Preparation method of iridovirus cell inactivated vaccine for Chinese giant salamanders, and application method thereof

A giant salamander iridescent virus and inactivated vaccine technology is applied in the field of virus cell inactivated vaccine preparation to achieve strong immune specificity, stable quality, and good immune protection effect

Inactive Publication Date: 2011-11-02
汉中天成生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no effective drug for th

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] (1) Take out the liver of giant salamander who is dying and confirmed to contain frog iridescent virus in its liver, and make tissue homogenate;

[0027] (2) Rapidly lyse the tissue homogenate with 20kHz ultrasonic waves on ice, centrifuge at 5000rpm for 30min, obtain the supernatant, and then filter it with a 0.4um sterile filter to collect the filtrate;

[0028] (3) The filtrate was centrifuged at 20000rpm for 60min, the supernatant was removed, the precipitate was dissolved with an appropriate amount of phosphate buffered saline (PBS), and then centrifuged at 20000rpm for 60min, then the virus titer of the precipitate was adjusted to reach 10 5 TCID 50 ;

[0029] (4) Grass carp gonad cells were inoculated in the ultra-clean bench of the aseptic room, at 25°C, 5% CO 2 In an incubator, use M199 medium containing 10% calf serum, with a pH of 7.0-7.4, culture for 48-72 hours, and harvest the cell culture medium. In this process, in addition to grass carp gonad cells, ...

Embodiment 2

[0034] Vaccinations for juvenile salamanders:

[0035] Get 100ml of the vaccine obtained in Example 1 in a water basin, dilute it with 5L of virus-free sterile culture water, put 100 juvenile salamanders with an opening of more than 60 days and fully developed digestive tract or adult salamanders below 0.25kg into it for Soak in a bath for 60 minutes, shake the basin every 10 minutes during the immersion to increase oxygen, and then put them into sterilized breeding ponds. According to their growth, a booster immunization will be carried out one month later according to the above bathing method.

Embodiment 3

[0037] Vaccinations for adult salamanders:

[0038] For adult salamanders below 0.25kg, the vaccine immunization can be completed according to the method described in Example 2.

[0039] For adult salamanders weighing more than 0.25kg, the method of intramuscular injection of vaccine is used for immunization, and the injection dose is 0.1ml / kg body weight. The specific steps are as follows:

[0040] (1) the water in the breeding pond is drained, and the body weight of the giant salamander is weighed;

[0041] (2) Gently wipe the injection area of ​​the giant salamander caudal muscle with 70% alcohol cotton ball;

[0042] (3) Use the vaccine obtained in Example 1 to inject intramuscularly according to the weight of the giant salamander.

[0043] (4) Put the vaccinated giant salamander into the breeding pond, add the breeding water again, and manage it according to the routine. One month later, the other side of the caudal muscle of the giant salamander was injected with the ...

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Abstract

The invention discloses a preparation method of an iridovirus cell inactivated vaccine for Chinese giant salamanders, and an application method thereof. The vaccine is prepared by the following method: 1, sterilely acquiring the liver of a moribund giant salamander containing frog iridovirus, preparing a tissue homogenate; 2, quickly cracking the tissue homogenate by ultrasonic wave of 20kHz on an ice surface, filtering; 3, centrifugally separating the filtrate to obtain pure virus precipitate, then regulating the virus titer to 105TCID50; 4, culturing the virus cells, collecting a cell culture solution; and 5, and inactivating the cell culture solution by formaldehyde with the final concentration of 0.4% at 37 DEG C for 5-10 hours, thus obtaining the iridovirus cell inactivated vaccine for Chinese giant salamanders. The vaccine is effective when preserved at 4 DEG C within one year. For young giant salamanders and adult giant salamanders below 0.25kg in body weight, dipping bath with the dilute solution of the vaccine is adopted for immunization, and for adult giant salamanders above 0.25kg in body weight, intramuscular injection is adopted for immunization. The vaccine has very strong immunologic specificity, and the immunization rate can reach 80-93%.

Description

technical field [0001] The invention relates to a preparation method and a use method of a virus cell inactivated vaccine, in particular to a preparation method and a use method of a Chinese giant salamander iridescent virus cell inactivation vaccine. Background technique [0002] Giant salamander, commonly known as giant salamander, is an amphibian with high scientific research, nutrition, medicinal, cultural and ornamental value. In recent years, the farming of giant salamanders has become a new sunrise industry, which has brought significant economic benefits to farmers and promoted the development of the entire industrial chain. However, with the development of intensive farming of giant salamanders and the continuous expansion of breeding scale, diseases of giant salamanders are also constantly occurring, and the symptoms are becoming more and more complicated, and the incidence rate is increasing year by year, especially with swollen limbs, swollen head, and body surfa...

Claims

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Application Information

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IPC IPC(8): A61K39/12C12N7/00C12N7/04A61P31/20C12R1/93
Inventor 董武子杨长明张驰
Owner 汉中天成生物工程有限公司
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