Application of biapenem in preparation of medicine for preventing and treating bovine enterovirus infection
A technology of biapenem and enterovirus, applied in the field of application of biapenem in the preparation of drugs for preventing and treating bovine enterovirus infection
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Embodiment 1
[0050] Example 1 Virus TCID 50 Determination of
[0051] MDBK cells (preserved by Dairy Cow Research Center, Shandong Academy of Agricultural Sciences) were digested and divided into 3 × 10 cells per well. 5 Cells / mL were seeded into 96-well cell culture plates, placed in 37°C, 5% CO 2 After being cultivated into monolayer cells in the cell incubator, discard the cell growth solution in the well, and the virus dilution solution of bovine enterovirus serial 10-fold dilution (dilution degree is 10 -1 ~10 -10 ) inoculated in a 96-well plate full of monolayer cells, 100 μL per well, placed in 37°C, 5% CO 2 Continue to culture in the incubator, observe the CPE of the cells day by day, and record the number of cells with pathological changes in detail. At the same time, a normal cell control group and a blank control group were set up, with 8 replicates in each group, and the results were judged when no further cell lesions occurred. Cytopathic wells are cell wells correspondin...
Embodiment 2
[0062] Example 2 Toxicity test of biapenem on MDBK cells:
[0063] MDBK cells are susceptible cells to bovine enterovirus. Therefore, the cytotoxicity of biapenem to MDBK cells was first detected, and the specific experimental steps were as follows:
[0064] (1) Inoculate 100 μL cells (MDBK 3×10 4 pieces / hole).
[0065] (2) After culturing for about 12 hours, the next step of drug addition analysis was carried out. Discard the medium, add 100 μL of 2% FBS DMEM containing different drug concentrations to each well, and make 3 parallels for each concentration. At the same time, control wells: add 100 μL 2% FBS DMEM medium. Zero well: no cells are plated.
[0066] (3) At 37°C, 5% CO 2 After culturing under the conditions for 48 hours, operate according to the instructions of the CCK-8 kit, and measure the OD value at 450nm with a microplate reader.
[0067] (4) 37°C, 5% CO 2 After culturing for 4 h under the same conditions, the absorbance was measured at 450 nm. A450nm ...
Embodiment 3
[0070] Example 3 Inhibition experiment of biapenem on bovine enterovirus:
[0071] (1) Inoculate 3×10 in each well of a 96-well plate 4 MDBK cells, 37°C, 5% CO 2 Cultivate overnight in an incubator;
[0072] (2) Discard the medium and add 100 μL 100TCID to each well 50 The bovine enterovirus dilution solution (use 2% FBS DMEM to add the virus dilution solution after the cells are overgrown, according to the initial concentration of 100 μ M, double the concentration gradient dilution and dosing, 5% CO 2 Cultivated in an incubator;
[0073] (3) After 48 hours, operate according to the instructions of the CCK-8 kit, and measure the OD value at 490 nm with a microplate reader.
[0074] (4) analysis data, virus inhibition rate (%)=(drug treatment group D450nm value-virus control group D450nm value) / (normal cell control group D450nm value-virus control group D450nm value) * 100%, get with GraphPad Prism5 software The half effective concentration of the compound (EC 50 )value. ...
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