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A kind of method for suspension culturing porcine circovirus type 2 in bioreactor

A bioreactor, porcine circovirus technology, applied in biochemical equipment and methods, viruses, microorganisms, etc., can solve the problems of high downstream purification process requirements, hidden dangers of virus vaccine quality, affecting enterprise production efficiency, etc., to reduce production. Cost, virus titer and high expression protein content, the effect of avoiding exogenous contamination

Active Publication Date: 2020-06-30
GUANGZHOU QIZHI BIOLOGICAL ENG EQUIP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention has the advantages of convenient operation, small operating space, precise control of process parameters, etc., but there are significant differences between different batches of serum, poor amplification reproducibility, high requirements for downstream purification processes, and complex components in serum are prone to lead to viruses and other diseases. Vaccine quality risks, and the global supply of serum is tightening, greatly affecting the production efficiency of enterprises
[0005] At the same time, as the above two Chinese patent applications CN103614344A and CN101289655 both use PK15 cells to cultivate porcine circovirus type 2 in vitro, but porcine circovirus type 2 is difficult to cultivate in vitro PK15 cells, the proliferative ability is weak, and the titer Lower, cause the porcine circovirus type 2 virus of prior art and its vaccine to exist titer lower, the defective that cost is higher

Method used

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  • A kind of method for suspension culturing porcine circovirus type 2 in bioreactor
  • A kind of method for suspension culturing porcine circovirus type 2 in bioreactor
  • A kind of method for suspension culturing porcine circovirus type 2 in bioreactor

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Embodiment 1

[0034] Embodiment 1 A kind of method of bioreactor suspension culture porcine circovirus type 2

[0035] Embodiment 1 method steps of the present invention are as follows:

[0036] S1. Preparation of cells: Take the 6th generation of Sf9 cells in a shake flask, add 2 / 3 of the nutrient solution of the shake flask volume, the inoculation amount is 0.1% of the nutrient solution volume, set the rotation speed at 30rpm, and the temperature at 26°C, and rotate for 48 hours ;

[0037] S2. Bioreactor culture: when the number of cells reaches 2×10 5 Inoculate Sf9 cells in a bioreactor for suspension culture at cells / ml, set the stirring speed at 80 rpm, temperature at 26°C, pH 6.15, dissolved oxygen at 50%, and continuously culture for 72 hours;

[0038] S3. Virus cultivation: when the cell count reaches 5×10 6 When cells / ml, inoculate the virus seed, the inoculum amount of the virus seed is 0.25% of the total volume of the cells and the culture medium, supplement the nutrient sol...

Embodiment 2

[0041] Embodiment 2 A kind of method of bioreactor suspension culture porcine circovirus type 2

[0042] Embodiment 2 method steps of the present invention are as follows:

[0043] S1. Preparation of cells: Take the 6th generation of Sf9 cells in a shake flask, add 2 / 3 of the nutrient solution of the shake flask volume, the inoculation amount is 0.05% of the nutrient solution volume, set the rotation speed at 20rpm, the temperature at 25°C, and rotate for 45 hours ;

[0044] S2. Bioreactor culture: when the number of cells reaches 2×10 5 Inoculate Sf9 cells in a bioreactor for suspension culture at cells / ml, set the stirring speed at 100 rpm, temperature at 25°C, pH 5.8, dissolved oxygen at 60%, and continuously culture for 70 hours;

[0045] S3. Virus cultivation: when the cell count reaches 5×10 6 When cells / ml, inoculate the virus seeds, the inoculum amount of the virus seeds is 0.08% of the total volume of the cells and the culture medium, supplement the nutrient solu...

Embodiment 3

[0048] Embodiment 3 A kind of method of bioreactor suspension culture porcine circovirus type 2

[0049] Embodiment 3 method steps of the present invention are as follows:

[0050] S1. Preparation of cells: Take the 6th generation of Sf9 cells in a shake flask, add 2 / 3 of the nutrient solution of the shake flask volume, the inoculation amount is 0.2% of the nutrient solution volume, set the rotation speed at 50rpm, and the temperature at 28°C, and rotate for 50 hours ; S2, bioreactor culture: when the number of cells reaches 2×10 5 Inoculate Sf9 cells in a bioreactor for suspension culture at cells / ml, set the stirring speed at 50 rpm, temperature at 28°C, pH 6.5, dissolved oxygen at 80%, and continuously culture for 75 hours;

[0051] S3. Virus cultivation: when the cell count reaches 5×10 6 When cells / ml, inoculate the virus seeds, the inoculum amount of the virus seeds is 0.35% of the total volume of the cells and the culture medium, supplement the nutrient solution of ...

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Abstract

The invention belongs to the technical field of veterinary biological products, and in particular relates to a method for culturing porcine circovirus type 2 in suspension in a bioreactor. The method steps include: S1, cell preparation; S2, bioreactor cultivation; S3, virus culture; S4, virus harvest. Compared with the prior art, the method of the present invention has the advantages of large production scale, high efficiency, low cost, etc., and at the same time adopts serum-free medium to cultivate the virus, and the obtained virus titer is high.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a method for suspension culturing porcine circovirus type 2 in a bioreactor. Background technique [0002] Porcine circovirus (PCV) was first discovered by Tischer in the 1970s. Porcine circovirus is divided into two genotypes, porcine circovirus type 1 and porcine circovirus type 2, of which porcine circovirus type 2 is pathogenic It can cause porcine multisystemic failure syndrome (PMWS) after weaning, and it is also the main cause of porcine dermatitis and nephrotic syndrome (PDNS) in finishing pigs. ), hyperplastic and necrotizing pneumonia (PNP) are closely related. Because the disease is an immunosuppressive disease, it can cause severe secondary infection and endanger the health of pigs. It is another major disease that seriously endangers the global pig industry after porcine reproductive respiratory syndrome. The infection rate of cyc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00
CPCC12N7/00C12N2750/10051
Inventor 尹顺义靳松松赵辉蒋东阳伍活镰任政华
Owner GUANGZHOU QIZHI BIOLOGICAL ENG EQUIP
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