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Inactivated vaccine for ictalurus punctatus hemorrhage disease, as well as preparation method and application thereof

A channel catfish and inactivated vaccine technology, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, antiviral agents, etc., can solve the problem of no channel catfish hemorrhagic disease vaccine, etc., and achieve the protection rate of fish immunity. , The effect of complete antigenic protein and good immunogenicity

Active Publication Date: 2013-09-04
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no related report on the channel catfish hemorrhagic disease vaccine. The present invention provides for the first time an efficient, safe and mass-producible inactivated channel catfish hemorrhagic disease vaccine and its preparation method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The preparation method of channel catfish reovirus CCRV-730, the steps are:

[0038] A. CCK cell culture: take CCK cells preserved in liquid nitrogen (established in our laboratory, CCTCC C201198), incubate at 37°C for 2-3 minutes to melt, add MEM culture medium containing 10% (V / V) calf serum (5ml / T 25 culture flask, pH 7 or 7.2 or 7.4 or 7.5), transfer to the culture flask and culture at 28°C. Let stand for 6 or 7 or 8 hours, and replace with fresh culture medium after the cells adhere to the wall. After the cells are cultured for 2 or 3 days, and the bottom of the culture bottle is covered, 0.1%-0.5% (W / V) trypsin is used to digest the adherent cells for subculture. The MEM culture medium is Eagle's MEM cell culture medium produced by Sigma-aldrich or Gibco or other companies.

[0039] B. CCRV-730 virus culture and harvest: When the CCK subculture cells are covered at the bottom of the bottle, remove the culture medium, and then add the infected CCRV-730 virus sus...

Embodiment 2

[0043] The preparation method of channel catfish haemorrhagic disease inactivated vaccine, its steps are:

[0044] A. Inactivation of CCRV-730 virus: take the purified virus liquid of CCRV-730 stored at -80°C and melt it at 4°C, and add the inactivator β-3, which is stored at -20°C, at a concentration of 0.025% (V / V). lactone, mixed quickly and placed at 4°C to inactivate the virus. After 24 hours of inactivation, the inactivated virus liquid was incubated in a water bath at 37° C. for 2 hours to hydrolyze β propiolactone.

[0045] B. Dilute the inactivated vaccine with fish normal saline (0.65%) to a titer of 10 5 TCID 50 / ml, the channel catfish cell culture β propiolactone inactivated vaccine that can be used directly was obtained.

[0046] C. Safety inspection of inactivated vaccines:

[0047] 1) Inactivation effect: take the vaccine prepared above, inoculate CCK cells according to the above virus propagation method, and set up a negative control at the same time, obse...

Embodiment 3

[0051] The protective effect evaluation of channel catfish haemorrhagic disease inactivated vaccine, the specific steps are:

[0052] A. Small-scale experiment in the laboratory: after healthy channel catfish species (body length 8-10 cm) were reared indoors for 10 days, each group was injected with 0.2 ml of inactivated vaccines of different dilutions, and at the same time, another group was injected with MEM cell culture medium. A batch of fish was used as a negative control, and after feeding at 28°C for 10 to 12 days, a part of fish in each group was randomly taken out for serum neutralization test to measure the serum neutralizing antibody level of immune fish and control fish; another part of fish in each group was Attack with virulence, and measure its immune protection rate. After testing, the titer of each injection of 0.2ml is 10 5 TCID 50 The β propiolactone inactivated vaccine per ml had the best immune effect, and the neutralizing antibody titer was 1:1024; the ...

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Abstract

The invention discloses an inactivated vaccine for ictalurus punctatus hemorrhage disease as well as a preparation method and application thereof. The preparation method comprises the following steps: A, culturing CCK (cholecystokinin) cells; B, culturing, harvesting and storing CCRV-730 (channel catfish reovirus-730) virus; C, detecting titer TCID (tissue culture infectious dose) 50 of a virus solution, wherein the virus titer is the titer of the inactivated vaccine; D, inactivating CCRV-730 virus with beta propiolactone, incubating and hydrolyzing the beta propiolactone at 37 DEG C; and E, diluting the inactivated vaccine to a certain titer with normal saline for fish to obtain the ictalurus punctatus cell cultivated inactivated vaccine which can be directly used. The inactivated vaccine is applied to prevention of the ictalurus punctatus hemorrhage disease, is good in inactivation effect, furthest avoids destruction of an inactivator to antigenic protein, and is good in immune effect. Furthermore, the toxicity of the inactivator is eliminated, and the safety of the vaccine is improved. The vaccine has the advantages of good immune effect, safety in use, low production cost, high production efficiency and the like.

Description

technical field [0001] The invention relates to the field of inactivated vaccines for aquaculture fish, in particular to an inactivated vaccine for channel catfish hemorrhagic disease, and also relates to a method for preparing an inactivated vaccine for channel catfish hemorrhagic disease, and also relates to a channel catfish hemorrhagic disease inactivated vaccine Use of live vaccines. Background technique [0002] Channel catfish (Channel catfish) was introduced to my country from the United States in 1984. Because of its rich nutrition and delicious meat, it has been welcomed by domestic consumers and has gradually become one of the main varieties of aquatic products exported in my country. Breeding in many provinces and cities. With the expansion of channel catfish breeding scale in my country and the improvement of intensive farming, the hazards of diseases are also increasing. Especially in recent years, fish farms in many areas have experienced explosive hemorrhagic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/15C12N7/00A61P31/14C12R1/93
Inventor 徐进曾令兵范玉顶周勇肖艺
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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