Application of chloroquine in the preparation of anti-herpes virus medicine
A technology of herpes virus and chloroquine, which is applied in the field of application of chloroquine to gamma-herpes virus and related diseases, and can solve the problems of vaccine research and development stage and the absence of anti-gamma-herpes virus drugs
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Embodiment 1
[0024] 1. Take well-grown BCBL1 cells and inoculate them in 6-well transparent flat-bottomed plates, 2×10 per well 6 Cells were divided into induced group and non-induced group. The medium used is a complete medium: high-glucose DMEM, 10% fetal bovine serum and 1% double antibody, and the culture condition is 5% carbon dioxide, 37°C;
[0025] 2. After 3 hours, the induction group was added with inducer TPA, the final concentration was 20ng / mL;
[0026] 3. Chloroquine was added after 3 hours of induction, and the final concentrations were 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM;
[0027] 4. After adding the drug, culture for 48 hours, harvest the cells at 1000 rpm, and 10 minutes;
[0028] 5. Carry out Western blot experiments, the results are as follows: figure 1 It was shown that the expression of KSHV-related proteins RTA, ORF45, K8, and ORF64 in BCBL1 cells was inhibited by chloroquine after 3 hours of induction, and the inhibitory effect was concentration-dependent. This s...
Embodiment 2
[0030] 1. Take well-grown iSLK-219 cells, inoculate them in 6-well transparent flat-bottomed plates at a ratio of 1:4, and divide the cells into induced and non-induced groups.
[0031] 2. After 12 hours, the cells adhered to the wall, and the induction group was added with inducers Dox and NaB, the final concentrations were 1 μg / mL and 0.3 μM, respectively;
[0032] 3. Chloroquine was added after 3 hours of induction, and the final concentrations were 0 μM, 2 μM, 5 μM, 8 μM, 10 μM, 15 μM, 20 μM, and 25 μM;
[0033] 4. After adding the drug for 48 hours, scrape the cells out of the plate with a cell scraper, rinse with cold PBS, collect the cells by centrifugation at 1000rpm, 10min, and 4°C;
[0034] 5. Carry out western blot experiment, the experimental results are as follows: figure 2 Shown: the expression of KSHV-related proteins RTA, ORF45, and K8 in iSLK-219 cells was inhibited by chloroquine, and the inhibitory effect was concentration-dependent.
Embodiment 3
[0036] 1. Take the well-growing cell line BCBL1 and plant it in a 48-well plate. The amount of cells used is 1×10 5 / hole, the cells are divided into non-induced group (3 holes) and induction group (15 holes);
[0037] 2. After 3 hours, the induction group was added with inducer TPA, the final concentration was 20ng / mL;
[0038] 3. After 3 hours of induction, add corresponding concentrations of chloroquine, with concentrations of 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM, respectively, with 3 wells for each concentration;
[0039] 4. Harvest the cells after 96 hours, weigh 10,000 g, and take the supernatant at 4°C;
[0040] 5. Extract extracellular viral DNA, the method is as follows:
[0041] a. Take 200 μL supernatant, add 2 μL DNase I, 20 μL 10×DNase I buffer, and incubate in a 37°C incubator for 1 hour;
[0042] b. Add 10 μL of 0.5M EDTA to the above liquid, mix well, and terminate the reaction;
[0043] c. Inactivate in a water bath at 80°C for 10 minutes;
[0044] d. Add...
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