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Anti-rabbit D-type pasteurella multocida egg yolk antibody and preparation method thereof

A pasteurella, egg yolk antibody technology, applied in the preparation method of peptides, antibodies, antibacterial drugs, etc., can solve the problem of no cross immune protection effect of serotype D strains, and achieve the prevention of type D Pasteurella multocida. Infected rabbits, simple operation, high titer effect

Pending Publication Date: 2021-06-11
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently only an inactivated vaccine against serotype A in China
The vaccine can only prevent and control serotype A strains, but has no cross-immunity protection against serotype D strains

Method used

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  • Anti-rabbit D-type pasteurella multocida egg yolk antibody and preparation method thereof
  • Anti-rabbit D-type pasteurella multocida egg yolk antibody and preparation method thereof
  • Anti-rabbit D-type pasteurella multocida egg yolk antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Isolation, Identification and Screening of Rabbit Pasteurella Type D

[0021] 1) Aseptically collect lung samples from dead rabbits with respiratory diseases, inoculate them on brain-heart infusion agar plates containing 5% (V / V) defibrinated sheep blood, incubate them upside down at 37°C for 24-48 hours, pick round, smooth, Translucent, non-hemolyzed, small colonies with a diameter of less than 1.0 mm and neat edges, were continuously purified for 3 times on a 5% (V / V) defibrinated sheep blood brain-heart infusion agar plate to obtain a purified culture.

[0022] 2) Take the above-mentioned pure culture, spread it evenly on a glass slide, and observe the bacterial morphology with a microscope after Gram staining. Pick Gram-negative small bacilli.

[0023] 3) Extract the genomic DNA from the pure cultures screened in steps 1) and 2) respectively, and use kmt1 gene and wxya Gene primers were used to amplify the isolates kmt1 gene and wxya Gene. kmt1 The upstre...

Embodiment 2

[0027] Preparation and Safety Test of Rabbit Pasteurella Type D Inactivated Vaccine

[0028] 1. Preparation of Rabbit Pasteurella Type D Inactivated Vaccine

[0029] Streak inoculate Pasteurella multocida PmD01 on a brain-heart infusion agar plate containing 5% (V / V) defibrated sheep blood, pick a single colony and transfer it to the brain-heart infusion liquid medium, 37°C 180rpm Expand the culture for 24 hours as the seed solution. Inoculate the seed liquid into the brain heart infusion liquid medium at 0.1% (V / V), incubate at 37°C 180rpm for 24 hours, add formaldehyde to a final volume concentration of 0.2%, inactivate at 37°C 100rpm for 24 hours, that is inactivation Bacterial liquid, the content of antigen in the inactivated bacterial liquid at this time is 4.0×10 10 -2.0×10 11 CFU / mL. Take 20 mL and 50 mL of inactivated bacteria liquid, add 20 mL of complete Freund's adjuvant and 50 mL of incomplete Freund's adjuvant respectively, and mix well to obtain 40 mL of com...

Embodiment 3

[0036] The preparation of yolk antibody comprises the following steps:

[0037] 1. Take 30 healthy egg-laying Jinghong No. 1 laying hens and inject 1 mL (0.5 mL per point) into the neck subcutaneously at 2 points with inactivated Pasteurella multocida rabbit type D vaccine (the antigen content is 1.0×10 11 CFU / mL), a total of 4 times of immunization, the time interval between two adjacent immunizations is 14 days, the first immunization with complete Freund's adjuvant inactivated vaccine (prepared in Example 2), the second immunization, the third Inactivate the vaccine with incomplete Freund's adjuvant (prepared in Example 2) for the immunization and the 4th immunization, start collecting eggs 7 days after the 4th immunization, and collect highly immune eggs with yolk antibody titer higher than 1:64;

[0038] 2. Wash the high-free eggs with water, dry them, disinfect the surface with 75% alcohol, take out the egg yolk, pass through 3 layers of sterilized gauze to remove the v...

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Abstract

The invention discloses an anti-rabbit D-type pasteurella multocida egg yolk antibody and a preparation method thereof. The preparation method comprises the following steps: (1) immunizing a healthy laying hen by using a rabbit D-type pasteurella multocida inactivated vaccine to obtain a hyper-immune egg; (2) taking the egg yolk of the hyper-immune egg, removing a yolk membrane and uniformly stirring the yolk; (3) adding sterilized double distilled water into the egg yolk, uniformly stirring to obtain egg yolk liquid, adjusting the pH value of the egg yolk liquid, removing lipid, and then adding ammonium sulfate to precipitate an egg yolk antibody; and (4) dialyzing, inactivating and filtering the product obtained in the step (3) to obtain the egg yolk antibody. The preparation method of the anti-rabbit D-type pasteurella multocida egg yolk antibody disclosed by the invention is simple in steps and simple and convenient to operate, and the egg yolk antibody prepared by the method has a very good prevention effect on infection of rabbit D-type pasteurella multocida.

Description

technical field [0001] The invention relates to an anti-rabbit Pasteurella multocida type D egg yolk antibody and a preparation method thereof, belonging to the field of veterinary biotechnology. Background technique [0002] Pasteurellosis in rabbits is an infectious disease caused by Pasteurella multocida infection in rabbits. It is distributed all over the world and is one of the important diseases affecting the development of rabbit industry. The serotypes of Pasteurella multocida are complex. According to the capsular antigens of the strains, they can be divided into serotypes A, B, D, E, and F. There is no cross-protection between the serotypes. Epidemiological surveys showed that serum A and D strains were the main epidemic strains in domestic rabbit populations in recent years. The most effective means of prevention and control of pasteurellosis in rabbits is vaccination. However, there are currently only inactivated vaccines against serotype A in China. The vacci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C07K16/02C07K1/36C07K1/34C07K1/30A61K39/40A61P31/04
CPCC07K16/1242C07K16/02A61P31/04C07K2317/11A61K2039/505A61K2039/552Y02A50/30
Inventor 王锦祥谢喜平孙世坤陈岩锋陈冬金桑雷
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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