Japanese encephalitis virus suspension culture method

A Japanese encephalitis virus, suspension culture technology, applied in the direction of virus, virus/phage, positive-sense single-stranded RNA virus, etc., can solve the problems of large area of ​​JEV virus, low production of JEV virus, unstable quality, etc. Large production scale, small footprint, and low cell damage

Active Publication Date: 2017-06-09
TIANJIN RINGPU BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims at the technical defect of prior art, provides a kind of Japanese encephalitis virus suspension culture method, to solve the technical problem of low output, unstable quality of JEV virus production method of prior art
[0005] Another technical problem to be solved by the present invention is that the large-scale cultivation method of JEV virus in the prior art occupies a larger technical problem and the production cost is higher

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] (1) Microcarrier preparation: Take 60g of microcarrier cytodex1 and add it to the bioreactor, add PBS (pH value 7.0) at a ratio of 200ml / g and soak for 2.5h, remove the PBS, add PBS again, stir at 10r / min, soak for 2h, discard PBS.

[0034] Add PBS at a ratio of 100ml / g, 121°C, high pressure for 30min, discard PBS after cooling, wash with medium twice, add fresh medium, cool to 8°C, equilibrate for 10h, and set aside.

[0035] (2) The sterilized microcarriers were equilibrated to 28°C, and the culture medium was supplemented with a microcarrier concentration of 2g / L; the Vero cells were mixed with 5×10 5 Inoculate at the ratio of cell / L, stir and mix at 45r / min, let stand for 2h, repeat twice to make the cells fully distributed.

[0036] (3) After the cells and the microcarriers are mixed, adjust the temperature to 37°C, stir at 45r / min, cultivate with 50% air saturation and control the pH value of 7.2, and add glucose and glutamine feed (50 times concentrated solution...

Embodiment 2

[0039] (1) Microcarrier preparation: Take 90g of microcarrier cytodex1 and add it to the bioreactor, add PBS (pH value 7.2) at a ratio of 300ml / g and soak for 2h, remove PBS, add PBS again, stir at 15r / min, soak for 1h, discard PBS .

[0040] Add PBS at a ratio of 150ml / g, 121°C, high pressure for 30min, discard the PBS after cooling, wash with medium for 3 times, add fresh medium, cool to 6°C, equilibrate for 12h, and set aside.

[0041] (2) The sterilized microcarriers were equilibrated to 25°C, and the culture medium was supplemented with a microcarrier concentration of 3g / L; the Vero cells were 7×10 5 Inoculate at the ratio of cell / L, stir and mix at 50r / min, let stand for 2.5h, repeat 3 times, so that the cells are fully distributed.

[0042] (3) After the cells and the microcarriers are mixed, adjust the temperature to 37°C, stir at 55r / min, cultivate with 48% air saturation and control the pH value of 7.4, and add glucose and glutamine feed (70 times concentrated solut...

Embodiment 3

[0045] (1) Microcarrier preparation: Take 75g of microcarrier cytodex1 and add it to the bioreactor, add PBS (pH value 7.2) at a ratio of 250ml / g and soak for 3h, remove the PBS, add PBS again, stir at 12r / min, soak for 1.5h, discard PBS.

[0046] Add PBS at a ratio of 130ml / g, 121°C, high pressure for 30min, discard the PBS after cooling, wash with medium for 3 times, add fresh medium, cool to 4°C, equilibrate for 12h, and set aside.

[0047] (2) The sterilized microcarriers were equilibrated to 30°C, the culture medium was supplemented, and the concentration of the microcarriers was 2.5g / L; the vero cells were 5.5×10 5 Inoculate at the ratio of cell / L, stir and mix at 50r / min, let stand for 2.5h, repeat 3 times, so that the cells are fully distributed.

[0048] (3) After the cells and the microcarriers are mixed, adjust the temperature to 37°C, stir at 50r / min, cultivate with 50% air saturation and control the pH value of 7.1, and add glucose and glutamine feed (60 times conc...

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PUM

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Abstract

The invention provides a Japanese encephalitis virus suspension culture method. The technical scheme is developed on the basis of an animal cell microcarrier culture technology. As a microcarrier provides a larger surface area for the growth and reproduction of a monolayer adherent cell, a homogenic suspension culture system is provided for cell growth, the microcarrier is utilized to serve as physical support for cell colonization, and together with optimization of culture conditions, the cell and virus culture densities are improved. The Japanese encephalitis virus suspension culture method realizes large-scale culture of the Japanese encephalitis virus and provides lots of high-quality virus antigens. Moreover, the shortcomings that traditional spinner cultivation animal primary cell production is low in single-batch yield, great in batch difference, unstable in product quality and high in production cost and pollution probability are overcome. Besides, the Japanese encephalitis virus suspension culture method can achieve continuous culture, is small in land occupation, large in production scale and small in cell damage, and the shortcomings that a traditional process is large in land occupation, discontinuous in culture and high in production cost are overcome.

Description

technical field [0001] The invention relates to the technical field of veterinary biological products, further relates to the preparation technology of antigens, in particular to a Japanese encephalitis virus suspension culture method. Background technique [0002] Japanese encephalitis is a zoonotic insect-borne infectious disease caused by Japanese encephalitis virus (JEV) belonging to the family Flaviviridae and the genus Flavivirus. Humans, pigs, and equine animals are all susceptible to the virus, and pigs are the most harmful. The main manifestations of Japanese encephalitis in pregnant sows are abortion, stillbirth, weak piglets, orchitis in boars, etc., which often cause serious economic losses to the pig industry. [0003] The manufacturing process of traditional inactivated vaccines is simple, easy to store, and safe, but the injection dose is large, requiring repeated injections, and the immune effect is poorer than that of attenuated live vaccines, so it is rare...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/02
CPCC12N7/00C12N2770/24251
Inventor 王尚尚吕茂杰王甜苏建东杨保收
Owner TIANJIN RINGPU BIO TECH
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