Application of inauhzin in preparation of medicine for preventing foot-and-mouth disease virus infection
A technology of foot-and-mouth disease virus and medicine, applied in the field of application
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Embodiment 1
[0027] Toxicity test of Inauhzin on IBRS-2 cells:
[0028] The cytotoxicity of Inauhzin (purchased from MCE Company) to IBRS-2 cells was determined by MTS method (MTS detection kit was purchased from Abcam Company). After spreading to the 96-well plate IBRS-2 cells grew to a monolayer, the supernatant culture medium of the cells was discarded, washed 3 times with fresh DMEM (purchased from Gibco), and finally added with FBS containing 2% fetal bovine serum (purchased from 100 μL of Inauhzin diluted in DMEM culture fluid from Gibco Company) was used as a negative control well with the corresponding concentration of the Inauhzin preparation solution DMSO, and as a cell control well without any treatment. Place at 37°C for 72 hours, discard the upper cell culture medium, wash with fresh DMEM three times, then add 100 μL of fresh DMEM, and add 20 μL of MTS solution to each well. After incubation at 37°C for 4 h, measure the absorbance value at 490 nm on a microplate reader, accor...
Embodiment 2
[0031] Evaluation of the anti-foot-and-mouth disease virus activity of Inauhzin on IBRS-2 cells
[0032] Spread the well-grown IBRS-2 cells on DMEM complete medium containing 10% fetal bovine serum FBS to a 96-well plate. After the IBRS-2 cells grow to a monolayer, discard the culture medium on the upper layer of the cells and wash with fresh DMEM. 3 times, inoculated with 100TCID50O / MY98 / BY / 2010 (saved and provided by the National Foot-and-Mouth Disease Reference Laboratory). After 1 hour, remove the virus solution, wash with fresh DMEM for 3 times, add 100 μL of Inauhzin diluted with DMEM culture solution containing 2% FBS, and use the corresponding DMSO concentration of the prepared solution of Inauhzin as the virus control well. Viruses were used as cell control wells. Put them in 37°C for 48 hours, discard the upper cell culture medium, wash with fresh DMEM three times, then add 100 μL of fresh DMEM, and add 20 μL of MTS solution to each well. After incubation at 37°C f...
Embodiment 3
[0041] Indirect Immunofluorescence Detection of FMDV Protein Expression in Infected Cell Group
[0042] Set the density to 3×10 5 Spread IBRS-2 cells per well on a 12-well plate. After the IBRS-2 cells grow to a monolayer, discard the culture medium on the upper layer of the cells, wash 3 times with fresh DMEM, and inoculate 100TCID 50 O / MY98 / BY / 2010. After 1 hour, remove the virus solution, wash 3 times with fresh DMEM, add 100 μL of Inauhzin diluted with DMEM culture solution containing 2% FBS, and use the corresponding DMSO concentration of the prepared solution of Inauhzin as the virus The control wells were cultured at 37°C for 12 hours. Discard the upper cell culture medium, wash with PBS twice, fix the cells with 4% paraformaldehyde for 15 min, discard the paraformaldehyde, add methanol for 5 min, rinse with PBS for 3 times, each time for 5 min, add blocking solution (10% FBS, 0.3% TritonX-100, 89.7% PBS) to block for 10min, then add the primary antibody diluted with ...
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