Method for preparing avian influenza virus and inactivated vaccine thereof with Vero passage cells
A technology of avian influenza virus and subcultured cells, which is applied in the field of preparation of avian influenza virus inactivated vaccines and inactivated vaccines using VERO subcultured cells. The effect of production costs
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Embodiment 1
[0034] Embodiment 1 utilizes VERO passage cell to produce influenza virus inactivated vaccine
[0035] 1. Preparation of immobilized trypsin by chitosan nanoparticle embedding method
[0036] (1) Weigh chitosan with a degree of deacetylation of 85%, dissolve it with 1% acetic acid to make a chitosan solution with a concentration of 0.5-2 mg / mL, and pass it through a 0.22 μm filter. Weigh trypsin, dissolve it in deionized water to make a trypsin solution with a concentration of 500 μg / mL, and pass it through a 0.22 μm filter. Weigh sodium tripolyphosphate, dissolve it with deionized water to make a sodium tripolyphosphate solution with a concentration of 1.0-2.0 mg / mL, and pass it through a 0.22 μm filter.
[0037](2) Take 5 mL of chitosan solution, add 2.5-7.5 mL of 500 μg / mL trypsin solution dropwise, and stir magnetically for 3 minutes to obtain solution A;
[0038] (3) Solution A was magnetically stirred at 900-1300 r / min for 30 seconds at room temperature and under steri...
Embodiment 2
[0057] Embodiment 2 Utilize the vaccine produced by the method of the present invention to compare with the vaccine produced by chicken embryo
[0058] In order to verify the production performance of the new method, so while utilizing chicken embryos to prepare avian influenza inactivated vaccine (prepared according to the method described in Experimental Example 1.), VERO cells were also used to prepare 10 batches of inactivated vaccine (according to Example 1 Described method preparation.), now the comparison result of some of them indicators is summarized as follows, as described in table 1-6:
[0059] Table 1 Comparison of agglutination value of semi-finished erythrocytes by two production methods
[0060]
[0061] Table 2 Semi-finished product EID of two production methods 50 Compare (lg EID 50 )
[0062] batch 1 2 3 4 5 6 7 8 9 10 Embryo 7.4 7.5 7.7 8.0 8.2 8.4 8.0 6.8 6.9 8.1 cell seed...
experiment example 1
[0073] Experimental example 1 utilizes chicken embryo to produce influenza virus
[0074] 1. Preparation of H9 subtype avian influenza virus HY strain virus solution
[0075] (1) Inoculation: get the H9 subtype avian influenza virus HY strain as the seed virus for production, and properly dilute it with sterilized physiological saline (10 -3 ~10 -4 ), inoculate 0.1 mL into the allantoic cavity of each embryo, seal the pinhole after inoculation, and continue incubation at 36-37°C without turning the eggs.
[0076] (2) Incubation and observation of chicken embryos: After the chicken embryos were inoculated, the chicken embryos that died 48 hours ago were discarded. After that, the eggs were illuminated once every 6 hours, and the dead chicken embryos were taken out at any time until 96 hours, all the embryos were taken out, and placed in 2-8°C to cool for 4-24 hours.
[0077] (3) Harvesting of virus fluid: Take out the cooled chicken embryo, disinfect the air cell part with t...
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