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Preparation method of duck reovirus yolk antibody

A technology for reovirus and egg yolk antibody is applied in the field of preparation of duck reovirus egg yolk antibody, which can solve the problems of long time and complicated operation process, and achieve the effects of short time consumption, simplified operation process and simple steps.

Active Publication Date: 2019-12-20
广东渔跃生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The operation process is complex and time-consuming

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  • Preparation method of duck reovirus yolk antibody

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preparation example Construction

[0017] (1) Preparation of passaged cell line LMH cells: LMH cells were digested with 0.025% EDTA-trypsin to disperse and passaged, and cultured in DMEM containing 5-10% newborn bovine serum and appropriate amount of double antibody at 37°C, 5% CO 2 Cultivated in an incubator;

[0018] (2) Seed virus propagation: inoculate duck reovirus seed into the LMH cells prepared in step 1 at a final volume of 1:200, absorb the virus solution at 37°C for 30 minutes, and discard the virus solution after using 1-2 % newborn bovine serum, appropriate amount of double antibody and 2mM glutamine in DMEM medium at 37°C, 5% CO 2 Cultivate to 58±2 hours;

[0019] (3) Virus collection, concentration and purification: when LMH cells have 80% cytopathic changes, the harvested cell venom is repeatedly frozen and thawed twice at -20°C, centrifuged at 5000rpm, 4°C for 10min, and the supernatant, i.e. the virus stock solution, is collected. The titer of the clear virus solution can be as high as 107.0...

Embodiment 1

[0028] Preparation:

[0029] (1) Use duck reovirus inactivated vaccine to immunize 100-day-old Hailan brown chickens to obtain high-immune eggs, specifically: subcutaneously inject 1 mL of duck reovirus inactivated vaccines into 100-day-old Hailan brown chickens Immunization, a total of 3 times of immunization, the time interval between two adjacent immunizations is 21 days, and the eggs are collected 5 days after the last immunization, that is, high-immunity eggs;

[0030] (2) Disinfect the surface of the high-free eggs with 75% alcohol, wash them with water, separate the egg yolks, pass through a 60-mesh nylon sieve to remove the vitelline membrane and frenulum egg yolks, and break them to obtain egg yolk liquid;

[0031] (3) The egg yolk liquid obtained in step (2) is mixed with purified water at a volume ratio of 1:1, and after stirring evenly, adding citric acid and 4% polyanion cellulose with a weight to volume ratio, after fully stirring, Place at 5°C for 6h, centrifug...

Embodiment 2

[0034] Preparation:

[0035] (1) Use duck reovirus inactivated vaccine to immunize 100-day-old Hailan brown chickens to obtain high-immune eggs, specifically: subcutaneously inject 1 mL of duck reovirus inactivated vaccines into 100-day-old Hailan brown chickens Immunization, a total of 3 times of immunization, the time interval between two adjacent immunizations is 21 days, and the eggs are collected 5 days after the last immunization, that is, high-immunity eggs;

[0036] (2) High-free eggs are surface-sterilized with 75% alcohol, and the yolk is separated, passed through a 60-mesh nylon sieve to remove the yolk membrane and frenulum, and then placed in a colloid mill to grind for 5 minutes to obtain egg yolk liquid;

[0037] (3) The egg yolk liquid obtained in step (2) is mixed with purified water at a volume ratio of 1:3, and after stirring evenly, adding citric acid and 1% polyanionic cellulose with a weight volume ratio, after fully stirring, Place at 1°C for 6h, centri...

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Abstract

The present invention discloses a preparation method of a duck reovirus yolk antibody. The preparation method comprises the following steps: (1) utilizing duck reovirus inactivated vaccine immune childbearing-age hens to obtain high immune eggs; (2) collecting egg yolk of the high immune eggs and after removing yolk membranes and ribbons, utilizing a colloid mill to conduct crushing to obtain eggyolk liquid; (3) mixing the egg yolk liquid obtained in the step (2) with purified water in a volume ratio of 1:(1-3), after conducting even stirring, adding citric acid with a final concentration weight-volume ratio of 1-3% of and polyanionic cellulose with a final concentration weight-volume ratio of 1-4%, after sufficient stirring, placing at low temperature for 3-6 his conducted, and centrifuging at 1-5 DEG C is conducted to collect supernatant; and (4) filtering and sterilizing the collected supernatant in the step (3) to obtain egg yolk antibody liquid. The disclosed preparation methodof the duck reovirus yolk antibody is simple in steps, high in efficiency and yield, low in addition and economical and applicable.

Description

technical field [0001] The invention relates to a preparation method of duck reovirus yolk antibody. Background technique [0002] Duck reovirus is currently a research hotspot in the world. It is pathogenic to ducks. The main lesions are spleen necrosis and liver hemorrhage. The disease can directly cause more than 30% of ducklings within 15 days of age in duck farms In addition, it is often co-infected with Limeris anatipestifer, Escherichia coli and duck astrovirus in clinical practice, resulting in obvious respiratory diseases, arthritis, severe egg production decline and deformed eggs in laying ducks. . Therefore, the disease has become one of the major diseases that seriously affect the production performance of duck farms. [0003] Yolk antibody (IgY) is the immunoglobulin in the blood of the mother poultry that is transferred to the yolk to have a protective effect on the offspring after the mother poultry is stimulated by a foreign antigen. It is a specific antibo...

Claims

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Application Information

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IPC IPC(8): C07K16/10C07K16/02
CPCC07K16/10C07K2317/11
Inventor 谢秉超黄淑芬严悌昆张毓金
Owner 广东渔跃生物技术有限公司
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