Application of Dapivirine in the Preparation of Drugs for Inhibiting Cell Infection by Fish Viral Hemorrhagic Septicemia Virus
A technology of viral hemorrhage and cell infection, applied in the field of dapivirine
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Embodiment 1
[0017] Example 1 Cell Viability Detection Research on the Protective Effect of Dapivirine on Cells
[0018] 1. Cell Lines and Cell Culture
[0019] The cell line used in the experiment was FHM (Fathead minnow), the medium was M199 (10% FBS culture medium and 2% FBS maintenance medium), and the cells were cultured in a constant temperature incubator at 28°C.
[0020] 2. Cell viability detection of dapivirine cytotoxicity to FHM
[0021] Cell Counting Kit-8 was used to detect the toxicity of dapivirine to FHM. The FHM cells in the logarithmic growth phase were made into a suspension and inoculated in a 96-well plate with 100 μl per well, cultured at 28°C for 24 hours to a cell coverage density of 80-90%, and replaced with different concentrations (0.5, 1, 2, 3, 5, 7, 9, 11 μM) dapivirine maintenance culture solution, 0.05% DMSO was used as a negative control and 4 replicate wells were set up for each group, and incubated at 28° C. for 48 hours. Then replace 100 μl of fresh ma...
Embodiment 2
[0026] Example 2 qRT-PCR detection of anti-VHSV activity of dapivirine
[0027] 1. Drug handling
[0028] The FHM cells in the logarithmic growth phase were made into a suspension, inoculated in a 12-well plate with 1ml per well, cultured at 28°C for 24h to a cell coverage density of 80-90%, and replaced with fresh maintenance medium, followed by Four different drug treatments were used to detect the anti-VHSV ability of dapivirine.
[0029] (1) The drug is added to the cells before virus infection
[0030] Add dapivirine at a concentration of 2 μM to the cell maintenance medium, incubate at 28°C for 4 h, and then inoculate with a final concentration of 10 1.5 TCID 50 VHSV was incubated at 28°C for 24h and 36h, digested with 0.25% trypsin and collected in RNase-free centrifuge tubes. 0.05% DMSO was used as a negative control, and 8 μM ribavirin was used as a positive control.
[0031] (2) The drug and the virus are added to the cell together
[0032] Dapivirine at a conc...
Embodiment 3
[0049] Embodiment 3qRT-PCR detects the impact research of dapivirine on the VHSV infection process
[0050] 1. Drug handling
[0051] The FHM cells in the logarithmic growth phase were made into a suspension, inoculated in a 12-well plate with 1ml per well, cultured at 28°C for 24h to a cell coverage density of 80-90%, replaced with fresh maintenance medium, and then carried out Four different drug treatments were used to explore the effect of dapivirine on the course of VHSV infection.
[0052] (1) Detect the effect of dapivirine pretreatment cells on VHSV infection
[0053] Add dapivirine at a final concentration of 2 μM to the maintenance culture medium, incubate at 28°C for 4 h, then discard the culture medium and wash with PBS for 3 times; add 10 1.8 TCID 50 The maintenance culture medium of VHSV was incubated at 28°C for 4h, then the culture medium was discarded, washed 3 times with PBS, fresh maintenance medium was added, and incubated at 28°C for 20h. Use 0.25% tr...
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