Serum-free full suspension culture method of duck tempusu viruses and application of duck tempusu viruses in vaccines

A technology of duck tambusu virus and serum-free culture solution, which is applied in the directions of vaccines, viruses, antiviral agents, etc., can solve the problem that cannot fully satisfy the large-scale low-cost culture of duck tambusu virus and the preparation of duck tambusu virus vaccine. Difficulties, economic losses in the duck industry, etc., to reduce the risk of virus spread, clear and controllable ingredients, and save operating costs

Inactive Publication Date: 2020-11-24
WENS FOOD GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The clinical symptoms of ducks infected with the virus are leg numbness, decreased feed intake, sudden drop in egg production, and neurological symptoms. The earliest onset age of ducklings is 7 days old, and the incidence is high during the laying period. Commercial ducks and laying ducks develop after recovery. Bad and poor recovery of egg production rate, causing huge economic losses to the duck industry
At present, duck embryos, duck embryo fibroblasts, and Vero cells are mainly used for in vitro culture in the existing technology, but they cannot fully meet the needs of large-scale and low-cost culture of duck Tembusu virus.
In addition, in the prior art, the titer of virus liquid obtained by cultivating duck Tembusu virus is low, which has caused great difficulties for the preparation of duck Tembusu virus vaccine

Method used

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  • Serum-free full suspension culture method of duck tempusu viruses and application of duck tempusu viruses in vaccines
  • Serum-free full suspension culture method of duck tempusu viruses and application of duck tempusu viruses in vaccines

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Embodiment 1

[0033] A serum-free full suspension culture method of duck Tembusu virus, comprising the following steps:

[0034] Step 1. Strain adaptation: The duck Tembusu virus strain was continuously passaged in the suspension BHK-21 cell line for 5 generations, and the TCID was measured using the adherent BHK-21 cells 50 , with a poison price of 10 7.67 TCID 50 / mL, save the virus liquid as the basic strain;

[0035] Step 2. Cell culture: Suspended BHK-21 cells were resuscitated and scaled up in a bioreactor with serum-free culture medium; the culture conditions were temperature 37°C, pH 7.0, DO 60%, stirring speed 70r / min, and cell density up to 1.0 ×10 7 cells / mL, add serum-free medium and dilute the cell density to 2.0×10 6 cells / mL to continue culturing.

[0036] Step 3, virus inoculation: after the cell culture volume reaches 5L, when the density of suspended BHK-21 cells reaches 1.0×10 7 When cells / mL, use serum-free medium to dilute the cells to a cell density of 6.0×10 5 ...

Embodiment 2

[0043] A serum-free full suspension culture method of duck Tembusu virus, comprising the following steps:

[0044] Step 1. Strain adaptation: The duck Tembusu virus strain was continuously passaged in the suspension BHK-21 cell line for 6 generations, and the TCID was measured using the adherent BHK-21 cells 50 , with a poison price of 10 8.33 TCID 50 / mL, save the virus liquid as the basic strain;

[0045] Step 2. Cell culture: Suspended BHK-21 cells were resuscitated and scaled up in a bioreactor using serum-free culture medium; the culture conditions were temperature 37°C, pH 7.1, DO 50%, stirring speed 60r / min, and cell density up to 9.0 ×106 cells / mL, add serum-free medium and dilute the cell density to 1.0×10 6 cells / mL to continue culturing.

[0046] Step 3, virus inoculation: after the cell culture volume reaches 5L, when the density of suspended BHK-21 cells reaches 9.0×10 6 When cells / mL, use serum-free medium to dilute the cells to a cell density of 2.0×10 6 c...

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Abstract

The present invention relates to a serum-free full suspension culture method of duck tempusu viruses and an application of the duck tempusu viruses in vaccines. The serum-free full suspension culturemethod comprises the following steps: a duck tambusu virus strain is continuously passaged to virus-adapted cells in a suspension BHK-21 cell line and the adapted virus strain with the highest virus content is selected as a basic virus strain; after the suspension BHK-21 cells are resuscitated, a serum-free culture solution is used in a bioreactor for amplified culture; the suspension BHK-21 cellsafter the amplified culture are inoculated with the basic virus strain for continuous culture; glucose monitoring is performed every day after virus inoculation, when glucose in the culture solutionis 1-5 mmol/L, a nutrition package is supplemented; 48 hours after the virus inoculation, samples are taken every other 6 hours to calculate cell viability and when the cell viability is less than 50%, the virus culture solution is harvested; and the virus culture solution is repeatedly frozen, thawed, centrifuged and stored. The suspension BHK-21 cell culture is used, the viruses have good adaptability, virus content is high, operating cost is low, product quality is stable, and the method is easy to operate and has feasibility of large-scale industrial production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a serum-free full-suspension culture method of duck Tembusu virus and its application in vaccines. Background technique [0002] Duck Tembusu virus (DTMUV) is a single-stranded positive-sense RNA virus that can cause duck egg production decline, belonging to the Flavivirus genus of the family Flaviviridae. The clinical symptoms of ducks infected with the virus are paralysis of feet, decreased feed intake, sudden drop in egg production and neurological symptoms. The earliest age of onset of ducklings is 7 days old, and the incidence is high during the laying period. Commercial ducks and laying ducks develop after recovery. Bad and poor recovery of egg production rate, causing huge economic losses to the duck industry. At present, duck embryos, duck embryo fibroblasts, and Vero cells are mainly used for in vitro culture in the prior art, but they cannot fully meet the need...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N5/071A61K39/12A61P31/14
CPCC12N7/00C12N5/0686A61K39/12A61P31/14C12N2770/24151C12N2770/24134A61K2039/5252A61K2039/552
Inventor 蒋天华周庆丰严专强温燕兰靖宇
Owner WENS FOOD GRP CO LTD
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