Optimized technical method for proliferation of Coxsackie virus A16

A technology of coxsackie virus and process method, applied in the optimized process field of amplifying coxsackie virus A16 type, achieving good cost performance and application prospect, reducing purification difficulty, high repeatability and stability

Inactive Publication Date: 2016-12-14
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hydrolyzed milk protein is a safe and effective medium additive, which has been widely used, but there is no report on its use in the process of amplifying CVA16 virus

Method used

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  • Optimized technical method for proliferation of Coxsackie virus A16
  • Optimized technical method for proliferation of Coxsackie virus A16
  • Optimized technical method for proliferation of Coxsackie virus A16

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Bioreactor: Hangzhou Anpu Bioengineering Co., Ltd. 10L torrent bioreactor with a working volume of 8L, model: AP20sc;

[0031] Microcarrier: 150 grams of Fibra disk paper carrier (Hangzhou Anpu Bioengineering Co., Ltd.);

[0032] Virus: Coxsackie virus CVA16 type, Zhejiang strain, subtype B1; inoculation MOI value is about 0.5;

[0033] Bioreactor cell culture: 150g of sterilized Fibra disk carrier has been added to the 10L reactor, soaked overnight in phosphate buffer solution PBS with pH 7.2, after discarding, add 8L of cell growth medium, and inoculate VERO cells, the inoculation amount is (2.1±0.3)×10 9 cells to be cultured. The culture parameters are set as follows: pH 7.1-7.5, temperature 37° C., dissolved oxygen 50-80%, stirring speed 40-55 revolutions per minute (rpm). Samples were taken regularly every day to measure glucose consumption, so as to estimate cell growth. Under stable conditions, glucose consumption was linearly related to cell density. The med...

Embodiment 2

[0040] Bioreactor: NBS 5L cell reactor, model: Bioflo310 (NBS Company, USA), working volume is 4L;

[0041] Microcarrier: 150 grams of Fibra disk paper carrier (NBS company in the United States);

[0042] Virus: Coxsackie virus A16 (CVA16) subtype C4 (Zhejiang strain); the MOI of inoculation is about 0.5;

[0043] Cell culture: 150g of sterilized Fibra disk carrier has been added to the 5L reactor, soaked overnight in phosphate buffer solution PBS with pH 7.2, discarded and added to 4L of cell growth medium as a working volume, inoculated with VERO cells, inoculum volume 2.3×10 9 cells to be cultured. The culture parameters are set as: pH 7.1-7.5, temperature 37° C., dissolved oxygen 50-80%, stirring speed 40-50 rpm. Samples were taken regularly every day to measure glucose consumption, so as to estimate cell growth. Under stable conditions, glucose consumption was linearly related to cell density. The liquid change mode adopts continuous perfusion, 3L for 24-48 hours, 3L ...

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Abstract

The invention discloses an optimized technical method for proliferation of Coxsackie virus A16. The method includes: taking flaky fibers as carriers, proliferating African green monkey kidney cells in a bioreactor, adopting an 8v / v% serum containing DMEM (Dulbecco modified eagle medium) culture medium in a cell culture phase, changing into a serum-free DMEM culture medium in 5-6 days of cell culture, adopting a 3-5% serum containing DMEM culture medium after virus inoculation and adsorption, adopting a 0.5w / v% lactoalbumin hydrolysate added serum-free culture medium in a virus harvesting phase, statically re-adsorbing for 3h, continuing to culture, and replenishing 2.0g / L glucose about every 24h. The method is high in repeatability and stability, high virus titer can be achieved, and subsequent purification difficulty can be lowered while requirements of biological products are met. In addition, the method is high in application value and applicable to bioreactor based Coxsackie virus A16 proliferation with flaky fiber carriers.

Description

technical field [0001] The invention relates to the technical field of virus culture, in particular to an optimized process method for amplifying Coxsackie virus A16 type. Background technique [0002] Coxsackievirus A16 (CVA16) is one of the main pathogens of HFMD. It has been found that HFMD caused by CVA16 infection can also cause more serious complications. Therefore, it is necessary to develop CVA16 virus-related vaccines. [0003] At present, the large-scale culture technology of green monkey kidney (Vero) cells is relatively mature, but there are few reports on the cultivation of CVA16 virus. At present, some progress has been made in cultivating CVA16 with Cytodex1 as a carrier, but it has not been reported on the fibradisk carrier. . [0004] To establish and improve this virus culture process, we must first have a comprehensive understanding and mastery of the physiological and growth characteristics of VERO cells, and have a more in-depth and detailed grasp of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00
CPCC12N7/00C12N2770/32051
Inventor 李兰娟陈科达张严峻吴晓鑫
Owner ZHEJIANG UNIV
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