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Duck reovirus vaccine and preparation method thereof

A reovirus and vaccine technology, which is applied in the field of duck reovirus vaccine and its preparation, can solve the problems of economic loss in the duck industry, achieve stable quality, good immune effect, and great application prospects

Active Publication Date: 2019-05-07
广东渔跃生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no duck reovirus vaccine on the market, and there is no related drug to control the disease caused by the virus. The occurrence of duck reovirus infection often causes heavy economic losses to the duck industry.

Method used

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  • Duck reovirus vaccine and preparation method thereof
  • Duck reovirus vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Preparation:

[0026] (1) Digest and disperse BHK-21 cells with trypsin, add 5% newborn bovine serum in DMEM culture solution in a spinner bottle at 37°C, 5% CO 2 After culturing to a monolayer of cells, the original culture medium was discarded, and the cells were washed 3 times with serum-free DMEM medium for duck reovirus inoculation.

[0027] (2) Virus inoculation and cultivation: Inoculate the duck reovirus seed into the cells prepared in step ① according to the final volume of 1:100, and absorb the virus solution at 37°C for 30 minutes. DMEM culture solution of bovine serum at 37°C, 5% CO 2 Culture until the cells are terminated when lesions appear.

[0028] (3) Virus collection, concentration and purification and determination of virus content: the collected cell virus liquid was repeatedly frozen and thawed twice at -20°C, centrifuged at 4°C and 5000rpm for 10min, and the supernatant after centrifugation was used for ultrafiltration and concentration . After...

Embodiment 2

[0031] Preparation:

[0032] (1) Digest and disperse the BHK-21 cells with trypsin, add 10% newborn calf serum to the DMEM culture solution in a microcarrier reactor at 37°C and 5% CO 2 After culturing to a single layer of cells, the original culture medium was discarded, and the cells were washed 3 times with serum-free DMEM medium for duck reovirus inoculation.

[0033] (2) Virus inoculation and cultivation: inoculate the duck reovirus seed into the cells prepared in step ① according to the final volume of 1:1000, and absorb the virus liquid after 60 minutes at 37°C, and then use 2% newborn bovine Serum in DMEM medium at 37°C, 5% CO 2 Culture until the cells are terminated when lesions appear.

[0034] (3) Virus collection, concentration and purification: freeze-thaw the above-mentioned inoculated diseased cells, collect the supernatant after centrifugation to obtain the virus stock solution; concentrate the above-mentioned virus solution by ultrafiltration to obtain the s...

Embodiment 3

[0037] Preparation:

[0038] (1) Digest and disperse the BHK-21 cells with trypsin, add 7% newborn bovine serum to the DMEM culture solution in a microcarrier reactor at 37°C and 5% CO 2 After culturing to a single layer of cells, the original culture medium was discarded, and the cells were washed 3 times with serum-free DMEM culture medium containing 2 mM glutamine for duck reovirus inoculation.

[0039] (2) Virus inoculation and cultivation: Inoculate the duck reovirus seed into the cells prepared in step ① according to the final volume of 1:500, and absorb the virus liquid after 45 minutes at 37°C, and use 1.5% newborn bovine Serum in DMEM medium at 37°C, 5% CO 2 Culture until the cells are terminated when lesions appear.

[0040] (3) Virus collection, concentration and purification: freeze-thaw the above-mentioned inoculated diseased cells, collect the supernatant after centrifugation to obtain the virus stock solution; concentrate the above-mentioned virus solution by ...

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Abstract

The invention discloses a duck reovirus vaccine. The inactivated vaccine is obtained by inoculating duck reovirus virus seed on continuous cell line BHK-21 cells. A Reed-Muench method is used to determine that the virus titer can reach 107.0-107.5 TCID50 / 0.1mL. The stable production and high titer of antigen are the most important factors in the preparation of vaccines. The duck reovirus proliferation is carried out by using BHK-21 cells, so that the virus titer is 10 times or above as high as that of a conventional duck embryo method and a primary cell method. If the virus proliferation is carried out by using a bioreactor, the virus titer can reach 100-time level. The duck reovirus inactivated vaccine disclosed by the invention has the advantages of high yield, stable quality, good immune effect and huge application prospect, and immune ducks can generate a high-level serum neutralizing antibody.

Description

technical field [0001] The invention relates to a duck reovirus vaccine and a preparation method thereof. Background technique [0002] Since 1997 in my country, a disease characterized by white necrotic spots on duck liver has broken out in provinces such as Guangdong, Guangxi, Zhejiang, Fujian, Henan and Hubei. The mortality rate of this disease is very high when encountering mixed infection or stress. Usually, the disease mainly occurs in Muscovy ducks aged 7 to 45 days, the pathogenicity rate is as high as 20% to 90%, and the mortality rate is 10 to 50%. Infected ducks often manifested as abdominal sea, soft feet, appendages or toe joints swelling in varying degrees, and ducks that were resistant became stiff ducks; necrosis of liver, spleen, pancreas, kidneys and intestines could be seen in necrosis. This new disease is characterized by irregular necrosis of the liver, bleeding spots / points, and hemorrhage in the myocardium and supracavitary bursa. It is commonly know...

Claims

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Application Information

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IPC IPC(8): A61K39/15A61P31/14C12N7/00C12N7/02
Inventor 张毓金严悌昆谢秉超黄淑芬张桂平
Owner 广东渔跃生物技术有限公司
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