Application of clinafloxacin or derivative thereof in preparation of HSV (herpes simplex virus) inhibitor
A technology of derivatives and inhibitors is applied in the application field of clinfloxacin or its derivatives in the preparation of HSV inhibitors, and achieves the effects of high safety and low cytotoxicity
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Embodiment 1
[0019] The present embodiment inoculates the virus and tests the drug to inhibit the growth of the virus, and the specific process is:
[0020] S1: Take the well-grown human neuroblastoma cell line and inoculate it in a 24-well transparent flat bottom plate, with 3 × 10 cells per well 5 cells. Use complete medium: high glucose DMEM, 10% fetal bovine serum and 1% double antibody (penicillin 100U / ml, streptomycin 100μg / ml), 5% CO 2 , 37 ℃ incubation;
[0021] S2: After 24h adherence, add 200 μL of 0.1MOI virus solution (DMEM medium containing live virus particles and 1% double antibody) to 24 wells, 5% CO 2 , 37 ℃ condition adsorption 1h. Discard the supernatant, gently wash the wells twice with PBS, add 1 mL of DMEM medium containing 1% double antibody, and add clinfloxacin hydrochloride with different concentration gradients (final concentrations are 0 μM, 5 μM, 10 μM, 50 μM, 100 μM respectively). , 200 μM), and mixed well, 5% CO 2 , Continue to culture at 37°C;
[0022]...
Embodiment 2
[0024] In this example, the virus titer experiment was carried out, and the principle of the improved virus plaque reduction test was adopted. 1.2% RC-591 was used as the overlay medium, and plaques were counted after fixing, staining and other steps to obtain plaques. The specific process for:
[0025] S1: Monkey kidney Vero cells were plated at 5 × 10 5 Inoculated in a flat-bottom 96-well plate per well, cultured with complete medium: DMEM+10% fetal bovine serum+1% double antibody;
[0026] S2: When the cells at the bottom of the well have just grown into a monolayer, the virus solution (the cell supernatant frozen in Example 1) is serially diluted with DMEM, and then 60 μL per well is inoculated into the well, 5% CO 2 , Incubate at 37°C, let the virus adsorb for 1h;
[0027] S3: After the virus adsorption is completed, directly add the covering medium containing 1.2% RC-591 to the well, 5% CO 2 , 37°C for 96h;
[0028] S4: Take the 96-well plate out of the incubator, an...
Embodiment 3
[0035] In this embodiment, the relative amount of viral DNA is measured by fluorescence quantitative PCR after adding different concentrations of clinfloxacin hydrochloride, and the specific process is as follows:
[0036] S1: Extract the DNA in each supernatant sample of 200 μL of the cell cryopreservation solution in Example 1 with a viral DNA extraction kit according to the operating instructions, and use it as a template for fluorescence quantitative PCR;
[0037] S2: Take 2 μL of DNA template and add it to the reaction system according to the instructions for installing the fluorescence quantitative PCR kit. PL 5'-ATCAACTTCGACTGGCCCTTC-3' (SEQ ID NO: 1) and PR,5'-CCGTACATGTCGATGTTCACC-3' (SEQ ID NO: 2) were used as upstream and downstream primers, and SYBR Green was a fluorescent dye;
[0038] S3: Put the reaction plate into the fluorescence quantitative PCR instrument, and set the program: 95°C pre-denaturation for 3 minutes, 95°C for 20s denaturation, 55°C for 20s annea...
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