Application of imidazolidin in the preparation of medicines for preventing and treating bmnpv-infected silkworm cells
A technology of imidazolone and silkworm, which is applied in the application field of imidazolone in the preparation of medicine for preventing and controlling BmNPV infection of silkworm cells
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Embodiment 1
[0020] 1. Construction of BmNPV carrying egfp reporter gene
[0021] The egfp fragment was cloned into the BamHI (Takara) and XhoI (Takara) sites of pFastBac1 (Invitrogen), and the donor plasmid pFastBac-egfp was constructed, and BmBac-egfp was obtained by transposition ( figure 1 ), after the BmBac-egfp DNA was transfected into BmN (bombyx mori ovary cells) cells, the expression of green fluorescence could be observed under a fluorescent microscope. The cell supernatant was used to infect BmN cells, and the virus was harvested 72 hours after infection, and the virus titer was determined by the terminal dilution method, and the virus was stored in a refrigerator at 4°C in the dark.
[0022] 2. Inoculation of cells and preparation of storage solution of imidazolidin
[0023] (1) Inoculate about 10 cells in a 35mm cell culture dish (Corning) respectively. 5 Add 2 mL of 10% FBS TC100 insect cell culture medium to each cell dish of BmN cells.
[0024] (2) Culture at 27°C for ab...
Embodiment 2
[0033] When the final concentration of imidazolidin is 1 μg / mL, the prevention and treatment of BmNPV infection carrying the egfp reporter gene:
[0034] (1) Take 10 of each inoculation 5 For 3 culture dishes of cells, add 2 mL of TC100 insect cell culture medium with 10% FBS to each dish. The old medium was removed, and 2 μL of 10 mg / mL imidazolidin stock solution and 2 mL of 10% FBS TC100 medium were added respectively, incubated at 27° C. for 30 min, and then BmBac-egfp virus with MOI=10 was added.
[0035] (2) After 72 hours, place 3 dishes in a fluorescent inverted microscope to observe the expression of green fluorescent protein, see figure 2 (T1).
[0036] (3) Take 10 μL of cell culture solution, and measure the virus titer by terminal dilution method.
[0037] The results showed that when cells were treated with 1 μg / mL imidaclomidone, the cells infected by BmNPV did not produce fluorescence, and completely inhibited, and the virus titer was 0 ( image 3 T1).
Embodiment 3
[0039] When the final concentration of imidazolidin is 0.25 μg / mL, the prevention and treatment of BmNPV infection carrying the egfp reporter gene:
[0040] (1) Take 10 of each inoculation 5 For 3 culture dishes of cells, add 2 mL of TC100 insect cell culture medium with 10% FBS to each dish. The old medium was removed, and 0.5 μL of 10 mg / mL imidazolidin stock solution and 2 mL of 10% FBS TC100 medium were added respectively, incubated at 27° C. for 30 min, and then BmBac-egfp virus with MOI=10 was added.
[0041] (2) After 72 hours, place 3 dishes in a fluorescent inverted microscope to observe the expression of green fluorescent protein, see figure 2 (T0.25).
[0042] (3) Take 10 μL of cell culture solution, and measure the virus titer by terminal dilution method.
[0043] The results showed that when cells were treated with 0.25 μg / mL imidazolidin, the fluorescence of BmNPV-infected cells was significantly reduced compared with the control, and the virus titer Log 10 ...
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