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Method for culturing duck plague virus

A duck plague virus and culture method technology, applied in the direction of virus, virus/bacteriophage, double-stranded DNA virus, etc., can solve the problems of growing cells, lesions, and methods without duck plague virus, and achieve the effect of good growth and improved safety

Active Publication Date: 2019-01-18
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

Studies have shown that infectious bursal virus, chicken Marek's virus, and Newcastle disease virus can all grow well on DF-1 cells, but common duck plague virus strains cannot directly grow on DF-1 cells and produce cells. Lesions, there is no report on the method of cultivating duck plague virus in DF-1 cell line

Method used

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  • Method for culturing duck plague virus
  • Method for culturing duck plague virus
  • Method for culturing duck plague virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1 The establishment of duck plague virus culture method

[0061] 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 inoculated group was inoculated with DPV F65 generation poison (the duck plague virus liquid DPV F65 generation poison of CEF cells passaged weakened by volume ratio 5% (ie 300ul), 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. After the adsorption was completed, the maintenance solution was added. At the same time, the CEF cell solution was set up to inoculate DF-1 cells as a blank control group. Viruses are the same.

[0062] Observe the cell pathology every day, if...

Embodiment 2

[0065] Embodiment 2 Determination of virus content

[0066] The DPV F80~F100 different generations virus liquids harvested in Example 1 were serially diluted 10 times with serum-free M199, and 4 dilutions were taken to inoculate 96-well micro-cell culture plates of CEF cells that had grown into a good monolayer, respectively. Inoculate 6 wells for each dilution, 0.1 ml per well, and set normal cell control 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.

[0067] Table 1 Detection results of virus content

[0068]

Embodiment 3

[0069] Example 3 Specific identification of DPV F80, F86, and F95 generations produced by DF-1 cells

[0070] 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 into 6 cell wells (48-well cell plate) that had grown into a single layer of CEF, 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.

[0071] The preparation method of the anti-duck plague virus specific serum used in the present embodiment is:

[0072] (1) Preparation method of anti-duck plague virus specifi...

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Abstract

The invention provides a method for culturing duck plague virus, which belongs to the technical field of virus culture. The invention adopts chicken embryo fibroblast cell line DF-Duck plague virus (DPV) was adapted to DF by inoculating DPV with overdose and adding chicken embryo allantoic fluid (CEAF) in the cell fluid, and was passed blindly from generation to generation. 1 cell line and producecytopathic effect. The duck plague virus culture method of the invention is simple in operation, and DF-1 Duck plague virus (DPV) grew well in cell line culture, the virus content was 107.4-108.0 TCID 50 / ml, which was 0.25% higher than that in CEF cell culture. 0.75 titer, avoiding the potential safety hazard of exogenous virus contamination in CEF cell culture, and due to DF-After the establishment of the cell library, the stability and sustainability of the production are ensured, and the production cost of duck plague virus in the process of culture is greatly reduced, which can improve the safety and reduce the cost, and the economic benefit is remarkable.

Description

technical field [0001] The invention relates to the technical field of biological product preparation, in particular to the technical field of virus cultivation, in particular to a method for cultivating duck plague virus using chicken embryo fibroblast cell line DF-1 as a host. 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. Inoculation of duck plague virus vaccines among ducks is one of the effective measures to prevent duck plague. The production of duck plague virus vaccines requires the cultivation of a large amount of duck plague virus liquid. Chicken embryo fibroblast primary cells (CEF) are the cells currently used...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00
CPCC12N7/00C12N2710/16051
Inventor 胡美容林德锐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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