Application of Tripterygium in the Preparation of Drugs for Inhibiting the Replication of Marek's Disease Virus
A technology for Marek's disease and tripterygne, applied in antiviral agents, pharmaceutical formulations, plant raw materials, etc., to achieve enhanced effects, broad application prospects, good economic value and research significance
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Embodiment 1
[0074] Tripterine inhibits the enzymatic activity of UL36 encoded by MDV.
[0075] Enzyme reaction system: 1.5nM UL36 enzyme in 100μL reaction system, 1× reaction buffer (50mM HEPESpH7.8, 0.1mg / mL BSA, 0.5mM EDTA, 1mM DTT), add reaction fluorescent substrate Ub-AMC,37 Incubate at °C, and measure the change in fluorescence intensity on a fluorescence spectrophotometer. The reaction system with 2 μM tripterine was added as "inhibitor group", and the reaction system without tripterine was added with the same amount of solvent DMSO as "no inhibitor group". The result is as figure 1 , UL36 in the "no inhibitor group" hydrolyzed Ub-AMC normally and produced a fluorescent signal, while the activity of UL36 in the "inhibitor group" was inhibited, and the fluorescent signal did not increase. This result shows that tripterine inhibits the enzymatic activity of UL36 encoded by MDV.
Embodiment 2
[0077] Preparation of CEF cells and infection with MDV:
[0078] 1. Take SPF chicken embryos aged 9-11 days, wipe the chicken embryos with alcohol cotton, and wipe and disinfect from the air chamber to the surrounding. Then put the chicken embryo into the ultra-clean workbench with the air cell facing up. Carefully tap the air cell with autoclaved forceps and clip off the eggshell.
[0079] 2. Use new sterile tweezers to uncover the allantoic chorion, and discard the egg shell and vitelline membrane. Hold the head of the embryo with sterilized forceps and gently transfer it to the plate.
[0080] 3. Fix the head with sterilized tweezers, cut off the head, limbs, and organs with sterilized scissors, and rinse repeatedly with sterilized PBS until colorless.
[0081] 4. Use sterilized scissors to cut the tissue pieces until there are no large particles of tissue, add 8 mL of sterilized PBS to wash, let stand for 1 min, and discard the suspension mixed with red blood cells and ...
Embodiment 3
[0098] Tripteryglide inhibits the replication of MDV and the occurrence of MD at the level of chicken MD disease model:
[0099] The specific operation is: take 1-day-old SPF chickens, according to the MDV titer of 1000PFU, the way of attacking the virus is intraperitoneal inoculation, and these challenged chickens are treated with intraperitoneal injection of tripterine every day, and the experiment is divided into 0.01, 0.05, 0.1, 0.15 , 0.2, 0.3 and 0.5, a total of 7 groups, no drug treatment, only injected with the same amount of DMSO as the control group. 10 chickens per group. 20 days after the challenge, blood was collected from the blood vessels under the wings, T lymphocytes were separated, and the quantitative analysis of the MDV gene copy number was carried out according to the number of 200,000 cells, as shown in Figure 7 . The MD incidence rate of each group chicken in 50 days after the statistical challenge, such as Figure 8 . 50 days after the challenge or...
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