Application of chlorhematin in preparing drug for resisting porcine reproductive and respiratory syndrome virus
A technology for hemin and respiratory syndrome, which is applied to the application field of hemin and the preparation of anti-porcine reproductive and respiratory syndrome virus drugs, can solve the problems that vaccines cannot provide effective and sustainable disease control, and the like, Achieve the effect of mature production process, obvious effect and easy transportation
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Embodiment 1
[0031] Example 1 The effect of hemin on the growth and apoptosis of MARC-145 cells
[0032] S1. Cell Viability Assay Experiment
[0033] AlamarBlue is a metabolic indicator for living cells. Under the enzymatic reduction reaction of mitochondria, it will produce measurable fluorescent metabolites, and the growth and viability of cells can be monitored by measuring their fluorescence intensity.
[0034] MARC-145 cells in the logarithmic growth phase were diluted with DMEM medium containing 10% fetal bovine serum and seeded in 96-well plates, 100 μl per well. When the cells were cultured to reach 60-70% confluence, the test drug hemin was added. Set up treatment groups with different concentrations of hemin (the concentrations of hemin were 5 μM, 10 μM, 50 μM, and 100 μM) and a DMSO control group (the concentration of DMSO: 5%), with 6 replicate wells in each group. Set the test time according to the needs of the experiment, replace the culture solution of the cells to be test...
Embodiment 2
[0038] The influence of embodiment 2 hemin on PRRSV viral replication
[0039] S1. Collection of samples
[0040] PRRSV strain CH-1a was inoculated into 80% confluent MARC-145 cells, adsorbed at 4°C and 37°C for 1 h each; the virus liquid was aspirated, and hemin (5 μM, 10 μM, 50 μM) and 2% DMEM maintenance medium with fetal bovine serum; at the same time, DMEM maintenance medium containing 5% DMSO and 2% fetal bovine serum was used as a control, and cells and supernatant were collected at 36 hpi.
[0041] S2. Relative expression of PRRSV N gene in cells detected by qPCR
[0042] Total RNA was extracted using RNAizol reagent from Invitrogen. For adherent cultured MARC-145 cells, remove the culture medium, wash twice with 1×PBS, and then add an appropriate amount of RNAizol to lyse the cells. Transfer the lysate to a 1.5 ml RNase-free EP tube and shake and mix well. After standing at room temperature for 5 minutes, add chloroform according to the ratio of 1ml RNAizol to 200μ...
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