Application of Cycloviral Buxus d against Dengue Virus
A technology of cyclovir buxus and dengue virus, which is applied in the field of biomedicine to achieve the effect of inhibiting replication
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Embodiment 1
[0024] Example 1 Screening of natural small molecule drugs against DENV
[0025] 13 natural compounds that significantly inhibit DGL2 replication were screened out of 143 natural products with a single component from Selleckchem. Among them, some have been reported to have antiviral effects, such as Cyclosporine can block the interaction between host cyclophilin and NS5 protein, baicalein can inhibit the replication of dengue virus, tripterygium wilfordii and powder Triptolide and tetrandrine can activate COX-2, NF-kappaB and AP-1 to resist dengue virus. The present invention discovers the effect of Cyclovirobuxin (Cvb D) on the replication of DENV.
[0026] The present invention selects the dengue virus replicon system to screen anti-dengue virus drugs, specifically, selects the replicon system comprising Gaussialuciferase (Gluc), although its molecular structure is small but the signal intensity is high, and Gluc can be secreted in the culture medium, Facilitates detection...
Embodiment 2Cvb
[0037] Example 2 Cvb D does not affect DENV adsorption and invasion into host cells
[0038] Treat the cells with Cvb D for 24 hours in advance, and then add DENV for 1 hour at 4°C. At this time, the virus will only adsorb to the cell surface but not enter the cell interior. PBS will wash off unbound virus, and use quantitative PCR to detect the binding to the cell surface. amount of virus. At 37°C, the virus may be endocytosed by the cells in about 90 minutes. In this experiment, the dengue virus bound to the cell surface was treated with trypsin, and then the cells were lysed, and the DENV entering the cells was detected by quantitative PCR. result Figure 5 , there is no significant difference, so it shows that Cvb D does not affect dengue virus adsorption and cell invasion.
Embodiment 3C
[0039] Example 3Cvb D inhibits the replication of DENV
[0040] After DENV infects the body, it first proliferates in the capillary endothelial cells, and the dengue fever caused by the systemic capillary endothelial cells is mainly characterized by extensive swelling, enhanced permeability, and slight skin hemorrhage. Therefore, the present invention selects human umbilical vein endothelial cells (HUVEC) for related research.
[0041] After the HUVEC cells were infected with DENV-2, different concentrations of Cvb D were added for treatment, and the cells were collected after 24 hours of virus infection. Using Promega's Cell Titer- Luminescent Cell Viability Assay reagent was used to measure cell viability, and Cvb D at 2.5-20 μM did not affect cell growth ( Image 6 A). HUVEC cells were used to infect DENV-2, and then different concentrations of Cvb D were added, and the cells were collected after 24 hours. It was found by RT-PCR and Western blot detection that Cvb D si...
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