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Completely inactivated vaccine for preventing and treating spinach frog skin rot and preparation method thereof

A technology for inactivated vaccines and rotten skin diseases, applied in skin diseases, bacterial antigen components, antibacterial drugs, etc., can solve problems such as lack of uniformity, and achieve the effects of low production cost, good promotion and use value, and convenient use.

Active Publication Date: 2016-08-17
ZHEJIANG NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Rotten skin disease is very harmful to the breeding industry of spiny-breasted frog, but so far, there is no unified conclusion for the pathogen of the disease. Staphylococcus), Proteus mirabilis, etc.

Method used

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  • Completely inactivated vaccine for preventing and treating spinach frog skin rot and preparation method thereof
  • Completely inactivated vaccine for preventing and treating spinach frog skin rot and preparation method thereof
  • Completely inactivated vaccine for preventing and treating spinach frog skin rot and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, screening, identification of pathogenic bacteria, carry out the following steps successively:

[0038] 1) Rinse the body surface of the diseased and dying frog with sterile water, disinfect with 75% (volume%) alcohol, and take the deep skin of the head, legs, and the rotten skin around the excretory hole and the liver tissue of the diseased frog. , respectively, were coated in LB solid medium and incubated at 30°C for 24h. Colonies of different morphologies were picked for further streaking until pure cultures were obtained.

[0039] LB solid medium is a conventional medium, as follows: (both mass concentration g / L): peptone 20g, K 2 HPO 4 .3H 2 O2.5g, MgSO 4 0.73g, glycerol 15.0ml, agar 15.0g, distilled water to 1.0L; pH 7.0, 121℃, 15min sterilization.

[0040] 2), using TaKaRa 16S rDNA Bacterial Identification PCR Kit (purchased from Dalian Bao Bioengineering Co., Ltd.) to extract the genomic DNA of each strain from the single colony obtained in the...

Embodiment 2

[0044] Example 2. Regression infection experiment

[0045] Follow these steps in order:

[0046] 1), inoculate the above-obtained main pathogenic bacteria (that is, Acinetobacter genus) in LB liquid medium, after 30 ℃ of expanded culture 16h, prepare OD with sterile physiological saline 600 The mixed infection solution with values ​​of 0.1 and 0.2. There were 10 healthy frogs in the experimental group and the control group. After alcohol disinfection, the experimental group was injected with the bacterial solution, and the control group was injected with the same amount of normal saline (Table 1). Cultivated in a 25°C culture tank, observed and recorded the incidence, dissected the dying frogs, and isolated and identified pathogenic bacteria (Table 2).

[0047] Table 1 Regression infection experiment

[0048]

[0049] The results of regression infection showed that Acinetobacter strains had strong pathogenicity to P. spinosa, and the mortality rate of P. spinosa in the e...

Embodiment 3

[0054] Example 3. Specific immune verification

[0055] 1), with Acinetobacter jelly, Acinetobacter lwoutii, and Acinetobacter johnsonii as the main pathogenic bacteria, carry out expanded culture.

[0056] Prepared to OD with sterile water 600 270mL of mixed infection solution with a value of 0.2. Remarks: Acinetobacter agarinii, Acinetobacter lwoutii, and Acinetobacter johnsonii have the same concentration in the mixed infection solution.

[0057] 2), get 8 healthy frogs, soak in the infection bacterial liquid, soak for 3 days in total, and set the control group of equal amount to soak in sterile water in the same way.

[0058] 3) After soaking, random sampling was performed to collect serum, and an immunoglobulin IgG content assay kit was used to determine the antibody IgG content in the experimental group and the control group.

[0059] Results: The antibody content in the experimental group was generally higher than that in the healthy frogs in the control group. There...

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Abstract

The invention discloses a fully inactivated vaccine for preventing and curing giant spiny frog canker. A preparation method of the fully inactivated vaccine comprises the following steps: (1) taking acinetobacter junii, acinetobacter lwoffii and acinetobacter johnsonii as pathogenic bacteria, and treating each pathogenic bacterium in such a way that a formaldehyde solution is added into a pathogenic bacterium suspension with the OD600 value of 0.2 to prepare a vaccine with the formaldehyde concentration of 0.2% and the vaccine is inactivated in an incubator at 37 DEG C for 96 h, thus obtaining the formaldehyde inactivated vaccines of the three pathogenic bacteria respectively; (2) washing and then centrifuging the formaldehyde inactivated vaccines of the three pathogenic bacteria so as to obtain the treated inactivated vaccines of the three pathogenic bacteria respectively; and (3) mixing the treated inactivated vaccines of the three pathogenic bacteria at the volume ratio of 1:1:1, and then concentrating the mixture to 1 / 5 of the original total volume to obtain the fully inactivated vaccine for preventing and curing giant spiny frog canker. The fully inactivated vaccine provided by the invention can be used for preventing and curing giant spiny frog canker and has high popularization and application values.

Description

technical field [0001] The invention provides a preparation method and application of a fully inactivated vaccine for preventing and treating Rana spinosa. Background technique [0002] Frog breeding is considered to be one of the most potential breeding projects at present, and it is developing rapidly in the area south of the Yangtze River in China. However, the disease seriously affected its further large-scale development. At present, the application of immunological technology to prevent and control bacterial diseases of aquatic animals has received more and more attention from aquatic animal disease control workers in various countries. Compared with antibiotics, the development of "efficient, practical, low-cost, and safe" vaccine prevention and control technologies has gradually become a major issue in the prevention and control of international aquatic diseases. [0003] The immunization method of vaccines largely determines the use and promotion of vaccines. Zho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/02A61P17/00A61P31/04
Inventor 郑荣泉宋婷婷程宏毅俞佳佳杨沙吴章文蒋欢乐孙玉婷陈一嘉
Owner ZHEJIANG NORMAL UNIVERSITY
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