Method for producing porcine pseudorabies gE gene deletion virus vaccine by using micro-carrier suspension culture cells

A technology of suspension culture and porcine pseudorabies, which is applied in the direction of microcarriers, biochemical equipment and methods, viruses, etc., can solve the problems of high price and increased cost of virus vaccines, increase the specific surface area, promote cell adherent growth, reduce The effect of production costs

Active Publication Date: 2017-09-26
广州渔跃生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commercialized microcarriers used in cell culture to produce virus vaccines in my country mainly include: CytodexⅠ, CytodexⅡ, CytodexⅢ of GE Com

Method used

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  • Method for producing porcine pseudorabies gE gene deletion virus vaccine by using micro-carrier suspension culture cells
  • Method for producing porcine pseudorabies gE gene deletion virus vaccine by using micro-carrier suspension culture cells
  • Method for producing porcine pseudorabies gE gene deletion virus vaccine by using micro-carrier suspension culture cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Chitosan microcarrier preparation and performance testing

[0035] Component (weight)

Group A

Group B

Group C

Group D

Chitosan (g)

200

200

200

200

Polyhydroxyalkanoate (g)

60

70

80

/

[0036] Preparation of group A chitosan microcarriers:

[0037] Get chitosan 200g and be dissolved in the dilute acetic acid of 2% (v / v), magnetic stirring makes the chitosan sol solution that concentration is 12% (m / v), adds 3% (m / v) ammonium acetate, Stir evenly, add 60g of polyhydroxyalkanoate, ultrasonic treatment for 25min, then seal in a high-pressure steam boiler, treat at 250°C for 3.5h, cool to room temperature, centrifuge to collect the precipitate, wash with deionized water 3 times, at 60°C Under vacuum drying 5h, obtain the chitosan microsphere that the surface contains polyhydroxyalkanoate, soak the chitosan microsphere that obtains in 1% (m / v) glutaraldehyde solution, react 2h under normal temperat...

Embodiment 2

[0045] Embodiment 2 The effect of chitosan microcarrier on the growth of BHK-21 cells

[0046] The chitosan microcarriers prepared in Example 1A and Group D and the commercially available Cytodex I microcarriers were used to carry out suspension culture of BHK21 cells, specifically:

[0047] (1) Take the BHK21 cell species out of the liquid nitrogen tank for recovery, add it to DMEM medium containing 10% newborn bovine serum, and store it at 37°C, 5% CO 2 cultured until it grows into a good single layer, then digested with an appropriate amount of trypsin digestion solution containing 0.02% EDTA, digested at 37°C for 6min, and adjusted the cell density to 4.5×10 with DMEM medium containing 10% newborn bovine serum. 5 cell suspension per mL;

[0048] (2) Inoculate the cell suspension obtained in step (1) into a stirred bioreactor, using 1.5% (m / v) FBS, 1% (m / v) D-glucosamine, 3.5mmol / L glutamine The DMEM culture fluid of amide, 6% (m / v) growth promoter, 1.0% (v / v) double anti...

Embodiment 3

[0053] Example 3 Production of porcine pseudorabies gE gene deletion virus by microcarrier suspension culture BHK-21 cells

[0054] (1) Take the BHK21 cell species out of the liquid nitrogen tank for recovery, add it to DMEM medium containing 10% newborn bovine serum, and store it at 37°C, 5% CO 2 cultured until it grows into a good single layer, then digested with an appropriate amount of trypsin digestion solution containing 0.02% EDTA, digested at 37°C for 6min, and adjusted the cell density to 4.5×10 with DMEM medium containing 10% newborn bovine serum. 5 cell suspension per mL;

[0055] (2) Inoculate the cell suspension obtained in step (1) into a stirred bioreactor, using 1.5% (m / v) FBS, 1% (m / v) D-glucosamine, 3.5mmol / L glutamine The DMEM culture fluid of amide, 6% (m / v) growth promoter, 1.0% (v / v) double antibody carries out microcarrier suspension culture, wherein, described growth promoter is made of keratan sulfate oligosaccharide and active mineral The yeast poly...

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Abstract

The invention relates to a method for producing a porcine pseudorabies gE gene deletion virus vaccines by using a BHK cell line. The method comprises the following steps: resuscitating BHK21 cells and performing micro-carrier culture by adopting a low-serum culture medium; then replacing the low-serum culture medium with a cell maintenance solution, performing continuous perfusion culture, inoculating when the cell density is maintained at (1.5-3)*10<7> pcs/ml and continuously harvesting virus liquid for six days after 48 hours; concentrating, inactivating and sterilizing the harvested virus liquid to prepare the finished product vaccine. The method provided by the invention has the advantages that the vaccine is high in potency and stronger in mmunogenicity and can achieve a good immune effect without adding an immunoenhancer and promote the secretion of neutralizing antibodies in vivo after immunization; the immune protective rate achieves 100 percent, so that the vaccine efficacy evaluation criteria is fully reached.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a method for producing porcine pseudorabies gE gene-deleted virus vaccine by microcarrier suspension culture of BHK cells. Background technique [0002] Porcine pseudorabies virus (PRV) belongs to herpesviridae aherpesviridae subfamily vesicular virus genus porcine herpesvirus type I, and pigs are the only natural host of the virus, causing pseudorabies in pigs. The disease is mostly outbreaks in pig herds, mainly harming sows, causing abortion or vertical transmission to piglets, resulting in a large number of deaths of newborn piglets, which has brought huge economic losses to the pig industry in my country and even the world. [0003] Currently, there is no effective drug for the treatment of porcine pseudorabies, so vaccination has become the main measure to control the occurrence and prevalence of the disease. Currently widely used in the ...

Claims

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Application Information

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IPC IPC(8): A61K39/245A61P31/22C12N5/071C12N5/02
CPCA61K39/12A61K2039/5252A61K2039/552A61K2039/575C12N5/0686C12N2501/90C12N2501/998C12N2531/00C12N2710/16734
Inventor 严悌昆张毓金黄淑芬敖艳华
Owner 广州渔跃生物技术有限公司
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