Epidemic encephalitis B and enterovirus 71 (EV71) combined vaccine
A combined vaccine, EV71 technology, applied in the field of preventive Japanese encephalitis EV71 combined vaccine and its preparation, can solve problems such as difficult to achieve 100% safety and increase compliance
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Embodiment 1
[0038] Example 1 Bioreactor perfusion culture and harvest of Japanese encephalitis virus
[0039] Take Vero cells that have grown to a monolayer cultured in a 15L flask or cell factory, trypsinize them into single cells, count the cells, and press 3 × 10 5 Cells / ml were inoculated into a bioreactor containing 5g / L Cytodex-1 microcarriers, and the culture medium was DMEM medium containing 10% newborn bovine serum, NaHCO 3 Adjust pH to 7.2, culture at 37°C, start 2 volumes / day perfusion culture after culturing for 2 days, the cell growth medium in the perfusion culture stage is DMEM medium containing 2% newborn calf serum, NaHCO 3 The pH was adjusted to 7.2, and the maintenance medium was changed on the 6th day. The maintenance medium was 199 medium containing 0.1% human serum albumin and NaHCO. 3 Adjust pH to 7.4. According to the optimized screening of the optimal inoculation concentration, culture conditions and harvest time of the virus, we selected the virus cell ratio to...
Embodiment 2
[0040] Example 2 Virus inactivation verification
[0041] 1) JE vaccine inactivation verification
[0042] Verification method: Inoculate 8 mice with 12-14 grams of the test product in the brain, each 0.03ml, and at the same time inoculate 0.5ml intraperitoneally, which is the first generation; 7 days later, 3 mice of the first generation were killed, and the brains were taken for After 7 days, 3 mice of the second generation were killed, and 6 mice were inoculated with 12-14 g per hour in the same method, which was the second generation. Three generations. After inoculation, observation was performed daily for 14 days, and those who died within 3 days were not counted (the number of dead animals should not exceed 20% of the total number of animals used in the experiment). Except for the sacrifice and non-specific death after inoculation, all mice in each generation survived as qualified.
[0043] 2) EV71 vaccine inactivation verification
[0044] Take the inactivated virus...
Embodiment 3
[0045] Example 3 titer determination
[0046] 1) Immunize Kunming mice with JE EV71 combined vaccine and reference vaccine according to the prescribed procedures, stimulate their immune response to produce antibodies, collect blood after expiration, separate serum, neutralize with Japanese encephalitis virus, and neutralize by plaque reduction Experiment to calculate the titer of the vaccine to be tested.
[0047] 2) SD rats were immunized with JE EV71 combined vaccine, immunized twice with an interval of 7 days, blood was collected on the 14th day after primary immunization, serum was separated, neutralized with EV71 virus, and the antibody titer was detected by microneutralization test.
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