collected peritoneal lavage fluid at 1000 r / min for 10 minutes, discard the supernatant, resuspend with cell culture medium and count the cells. Adjust the cell concentration and inoculate into a cell 6-well plate. Incubate in a 37°C incubator for 4-6 hours. After the macrophages are fully adhered, discard the supernatant, replace it with new cell culture medium, and continue incubation in the incubator.

[0064] 2) Infection: Replace the pure culture medium before infection, and add HSV-1 at a dose of MOI = 10 for a specific time.

[0065] 3) Sample collection: Add 200 μL of cell lysis solution (Biyun Tian, P0013, add PMSF, cocktail, NaF and sodium metavanadate to inhibit the degradation of phosphorylated proteins), lyse at 4°C for 40 minutes, blow and transfer to a new ep tube, 13200 r, 4°C centrifuge for 15 minutes, take 150 μL of supernatant, add loading, cook the sample at 100°C for 10 minutes.

[0066] 4) Electrophoresis: Prepare 10% separation gel (according to the instructions), and suck out the residual gel in the hole before loading; load 3 μL of marker protein, 10 μg of sample, calculate the corresponding volume according to the protein concentration, and use loading buffer to make up the highest volume; concentrate gel 80 mV, 30 min; separation gel 110-120 mV, 80 min.

[0067] 5) Membrane transfer: Cut 4 cm * 8 cm PVDF membrane and activate it in methanol in advance. Install the membrane transfer clamp in the order of black clamp-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-white clamp, 100V, 1KD / min, set the membrane transfer time.

[0068] 6) Blocking: 5% skimmed milk blocking for 1 hour, TBST (regular membrane washing solution) washing three times, each for 5 minutes.

[0069] 7) Incubate primary antibody: incubate primary antibody at 4°C overnight.

[0070] 8) Wash with TBST for 3 times, 5 min each time, incubate secondary antibody (secondary antibody is the antibody combined with primary antibody, which has an enzyme that can react with the substrate in the luminescence solution), 60 min, wash with TBST for 3 times, 5 min each time.

[0071] 9) Develop.

[0072] Example 3: Deletion of H1.2 can enhance the body's immune response to infection

[0073] (1) Deletion of H1.2 can reduce the damage caused by viral infection to the lung (Figure 3-A).

[0074] 1) Sampling: 6-8w wild type male mice and H1.2 knockout mice were injected intraperitoneally with HSV-1 at a dose of PFU 10 8 / each, and sacrificed by decapitation 48 hours later. The right lung tissue was taken for sectioning and fixed with a fixing solution.

[0075] 2) Wash the fixing solution with PBS, add hematoxylin dropwise, and control the staining time according to the tissue condition and dye condition (generally about 5 min, as the standing time of hematoxylin increases, the effect of hematoxylin staining will increase, and the time should be appropriately shortened).

[0076] 3) Wash with water until the tissue turns blue-purple, add 1% hydrochloric acid ethanol dropwise and differentiate for 2s, and wait for the tissue to turn red.

[0077] 4) Wash the hydrochloric acid ethanol with water until the tissue turns blue, and then stain with eosin. Control the staining time according to the tissue condition and dye condition (generally about 2s-8s), wash the dye with water, and cover the tissue with glycerol. Confirm the staining condition under a microscope. If the staining is good, proceed to mounting.

[0078] 5) Observe the section under an optical microscope.

[0079] (2) Deletion of H1.2 can reduce the titer of virus in the infected tissue (Figure 3-B).

[0080] 1) Sampling: 6-8w wild type male mice and H1.2 knockout mice were injected intraperitoneally with HSV-1 at a dose of PFU 10 8 / each, and sacrificed by decapitation 48 hours later. The right lung tissue was taken for sectioning and fixed with a fixing solution.

[0081] 2) Prepare the vero cells in advance, filter the virus solution obtained in 1) with a 0.22μL filter, and dilute it by a factor of 10.

[0082] 3) Absorb the medium in the culture plate, add the diluted virus liquid in 2), 8 duplicate wells for each gradient. Leave a row of wells to add new medium as a control.

[0083] 4) After 5 days of culture in the cell incubator, observe the number of wells with lesions in each gradient under the microscope, and calculate the virus titer according to the TCID50 method.

[0084] (3) Deletion of H1.2 can enhance the expression of IFN in the peripheral blood of mice (Figure 3-C).

[0085] 1) Blood was collected from wild-type mice or knockout mice (6-8 weeks old, male) 48 hours after injection of HSV-1.

[0086] 2) Sample collection: Anesthetize the mouse, which can be done by using anesthetics for general anesthesia; fix the mouse, before blood collection, use the left thumb and index finger to hold the skin between the two ears of the mouse and fix it, gently press the two sides of the neck, make the orbital venous sinus congest, and the eyeball protrudes outward; blood collection operation, hold the blood collection tube (or capillary tube) with the right hand, insert the tip between the inner corner of the eye and the eyeball, gently stab towards the throat, stop when resistance is felt, rotate the blood collection tube to cut the venous sinus, and blood will flow into the blood collection tube; hemostasis and treatment, after blood collection, remove the blood collection tube, relax the left hand, and use a dry cotton ball to stop bleeding; mice weighing 20-30 g can be collected 0.2-0.3 mL of blood at a time.

[0087] 3) The blood sample was left at room temperature for 30 min, centrifuged at 4000 r for 10 min at room temperature, and the upper serum was transferred to a new ep tube.

[0088] 4) The Elisa assay used an enzyme-linked biological mouse IFN-β kit (mL063095), and the operation and data processing were performed according to the instructions.

[0089] Example 4: Sp1-H1.2 axis is involved in immune regulation during infection

[0090] (1) During infection, cells spontaneously down-regulate the expression of H1.2 transcription level (Figure 4-A).

[0091] 1) Take the cells: ①Three days before the experiment, inject 1 mL of 3% thioacetate broth into the mouse's abdominal cavity to obtain more macrophages. Sterilize metal instruments such as ophthalmic scissors, ophthalmic forceps, etc. with high pressure. Centrifuge tubes are irradiated and disinfected. The mice are fasted for 8 hours, and the ultraclean workbench is disinfected with ultraviolet light for 30 minutes before the experiment. ②Euthanize the mouse by cutting off its neck, immerse the whole mouse in 70% alcohol for 3-5 seconds, and place it on a stainless steel tray on its back. Inject normal saline or cell culture medium into the abdominal cavity, rub the mouse's abdomen several times, and let it stand for 3-5 minutes to allow the liquid to flow freely in the abdominal cavity. ③Dissect and expose the peritoneum, use forceps to lift the lower abdominal skin and separate it from the peritoneum. Cut a small opening in the skin at the point where the forceps are extracted, then cut the entire abdominal skin. Hold the skin on both sides and slowly tear it apart to expose the peritoneum. Extract the peritoneal fluid by pinching the mouse's skin on one side and squeezing the internal organs. Create a cavity in the abdominal cavity, and insert a syringe needle into the gap to extract the peritoneal fluid. Then inject the peritoneal fluid into a centrifuge tube. ④Centrifuge the collected peritoneal lavage fluid at 1000 r / min for 10 minutes, discard the supernatant, resuspend with cell culture medium, and count the cells. Adjust the cell concentration and inoculate into a cell 6-well plate. Incubate in a 37°C incubator for 4-6 hours. After the macrophages are fully adhered, discard the supernatant, replace it with new cell culture medium, and continue incubation in the incubator.

[0092] 2) Infection: Replace the pure culture medium before infection, and add HSV-1 at a dose of MOI = 10 for a specific time.

[0093] 3) Sample collection: Add 1 mL of TRIZOL (all EP tubes, washing heads, etc. are RNAase free), mix well by blowing, and transfer to a new EP tube.

[0094] 4) Add 200 μL of chloroform, cover the tube cap, shake vigorously for 15 seconds, and let it stand at room temperature for 5 minutes. Centrifuge at 12000 r for 15 minutes. The sample will be divided into three layers. Take 400 μL of the upper aqueous phase, add 400 μL of isopropanol, let it stand at room temperature for 10 minutes, centrifuge at 10000 r for 10 minutes, and discard the supernatant.

[0095] 5) Wash the RNA precipitate with 75% ethanol, centrifuge at 10000 r for 5 minutes, discard the supernatant, and repeat 3 times.

[0096] 6) Let the RNA precipitate dry at room temperature, add an appropriate amount of RNase-free water, and dissolve the RNA by sucking and beating a few times with a gun head. Detect the RNA concentration and purity.

[0097] 7) Reverse transcription: Use the HiScript IV RT SuperMix for qPCR (R423-01) kit from Vazyme Company, and follow the instructions to reverse transcribe into cDNA.

[0098] 8) qPCR: 0.2 μL of upper and lower primers + 1 μL of cDNA + 3.6 μL of ddH2O + 5 μL of 2X sybr were loaded, the reagent was ChamQ Blue Universal SYBR qPCR Master Mix of Vazyme Company, the instrument was Roche 480, and the data was exported.

[0099] (2) Sp1 affects the transcription level of H1.2 during infection (Figure 4-B).

[0100] 1) Transfection: L929 cells were transfected with exogenous Sp1 or control by cationic liposome, the liquid was changed after 4-6 hours, the cells were starved after 36 hours, and then infected.

[0101] 2) Sample collection: 1 mL of TRIZOL was added (all EP tubes, washing heads, etc. were RNAase free), and then transferred to a new EP tube after mixing.

[0102] 3) Reverse transcription: the reagent was HiScript IV RT SuperMix for qPCR (R423-01) kit of Vazyme Company, and the cDNA was reversely transcribed according to the kit instructions.

[0103] 4) qPCR: 0.2 μL of upper and lower primers + 1 μL of cDNA + 3.6 μL of ddH2O + 5 μL of 2X sybr were loaded, the reagent was ChamQ Blue Universal SYBR qPCR Master Mix of Vazyme Company, the instrument was Roche 480, and the data was exported.

[0104] (3) Sp1 participates in the antiviral immunity of cells under viral infection (Figure 4-C).

[0105] 1) Transfection: L929 cells were transfected with exogenous Sp1 or control by cationic liposome, the liquid was changed after 4-6 hours. The cells were starved after 36 hours, and then infected.

[0106] 2) Sample collection: 200 μL of cell lysis solution (Biyun Tian, P0013, PMSF, cocktail, NaF and sodium metavanadate were added to inhibit the degradation of phosphorylated proteins) was added, 4-degree lysis for 40 minutes, and then transferred to a new EP tube by blowing, 13200 r, 4-degree centrifugation for 15 minutes. 150 μL of supernatant was taken, loading was added, and the sample was boiled at 100°C for 10 minutes.

[0107] 3) Electrophoresis.

[0108] 4) Development.

[0109] Example 5: Plicamycin as an anti-infective drug for use in antiviral immunity

[0110] (1) Activation of IFN signaling pathway upon infection with HSV-1 in the presence of Plicamycin (Figure 5-A).

[0111] 1) Cell collection: ① Three days before the experiment, inject 1 mL of 3% thioglycolate broth into the abdominal cavity of the mice to obtain a greater number of macrophages, sterilize metal instruments such as ophthalmic scissors, ophthalmic forceps, etc. by autoclaving, and sterilize the centrifuge tubes by irradiation. The mice were fasted for 8 h, and the ultraclean bench was sterilized by ultraviolet light for 30 min before the experiment. ② Euthanize the mice by decapitation, immerse the whole mouse in 70% alcohol for 3-5 seconds, and place it on a stainless steel tray on its back. Inject normal saline or cell culture medium into the abdominal cavity, rub the mouse's abdomen several times, and let it stand for 3-5 min to allow the liquid to flow freely in the abdominal cavity. ③ Dissect and expose the peritoneum, use forceps to lift the lower abdominal skin, and separate it from the peritoneum. Cut a small opening in the skin at the point where the forceps are lifted, and then cut the entire abdominal skin. Gently tear the skin on both sides to expose the peritoneum. Extract the abdominal fluid by pinching one side of the mouse's skin and squeezing the internal organs, creating a cavity in the abdominal cavity. Insert the needle of a syringe into the gap to extract the abdominal fluid, and then inject it into a centrifuge tube. ④ Centrifuge the collected peritoneal lavage fluid at 1000 r / min for 10 min, discard the supernatant, resuspend in cell culture medium, and count the cells. After adjusting the cell concentration, inoculate the cells into a 6-well plate and incubate in a 37°C incubator for 4-6 h. After the macrophages have fully adhered, discard the supernatant, replace it with fresh cell culture medium, and continue incubation in the incubator.

[0112] 2) Treat the cells with DMSO or Plicamycin (100 nM), and 12 hours later, add HSV-1 for infection.

[0113] 3) Sample collection: Add 200 μL of cell lysis solution (Bi Yun Tian, P0013, with PMSF, cocktail, NaF, and sodium metavanadate to inhibit the degradation of phosphorylated proteins), lyse at 4°C for 40 min, blow and transfer to a new ep tube, centrifuge at 13200 r for 15 min at 4°C. Take 150 μL of supernatant, add loading, and boil the sample at 100°C for 10 min.

[0114] 4) Electrophoresis.

[0115] 5) Development.

[0116] (2) Plicamycin promotes the transcription level of IFN-β in the infected state by affecting H1.2 (Figures 5-B, C).

[0117] 1) Take cells: ①Three days before the experiment, inject 1 mL of 3% thioacetate broth into the mouse's abdominal cavity to obtain more macrophages. Sterilize metal instruments such as ophthalmic scissors, ophthalmic forceps, etc. with high pressure, and irradiate the centrifuge tube for disinfection. The mice were fasted for 8 hours, and the ultraclean workbench was disinfected with ultraviolet light for 30 minutes before the experiment. ②The mouse was sacrificed by cutting off the neck, and the whole mouse was immersed in 70% alcohol for 3-5 seconds, then placed on a stainless steel tray on its back, and injected with normal saline or cell culture medium into the abdominal cavity. Rub the mouse's abdomen several times, and let it stand for 3-5 minutes to allow the liquid to flow freely in the abdominal cavity. ③Dissect and expose the peritoneum, use forceps to lift the lower abdominal skin and separate it from the peritoneum. Cut a small opening in the skin at the point where the forceps are extracted, then cut the entire abdominal skin. Hold the skin on both sides and slowly tear it apart to expose the peritoneum. Extract the peritoneal fluid by pinching the mouse's skin on one side and squeezing the internal organs to create a cavity in the abdominal cavity. Insert the needle of a syringe into the gap to extract the peritoneal fluid, then inject it into a centrifuge tube. ④Centrifuge the collected peritoneal lavage fluid at 1000 r / min for 10 minutes, discard the supernatant, resuspend with cell culture medium and count the cells. Adjust the cell concentration and inoculate into a cell 6-well plate, incubate in a 37°C incubator for 4-6 hours. After the macrophages are fully adhered, discard the supernatant, replace it with new cell culture medium, and continue to culture in the incubator.

[0118] 2) DMSO or Plicamycin (100 nM) treated cells, 12 hours later, HSV-1 was added for infection.

[0119] 3) Sample collection: Add 200 μL cell lysis solution (Biyun Tian, P0013, add PMSF, cocktail, NaF and sodium metavanadate to inhibit degradation of phosphorylated proteins), lyse at 4°C for 40 minutes, blow and transfer to a new ep tube, 13200 r, 4°C centrifuge for 15 minutes. Take 150 μL supernatant, add loading, boil the sample at 100°C for 10 minutes.

[0120] 4) Reverse transcription: Use the HiScript IV RT SuperMix for qPCR (R423-01) kit from Vazyme Company, follow the kit instructions to reverse transcribe into cDNA.

[0121] 5) qPCR: Follow the system of 0.2 μL upstream and downstream primers + 1 μL cDNA + 3.6 μL ddH2O + 5 μL 2X sybr, use ChamQ Blue Universal SYBR qPCR Master Mix from Vazyme Company, use Roche 480 instrument, and export data.

[0122] (3) Plicamycin treatment of mice after infection can reduce the damage of virus to tissues (Figure 5-D).

[0123] 1)Material: 6w wild type male mice were infected with HSV-1 (PFU 10 8 / each) or PBS for 24 hours, then treated with Plicamycin or control (0.2mg / kg, physiological saline dilution, intraperitoneal injection). 24 hours later, the right lung tissue was taken out, sectioned and fixed.

[0124] 2) PBS was used to wash the fixative, then hematoxylin was added for staining, and the staining time was controlled according to the tissue and dye (generally about 5 minutes, the staining effect of hematoxylin will be enhanced with the increase of the standing time of hematoxylin, and the time should be appropriately shortened).

[0125] 3) Water was used to wash until the tissue was blue-violet, then 1% hydrochloric acid ethanol was added for differentiation for 2 seconds, and the tissue was red.

[0126] 4) Water was used to wash the hydrochloric acid ethanol until the tissue was blue, then eosin was added for staining, and the staining time was controlled according to the tissue and dye (generally about 2-8 seconds). The dye was washed with water, and the tissue was covered with glycerol, and the staining condition was confirmed under a microscope. If the staining was good, the tissue was sealed.

[0127] 5) The section was observed under an optical microscope.

[0128] The specific embodiment is only an explanation of the application, and is not a limitation of the application. Those skilled in the art can make modifications to the embodiment without creative contribution after reading the specification, and the application is protected by the patent law as long as it is within the scope of the claims.

Claims

1. Application of H1.2 as a target in the preparation of drugs that enhance antiviral immunity and antiviral infection.

2. The application according to claim 1, characterized in that, H1.2 enhances antiviral immunity and antiviral infection by regulating cGAS.

3. Application of substances that inhibit H1.2 expression during infection in the preparation of drugs that enhance antiviral immunity and antiviral infection.

4. The application according to claim 3, characterized in that, The substance that inhibits the expression of H1.2 during infection is a small molecule inhibitor of Sp1.

5. The application according to claim 4, characterized in that, The substance that inhibits the expression of H1.2 during infection is Plicamycin.

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