Seed selection of high-immunogenicity rabies virus fixed strain and application thereof in vaccine development
A rabies virus, high-immunity technology, applied in the direction of antiviral agents, viruses/phages, medical preparations containing active ingredients, etc. and other problems, to achieve the effect of high immunogenicity, good immune protection effect and high safety
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Embodiment 1
[0040] Virus strain G 0 preparation of
[0041] Suspend the dry powder of lyophilized rabies virus fixed strain 4aG in water for injection, dilute it 10 times with maintenance solution A (M199 medium containing 0.5% bovine blood albumin, pH7.0-8.0), and inoculate guinea pigs weighing 45-50g in the brain cavity , 0.1ml / rat; 96 hours later, the brains were aseptically removed, and the three brains were combined, and 12ml of maintenance solution A was added, and the homogenizer was used to make a homogenate; high-speed centrifugation (10000×G, 30 minutes, 4°C) to remove the precipitate; The supernatant was placed on a sucrose layer with a density gradient of 30-60% for ultracentrifugation (80000-100000×G, 240 minutes, 4°C), and the solutions were collected in groups, and the sucrose content and antigen content of each component solution were measured; Combine the components with a sucrose concentration of 45-55%, put them into a dialysis bag with a molecular cut-off of 30K, dial...
Embodiment 2
[0043] Virus strain GV 1 preparation of
[0044] For Vero cells adherently cultured for 24-72 hours, digest with trypsin and use maintenance solution B (M199 medium with 0.01%-0.1% trypsin, 0.1-1mM EDTA, 0.5-1% bovine serum albumin, pH7.0- 8.0) Made with a density of 4×10 6 The cell suspension of cells / ml; Add the G prepared in Example 1 in an amount of 1:1-1:100 by virus: cells 0 Virus suspension, place at 37°C, shake regularly; inoculate cell culture flasks after 30 minutes, the inoculum size is 5×10 5 -2×10 6 cells / ml, add culture solution A (M199 medium containing 0.5% bovine albumin, pH7.0-8.0), and culture at 37°C; remove culture solution A after 48 hours and add maintenance solution B (containing 0.5 % bovine serum albumin M199 medium, pH7.8), adherent culture under the condition of 30-34°C; harvest the culture medium after 144 hours, carry out sucrose density gradient ultracentrifugation purification and concentrated virus according to the same method as in Example...
Embodiment 3
[0046] Virus strain GNV 1 preparation of
[0047] The GV prepared in embodiment 2 1 Virus suspension, inoculated nude mice with a body weight of 8-10g in the brain cavity, and the inoculated dose was 20ul / only, and the brain was aseptically taken after 72-96 hours, and the virus suspension was prepared according to the same method as in Example 1, numbered GNV 1 . The virus titer determined by the mouse brain cavity method was 8.02 LogLD 50 / ml.
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