Method for screening influenza virus resisting medicines
An influenza virus, active technology, applied in the field of drug screening, can solve the problem of high toxicity and side effects of host cells, and achieve the effect of solving the problem of drug resistance
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[0017] Example 1
[0018] 1. Construction of expression vector;
[0019] PCR was used to amplify the Rluc gene fragment in the eukaryotic expression vector pRluc-N2 (PerkinElmer). Primers used: Upstream primer 5'-GATTCAATTCAGCTACCATAATGATGCAGAGAGGCAATTTTGGTAC-3', downstream primer 5'-CAAAATTGCCTCTCTGCATCATTATGCTAGCTGAATTG-3'.
[0020] reaction system:
[0021]
[0022]
[0023] Reaction conditions: 94°C for 45s, 68°C for 90s, a total of 30 cycles.
[0024] The Rluc fragment was inserted into the cloning site BsmB I of the influenza virus M fragment expression vector pWSN-M (gifted by Dr. Kawaoka) to obtain the plasmid pFLU-Rluc, which expresses the fluorescent protein.
[0025] 2. Cell culture
[0026] Take 293T cells for culture. After the cells have grown up in the culture flask, discard the old medium and digest it with a digestion solution containing 0.25% trypsin and 0.02% EDTA. When the cells become round, discard the digestion solution, immediately add 10% FBS-containing high-sug...
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