Method for subculturing and purifying mumps virus by dilution method
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A technology of mumps virus and dilution method, which is applied in the direction of biochemical equipment and methods, virus, recovery/purification, etc., can solve the problems of no specific method, traditional technology, and the activity of virus seeds is not particularly ideal, and achieve virus drop Increased speed and good stability
Pending Publication Date: 2022-05-10
SINOVAC DALIAN VACCINE TECH
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Problems solved by technology
[0002] In the process of vaccine research and development and production, especially for attenuated live vaccine products, the quality of virus seeds is particularly critical, such as chickenpox, mumps, measles and other vaccines, the virus seeds of which are prepared by continuous attenuation. Due to the relatively long history of the isolation and cultivation of virus species, the establishment of seed banks, and the relatively traditional technology, some virus species are not particularly ideal in terms of activity.
There is no specific method for the purification of existing poisonous species, and there is no relevant scientific and systematic experimental research
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Embodiment 1
[0022] A method for passaging and purifying mumps virus by dilution method, mainly comprising the following steps:
[0023] (1) Cell preparation:
[0024] Incubate SPF eggs, take chicken embryos aged 9-11 days, after disinfection, dissect and collect embryo bodies, cut them into pieces, add trypsin solution to digest until fluffy and transparent, blow repeatedly with a straw, filter, collect the filtrate to obtain a cell suspension, and cell The suspension was inoculated into a T25 cell culture flask, and cultured statically at 36.5°C for about 48 hours. After the cells grew to a monolayer, a monolayer of chicken embryo cells was obtained for virus inoculation.
[0025] (2) Virus dilution
[0026] Mumps virus (purchased from ATCC) was serially diluted, and the diluent was Earle's solution containing newborn bovine serum and 0.2% hydrolyzed milk protein, and the dilution gradient was 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 . The specific compone...
Embodiment 2
[0041] Study on Stability of Purified Mumps Virus
[0042] The stability test includes the following steps: store the mumps virus seeds below -70°C, and store them at 0 days, 1 month, 3 months, 6 months, 12 months, 18 months, 24 months, 30 months, respectively. Take it out at one month and check the virus titer. The stability results of the purified mumps virus are shown in the table below.
[0043] Table 2. Stability of purified mumps virus
[0044]
[0045] It can be seen from the above table that the mumps virus obtained by the method of the present invention has good stability.
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Abstract
The invention discloses a method for subculturing and purifying mumps virus by a dilution method, and belongs to the technical field of attenuated live vaccines. Comprising the following steps: gradually diluting mumps virus mother liquor to 10 <-1 >-10 <-9 > dilution, respectively inoculating obtained virus solutions with 10 <-5 >-10 <-9 > dilution to a cell matrix, culturing at 32-37.0 DEG C, harvesting virus culture supernatant with cell fusion lesion, and detecting virus titer; diluting the harvested viruses step by step again until the dilution degree is 10 <-1 > to 10 <-9 >; culturing at 32-37.0 DEG C, harvesting a virus culture supernatant with obvious cell fusion lesion, and detecting virus titer; and if the virus titer is lower than 6.5 lgCCID50/ml, repeating the operation steps until high-quality virus seeds are obtained. The virus titer of the virus seeds obtained by the method is obviously improved, the stability is good, the gene is not changed, and the effect of improving the quality of attenuated live vaccines is obvious.
Description
technical field [0001] The invention belongs to the technical field of attenuated live vaccines, in particular to a method for passaging and purifying mumps virus by a dilution method. Background technique [0002] In the process of vaccine research and development and production, especially for attenuated live vaccine products, the quality of virus seeds is particularly critical, such as chickenpox, mumps, measles and other vaccines, the virus seeds of which are prepared by continuous attenuation. Due to the relatively long history of the isolation and cultivation of virus species, the establishment of seed banks, and the relatively traditional technology, some virus species are not particularly ideal in terms of activity. There is no specific method for the purification of existing poisonous species, and there is no relevant scientific and systematic experimental research. Contents of the invention [0003] In order to solve the above technical problems, the present inv...
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