A method for producing porcine pseudorabies virus antigen by whole suspension cell culture

A porcine pseudorabies virus and cell culture technology, applied in the directions of virus antigen components, biochemical equipment and methods, viruses, etc., can solve the problems of producing porcine pseudorabies virus antigens, difficult to achieve expansion culture, low antigen titers, etc. Conducive to cell absorption, improved product quality, and easy to expand the effect of culture

Active Publication Date: 2018-01-23
广东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

Among them, the antigen titer of the semi-finished product produced by the spin bottle process is low, only 10 5.0-6.8 TCID 50 / mL, high concentration is required, and the production cycle is long, it takes 10 days
However, although the microcarrier suspension culture process has been improved compared with the spinner bottle process, there is still a large room for improvement, and its production cycle still needs 6 to 7 days, and the cost is high, so it is difficult to achieve expanded culture.
At present, there is no relevant report on the production of porcine pseudorabies virus antigen by using the whole suspension cell culture technology

Method used

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  • A method for producing porcine pseudorabies virus antigen by whole suspension cell culture
  • A method for producing porcine pseudorabies virus antigen by whole suspension cell culture
  • A method for producing porcine pseudorabies virus antigen by whole suspension cell culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 Keratan sulfate oligosaccharide preparation and component analysis

[0036]

[0037] Preparation of Group A:

[0038] Take 50g of keratan sulfate, dissolve it in 300ml of 0.1M acetate buffer (pH6.0), add 25U of mixed enzyme, and degrade at 37°C for 24h. After the reaction, 2 times the volume of ethanol was added, stirred, left overnight at room temperature, centrifuged at 4000 rpm for 15 min, and the supernatant (supernatant A) was taken. Add 300ml of distilled water to the precipitate to dissolve, add 3 times the amount of ethanol, stir, leave at room temperature overnight, centrifuge at 4000rpm for 15min, and take the supernatant (supernatant B). Mix supernatant A and supernatant B, concentrate under reduced pressure, use a Bio-Gel-P-2 column (3.6╳134cm), use distilled water as a solvent, perform gel filtration, and freeze-dry the filtrate.

[0039] The preparation of keratan sulfate oligosaccharides in groups B-D refers to group A.

[0040] Composi...

Embodiment 2

[0044] The influence of embodiment 2 keratan sulfate oligosaccharides on BHK-21 cell growth

[0045] BHK21 cells were cultured with keratan sulfate oligosaccharides prepared in Example 1A-D groups, specifically:

[0046] Take the BHK21 cells out of the liquid nitrogen tank and add them to the DMEM medium containing 10% newborn calf serum, at 37°C, 5% CO 2 cultured until it grows into a good monolayer, then digested with an appropriate amount of trypsin digestion solution containing 0.02% EDTA, digested at 37°C for 6 min, and adjusted the cell density to 5×10 with DMEM medium containing 10% newborn bovine serum. 5 cells / mL of cell suspension, inoculate in shake flasks, add nutrient solution containing 2% newborn bovine serum at 37°C, 5% CO 2 The culture was carried out in the incubator, and the rotation speed was set to 150rpm / min for suspension culture until the cell density reached 4×10 6 each / mL, wherein, the nutrient solution containing 2% neonatal bovine serum contains 1...

Embodiment 3

[0050] Example 3 Production of Porcine Pseudorabies Virus Antigen by Full Suspension Cell Culture

[0051] (1) Resuscitate the seed cell BHK21, and use a shaker flask for suspension culture expansion: take the BHK21 cell species out of the liquid nitrogen tank for resuscitation, add it to DMEM medium containing 10% newborn calf serum, and store it at 37°C, 5% CO 2 cultured until it grows into a good monolayer, then digested with an appropriate amount of trypsin digestion solution containing 0.02% EDTA, digested at 37°C for 6 min, and adjusted the cell density to 5×10 with DMEM medium containing 10% newborn bovine serum. 5 cells / mL of cell suspension, inoculated in shake flasks, added nutrient solution containing 3% newborn bovine serum at 37°C, 5% CO 2 The culture was carried out in the incubator, and the rotation speed was set to 150rpm / min for suspension culture until the cell density reached 4×10 6 each / mL, wherein, the nutrient solution containing 3% neonatal bovine serum...

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Abstract

The invention relates to a method of producing a porcine pseudorabies virus antigen by a fully suspended cell culture. The method comprises the following steps: 1) recovering a BHK21 cell, performing suspension culture amplification with a shake flask, and inoculating the BHK21 cell to a reactor for suspended culture with a low-serum culture medium to obtain a suspension culture cell sap; 2) replacing the low-serum culture medium in the suspension culture cell sap with a serum-free culture medium for further cultivation, and performing exposure for virus culture; 3) continuously cultivating the BHK21 suspended cell for 96-144h till dead cell quantity reaches 90% above, harvesting the cell suspended liquid, freeze thawing, centrifugalizing and collecting the supernatant to obtain a virus liquid; and 4) concentrating, deactivating and degerming the virus liquid to obtain the porcine pseudorabies virus antigen. A porcine pseudorabies virus antigen vaccine obtained by the method is high in titer which is stabilized at 108.5TCID50 / mL above, and the porcine pseudorabies virus antigen vaccine is high in purity and stable in quality, and the product quality is remarkably improved.

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 virus antigen by whole suspension cell culture. Background technique [0002] In vitro cell culture production of virus is the most commonly used method at home and abroad. Among them, the traditional spinner bottle cell culture process has disadvantages such as low cell density, low virus yield, high production cost, and high labor intensity. It has gradually become an emerging cell suspension culture technology. replaced. Cell suspension culture technology makes up for many deficiencies of traditional spinner bottle culture technology with its advantages of automation, scale, cell culture simplification, safe operation, and batch-to-batch uniformity, and solves the problems of labor, cost, site, and production in the actual production process. cycle, raw material control, unstable quality and many o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K39/245A61P31/22
CPCA61K39/12A61K2039/552C12N7/00C12N2710/16734C12N2710/16751
Inventor 张毓金严悌昆敖艳华黄淑芬
Owner 广东渔跃生物技术有限公司
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