Method for producing porcine pseudorabies gE gene deletion virus inactivated vaccine

A porcine pseudorabies, gene deletion technology, applied in the fields of biochemical equipment and methods, vaccines, viruses, etc., can solve the problems of lack of in-depth research, hidden dangers of vaccine safety, and long virus liquid time, so as to shorten production time and improve immunity. effect, the effect of improving growth activity

Active Publication Date: 2017-09-08
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

[0005] At present, the research on porcine pseudorabies virus gene-deleted vaccines is mainly aimed at how to construct and screen strains of pseudorabies virus gene-deleted strains, but there is no in-depth study on the culture conditions of the corresponding strains, and most of them adopt conventional culture conditions , as the publication number is the Chinese patent application of CN 1244692 C " a kind of pseudorabies TK - / gE - / gI - Gene deletion marker live vaccine and preparation method", the constructed gene deletion pseudorabies virus strain was cultured in primary chicken embryo fibroblasts in a spinner bottle, and the cell maintenance solution used was DMEM solution containing 2% newborn bovine serum to prepare The virus titer in the virus fluid was only 10 6.3 TCID 50 / mL, and it takes a long time to harvest the virus liquid
At the same time, because its cell maintenance solution contains newborn bovine serum, it will cause serum residues, cause allergies, and bring certain safety hazards to the use of vaccines.

Method used

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  • Method for producing porcine pseudorabies gE gene deletion virus inactivated vaccine
  • Method for producing porcine pseudorabies gE gene deletion virus inactivated vaccine
  • Method for producing porcine pseudorabies gE gene deletion virus inactivated vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1 Keratan sulfate oligosaccharide preparation and component analysis

[0030]

[0031] Preparation of Group A:

[0032] 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). Supernatant A and supernatant B were mixed, concentrated under reduced pressure, and used Bio-Gel-P-2 column (3.6 ╳ 134cm), using distilled water as solvent, carry out gel filtration, and freeze-dry the filtrate to get final product.

[0033] The preparation of keratan sulfate oligosaccharides in groups B-D...

Embodiment 2

[0038] The influence of embodiment 2 keratan sulfate oligosaccharides on BHK-21 cell growth

[0039] BHK21 cells were cultured with keratan sulfate oligosaccharides prepared in Example 1A-D groups, specifically:

[0040] (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 monolayer, 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 2.26×10 with DMEM medium containing 10% newborn bovine serum. 5 cell suspension per mL;

[0041] (2) Put the cell suspension obtained in step (1) into a spinner bottle, add cell growth solution, the volume ratio of the cell suspension to the cell growth solution is 1:10, and the cell growth solution contains 4 mmol / L glutamine, 0.8mg / L dilinoleoylphosphatidylcholine, 4% (m / v) D-glucosamine, 2% (m...

Embodiment 3

[0047] Example 3 Production of porcine pseudorabies gE gene deletion virus with BHK-21 cells

[0048] (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 monolayer, and then digested with an appropriate amount of trypsin digestion solution containing 0.02% EDTA, digested at 37°C for 6 minutes, and adjusted the cell density to 3.24×10 with DMEM medium containing 10% newborn bovine serum. 5 cell suspension per mL;

[0049] (2) Put the cell suspension obtained in step (1) into a spinner bottle, add cell growth solution, the volume ratio of the cell suspension to the cell growth solution is 1:10, and the cell growth solution contains 6 mmol / L glutamine, 0.4mg / L dilinoleoylphosphatidylcholine, 4% (m / v) D-glucosamine, 3% (m / v) growth promoter, 1.0% (v / v) double antibody The DMEM culture solution was placed on a bottle spinner and c...

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Abstract

The invention relates to a method for producing a porcine pseudorabies gE gene deletion virus inactivated vaccine by using a BHK cell line. The method comprises the following steps: thawing BHK21 cells, inoculating the cells into a spinner bottle for culturing until the cells grow into sheets, inoculating a porcine pseudorabies gE gene deletion virus, continuously culturing, collecting the cell venom when cytopathy CPE reaches 90% or higher, and concentrating, inactivating and degerming the collected venom, thereby obtaining a finished product. The porcine pseudorabies gE gene deletion virus liquor collected by the vaccine production method provided by the invention has high virus content of more than or equal to 107.2TCID50 / mL and has relatively high immunogenicity, an excellent immune effect can be achieved without adding any immunopotentiator, in-vivo secretion of neutralizing antibodies can be promoted after immunization, the immune protective rate reaches 100%, and the vaccine efficacy evaluation standard is completely met.

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 inactivated vaccine with BHK cell line. 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 market is the nat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/245A61P31/22C12N5/071
CPCA61K39/12A61K2039/5252A61K2039/552C12N5/0686C12N2500/34C12N2501/998C12N2710/16734
Inventor 张毓金严悌昆黄淑芬敖艳华张桂平
Owner 广东渔跃生物技术有限公司
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