Application of peroxisome accelerant in resisting EV71 virus infection

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of a peroxisome accelerant in resisting EV71 virus infection. By researching the interaction between EV71 and peroxisome in a host cell, it is found that EV71 virus can induce autophagy of peroxisome, and autophagy of peroxisome has a promoting effect on replication of EV71 virus. Meanwhile, the EV71 infection is found to cause the increase of ROS by changing the position of catalase; both the calase and the ROS inhibitor can effectively inhibit the replication of the EV71 virus and reduce the autophagy of peroxisome in cells. Research results prove that the peroxisome plays an important role in resisting EV71 virus replication and infection, and a clue is provided for developing a new treatment strategy aiming at the EV71 virus.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an application of a peroxisome promoter in resisting EV71 virus infection. Background Art

[0002] Enterovirus 71 (EV71) is a member of the RNA virus family and the enterovirus genus, and is one of the main pathogens that cause hand, foot and mouth disease. EV71 is common in infants and young children under the age of 5, and infection may lead to serious neurological complications or even death. In addition, EV71 can also cause serious central nervous system diseases such as aseptic meningitis, encephalitis, poliomyelitis-like paralysis, cardiopulmonary failure, liver failure, neurogenic pulmonary edema, etc. At present, people lack understanding of the infection mechanism of EV71, and there is also a lack of corresponding effective prevention and treatment methods for hand, foot and mouth disease caused by EV71.

[0003] Some existing technologies have studied drugs or vaccines against EV71 virus. For example, patent CN112138032A discloses the use of Pien Tze Huang and its preparations in the preparation of drugs for the prevention and treatment of enterovirus EV71 infection. Its research shows that Pien Tze Huang significantly inhibits cytopathic effect, inhibits viral replication, and reduces the viral particle load during enterovirus EV71 infection, and has strong anti-EV71 virus activity. For another example, patent CN113082028B discloses the use of an Alpelisib compound in the preparation of anti-EV71 virus drugs. Its research found that BYL719 has anti-EV71 virus activity, which is specifically manifested in that it can inhibit the cytopathic effect caused by EV71 virus, enhance the survival rate of infected cells, inhibit the replication and proliferation of EV71 virus in cells, and reduce the yield of progeny viruses.

[0004] Peroxisomes are important organelles involved in cellular lipid metabolism, ROS production, etc., and play a key role in the replication of many viruses, but their association with EV71 replication is still unclear. Summary of the invention

[0005] In view of this, the present invention has conducted an in-depth study on the interaction between EV71 and peroxisomes in host cells and found that peroxisomes play an important role in EV71 virus replication and infection, and peroxisomes are a new target for resisting EV71.

[0006] One of the purposes of the present invention is to provide a peroxisome promoter for use in the preparation of drugs for resisting EV71 virus infection.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] Application of peroxisome promoter in the preparation of drugs for resisting EV71 virus infection.

[0009] Preferably, the peroxisome promoter includes any one or more of an autophagy inhibitor, a ROS inhibitor, and a H2O2 enzyme.

[0010] Preferably, the ROS inhibitor comprises N-Acetylcysteine.

[0011] Preferably, the autophagy inhibitor includes any one or more of chloroquine and 3-methyladenine.

[0012] The second object of the present invention is to provide a peroxisome promoter for use in the preparation of a drug for treating hand, foot and mouth disease caused by EV71 virus.

[0013] To achieve the above object, the present invention adopts the following technical solutions:

[0014] Application of a peroxisome promoter in the preparation of a drug for treating hand, foot and mouth disease caused by EV71 virus.

[0015] Preferably, the peroxisome promoter includes any one or more of an autophagy inhibitor, a ROS inhibitor, and a H2O2 enzyme.

[0016] Preferably, the ROS inhibitor comprises N-Acetylcysteine.

[0017] Preferably, the autophagy inhibitor includes any one or more of chloroquine and 3-methyladenine.

[0018] The third object of the present invention is to provide a peroxisome promoter for use in the preparation of a drug for inhibiting EV71 virus replication.

[0019] To achieve the above object, the present invention adopts the following technical solutions:

[0020] Application of peroxisome promoter in the preparation of medicine for inhibiting EV71 virus replication.

[0021] Preferably, the peroxisome promoter includes any one or more of an autophagy inhibitor, a ROS inhibitor, and a H2O2 enzyme.

[0022] Preferably, the ROS inhibitor comprises N-Acetylcysteine.

[0023] Preferably, the autophagy inhibitor includes any one or more of chloroquine and 3-methyladenine.

[0024] A fourth object of the present invention is to provide a peroxisome promoter for use in the preparation of a drug for inhibiting intracellular peroxisomal autophagy and / or inhibiting the transfer of H2O2 enzymes from peroxisomes to cytoplasm.

[0025] To achieve the above object, the present invention adopts the following technical solutions:

[0026] The invention discloses a use of a peroxisome promoter in the preparation of a drug for inhibiting intracellular peroxisome autophagy and / or inhibiting the transfer of H2O2 enzyme from peroxisome to cytoplasm.

[0027] Preferably, the cells include RD cells and / or 293T cells.

[0028] Preferably, the peroxisome promoter includes any one or more of an autophagy inhibitor, a ROS inhibitor, and a H2O2 enzyme.

[0029] Preferably, the ROS inhibitor comprises N-Acetylcysteine.

[0030] Preferably, the autophagy inhibitor includes any one or more of chloroquine and 3-methyladenine.

[0031] A fifth object of the present invention is to provide a peroxisome promoter for use in the preparation of a drug for promoting the expression of peroxisome membrane proteins, inhibiting the expression of EV71 viral structural proteins and / or inhibiting the expression of autophagy-related proteins.

[0032] To achieve the above object, the present invention adopts the following technical solutions:

[0033] The invention discloses a peroxisome promoter for preparing a drug for promoting the expression of peroxisome membrane proteins, inhibiting the expression of EV71 virus structural proteins and / or inhibiting the expression of autophagy-related proteins.

[0034] Preferably, the peroxisomal membrane protein includes PEX14, the autophagy-related protein includes LC3, and the EV71 virus structural protein includes VP1.

[0035] Preferably, the peroxisome promoter includes any one or more of an autophagy inhibitor, a ROS inhibitor, and a H2O2 enzyme.

[0036] Preferably, the ROS inhibitor comprises N-Acetylcysteine.

[0037] Preferably, the autophagy inhibitor includes any one or more of chloroquine and 3-methyladenine.

[0038] The beneficial effects of the present invention are:

[0039] 1. The present invention has conducted an in-depth study on the interaction between EV71 virus and peroxisome, and found that there is a significant interaction between EV71 and peroxisome, and peroxisome is expected to become a new target for resisting EV71. The present invention clarifies the importance of peroxisome in EV71 replication and reveals new mechanisms of viral infection, replication or release, providing valuable clues for the development of new antiviral strategies against EV71.

[0040] 2. The present study found that EV71 infection has a profound effect on the morphology and number of peroxisomes in cells, and that peroxisomes play a key role in the progression of viral infection, cellular metabolism, and pathological development.

[0041] 3. The present study confirms that EV71 virus can induce peroxisome autophagy, and peroxisome autophagy promotes the replication of EV71 virus.

[0042] 4. The present study confirmed that EV71 infection leads to an increase in ROS, and the mechanism is achieved by changing the location of catalase rather than the amount. In addition, catalase and ROS inhibitors can effectively inhibit the replication of EV71 virus and reduce intracellular peroxisomal autophagy. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The images were taken under a 4x microscope 24 hours after adding different virus concentrations of EV71 virus (0MOI, 0.5MOI, 1MOI, 2MOI) to RD cells; Figure 1 -A is a picture taken under a 4x microscope 24 hours after RD cells were added with 0 MOI EV71 virus; Figure 1 -B is a picture taken under a 4x microscope 24 hours after RD cells were added with 0.5MOI EV71 virus; Figure 1 -C is a picture taken under a 4x microscope 24 hours after RD cells were added with 1MOI EV71 virus; Figure 1 -D is a picture taken under a 4x microscope 24 hours after RD cells were added with 2MOI EV71 virus.

[0044] Figure 2 The RD cells were photographed under a 4x microscope after adding the same virus concentration of EV71 virus (1MOI) 0 hours, 8 hours, 16 hours and 24 hours later; Figure 2 -A is a picture taken under a 4x microscope 0 hours after RD cells were added with 1MOI EV71 virus; Figure 2 -B is a picture taken under a 4x microscope 8 hours after RD cells were added with 1MOI EV71 virus; Figure 2 -C is a picture taken under a 4x microscope 16 hours after RD cells were added with 1MOI EV71 virus; Figure 2 -D is a picture taken under a 4x microscope 24 hours after RD cells were added with 1MOI EV71 virus.

[0045] Figure 3 The graph shows the changes in protein expression of PEX14 and β-actin in RD cells 24 hours after the addition of EV71 virus at different virus concentrations (0MOI, 0.5MOI, 1MOI, 1.5MOI, 2MOI, and 2.5MOI).

[0046] Figure 4 The morphology and partial magnification of peroxisomes after 0 and 24 hours of adding EV71 virus (1MOI = 0.137μL of original concentration of EV71 virus) to RD cells, where: Figure 4 -A is the morphology of peroxisomes in RD cells after EV71 virus was added 0 hours later. Figure 4 -B is a partial magnified image of the peroxisome morphology in RD cells 0 hours after the addition of EV71 virus. Figure 4 -C is the morphology of peroxisomes in RD cells after adding EV71 virus for 24 hours. Figure 4 -D is a partial magnified image of the peroxisome morphology in RD cells 24 hours after the addition of EV71 virus.

[0047] Figure 5 Graph showing the percentage of cells with elongated morphology within 48 hours after addition of EV71 virus (1 MOI = 0.137 μL of original concentration of EV71 virus).

[0048] Figure 6 This is a statistical chart of the number of peroxisomes in each cell within 48 hours after the addition of EV71 virus (1 MOI = 0.137 μL of original concentration of EV71 virus).

[0049] Figure 7 This graph shows the changes in the expression levels of PEX14, LC3, VP1 and ACTIN proteins within 48 hours after the addition of EV71 virus (1 MOI = 0.137 μL of original concentration of EV71 virus) to RD cells.

[0050] Figure 8 This graph shows the changes in the expression levels of PEX14, LC3, VP1 and ACTIN proteins after adding different doses of EV71 virus (0, 0.5MOI, 1MOI, 1.5MOI, 2MOI, 2.5MOI) into RD cells for 24 hours.

[0051] Fig. 9This graph shows the changes in the expression levels of PEX14, LC3, VP1 and ACTIN proteins within 48 hours after the addition of EV71 virus (1 MOI = 0.137 μL of original concentration of EV71 virus) into 293T cells.

[0052] Fig.10 This graph shows the changes in the expression levels of PEX14, LC3, VP1 and ACTIN proteins after adding different doses of EV71 virus (0, 0.5MOI, 1MOI, 1.5MOI, 2MOI, 2.5MOI) into 293T cells for 24 hours.

[0053] Fig.11 This graph shows the changes in the expression levels of PEX14, LC3, VP1 and ACTIN proteins 24 hours after the addition of EV71 virus at a dose of 1.5 MOI and 20 μmol of Chloroquine into RD cells.

[0054] Fig.12 This graph shows the changes in the expression levels of PEX14, LC3, VP1 and ACTIN proteins in RD cells 24 hours after the addition of EV71 virus at a dose of 1.5 MOI and 100 μmol of 3-Methyladenine.

[0055] Fig.13 This graph shows the changes in the expression levels of PEX14, LC3, VP1 and ACTIN proteins in RD cells 24 hours after the addition of EV71 virus at a dose of 1.5 MOI and 100 μmol of Rapamycin.

[0056] Fig.14 This graph shows the changes in the expression levels of PEX14, LC3, VP1 and ACTIN proteins after 24 hours of adding EV71 virus at a dose of 1.5 MOI and 20 μmol of Chloroquine to 293T cells.

[0057] Fig.15 This graph shows the changes in the expression levels of PEX14, LC3, VP1 and ACTIN proteins after 24 hours of adding EV71 virus at a dose of 1.5 MOI and 100 μmol of 3-Methyladenine into 293T cells.

[0058] Fig.16 This graph shows the changes in the expression levels of PEX14, LC3, VP1 and ACTIN proteins in 293T cells after the addition of EV71 virus at a dose of 1.5 MOI and 100 μmol of Rapamycin for 24 hours.

[0059] Fig.17The fluorescence images of Hyper (hydrogen peroxide fluorescent probe) after adding EV71 virus (1MOI=0.137μL original concentration of EV71 virus) to RD cells at 0 and 24 hours, where: Fig.17 -A is the fluorescence image of Hyper after adding EV71 virus to RD cells for 0 hours. Fig.17 -B is the fluorescence image of Hyper 24 hours after adding EV71 virus to RD cells.

[0060] Fig.18 The graph shows the ratio response of EV71 virus (1MOI=0.137 μL original concentration of EV71 virus) added to RD cells at two wavelengths of 400 / 480 nm in different time periods within 48 hours.

[0061] Fig.19 This is a graph showing the protein expression levels of CATALASE and internal control (ACTIN) within 24 hours after EV71 virus (1 MOI = 0.137 μL of original concentration of EV71 virus) was added to RD cells.

[0062] Fig. 20 This is a graph showing the changes in the expression levels of PEX14, LC3, VP1 and ACTIN proteins 24 hours after the same dose of EV71 virus (1MOI=0.137μL of original concentration of EV71 virus), CATALASE and N-Acetylcysteine ​​were added to RD cells.

[0063] Fig.21 The EV71 virus (1MOI = 0.137 μL original concentration of EV71 virus) was added to RD cells. After 0 hours, the immunofluorescence images of DAPI / CATALASE / PEX14 and the merged images of the three and the local magnified images are shown. Fig.21 -A is the merge image of DAPI / CATALASE / PEX14 after EV71 virus was added to RD cells 0 hours later. Fig.21 -B is the immunofluorescence image of DAPI after EV71 virus was added to RD cells for 0 hours. Fig.21 -C is the immunofluorescence image of CATALASE after EV71 virus was added to RD cells 0 hours later. Fig.21 -D is the immunofluorescence image of PEX14 in RD cells 0 hours after the addition of EV71 virus. Fig.21 -E is Fig.21 -A partial enlarged view; Fig.21 -F is Fig.21 -A partial enlarged view of B; Fig.21 -G is Fig.21 -Partial enlarged view of C; Fig.21 -H is Fig.21 -A partial enlarged view of D.

[0064] Fig. 22 The EV71 virus (1MOI = 0.137 μL original concentration of EV71 virus) was added to RD cells. After 24 hours, the immunofluorescence image of DAPI / CATALASE / PEX14 and the merge image of the three and the local magnification image were obtained. Fig. 22 -A is the merge image of DAPI / CATALASE / PEX14 after EV71 virus was added to RD cells for 24 hours. Fig. 22 -B is the immunofluorescence image of DAPI after EV71 virus was added to RD cells for 24 hours. Fig. 22 -C is the immunofluorescence image of CATALASE after 24 hours of adding EV71 virus into RD cells. Fig. 22 -D is the immunofluorescence image of PEX14 in RD cells 24 hours after the addition of EV71 virus. Fig. 22 -E is Fig. 22 -A partial enlarged view, Fig. 22 -F is Fig. 22 -A partial enlarged view of B, Fig. 22 -G is Fig. 22 -A partial enlarged view of C. Fig. 22 -H is Fig. 22 -A partial enlarged view of D.

[0065] Fig.23 This graph shows the copy number of EV71 virus 24 hours after adding the same dose of EV71 virus (1 MOI = 0.137 μL of original concentration of EV71 virus), CATALASE and N-Acetylcysteine ​​to RD cells.

[0066] Fig.24 This is a graph showing the logarithmic function of EV71 virus TCID50 24 hours after the addition of the same dose of EV71 virus (1 MOI = 0.137 μL of original concentration of EV71 virus), CATALASE and N-Acetylcysteine ​​to RD cells. DETAILED DESCRIPTION

[0067] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0068] In the embodiment of the present invention, RD cells are derived from muscle (rhabdomyosarcoma); their morphology is spindle-shaped cells and large multinucleated cells, growing on the wall, with a small amount suspended. The cell type is tumor cell; the tumor type is sarcoma cell.

[0069] In the embodiment of the present invention, PEX14 is a peroxisomal membrane protein, LC3 is an autophagy-related protein, VP1 is an EV71 virus structural protein, and ACTIN is an internal reference protein.

[0070] In the examples of the present invention, 3-Methyladenine (3-methyladenine), Chloroquine (chloroquine), Rapamycin (rapamycin) and N-Acetylcysteine ​​were purchased from MCE (medchemexpress.cn), the item number of 3-Methyladenine is HY-19312, the item number of Chloroquine is HY-17589A, the item number of Rapamycin is HY-10219, and the item number of N-Acetylcysteine ​​is HY-B0215. PEX14 antibody, LC3 antibody, VP1 antibody and ACTIN antibody were all purchased from Abcam (https: / / www.abcam.cn / ), the item number of PEX14 antibody is ab183885, the item number of LC3 antibody is epr18709, the item number of VP1 antibody is HL1929, and the item number of ACTIN antibody is ab6276.

[0071] In the present example, EV71 TW-2231 strain (EV712231) (C2 subtype) was used to infect cells. RD (human rhabdomyosarcoma) cells and 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 1% non-essential amino acids, 1% L-glutamine, and 1% penicillin / streptomycin (all from Thermo-Fisher Scientific, Waltham, MA, USA).

[0072] In the embodiment of the present invention, the cell culture operation steps are as follows:

[0073] (1) Cell recovery

[0074] 1) Prepare complete culture medium: basic culture medium (MEM) + fetal bovine serum FBS (10%) + double anti-PS (1%).

[0075] 2) Recovery of RD and HEK293 cells: Take 5 mL of complete culture medium into a 15 mL centrifuge tube and preheat it in a 37°C water bath. Quickly take out the frozen RD and HEK293 cells from the liquid nitrogen tube, put them into a 37°C water bath, shake them to thaw quickly (about 1 min), then move the thawed cells and preheated culture medium into an ultra-clean workbench, add the thawed cells into a 15 mL centrifuge tube containing preheated culture medium, and centrifuge at 1000 rpm for 5 min.

[0076] 3) Aspirate and discard the supernatant to obtain the cell pellet, gently resuspend the cells in 2 mL of complete culture medium, add the cells to a culture dish, mark it, and culture it in a 37°C, 5% CO2 saturated incubator.

[0077] 4) After 24 hours, observe the adhesion of RD and HEK293 cells (those not attached are dead cells - for adherent cells), discard the old culture medium, add fresh preheated (room temperature or 37°C) complete culture medium, and continue culturing.

[0078] (2) Cell passaging

[0079] 1) When RD and HEK293 cells grow to 80%-90% confluence, discard the old culture medium, add 1mL sterile PBS to rinse once to remove the residual culture medium and serum (serum contains inhibitors of trypsin), then add 1mL 0.25% trypsin, and digest in a 37℃ incubator (about 1-2min, different cells have different digestion times). Gently flush with trypsin, if blank spots are observed, digestion is complete.

[0080] 2) Add 1 mL of complete culture medium (containing FBS) to stop digestion, tap gently to make the cells fall off into a single cell suspension, collect the cells in a 15 mL sterile centrifuge tube, and centrifuge at 1000 rpm for 5 min.

[0081] 3) Collect the cell pellet, resuspend it in complete culture medium, divide it into two (the ratio can be adjusted according to the cell growth rate), add them into two new culture dishes respectively, mark them, and culture them in an incubator.

[0082] (3) Cell cryopreservation

[0083] 1) According to the cell passaging method, digest and collect the cell pellet in an ultra-clean workbench and take a small amount of cells for counting.

[0084] 2) Resuspend the cells in 1 mL of pre-cooled freezing solution (90% complete medium + 10% DMSO) or serum-free cell freezing solution and add to a 1.2 mL cryotube at a density of 1*10 6 Pieces / mL.

[0085] 3) Place the sample in a cryopreservation box and store at -80°C overnight, then transfer to liquid nitrogen for long-term storage.

[0086] In the embodiment of the present invention, the western blot experiment is as follows:

[0087] (1) Protein extraction

[0088] 1) Preparation: Turn on the ice maker, turn on the low-temperature centrifuge (low temperature prevents protein denaturation), cell scraper, pointed printed tubes, 50mL centrifuge tubes (with BCA working solution), various guns and gun tips.

[0089] 2) Prepare lysis buffer, PBS (for external use), PMSF, SDS, and BCA working solution (solution A, solution B, protein standard).

[0090] 3) Take out RD and HEK293 cells, discard the supernatant, and wash with PBS.

[0091] 4) Add 1 mL of PBS (for easy scraping of cells) and scrape off the cells with a cell scraper and pipette them into a pre-cooled pointed tube.

[0092] 5) Centrifuge at 3000 rpm for 8 min, and carefully discard the supernatant (cells are at the bottom).

[0093] 6) Add 500uL of lysis buffer and 5uL of PMSF. Slightly shake and centrifuge at 14000rpm for 8min. Aspirate the supernatant (protein extract) into a new EP tube.

[0094] 7) Aspirate 2 μL of protein extract to detect protein concentration (3 replicate wells x 3 = 6 μL), add 5Xsds to the remaining protein extract, the amount added is protein extract ÷ 4 (480 ÷ 4 = 120 μL), mix with the pipette tip and place in a metal bath (100 degrees) for 10 minutes.

[0095] 8) Store in a -80 degree refrigerator.

[0096] (2) Concentration measurement

[0097] 1) In step 6 of protein extraction, centrifuge at 14000rpm for 8min, prepare BCA working solution (200μL per well, A:B=50:1). For example, if there are 6 wells of protein standards and 6 wells of protein samples (2 different concentrations), then prepare: 200x12=2400μL of BCA. However, in order to facilitate calculation and prevent BCA from being insufficient, prepare 3000μL (3mL) of BCA. Therefore, take 3000μL of A and 60μL of B and add them to a 50mL centrifuge tube.

[0098] 2) Add the standard samples at 0, 1, 2, 4, 8, 12, 16, and 20 to the standard wells of the 96-well plate, and add standard diluent to make up to 200 μL.

[0099] 3) Add 200 μL of BCA working solution to each well and place at 37°C for 30 min or directly place in a microplate reader for measurement.

[0100] 4) Calculate the concentration of the protein sample based on the standard curve and dilute it to a certain concentration so that the loading amount is the same, which can ensure that the total amount of protein in each well is the same when running the gel.

[0101] (3) Glue preparation

[0102] 1) Assemble the glass plates. Take one 1mm thick glass plate and one thin glass plate. Align the glass plates and put them into the rack. Clamp the two sides tightly, with the short glass on the outside. (To facilitate glue addition)

[0103] 2) Leak detection: take 1mL of anhydrous ethanol and put it between the two glass plates to observe whether there is leakage and whether the liquid surface is level. If it is level, remove the anhydrous ethanol.

[0104] 3) Preparation and addition of separation gel. Select the separation gel concentration according to the molecular weight of the target protein and follow the instructions (TEMED is toxic, so add it last and outdoors. Cover the lid after adding, invert and mix, and immediately draw up the gel with a large gun tip. (5 mL of separation gel, 2 mL of concentrated gel)

[0105] 4) Take 1mL of anhydrous ethanol and slowly add it to the separation gel. Let it stand at room temperature for 25-30 minutes. After the gel solidifies, take up the entire support and pour out the anhydrous ethanol. (1. If bubbles are generated when adding the separation gel, anhydrous ethanol will eliminate the bubbles. 2. Use filter paper to wash off the residual ethanol.)

[0106] 5) While waiting for the separation gel to solidify, you can prepare the concentrated gel. Do not add TEMED (to promote coagulation) first. After the separation gel solidifies, add TEMED and quickly add it between the glass plates. (Be careful not to hold the concentrated gel bottle with your hands)

[0107] 6) Gently insert the comb vertically with the written side facing you, and let it stand at room temperature for 20-30 minutes until the concentrated gel solidifies.

[0108] 7) Prepare electrophoresis buffer and transfer buffer: 14.4 g glycine, 3.03 g Tris (add pure water to the electrophoresis buffer to make up to 1000 mL, then add 1 g SDS. Add pure water to the transfer buffer to make up to 800 mL, then add 200 mL of methanol to 1000 mL).

[0109] (4) Electrophoresis

[0110] 1) Take out the protein sample from the -80℃ refrigerator and place it on an ice box. Take out the marker from -20℃ and dissolve it. Put the prepared gel into the inner tank of electrophoresis, pour in the new electrophoresis solution, and place the inner tank in the outer tank. (The short glass plate is placed inside, and the long glass plate is placed outside. Pay attention to check for leakage. If there is leakage, add electrophoresis solution in the outer tank until it is flush with the inner tank) (If only one gel is run, the other side needs to be leveled).

[0111] 2) Hold the comb at both ends with both hands and lift it up vertically, then add the sample according to the calculated amount. (Usually add 4μL of marker for 60-80 molecular weight, if the gel is not balanced, add 8-10μL of buffer) (Mix the sample before adding the sample / you can leave it for a few seconds, and wipe the tip of the gun with a paper towel every time you add the sample)

[0112] 3) Add electrophoresis solution (reusable) to the outer tank until the first black line from the bottom of the box (two pieces of gel), with black electrodes facing black and red electrodes facing red. Run the concentrated gel first at 80V for 30 minutes. (The current of one piece of gel is about 20mA). If there are bubbles in the electrophoresis solution in the inner tank, turn on the power.)

[0113] 4) Run the separation gel and adjust the voltage directly to 120V for 1H. After running, add the electrophoresis solution in the inner and outer tanks into the recovery bottle.

[0114] (Next time you run the glue, you can add it to the outer tank)

[0115] (5) Transfer

[0116] 1) Pour the transfer solution into an iron plate, take out the glass plate from the electrophoresis tank, rinse it with water, use a small board to separate the gel from the thick and thin plates, let the gel fall onto the thin glass plate, and soak it in the transfer solution.

[0117] 2) Take out a flat filter paper and place it on the table, put the thin glass plate with glue on the paper, and cut the glue with a ruler. Soak the cut glue in the transfer solution.

[0118] 3) Place the sandwich on an iron plate and soak it in the transfer solution. Cut the corresponding membrane according to the size of the gel, activate the membrane in methanol (which can be reused) for one minute, and then place it in the transfer solution. If it floats, the activation is successful.

[0119] 4) Place the sandwich in the order of (sponge, filter paper, glue, membrane, filter paper, sponge), with the white one on the bottom and the black one on the top, i.e. the membrane is on the white side and the glue is on the black side. (Note that there should be no bubbles between the membrane and the glue)

[0120] 5) Place the sandwich on the transfer tank and find another sandwich to balance it. (Red to white, black to black) 250mA, time: 1KD / MIN.

[0121] (6) Antibody application

[0122] 1) Preparation of blocking solution: TBST + 5% milk powder (20 mL TBST corresponds to 1 g milk powder) 5 mL per strip, add to the corresponding well in the incubation box. (2X TBS and ultrapure water are mixed 1=1 to make 1X TBS, 90 mL 1X TBS and 70 mL Twen-20 are made into TBST, milk powder is placed in a -20℃ refrigerator, and 1 membrane is equipped with 4-5 mL blocking solution)

[0123] 2) Blocking: Take out the transferred membrane, wash it in TBST first, then put it into the blocking solution with the protein side facing up, and incubate it at 37°C, 54 rpm, on a shaker for 1 hour.

[0124] 3) Apply 1 antibody, dilute the antibody and diluent according to the ratio, the target molecule and the internal reference each have their own antibody and diluent. Wash the blocked membrane in TBST first, then add the membrane and antibody, and place in a 4℃ refrigerator overnight.

[0125] 4) Apply secondary antibody, antibody: TBST = 1:10000, rabbit-derived. Incubate at 54 rpm for 1 hour. After incubation, wash three times with TBST, 10 minutes each time.

[0126] 5) Prepare the developer, A:B=1:1, and expose.

[0127] In the embodiment of the present invention, the immunofluorescence test is as follows:

[0128] (1) Principle

[0129] Since the fluorescence emitted by fluorescein can be detected under a fluorescence microscope, fluorescein emits bright fluorescence when irradiated with excitation light, the cells or tissues where the fluorescence is located can be seen, and the content can be determined using quantitative technology, thereby performing cellular qualitative and localization analysis of the antigen.

[0130] (2) Steps

[0131] Preparation before the experiment: prepare 10% blocking solution; 0.2% Triton X-100.

[0132] Day 1:

[0133] 1) Take a clean cover glass and soak it in 75% ethanol, blow it dry in a sterile clean bench, place the cover glass in a 24-well plate, and inoculate it with cell culture medium overnight to make it about 30%-60% full.

[0134] 2) After treating the cells according to the specific experimental purpose, aspirate the culture medium, wash twice with 0.5 mL PBS, add 0.25 mL fixative, and fix at room temperature for 15 minutes.

[0135] 3) Remove the fixative solution and rinse with PBS three times, 5 minutes each time, with gentle shaking on a shaker.

[0136] 4) Permeabilize with 0.2% TritonX-100 at room temperature for 5 min, rinse with PBS 3 times, 5 min each time, and shake gently on a shaker.

[0137] 5) Block with blocking solution (0.5 mL / well) for 60 min.

[0138] 6) Aspirate the blocking solution (do not wash), apply primary antibody (0.25 mL / well) for 60 min or overnight at 4°C, and shake gently on a shaker.

[0139] the next day:

[0140] 7) Remove the primary antibody and rinse with PBS three times, 5 minutes each time, with gentle shaking on a shaker.

[0141] 8) Discard PBS, add 0.25 mL / well of diluted fluorescent secondary antibody, incubate at room temperature in the dark for 40 min, and shake gently on a shaker.

[0142] 9) Recover the fluorescent secondary antibody, rinse with PBS three times, 5 min each time, and shake gently on a shaker.

[0143] 10) Add DAPI (50 μL / well) and incubate in the dark for 5 min. Rinse with PBS 4 times, 5 min each time, and wash away excess DAPI.

[0144] 11) Use filter paper to absorb the liquid on the slide, drop 1 drop of anti-fluorescence quenching sealing solution on the slide, cover it with the cover slip with cells, avoid bubbles, make the cells contact with the sealing solution, and observe the green fluorescence under a fluorescence microscope.

[0145] Note:

[0146] 1. It is recommended that cells be directly seeded into well plates and photographed using an inverted fluorescence microscope. This can avoid damage to the slices or cell slices during the final selection process and possible slippage during the final sealing process.

[0147] 2. Determine whether to add Triton for permeabilization based on the location of the antigen being detected. If the antigen epitope being detected is located in the outer white segment of the membrane protein, permeabilization is not required.

[0148] 3. Starting from adding fluorescent secondary antibody, try to avoid light in all subsequent steps.

[0149] 4. Shorten the observation time under the fluorescence microscope to 1-2 hours.

[0150] 5. Observe under a fluorescence microscope as soon as possible after staining. If this is not possible, store in a 4°C refrigerator away from light for one week.

[0151] 6. Too dark staining: the primary antibody concentration is too high; the staining test concentration is too high or the incubation time is too long; the staining agent concentration is too high or the incubation time is too long.

[0152] 7. When choosing an aldehyde fixative, keep it fresh. It is best to prepare it and use it immediately. Using an aldehyde fixative that is not fresh will increase the autofluorescence background.

[0153] Frequently asked questions

[0154] Weak or no signal: 1) Cell or tissue samples have been stored for too long; 2) The antibody concentration is not appropriate. Refer to the dilution concentration in the antibody manual and then explore according to the expression level of the sample; 3) The primary antibody incubation time is not appropriate. Overnight incubation at 4°C is recommended.

[0155] Example 1. Effects of EV71 virus infection on peroxisome morphology and number

[0156] In order to study the effect of EV71 virus infection on the morphology and quantity of peroxisomes, the present invention added EV71 virus (1MOI=0.137μL of original concentration of EV71 virus) to RD cells, and observed the changes in the morphology and quantity of peroxisomes after EV71 infection of cells using an electron microscope. Figure 1 to Figure 3 As shown in the figure, after adding different concentrations of EV71 virus (0MOI, 0.5MOI, 1MOI, 2MOI), 24 hours later, as the concentration of EV71 virus increased, the number of cytopathic changes in RD cells increased; and the peroxisomal membrane protein (PEX14) also decreased, proving that the peroxisomes in the cells were degraded. Figures 4 to 6 As shown in the figure, after EV71 virus infected RD cells, the morphology and number of intracellular peroxisomes changed significantly. After the virus invaded the cells, the peroxisome morphology gradually changed from the original regular round to an irregular elongated shape, and as the infection time prolonged, the number of peroxisomes in this irregular elongated shape continued to increase. At the same time, the overall number of peroxisomes began to steadily decline since the early stage of infection, and the longer the infection time, the more significant the reduction trend. This series of changes shows that EV71 infection has a profound impact on the morphology and number of intracellular peroxisomes, and is likely to play a key role in the process of viral infection, cell metabolism, and pathological development.

[0157] Example 2. Effects of virus dose and infection time on peroxisomes, cell autophagy and virus replication

[0158] This example further explores the effects of virus dose and infection time on peroxisomes, cell autophagy and virus replication. Figure 7 to Figure 10As shown in the figure, in the process of studying EV71 virus infection of cells, it was found that under the same virus dose conditions, within 48 hours, the expression of PEX14 protein gradually decreased, proving that the number of intracellular peroxisomes showed a gradually decreasing trend, while the expression of LC3 protein gradually increased, proving that the level of cell autophagy continued to rise, and the expression of VP1 protein gradually increased, proving that the amount of virus replication increased significantly. Moreover, within the same time frame, with the continuous increase in virus dose, the expression of PEX14 protein gradually decreased, the expression of LC3 protein gradually increased, and the expression of VP1 protein gradually increased, that is, the number of intracellular peroxisomes decreased, cell autophagy was enhanced, and the replication of EV71 virus increased. This reveals that there is a close connection between virus dose and infection time and related physiological processes and virus replication in cells.

[0159] Example 3. Association of autophagy with EV71 virus replication and peroxisomes

[0160] In order to further explore the intrinsic relationship between cellular autophagy, EV71 virus replication and peroxisomes, this experiment introduced autophagy inhibitors (chloroquine and 3-methyladenine) and autophagy activators (rapamycin). Figures 11 to 16 As shown in the figure, after adding the autophagy inhibitor, the expression of the viral structural protein VP1 was significantly reduced, the expression of LC3 decreased, that is, the level of cellular autophagy decreased significantly, and the expression of PEX14 protein increased, that is, the number of peroxisomes increased significantly. In contrast, after adding the autophagy activator, the expression of viral structural proteins increased significantly, the cellular autophagy activity was enhanced, and the number of peroxisomes decreased significantly. This series of experimental results further confirmed that the EV71 virus can induce peroxisomal autophagy, and peroxisomal autophagy promotes the replication of the EV71 virus.

[0161] Example 4. Effects of EV71 infection on ROS and related mechanisms

[0162] The effects of EV71 infection on ROS and related mechanisms were further explored. Figures 17 to 24As shown. After the addition of EV71 virus, the number of hyper was observed to increase significantly, and the 400 / 480 nm ratio response was enhanced, which strongly proved that the content of ROS (H2O2) in the cell system increased accordingly. Further studies found that the expression of catalase protein did not change, proving that the number of H2O2 enzymes did not change, but its position was significantly transferred from the peroxisome to the cytoplasm. When catalase (H2O2 enzyme) and N-Acetylcysteine ​​(ROS inhibitor) were further added, the replication level of EV71 was significantly reduced (VP1 expression decreased), the degree of cell autophagy also decreased (LC3 expression decreased), and the number of peroxisomes increased (PEX14 expression increased). These results show that EV71 infection leads to an increase in ROS (H2O2), and its mechanism is achieved by changing the location of catalase rather than the amount. At the same time, catalase and ROS inhibitors can effectively inhibit the replication of EV71 virus and reduce peroxisomal autophagy in cells.

Claims

1. Application of peroxisome promoters in the preparation of drugs against EV71 virus infection.

2. The use according to claim 1, characterized in that: The peroxisome promoter includes any one or more of an autophagy inhibitor, a ROS inhibitor, and a H2O2 enzyme.

3. The use according to claim 2, characterized in that: The ROS inhibitors include N-Acetylcysteine.

4. The use according to claim 2, characterized in that: The autophagy inhibitor includes any one or more of chloroquine and 3-methyladenine.

5. Use of the peroxisome promoter described in claim 1 in the preparation of a drug for treating hand, foot and mouth disease caused by EV71 virus.

6. Use of the peroxisome promoter described in claim 1 in the preparation of a drug for inhibiting EV71 virus replication.

7. Use of the peroxisome promoter according to claim 1 in the preparation of a drug for inhibiting intracellular peroxisomal autophagy and / or inhibiting the transfer of H2O2 enzyme from peroxisomes to cytoplasm.

8. The use according to claim 7, characterized in that: The cells include RD cells and / or 293T cells.

9. Use of the peroxisome promoter described in claim 1 in the preparation of a drug for promoting the expression of peroxisome membrane proteins, inhibiting the expression of EV71 virus structural proteins and / or inhibiting the expression of autophagy-related proteins.

10. The use according to claim 9, characterized in that: The peroxisomal membrane protein includes PEX14, the autophagy-related protein includes LC3, and the EV71 virus structural protein includes VP1.

Images