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Live duck plague vaccine and preparation method thereof

A technology for live and live duck plague vaccines, applied in biochemical equipment and methods, vaccines, veterinary vaccines, etc., can solve the problems of decreased live virus content of vaccines, no use of transportation and storage, and increased storage costs.

Active Publication Date: 2021-09-14
广东永顺生物制药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, studies have shown that infectious bursal virus, chicken Marek's virus, and Newcastle disease virus can all grow well on DF-1 cells, but there is no report on the method of cultivating duck plague virus in DF-1 cell lines
[0004] The preservation method of live duck plague vaccine in the prior art is to store it below -15°C for 24 months. It takes at least 2 months for the vaccine from production, inspection to delivery, and then from dealer to user. Once the storage temperature exceeds The "cold chain" of -15°C will cause the content of live virus in the vaccine to drop, affect the effect of the vaccine, and even cause immune failure
At the same time, this preservation condition increases the preservation cost to a certain extent, and does not utilize transportation and storage

Method used

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  • Live duck plague vaccine and preparation method thereof
  • Live duck plague vaccine and preparation method thereof
  • Live duck plague vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Preparation of duck plague virus liquid

[0056] After DF-1 cells were cultured to a single layer according to conventional methods, an inoculation group and a blank control group were established. The experimental group was inoculated with DPV F65 generation poison (the duck plague virus liquid DPV F65 generation poison that was weakened by CEF cells passaged) according to the volume ratio of 5% (ie 300ul), and the virus content was 10 7.5 TCID 50 / ml), at the same time, add 5% (ie 300ul) SPF chicken embryo allantoic fluid of 9-10 days old according to the volume of cell fluid 6ml, and place it at 37°C and 5% CO 2 Under the condition of adsorption for 60 minutes, the cell bottle was gently shaken every 15 minutes during the period, and the maintenance solution was added after the adsorption was completed. At the same time, the control group inoculated with CEF cells and DF-1 cells was set up as a blank control group. Viruses are the same.

[0057] Observe ...

Embodiment 2

[0060] Embodiment 2 Determination of virus content

[0061] The DPV F80~F100 virus fluids of different generations harvested in Example 1 were serially diluted 10 times with serum-free M199, and 4 appropriate dilutions were taken to inoculate the 96-well micro-cell culture plate of CEF cells that had grown into a good monolayer. , each dilution was inoculated into 6 wells, 0.1ml per well, and a normal cell control was set at the same time. Set at 37°C, containing 5% CO 2 After adsorption in the incubator for 1 hour, add 0.1ml of M199 maintenance solution containing 4% serum to each well, observe for 120-144 hours, and record the number of cytopathic (CPE) wells. Calculate TCID by Reed-Muench method 50 , the results showed that the virus content of DPV F80~F100 generations was stable, and the virus content reached 10 7.4 ~10 8.0 TCID 50 / mL.

[0062] Table 1 Detection results of virus content

[0063]

Embodiment 3

[0064] Example 3 Specific identification of DPV F80, F86, and F95 generations produced by DF-1 cells

[0065] The F80, F86, and F95 generation virus fluids harvested in Example 1 were diluted with serum-free M199 to contain 100 TCID 50 / 0.1ml, mixed with the same amount of anti-duck plague virus specific serum, neutralized at 37°C for 1 hour, inoculated 6 cell wells (48-well cell plate) that had grown into a CEF monolayer, 0.2ml per well, and set There were 6 wells each for virus control and normal cell control. Set at 37°C, containing 5% CO 2 After culturing in an incubator and observing for 120-144 hours, the results showed that both the neutralization group and the normal cell control group had no cytopathic changes, and the cells in the virus control group showed cytopathic changes.

[0066] The preparation method of the anti-duck plague virus specific serum used in the present embodiment is:

[0067] (1) Preparation method of anti-duck plague virus specific serum

[0...

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Abstract

The invention provides a live duck plague vaccine and a preparation method thereof, belonging to the technical field of biological product preparation. The invention uses the chicken embryo fibroblast cell line DF‑1 as the host of the duck plague virus to produce the duck plague virus liquid, and then adds a heat-resistant protective agent to the prepared virus liquid to prepare a duck plague virus live vaccine. The present invention adopts DF-1 to cultivate duck plague virus, which avoids the potential safety hazard of exogenous virus contamination in the CEF cell culture virus, and because DF-1 cells can be taken at any time, time and effort are saved, and the culture process of duck plague virus is greatly reduced The produced duck plague live vaccine has good safety, high immune efficacy, and complete immune protection against the virulent attack of duck plague. An improved heat-resistant protective agent is added to the vaccine, so that the duck plague live vaccine can be used Stored at 2-8°C for 24 months, the virus content and titer will not decrease, thereby reducing storage requirements, facilitating storage and transportation of live duck plague vaccines, and reducing production costs.

Description

technical field [0001] The invention relates to the technical field of biological product preparation, in particular to a live duck plague vaccine and a preparation method thereof. Background technique [0002] Duck plague (Duck Plague, DP) is an acute septic infectious disease caused by duck plague virus (Duck Plague Virus, DPV) in ducks, geese and various geese. DPV spreads rapidly, is widespread, and has a very high morbidity and mortality rate, causing huge economic losses to the duck industry. It is one of the most serious infectious diseases that endanger the duck industry. Immunization against duck plague virus vaccine is one of the effective measures to prevent duck plague virus. The production of duck plague virus vaccine requires the cultivation of a large amount of duck plague virus liquid. Chicken embryo fibroblast primary cells (CEF) are the cells currently used to propagate duck plague virus in my country. Although the country has stipulated that the chicken ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/245A61K9/19A61K47/42A61K47/26A61K47/18A61P31/22C12N7/00
CPCA61K9/0019A61K9/19A61K39/12A61K47/183A61K47/26A61K47/42A61K2039/5254A61K2039/552A61P31/22C12N7/00C12N2710/16334C12N2710/16351
Inventor 林德锐胡美容齐冬梅郑铁锁李嘉爱
Owner 广东永顺生物制药股份有限公司
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