Application of alspiramycin and / or imiquimod in preparation of medicine for treating VACV

Abstract

The invention relates to the technical field of medicines, in particular to application of aspiramycin and / or imiquimod in preparation of a medicine for treating VACV. Aspiramycin and imiquimod which are screened from medicines on the market have antiviral activity, and experimental data show that the Aspiramycin and the imiquimod have anti-VACV medicine activity. Aspiramycin and imiquimod (especially combined application) are used for preparing the medicine for treating VACV, so that the time and cost of preclinical research can be greatly reduced. As the action mechanism of the pox viruses is not unique to the VACV, the combined strategy has the potential to be expanded to infection treatment of other pox viruses (such as monkey pox virus MPXV) and even other DNA (Deoxyribose Nucleic Acid) viruses.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the use of aspirin and / or imiquimod in the preparation of medicaments for the treatment of VAV. Background Technology

[0003] Tecovirimat is currently the first-line antiviral drug for orthopoxvirus, working by inhibiting viral envelope formation. While effective, resistant strains have been reported, necessitating the search for alternative treatments. Cidofovir is a broad-spectrum antiviral drug effective against VAV, but its strong nephrotoxicity limits its clinical application.

[0004] Therefore, there is an urgent need in this field to find a new drug for the treatment of VACV. Summary of the Invention

[0005] The purpose of this invention is to provide the use of aspirin and / or imiquimod in the preparation of a medicament for the treatment of VACV.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The use of aspirin and / or imiquimod in the preparation of medicaments for the treatment of VACV.

[0008] Among them, the IC50 of the aspirin at the cellular level against VACV virus 50 The concentration ranged from 7.423 μM to 18.32 μM.

[0009] Among them, the imiquimod has an IC50 activity against VACV virus at the cellular level. 50 The concentration ranged from 305.6 μM to 308.1 μM.

[0010] Compared with the prior art, the beneficial effects of the present invention are:

[0011] The present invention screened aspirin and imiquimod from marketed drugs and found them to have antiviral activity. Furthermore, experimental data showed that aspirin (an ancient anti-syphilis arsenic agent) and imiquimod (an immunomodulator used to treat genital warts and skin cancer) have anti-VACV pharmacological activity.

[0012] The preparation of aspirin and imiquimod (especially in combination) for the treatment of VAV has the following significant advantages: (1) Aspirin may act directly on certain key proteins of the virus or host cells through its arsenic-containing structure, inhibiting the early replication stage of the virus (such as gene expression). Imiquimod, as a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, activates the host's natural immune system (such as stimulating the production of cytokines such as interferon), creating an antiviral state and inhibiting viral transmission. At the same time, it attacks different stages of the viral life cycle (directly killing the virus + activating immunity), making it more difficult for the virus to evade the drug's effects through a single mutation, which provides a superior strategy for preventing and overcoming viral resistance. (2) As "old drugs", their clinical safety, pharmacokinetics and toxicology data are relatively complete, which greatly reduces the time and cost of preclinical research. Imiquimod is a topical drug with high safety when used locally; although aspirin is an injectable drug, its dosage and side effects are controllable, which accelerates their transition to clinical application. (3) Since their mechanisms of action (immune activation and direct antiviral action) are not specific to VACV, this combined strategy has the potential to be extended to the treatment of infections caused by other poxviruses (such as monkeypox virus MPXV) and even other DNA viruses. Attached Figure Description

[0013] Figures 1-2 The results are from positive control experiments for Cidofovir (CDV) and Tecovirimat (TPOXX).

[0014] Figures 3-4 The results are from fluorescence experiments of alvespimycin (17-DMAG) and imiquimod (IMQ).

[0015] Figures 5-8 This is the result of qpcr.

[0016] Figures 9-12 Results of Western blot analysis (HeLa cells).

[0017] Figures 13-14 For WB results (Vero cells).

[0018] Figures 15-16 The results are from Example 11. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: VCV Amplification

[0021] (1) Cell Culture

[0022] Cell passage: Vero cells were grown to approximately 90% confluence in 10cm dishes. The old culture medium was discarded, and the cells were washed twice with 6mL PBS. 1mL trypsin was added, and the cells were incubated at 37°C for 2 minutes for digestion. Under a microscope, most cells were observed to have become rounded. The trypsin was discarded, and 1mL DMEM was added to terminate the digestion. The cells were centrifuged at 1000rpm for 3 minutes. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1mL of complete culture medium. One-third of the cells were then passaged.

[0023] Cell seeding: 0.05 × 10⁻⁶ cells per well in a 24-well plate. 6 The cells were seeded at a density of 100 cells / well, and then the 24-well plates were incubated overnight in an incubator at 37°C with 5% CO2.

[0024] (2) Viral infection

[0025] Aspirate the culture medium from the culture dish, add 6 mL of PBS to rinse the cells, repeat twice, then discard the PBS. Take 50 μL of 1xVACV stock solution and mix it thoroughly with 7 mL of FBS and PLS-free-DMEM, then add it to the culture dish. After virus adsorption for 1.5 h, discard the liquid in the culture dish, and incubate with 7 mL of 2% FBS and 1% PLS-complete DMEM for 48 h. Observe the virus infection status under a fluorescence microscope at 12 h, 24 h, and 48 h.

[0026] (3) Sample collection

[0027] After viral infection, seal the culture dish with sealing film and freeze at -80°C for 30 minutes, then shake on a shaker at room temperature for 15 minutes, repeating three times. Collect the thawed liquid and transfer it to a 15 mL centrifuge tube, centrifuge at 12,000 rpm for 15 minutes at 4°C. Connect a 0.45 μm filter to a syringe, pour the supernatant from centrifugation into the syringe, push the syringe to the bottom to filter, collect the filtrate, aliquot it into 1.5 mL centrifuge tubes, and store at -80°C.

[0028] (4) Viral amplification

[0029] Take 10 μL of 1xVACV stock solution and mix it with 1 mL of FBS and PLS-free-DMEM in a 1.5 mL centrifuge tube for later use. Rinse Vero cells in the culture dish with 6 mL of PBS, then add 3 mL of FBS, PLS-free DMEM and virus dilution solution for adsorption. After 1 hour, add 3 mL of 10% FBS and 1% PLS-DMEM and incubate overnight. Observe the virus infection status under a fluorescence microscope after overnight incubation. Collect and filter the virus-infected samples, repeating the process four times.

[0030] Example 2 Plaque Experiment

[0031] (1) Cell Culture

[0032] The steps are as described in step (1) of Example 1.

[0033] (2) Agar preparation

[0034] Add 0.15g of agar (white shark) and 10mL of deionized water to a 50mL Erlenmeyer flask, seal the flask, and sterilize it in an autoclave. After sterilization, place it in an oven for later use.

[0035] (3) Dilution of the original virus solution

[0036] Take 12 1.5 mL centrifuge tubes and add 900 μL of FBS and PLS-free-DMEM to each tube, labeling them for later use. Add 100 μL of 1xVACV stock solution to tube #1 and mix thoroughly. Then add 100 μL from tube #1 to tube #2 and mix thoroughly. Continue this process until all 12 tubes contain a 10⁻¹¹ concentration of VACV diluted solution.

[0037] (4) Viral infection

[0038] Remove the 12-well plate, discard the original culture medium, and rinse twice with PBS. Then, add the prepared VCV dilutions of various concentrations to each well sequentially, allowing adsorption to occur for 1.5 hours. Preheat 12 mL of 10% FBSDMEM in a 42°C water bath. Remove the agar from the oven and mix it with the preheated DMEM. Add 1 mL of agar along the wall of each well and allow it to solidify. After solidification, return the 12-well plate to the incubator for further incubation.

[0039] (5) Observe the condition of the lesion

[0040] The number of fluorescence particles in each well was observed and recorded under a fluorescence microscope 24 and 48 hours after infection.

[0041] (6) Cell fixation

[0042] Prepare a fixative by mixing 9 mL of anhydrous ethanol and 3 mL of acetic acid. Add 1 mL of fixative to each well and let stand at room temperature for 30 min. Discard the fixative, and gently scrape around the well wall with a 200 μL pipette tip to remove the agar. Rinse the remaining agar gently with deionized water. Add 1 mL of crystal violet dye to each well along the wall and stain at room temperature for 30 min. After staining, recover the crystal violet, rinse the well wall with deionized water to remove excess dye, and then invert the plate onto absorbent paper to air dry for 2-3 hours.

[0043] (7) Calculate the number of plaques

[0044] PFU=(10×10 -7 ) / 0.85=1.2×10 8

[0045] V=(1×10 6 ) / (1.2×10 8 ) = 0.83 × 10 -2 mL

[0046] Example 3 Drug Dilution

[0047] Prepare a 15 mM stock solution (4.19 mg / mL) by mixing 25 mg CDV with 5.97 mL deionized water and dissolving thoroughly. Prepare a 10 mM stock solution (3.76 mg / mL) by mixing 5 mg TPOXX with 1.3286 mL DMSO and dissolving thoroughly. Aliquot each solution into 1.5 mL centrifuge tubes and store at -80°C protected from light. Prepare a 5 mM stock solution (1.202 mg / mL) by mixing 50 mg IMQ with 41.6145 mL DMSO and allowing to stand at 60°C for 10 min. Vortex thoroughly three times until the IMQ powder is completely dissolved. Prepare a 10 mM stock solution (6.53 mg / mL) by mixing 5 mg 17-DMAG with 0.7655 mL DMSO and dissolving thoroughly. Aliquot each solution into 1.5 mL centrifuge tubes and store at -80°C protected from light.

[0048] Example 4: Positive Control Experiment with Cidofovir (CDV) and Tecovirimat (TPOXX)

[0049] (1) Cell Culture

[0050] Cell passage: HeLa cells were grown to approximately 90% confluence in 10cm dishes. The old culture medium was discarded, and the cells were washed twice with 6mL PBS. 1mL trypsin was added, and the cells were incubated at 37°C for 2 minutes for digestion. Under a microscope, most cells were observed to have become rounded. The trypsin was discarded, and 1mL DMEM was added to terminate the digestion. The cells were centrifuged at 1000rpm for 3 minutes. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1mL of complete culture medium. One-third of the cells were then passaged.

[0051] Cell seeding: 0.05 × 10⁻⁶ cells per well in a 24-well plate. 6 The cells were seeded at a density of 100 cells / well, and then the 24-well plates were incubated overnight in an incubator at 37°C with 5% CO2.

[0052] (2) Viral infection

[0053] Virus stock solution dilution: Prepare 10 1.5mL sterile centrifuge tubes and label them. Add 900μL of DMEM medium without serum-containing double antibiotics to each tube. Add 100μL of VCV stock solution to each centrifuge tube and mix well to prepare a 10-fold dilution of VCV for later use.

[0054] Infection: Vaccinia virus TinaTan strain (VTT, VCV) tagged with GFP was used for infection (MOI = 1 for HeLa; MOI = 0.05 for Vero). 100 μL of a 10x VTT-GFP dilution was added to 400 μL of serum-free, antibiotic-free DMEM medium and mixed thoroughly. The 24-well plate was removed, and the DMEM from each well of the experimental group was aspirated. The plates were washed twice with PBS, and then 500 μL of diluted medium was added. After virus adsorption for 1 hour, the DMEM from the experimental group wells was aspirated, and 500 μL of DMEM containing 2% FBS was added to each well for further incubation. The control group did not require virus infection.

[0055] (3) Drug treatment

[0056] ①CDV concentration dilution

[0057] Remove the aliquoted 15mM CDV from the -80℃ freezer and thaw it in an ice box. Take out six 1.5mL centrifuge tubes and add 400μL of 2% FBS-DMEM to each tube. Then add 0, 0.53μL, 1.06μL, 2.67μL, 5.33μL, and 10.67μL of CDV stock solution to each tube, respectively, to prepare CDV dilutions with concentrations of 0mM, 20mM, 40mM, 100mM, 200mM, and 400mM for later use.

[0058] ② TPOXX concentration dilution

[0059] Remove the aliquoted 10mM TPOXX from the -80℃ freezer and thaw it in an ice box. First, mix 10μL of the stock solution with 90μL of LDMSO, at which point the stock solution concentration becomes 1mM. Take four 1.5mL centrifuge tubes, add 400μL of 2% FBS-DMEM to each tube, and then add 0μL, 2μL, 4μL, and 6μL of the 1mM TPOXX stock solution respectively, thus preparing TPOXX dilutions with concentrations of 0mM, 5mM, 10mM, and 15mM for later use.

[0060] ③ Add medicine

[0061] 24 hours after viral infection, the prepared CDV dilution solutions of each concentration were added to the control group and the experimental group at a rate of 200 μL / well, and the groups were placed in an incubator for further incubation. The TPOXX group was treated in the same way.

[0062] ④ Observe the results of the lesion

[0063] The lesions were observed under a fluorescence microscope at 24h, 48h, and 72h after drug administration. The results are as follows: Figure 1-2 As shown.

[0064] Example 5: Fluorescence assays of alvespimycin (17-DMAG) and imiquimod (IMQ)

[0065] (1) Cell Culture

[0066] The steps are as described in step (1) of Example 4.

[0067] (2) Viral infection

[0068] The steps are as described in step (2) of Example 4.

[0069] (3) Drug treatment

[0070] ①Positive control group (CDV, TPOXX) concentration dilution

[0071] Remove the dispensed 15mM CDV and 10mM TPOXX stock solutions from the -80℃ freezer and thaw them in an ice box. Take 1.06μL of 15mM CDV and 0.8μL of 10mM TPOXX respectively and mix them with 800μL of 2% FBS-DMEM to prepare CDV and TPOXX dilutions with concentrations of 20μM and 10μM for later use.

[0072] ②17-DMAG concentration dilution

[0073] Remove the aliquoted 10mM 17-DMAG stock solution from the -80℃ freezer and thaw it in an ice box. Take out five 1.5mL sterile centrifuge tubes and label them. Add 800μL of 2% FBS-DMEM to each tube, followed by 0.08μL, 0.4μL, 0.8μL, 4μL, and 8μL of the stock solution, respectively, and mix well to prepare 17-DMAG dilutions with concentrations of 1μM, 5μM, 10μM, 50μM, and 100μM.

[0074] ③IMQ concentration dilution

[0075] Remove the aliquoted 5mM IMQ stock solution from the -80℃ freezer and thaw it in an ice box. Take out four 1.5mL sterile centrifuge tubes and label them. Add 800μL of 2% FBS-DMEM to each tube, followed by 1.6μL, 8μL, 16μL, and 32μL of the stock solution, respectively, and mix well to prepare IMQ dilutions of 10μM, 50μM, 100μM, and 200μM.

[0076] ④ Add medicine

[0077] Twelve days after infection, each concentration of the drug was added to two 24-well plates at a rate of 400 μL / well, and the plates were placed in an incubator for further incubation.

[0078] (4) Observe the lesion results

[0079] The lesions were observed under a fluorescence microscope 24 hours after drug administration, and the results were as follows: Figure 3-4 As shown.

[0080] Example 6 Alvespimycin (17-DMAG) and Imiquimod (IMQ) fluorescence assay 2 (i.e., Western blotting).

[0081] The concentrations of the positive controls CDV and TPOXX were adjusted to 50 μM and 20 μM, respectively, by adding 4 μL and 24 μL of 15 mM CDV stock solution and 1 mM TPOXX stock solution. A 12-well plate was used for plating, and the concentrations of 17-DMAG were 10 μM and 50 μM, respectively; the concentrations of IMQ were 50 μM and 200 μM. The experiment was repeated.

[0082] Example 7: qpcr experiment

[0083] (1) RNA / DNA extraction

[0084] RNA: RNA was extracted using the Trizol method from two 24-well plates used in the 17-DMAG and IMQ fluorescence experiments, yielding 16 RNA molecules (P1; including Mock, M-CDV, M-TPOXX, M-17-DMAG-100, M-IMQ-200, VTT-0, V-CDV, V-TPOXX, V-17-DMAG-5, V-17-DMAG-10, V-17-DMAG-50, V-17-DMAG-100, and V-IMQ-1) respectively. RNA samples were collected, including 0, V-IMQ-50, V-IMQ-100, V-IMQ-200, and 14 (P2, Mock, M-17-DMAG-50, M-IMQ-100, VTT-0, V-CDV, V-TPOXX, V-17-DMAG-1, V-17-DMAG-5, V-17-DMAG-10, V-17-DMAG-50, V-IMQ-10, V-IMQ-50, V-IMQ-100, V-IMQ-200). The concentration of each sample was determined and recorded using Nano Drop 2000. Thirty cDNA samples were obtained after reverse transcription (50℃, 5 min; 85℃, 2 min; 12℃) and stored at -20℃.

[0085] DNA: The well plate was frozen and thawed three times at -80°C. After thawing, DNA was extracted using a Boge virus DNA purification kit. The concentration of each sample was detected and recorded using Nano Drop 2000, and the samples were stored at -20°C.

[0086] (2) QPCR Mix system configuration

[0087] ①cDNA dilution

[0088] Take two rows of eight-tube strips, add 3 μL of cDNA (P1) and 40 μL of DEPC water to each tube, label them, and store them at -20°C for later use. The same treatment applies to the P2 generation.

[0089] ② Mix system configuration (P1 generation, plates 1 and 2, 8 samples per plate)

[0090] Take three 1.5 mL sterile centrifuge tubes and label them. Add 12.5 μL of H-TD8L-F and 12.5 μL of H-TD8L-R to the centrifuge tubes, then add 245 μL of SYBR and mix well. The amounts of primers TE3L and GAPDH added are the same as those for TD8L; the mixing system for the second plate is prepared in the same way.

[0091] ③ Mix system configuration (P2 generation, plates 3 and 4, 7 samples per plate)

[0092] Take three 1.5 mL sterile centrifuge tubes and label them. Add 11 μL of H-TD8L-F and 11 μL of H-TD8L-R to the centrifuge tubes, then add 225 μL of SYBR and mix well. The amounts of primers TE3L and GAPDH added are the same as those for TD8L; the mixing system for the fourth plate is prepared in the same way.

[0093] (3) Spotting

[0094] Add 6 μL of the prepared mixture of three different primers to each of the eight tubes. Then add 4 μL of diluted cDNA to each tube, and perform three replicates for each cDNA sample. Centrifuge to mix.

[0095] (4)qpcr

[0096] The QPCR conditions are: 95℃, 30s; 95℃, 10s; 55℃, 30s; 72℃, 30s.

[0097] Example 8 qpcr(2)

[0098] (1) cDNA dilution

[0099] Take 3 μL of each P1 generation cDNA and mix it with 40 μL of DEPC water; take 3.5 μL of each P2 generation cDNA and mix it with 40 μL of DEPC water for later use.

[0100] (2) Mix system configuration

[0101] Take four 1.5 mL sterile centrifuge tubes and label them. Add 100 μL of H-Hsp90-F and 100 μL of H-Hsp90-R to the centrifuge tubes, then add 500 μL of SYBR and mix thoroughly. Add the same amount of GAPDH as Hsp90. Add 4 μL of TD8L (2 μL each of the primers) and 20 μL of SYBR to the remaining two centrifuge tubes, and 4 μL of TE3L (2 μL each of the primers) and 20 μL of SYBR, respectively.

[0102] (3) Spotting

[0103] Add 6 μL of the prepared mixture of two different primers to each well of an eight-tube strip. Then add 4 μL of diluted cDNA, and perform three replicates for each cDNA sample. Centrifuge to mix. Spot TD8Lmix and TE3Lmix solutions into the last six wells of the second plate, and then add 4 μL of V-17-DMAG-50 cDNA.

[0104] (4)qpcr

[0105] The QPCR conditions are: 95℃, 30s; 95℃, 10s; 55℃, 30s; 72℃, 30s.

[0106] Example 9 qpcr(3)

[0107] RNA (P3; Mock, VTT-0, V-CDV, V-TPOXX, V-17-DMAG-1, V-17-DMAG-5, V-17-DMAG-10, V-17-DMAG-50, V-17-DMAG-100, V-IMQ-10, V-IMQ-50, V-IMQ-100, V-IMQ-200) was extracted using the Trizol method and 12 cDNA samples were obtained after reverse transcription.

[0108] (1) cDNA dilution

[0109] Take 6 μL of each P3 generation cDNA, mix with 80 μL of DEPC water, and put into an eight-tube bundle. Store at -20℃ for later use.

[0110] (2) Mix system configuration

[0111] Take four 1.5 mL sterile centrifuge tubes and label them. Add 10 μL of H-TD8L-F and 10 μL of H-TD8L-R to the centrifuge tubes, then add 100 μL of SYBR and mix thoroughly. The TE3L, HSP90, and GAPDH systems are prepared using the same method as TD8L; the four-primer mix system for the second plate is prepared using the same method.

[0112] (3) Spotting

[0113] The prepared mixture of four different primers was added to an eight-tube strip at a rate of 6 μL per well. Then, 4 μL of diluted cDNA was added to each well, and each cDNA sample was processed in triplicate. The mixture was then centrifuged and refluxed.

[0114] (4)qpcr

[0115] The QPCR conditions are: 95℃, 30s; 95℃, 10s; 55℃, 30s; 72℃, 30s.

[0116] (5) Summary of QPCR results

[0117] Both drugs inhibited VCV infection in HeLa cells. They also showed some effect in Vero cells, but the effect was not as good as the positive control tecoviride (TPOXX).

[0118] HeLaRNA (VACV-GFP infection for 12 h, drug treatment for 24 h, MOI = 1), results as follows Figure 5 As shown.

[0119] HeLaDNA (VACV-GFP infection for 12 h, drug treatment for 24 h, MOI = 1), results as follows Figure 6 As shown.

[0120] Vero RNA (VACV-GFP infection for 12 h, drug treatment for 24 h, MOI = 0.05), results as follows Figure 7 As shown.

[0121] Vero DNA (VACV-GFP infection for 12 h, drug treatment for 24 h, MOI = 0.05), results as follows Figure 8 As shown.

[0122] Example 10 WB Experiment

[0123] (1) Cell Culture

[0124] The steps are as described in step (1) of Example 4.

[0125] (2) Viral infection

[0126] The steps are as described in step (2) of Example 4.

[0127] (3) Drug treatment

[0128] In the positive control group, CDV and TPOXX concentrations were 50 μM and 20 μM, respectively. 17-DMAG concentrations were 10 μM and 50 μM; IMQ concentrations were 50 μM and 200 μM. Twelve hours after viral infection, 600 μL of drug diluent was added to each well. Another plate contained only the positive control, 50 μM 17-DMAG diluent, and 200 μM IMQ diluent. No drug was administered to the wells containing VCV.

[0129] (4) Observation of lesion results

[0130] Cellular pathogenesis was observed under a fluorescence microscope 12 and 24 hours after drug administration.

[0131] (5) Protein sample collection (operation on ice throughout)

[0132] Mix 12 μL of COCIK Tail with 1.2 mL of Promega 5x passive lysis buffer, and add 50 μL to each well of a 12-well plate. Lyse on ice for 5 min. After the cells are fully lysed, transfer the samples to centrifuge tubes and centrifuge at 12,000 rpm for 15 min at 4 °C. After centrifugation, collect the supernatant, add 20 μL of pre-thawed 6x Loading dye to each tube, vortex to mix thoroughly, and then in a 98 °C metal bath for 10 min. After the metal bath, store all 23 samples at -20 °C.

[0133] (6) Gel preparation and protein loading electrophoresis

[0134] Two 10% separating gels were prepared (1.9 mL deionized water, 1.7 mL 30% acrylamide, 1.3 mL 1.5 mol / L Tris-HCl (pH = 8.8), 0.05 mL 10% SDS, 0.05 mL 10% APS, and 0.004 mL TEMED). The mixtures were poured into gel plates and sealed with anhydrous ethanol, allowing them to solidify for 30 min. Simultaneously, two 5% stacking gels were prepared (3.4 mL deionized water, 0.83 mL 30% acrylamide, 0.63 mL 1.0 mol / L Tris-HCl (pH = 6.8), 0.05 mL 10% SDS, 0.05 mL 10% APS, and 0.005 mL TEMED). After the lower gel solidified, the anhydrous ethanol was discarded, the upper gel was added, a comb was inserted, and the gels were allowed to solidify at room temperature for 1 h.

[0135] After the upper gel solidifies, remove the comb, fix the gel plate to the electrophoresis clamp, and pour 1x Running Buffer (900mL deionized water mixed with 100mL 10x Running Buffer) into the electrophoresis tank. Divide the 23 protein samples into two groups. The loading order for the first group is: Mock, VTT-0, M-CDV-50, V-CDV-50, M-TPOXX-20, V-TPOXX-20, M-17-DMAG-50, V-17-DMAG-10, V-17-DMAG-50, M-IMQ-200, V-IMQ-50, V-IMQ-200; the second group omits V-CDV-50, but the rest are the same. Cover the tank, adjust the electrophoresis voltage to 80V, and after 30 minutes, adjust the voltage to 120V and continue electrophoresis for another 30 minutes.

[0136] (7) Transfer and sealing

[0137] Activate the PVDF membrane with methanol by shaking at room temperature for 3 minutes. After activation, wash away the methanol by shaking with deionized water for another 3 minutes. After electrophoresis, remove the gel plate, pry open the short glass plate, cut off the excess gel, and then transfer the gel to pre-cooled transfer buffer (700 mL deionized water, 200 mL methanol, 100 mL 10x transfer buffer). Place the gel and membrane in the order of filter paper-membrane-gel-filter paper, close the transfer clamp, and place the membrane in the transfer tank. Fill the tank completely with transfer buffer, ensuring it does not submerge the transfer clamp. Transfer at a constant voltage of 120V for 80 minutes under ice-water bath conditions.

[0138] After the transfer is complete, transfer the membrane to a clean small box, pour in the pre-prepared skim milk (1g milk powder + 20mL TBST, vortex thoroughly to mix), and seal on a shaker at room temperature for 1 hour.

[0139] (8) Antibody incubation

[0140] Discard the skim milk, add TBST until it covers the bottom of the filter paper, and wash on a shaker for 10 min at room temperature. Then discard the TBST and repeat three times. After washing, add the primary antibody (Proteintech, Cat No. 50430-2-AP GFP tag Rabbit; 1:2000 dilution) and incubate overnight at 4°C on a shaker. After the primary antibody incubation, recover the primary antibody, add TBST again until it covers the bottom of the filter paper, wash on a shaker for 10 min at room temperature, discard the TBST, and wash again. Repeat three times. Then add the secondary antibody (HRP, Goat Anti-Rabbit IgG; 1:5000 dilution) and incubate at room temperature for 1 h.

[0141] (9) Development

[0142] After incubation, the secondary antibody was recovered, and the membrane was washed three times with TBST. Two luminescent solutions (200 μL each) were poured into a small box at a 1:1 ratio. The TBST on the membrane was blotted dry with absorbent paper, and then the membrane was fully immersed in the luminescent solution. The exposure results were observed using a chemical imaging system.

[0143] (10) Adjustment of sample loading amount

[0144] Based on the first WB result and the imagej software, adjust the sample loading amount and repeat the above steps twice until the bands are clear and neat.

[0145] (11) Summary of WB results

[0146] HeLa cells:

[0147] ① First, two positive control drugs were run separately to ensure that these two concentrations of the drug could indeed inhibit VCV infection. The results were as follows: Figure 9 As shown.

[0148] ② Run 17-DMAG and IMQ separately to observe the trend of GFP to demonstrate the effects of the two drugs. The results are as follows: Figures 10-11 As shown.

[0149] ③ To compare the efficacy of the two drugs in HeLa cells, all drug concentrations were run on a gel. It was observed that IMQ(200) showed the best efficacy, as shown in the results below. Figure 12 As shown.

[0150] Vero cells:

[0151] ①17-DMAG also showed some effect in Vero cells, but the effect of IMQ was not obvious, as shown in the results. Figure 13 As shown.

[0152] ② When all the drugs were run on a gel, tecoviriprine showed the best effect. This phenomenon is consistent with the results of qPCR. Figure 14 As shown.

[0153] Example 11 CCK8 Experiment Determines IC 50

[0154] (1) Cell Culture

[0155] The steps are as in step (1) of Example 4; the cells are seeded in a 96-well plate.

[0156] (2) Viral infection

[0157] The steps are as described in step (2) of Example 4.

[0158] (3) Drug treatment

[0159] In HeLa cells, the concentrations of 17-DMAG were 5 μM, 10 μM, 50 μM, and 100 μM; and the concentrations of IMQ were 50 μM, 100 μM, 200 μM, and 500 μM. In Vero cells, the concentrations of 17-DMAG were 0.1 μM, 0.5 μM, 5 μM, and 10 μM; and the concentrations of IMQ were 100 μM, 200 μM, 400 μM, and 500 μM. The drug concentrations were added to 96-well plates at 48 h, 24 h, 12 h, and 0 h.

[0160] (4) OD value measurement

[0161] Add 10 μL of CCK8 solution to each well of the plate, incubate in an incubator for 40-60 min, and measure the absorbance at 450 nm using a microplate reader.

[0162] The results are as follows Figures 15-16 As shown.

[0163] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The use of aspirin and / or imiquimod in the preparation of medicaments for the treatment of VCV.

2. The use of aspirin and / or imiquimod according to claim 1 in the preparation of a medicament for treating VAV, characterized in that: The IC50 of aspirin at the cellular level against VAV virus 50 The concentration ranged from 7.423 μM to 18.32 μM.

3. The use of aspirin and / or imiquimod according to claim 2 in the preparation of a medicament for treating VAV, characterized in that: The IC50 of imiquimod at the cellular level against VAV virus 50 The concentration ranged from 305.6 μM to 308.1 μM.

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