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Method of utilizing stirred bioreactor to produce infectious Bursal disease virus

A bioreactor, stirring technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of low degree of automation, unstable quality, etc., achieve convenient operation, increase culture titer, and good The effect of operational flexibility

Inactive Publication Date: 2016-03-02
TIANJIN RINGPU BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the technical defects of the prior art, and provides a method for producing infectious bursal virus using a stirred bioreactor, so as to solve the technical problems of low automation and unstable quality of related methods in the prior art

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] (1) Choose a bioreactor as the means of cultivation: 10L German sartoriusstedim bioreactor.

[0040] (2) Select the microcarrier as the carrier for cell attachment and growth: microcarrier Cytodex1.

[0041] (3) Microcarrier cleaning and sterilization methods: 1) Weigh Cytodex1 microcarrier 3g / L, soak the microcarrier with 1LPBS at room temperature overnight; 2) wash with 1LPBS for 3 times; 3) soak the microcarrier with 1LPBS, and steam Sterilize for 30min.

[0042] (4) DF1 cells were selected as cells for seedling production.

[0043] (5) Passaging and culturing of cells for seedling preparation: the above-mentioned cells were digested and passaged with EDTA-trypsin (PBS containing 0.03% trypsin and 0.02% EDTA), and cultured with 90% DMEM / F12 culture medium and 10% newborn bovine serum. , 100IU / mL of penicillin sodium and streptomycin sulfate, and the pH value was adjusted to 7.2 for cell growth medium to continue culturing. The culture temperature is 37°C, and when...

Embodiment 2

[0049] (1) Choose a bioreactor as the means of cultivation: 10L German sartoriusstedim bioreactor bioreactor.

[0050] (2) Select the microcarrier as the carrier for cell attachment and growth: microcarrier Cytodex2.

[0051] (3) Microcarrier cleaning and sterilization methods: 1) Weigh Cytodex1 microcarrier 4g / L, soak the microcarrier with 1LPBS at room temperature overnight; 2) wash with 1LPBS for 3 times; 3) soak the microcarrier with 1LPBS, and steam Sterilize for 30min.

[0052] (4) DF1 cells were selected as cells for seedling production.

[0053] (5) Passaging and culturing of cells for seedling preparation: the above-mentioned cells were digested and passaged with EDTA-trypsin (PBS containing 0.03% trypsin and 0.02% EDTA), and cultured with 90% DMEM / F12 culture medium and 10% newborn bovine serum. , 100IU / mL of penicillin sodium and streptomycin sulfate, and the pH value was adjusted to 7.2 for cell growth medium to continue culturing. The culture temperature is 37°...

Embodiment 3

[0059] A method utilizing a stirred bioreactor to produce infectious bursal virus, comprising the following steps:

[0060] 1) In the stirred bioreactor, the sterilized microcarriers were mixed with the sterile cell growth solution in an amount of 3 g / L to obtain a mixed medium, and the density of DF1 cells inoculated therein was 0.6×10 6 cells / mL, DF1 cells were adsorbed and cultured on microcarriers, the culture temperature was 36°C, pH 7.2, dissolved oxygen was 40%, and the stirring speed was 30rpm;

[0061] 2) 48 hours after inoculating DF1 cells, discard the cell growth solution in the mixed culture medium, add cell maintenance solution, insert 0.3 MOI of infectious bursal virus, and continue to cultivate;

[0062] 3) 48 hours after virus inoculation, the virus liquid was harvested.

[0063] On the basis of the above technical solutions, the following conditions are met:

[0064] The total volume of the mixed medium in step 1) is 40% of the total volume of the stirred b...

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Abstract

The invention provides a method of utilizing a stirred bioreactor to produce an infectious Bursal disease virus. Bioreactor microcarrier cell culture technology is used to replace conventional roller bottle culture, so that the problems of low production efficiency, unstable product quality and low virus titer can be solved. On this basis, biological characteristics of the infectious Bursal disease virus and DF1 cells are combined, and proper conditions are matched from the perspectives of microcarrier adding amount, cell inoculation density, virus inoculation amount, cell density during virus inoculation, virus collection time and reactor operation parameters, so that virus culture efficiency is improved remarkably, and unit culture titer is improved by 10-100 times. In addition, compared with roller bottle culture, the method utilizing the bioreactor has the advantages that culture scale is large, and parameter control is comprehensive, so that systematic risk of being polluted is lowered, quality stability is improved, and the method has a wide application prospect.

Description

technical field [0001] The invention relates to the technical field of veterinary biological products, in particular to a method for producing infectious bursal virus using a stirred bioreactor. Background technique [0002] Infectious Bursal Disease (Infectious Bursal Disease, IBD) is a kind of highly contagious infectious disease caused by Infectious Bursal Disease Virus (Infectious Bursal Disease Virus, IBDV) of the family Avian biRNAvirus, which belongs to the genus Avian biRNAvirus. The disease mainly invades lymphatic tissues such as the bursa of Fabricius, the central organ of humoral immunity of chickens, and the harm to the poultry industry mainly includes two aspects. On the one hand, the disease itself can cause clinical morbidity or death of 3-week-old or older chicks; on the other hand, it can produce long-term immunosuppression on the chicken body, causing secondary diseases (mainly chicken Marek, Newcastle disease, E. coli disease) , Salmonellosis, etc.), res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12R1/93
Inventor 孟沙沙李亚杰杨保收付旭彬梁武
Owner TIANJIN RINGPU BIO TECH
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