Application of Danjie emodin in preparation of medicine for preventing and treating African swine fever virus
A technology of African swine fever virus and emodin, which is applied in the field of biomedicine, can solve the problem of no virus application and achieve good application prospects
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Embodiment 1
[0033] Example 1 Cytotoxicity of Emodin to PAM Cells
[0034] 1. Experimental method
[0035] 1. Preparation of emodin
[0036] Dissolve emodin in DMSO to make 20mM, dilute it with cell culture medium RPMI-1640 when used, wrap it in tinfoil after aliquoting, and store it at -20°C for later use.
[0037] 2. Effect of Danye-emodin on PAM cells
[0038] The PAM cells were seeded in 96-well plates, and after the cells adhered to the wall, the culture medium in the plate was discarded, and RPMI Medium 1640 medium with 10% FBS (containing penicillin 100 U / mL, streptomycin 50 μg / mL, amphotericin 0.25 μg / mL) dilute emodin to 0, 20, 40, 80, 160 and 320 μM, 100 μL / well, set blank cell control group, do 6 duplicate wells for each concentration, at 37 ° C, 5% CO 2 After culturing in the incubator for 48 hours, discard the supernatant, wash twice with PBS, add new culture solution, and then add CCK-8 reagent, 10 μL / well, continue to incubate in the dark for 1 hour, use a microplate read...
Embodiment 2
[0041] Effect of different concentrations of emodin of embodiment 2 on ASFV p30 protein expression in PAM cells
[0042] The p30 protein is a structural protein of African swine fever, which is expressed in the early stage and has a high content, so the p30 protein is selected as a marker for detecting African swine fever virus.
[0043] 1. Experimental method
[0044] Inoculate PAM cells in a 96-well plate, wait for the cells to adhere to the wall, discard the culture medium, inoculate ASFV (MOI=0.1), 100 μL / well, culture at 37°C for 2 hours, discard the virus supernatant, and wash twice with PBS , add 5 μM, 10 μM, 20 μM emodin culture solution respectively, 100 μL / well, at the same time, set the cell group as the negative control and the virus group as the positive control, and set three parallels for each test concentration. Placed at 37°C, 5% CO 2 After culturing in the incubator for 48 hours, discard the supernatant, add 100 μL of 4% paraformaldehyde to each well, fix a...
Embodiment 3
[0047] Effect of different concentrations of emodin of embodiment 3 on ASFV B646L gene expression in PAM cells
[0048]ASFV-B646L is the gene of p72 protein, the main structural protein of African swine fever virus. This gene is highly conserved and must be detected for virus detection.
[0049] 1. Experimental method
[0050] Inoculate PAM cells in a 24-well plate, discard the culture medium after the cells adhere to the wall, add ASFV (MOI=0.1), set a blank cell control group, 500 μL / well, culture at 37°C for 2 hours, discard the virus supernatant solution, washed twice with PBS, added different concentrations of emodin, set virus control group, 500 μL / well, placed at 37 ° C, 5% CO 2 Cultivate in the incubator for 48h. After 48 hours, the cell samples were repeatedly frozen and thawed three times at -80°C and 4°C, and the cell supernatant was collected.
[0051] Use the AxyPrep Body fluid Viral DNA / RNA Miniprep Kit kit (Axygen Company) to extract the genome of ASFV from t...
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