A medicine for treating influenza virus infection
A technology of influenza virus infection, influenza virus, applied in the direction of antiviral agents, pharmaceutical formulas, medical preparations containing active ingredients, etc.
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Embodiment 1、293
[0123] Example 1. Preparation of 293T-IAV-Luc cells
[0124] 1. Construction of pREP4-IAV-Luc plasmid
[0125] 1.1. The DNA fragment shown in SEQ ID NO.: 1 of SEQ ID NO. 1 in the artificially synthesized sequence table (see patent CN106562957A). Sequence 1 consists of a total of 1748 nucleotides, the 14-58th position is marked as fragment 1, the 59-1711 position is the firefly luciferase encoding gene (reporter gene), and the 1712-1734 position is marked as fragment 2. is the recognition site sequence of BsmBI. Wherein, fragment 1 and fragment 2 are both promoters of influenza virus NP protein. In the presence of influenza virus, the promoter located on the fusion plasmid can be activated, and firefly luciferase can be expressed.
[0126] 1.2, use the restriction endonuclease BsmBI to digest the DNA fragment shown in sequence 1SEQ ID NO.: 1 in the sequence table, after the recovery of the digested fragment, it is positively connected with the large skeleton fragment of the p...
Embodiment 2
[0129] Example 2. Compound NSC 13294 inhibits the replication of H1N1 subtype influenza virus
[0130] 1. Mix A / WSN / 33 strain, DMEM medium and compound NSC 13294 to obtain a mixed solution. The mixture contained 0.5MOI virus and 50μmol / L compound NSC 13294.
[0131] 2. The 293T-IAV-Luc cells prepared in Example 1 were evenly plated on a 96-well plate (20,000 cells per well), cultured at 37°C for 12 hours, discarded the supernatant, and washed the wells with PBS buffer. cell.
[0132] 3. After completing step 2, take the 96-well plate, add the mixture obtained in step 1 (MOI=0.5), incubate at 37° C. for 1 hour, and discard the supernatant.
[0133] 4. After completing step 3, take the 96-well plate, add DMEM medium containing 10% (volume ratio) fetal bovine serum and 50 μmol / L compound NSC 13294, and culture at 37°C for 12 hours. Cells in the wells were washed with PBS buffer.
[0134] 5. After completing step 4, take the 96-well plate, add the lysate in the luciferase acti...
Embodiment 3
[0142] Example 3. Compound NSC 13294 inhibits the activity of HA pseudovirus
[0143] 3.1 Preparation of HA pseudovirus
[0144] The 293T cells were seeded into cell culture dishes, and after reaching 80% density, 6 μg pNLLucE-R-HIV-Luc plasmid, 6 μg pEWSN-HA plasmid and 6 μg pCAGGS-NA plasmid were co-transfected with the help of transfection reagent PEI, and replaced with 10 μg pNLLucE-R-HIV-Luc plasmid after 6 hours. % (volume fraction) of fetal bovine serum in DMEM complete medium, transfer the entire culture system to a 15ml centrifuge tube after 48 hours, blow off the cells, freeze and thaw once and filter with a 0.22 μm filter membrane to collect the filtrate, which is the HA pseudovirus The virus solution was stored at -80°C.
[0145] 3.2. The compound NSC 13294 inhibits the activity of HA pseudovirus
[0146] 1. Spread 293T cells evenly on a 24-well plate (160,000 cells per well), culture at 37°C for 15 hours, and discard the supernatant.
[0147] 2. After completin...
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