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Cyprinidae herpes virusIII-resisting chicken egg-yolk antibody as well as preparation method and application of antibody

A chicken egg yolk antibody, herpes virus technology, applied in the direction of antiviral agents, antiviral immunoglobulins, applications, etc., can solve the problems of high cost of cell inactivated vaccines, residual virulence of attenuated vaccines, etc. Recovery rate, the effect of good capsule integrity

Inactive Publication Date: 2014-09-10
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, literature has reported the prevention and treatment application of cyprinidae herpesvirus type 3 attenuated live vaccine and cell inactivated vaccine, but there are safety problems such as residual toxicity in the above-mentioned attenuated vaccine; 4 TCID 50 / mL), resulting in the high cost of the above-mentioned cell-inactivated vaccine; and the generation of specific antibodies after the above-mentioned vaccine immunization also takes 3 to 4 weeks, and the death of the host within 1 to 2 weeks after the cyprinidae herpesvirus type 3 infects koi is more than 80%, so there is a need for a safe, cheap and fast-acting preventive drug to control the transmission of cyprinidae herpesvirus type 3

Method used

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  • Cyprinidae herpes virusIII-resisting chicken egg-yolk antibody as well as preparation method and application of antibody
  • Cyprinidae herpes virusIII-resisting chicken egg-yolk antibody as well as preparation method and application of antibody
  • Cyprinidae herpes virusIII-resisting chicken egg-yolk antibody as well as preparation method and application of antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Purification of Cyprinidae Herpes Virus Type 3

[0048] Cyprinidae herpes virus type 3 (purchased from the American Culture Collection, strain number: VR-1592 TM ) Inoculate the carp brain cell line (CCB). When the cytopathic effect reaches 90%, collect the culture, which is the culture of cyprinidae herpes virus type 3 proliferated by the CCB cell line. The 600mL culture is repeatedly frozen and thawed for 3 times. Speed ​​centrifugation: the parameters of low speed centrifugation is 1000g, 4℃ low speed centrifugation for 20min, cell debris is removed, the supernatant is collected, the parameters of ultracentrifugation is 45000g, 4℃ ultracentrifugation for 60min, precipitation virus, negative staining, observe the purified virus by transmission electron microscope Capsule integrity ( figure 1 ). The purified virus used BCA protein quantitative method to detect the protein concentration as an antigen. The results showed that more than 12 mg of Cyprinidae herpes ...

Embodiment 2

[0049] Example 2 Immunization of laying hens with purified cyprinid herpes virus type 3 as antigen

[0050] Use purified cyprinid herpes virus type 3 as the antigen to immunize the chickens to open laying hens. The first immunization is 100μg / bird, two weeks later 200μg / bird for the second immunization, and after two weeks, 300μg / bird for the third immunization . Immunization injection sites are multi-point injections under the chest muscle and neck. Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for the second and third immunizations. 10 days after each immunization, the immune eggs are collected using indirect Enzyme-linked immunosorbent assay was used to determine the yolk antibody titer.

Embodiment 3

[0051] Example 3 Determination of yolk antibody titer by indirect enzyme-linked immunosorbent assay

[0052] Purified Cyprinus herpes virus type 3 is used to coat the ELISA plate, the coating buffer is sodium carbonate-sodium bicarbonate buffer (0.05M, pH9.6), and the coating concentration is determined to be 0.4μg / mL by square titration , Coated overnight at 4°C; after washing the plate, add a 2-fold dilution (1:3200~1:51200) of the egg yolk of the immunized layer, and set up a negative control (purified non-immunized egg yolk antibody) and a blank control ( PBS), incubate at 37°C for 1 hour; after washing the plate, add HRP-labeled rabbit anti-chicken IgG, the antibody dilution ratio is determined by the square matrix titration test to be 1:10000, incubate at 37°C for 1 hour; add TMB color developing solution after washing the plate, React at 37℃ for 15min, add 2M H 2 SO 4 Stop the reaction and read the OD 450 Value: P / N value greater than 2.1 is defined as positive, and when t...

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Abstract

The invention discloses a preparation method of a cyprinidae herpes virusIII-resisting chicken egg-yolk antibody. The method is characterized in that the cyprinidae herpes virus III is used as an antigen to immunize a chicken egg; after the titer of the chicken egg-yolk antibody is determined, the immunized egg is collected, the egg yolk is separated, and the egg yolk is purified to obtain the cyprinidae herpes virusIII-resisting chicken egg-yolk antibody. The invention also discloses a cyprinidae herpes virusIII-resisting chicken egg-yolk antibody prepared through the method, an application of the cyprinidae herpes virusIII-resisting chicken egg-yolk antibody in preparation of drugs for detecting, preventing or treating relevant diseases caused by infecting the cyprinidae herpes virus III and an application of the cyprinidae herpes virus-resisting type-III chicken egg-yolk antibody in preparation of feed additive for preventing and / or treating relevant diseases caused by infecting the cyprinidae herpes virus. The cyprinidae herpes virusIII-resistinng chicken egg-yolk antibody prepared through the method is high in purity, easy to industrialize and capable of remarkably reducing the dead rate of koi caused by the infection of the cyprinidae herpes virus III.

Description

Technical field [0001] The invention belongs to the technical field of anti-cypripid herpes virus antibodies, and specifically relates to an anti-cypripid herpes virus type 3 chicken egg yolk antibody, and a preparation method and application thereof. Background technique [0002] Cyprinidae herpesvirus type 3, also known as Koi herpes virus, specifically infects Koi and common carp, and then causes Koi herpes virus disease. This disease is a second-class animal disease and can cause 80% to 100% mortality of the host. , A serious threat to the safety of koi and carp breeding industry. Since the outbreak of the disease in the late 1990s, the disease has been widespread worldwide, causing huge economic losses. In 2002, an outbreak caused by Cyprinidae herpesvirus type 3 was also reported in mainland China. The production of carp in my country ranks first in the world, and koi as an ornamental animal is also widely cultivated. Therefore, the need for the prevention and control of C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C07K16/02G01N33/569A61K39/42A61P31/22A23K1/16A23K20/147
Inventor 刘振兴柯浩马艳平郝乐马江耀梁志凌
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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