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The method of ibdv serum-free microcarrier suspension culture proliferation

A technology of serum-free culture and serum-free medium, which is applied in the field of bioengineering, can solve problems such as inability to carry out large-scale preparation, and achieve the effects of increasing virus titer, improving medium components, and strong immunogenicity

Inactive Publication Date: 2011-12-07
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the production method of inoculating IBDV in spinner bottles is widely used in China, and the culture volume is about 0.5L, which cannot be used for large-scale preparation.

Method used

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  • The method of ibdv serum-free microcarrier suspension culture proliferation
  • The method of ibdv serum-free microcarrier suspension culture proliferation
  • The method of ibdv serum-free microcarrier suspension culture proliferation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Domesticated Vero cells were cultured in serum-free microcarrier suspension

[0023] Acclimatization of Vero cells in serum-free microcarrier suspension culture includes the following steps:

[0024] 1. Domestication of Vero cells

[0025] The Vero cells were revived, and the sequential adaptation method was used to adapt the cells. During cell adaptation, at 1 x 10 5 cell / mL cell concentration for passage.

[0026] Detailed steps of the sequential adaptation method: Vero cells were revived and cultured in DMEM with 10% FCS, and then passaged in 199 medium with 5%, 2%, 1%, and 0.5% FCS in sequence, gradually reducing the serum concentration, and finally at passage 15 The cells were transferred to serum-free IVT culture, and the cell morphology was observed, such as figure 1 shown.

[0027] The acclimatized cells were slender and slender than the Vero cells cultured in 10% serum; after the cells were subcultured in 1 minute and 3 passages, the cells ...

Embodiment 2

[0036] Example 2 IBDV (chicken infectious bursal virus) propagates on Vero cells cultured in serum-free microcarrier suspension

[0037] 1. Initial Vero cells were cultured in suspension on serum-free microcarriers

[0038] Serum-free cultured Vero cells were digested with 0.25% trypsin, resuspended in fresh IVT medium, centrifuged at 1500rpm for 10min, and discarded the supernatant. Cells were divided into 3~4×10 5 The initial density of cell / mL was resuspended in virus maintenance solution: serum-free medium IVT containing 0.25% whey protein hydrolyzate, and entered into the reactor for culture.

[0039] Initial cell culture conditions: pH value 7.1, suspension culture stirring speed 35rpm, dissolved oxygen 30%, temperature 37°C;

[0040] 2. Proliferation of IBDV virus

[0041] After the cells are attached to the microcarrier, the IBDV virus is inoculated, and the sticky ball rate of the microcarrier reaches 40-50% when the virus is inoculated;

[0042] Virus inocu...

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Abstract

The invention provides a method for IBDV serum-free microcarrier suspension culture proliferation, comprising the following steps: 1) Acclimatization of Vero cells in serum-free culture; 2) IBDV virus propagation: inoculate the acclimatized cells into 0.25% whey The serum-free medium of the protein hydrolyzate was inoculated with the IBDV virus when the cells adhered to the microcarrier and started to grow, and the virus was harvested after 90 hours. The present invention domesticates the Vero cells introduced from ATCC, cultivates them with reduced serum, finally obtains Vero cells under the condition of serum-free microcarrier culture, improves the culture medium composition during IBDV virus proliferation at the same time, reduces the influence of exogenous viruses, and improves IBDV Virus titers under serum-free microcarrier culture conditions. The titer of the IBDV virus propagated by optimizing the virus propagation medium is higher than that of the IBDV propagated by the unoptimized medium and the IBDV virus titer propagated by chicken embryo fibroblasts; it is equivalent to the IBDV cultured by Vero cells with serum; the immunogenicity experiment shows that : The virus has strong immunogenicity and is suitable for production as a vaccine.

Description

technical field [0001] The invention relates to the propagation of chicken infectious bursal virus on the suspension culture Vero of serum-free medium and microcarrier, and belongs to the technical field of bioengineering. Background technique [0002] Chicken infectious bursal disease (Infectious Bursal Disease, IBD) is a highly contagious disease of chickens caused by infectious bursal virus (IBDV). In young chickens aged 3 to 6 weeks, the virus multiplies rapidly in the lymphocytes of the bursa, damages the B lymphocytes of the bursa, and causes severe immunosuppression. After 1980, the disease was introduced into my country and broke out in a large area and continued to be popular, which seriously affected the development of my country's chicken industry. Currently, vaccination is generally used to prevent the disease. [0003] At present, most of the IBDV vaccines produced in China use chicken embryo cultured virus or chicken embryo fibroblasts cultured with serum. Ad...

Claims

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Application Information

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IPC IPC(8): C12N7/00
Inventor 吴培培冯磊王伟峰侯继波
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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