Preparation method of rainbow trout infectious haematopoietic necrosis virus (IHNV) inactivated vaccine and application thereof

A technology of hematopoietic organ necrosis and inactivated vaccine, which is applied to the preparation of rainbow trout infectious hematopoietic necrosis virus (IHNV) inactivated vaccine and its application field, and can solve the problem of reducing the incidence of IHN infection in rainbow trout and other infections, the low efficiency of vaccine preparation, Vaccine inactivation effect is not good and other problems, to achieve the effect of good inactivation effect, reduce morbidity, reduce cycle and cost

Inactive Publication Date: 2018-06-05
CHENGDU ACAD OF AGRI & FORESTRY SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Existing farms generally take measures to destroy all diseased fish infected with IHN, and fully disinfect the breeding sites and equipment; however, this method cannot effectively reduce the incidence of IHN infected rainbow trout from the root.
At the same time, the preparation cost of the existing IHNV virus inactivated vaccine is high, the process is complicated, and the inactivation time is long, and the inactivation effect of the vaccine is not good at the same time, which leads to the low preparation efficiency of the IHNV virus inactivated vaccine.

Method used

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  • Preparation method of rainbow trout infectious haematopoietic necrosis virus (IHNV) inactivated vaccine and application thereof
  • Preparation method of rainbow trout infectious haematopoietic necrosis virus (IHNV) inactivated vaccine and application thereof
  • Preparation method of rainbow trout infectious haematopoietic necrosis virus (IHNV) inactivated vaccine and application thereof

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Experimental program
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Effect test

preparation Embodiment 1

[0055] The preparation method of IHNV virus inactivated vaccine is as follows:

[0056] S1. Recovery of FHM cells: Take 1 mL of FHM cells frozen in liquid nitrogen, place the cell liquid in a constant temperature water bath at 37°C for 2 minutes, and then centrifuge at 1000 r / min for 5 minutes, remove the supernatant, and obtain recovered FHM cells;

[0057] S2. Basic culture of FHM cells: Add 5 mL of DMEM medium containing 10% (volume fraction) FBS to the FHM cells obtained in step S1, and then transfer the cell liquid to T 25 Culture in a culture flask;

[0058] S3. FHM cell expansion culture: After the cells are 90%-100% full in step S2, remove the culture medium, rinse with D-PBS 2 times, use D-PBS 2-5mL each time; then take 0.5mL to 0.25% concentration Digest the adherent cells with trypsin for 1 min, then remove the trypsin; add 5 mL of DMEM medium containing 10% (volume fraction) FBS to the cell fluid, mix well, and transfer the cell fluid to T 75 In the culture flask, place t...

preparation Embodiment 2

[0064] The preparation method of IHNV virus inactivated vaccine is as follows:

[0065] S1. Recovery of FHM cells: Take 1mL of FHM cells frozen in liquid nitrogen, place the cell liquid in a constant temperature water bath at 37°C for 2 minutes, and then centrifuge at 850 r / min for 6 minutes, remove the supernatant, and obtain recovered FHM cells;

[0066] S2. FHM cell basic culture: Add 4 mL of DMEM medium containing 9% (volume fraction) FBS to the FHM cells obtained in step S1, and then transfer the cell fluid to T 25 Culture in a culture flask;

[0067] S3. FHM cell expansion culture: When the cells are 90%-100% full in step S2, remove the culture medium, rinse with D-PBS 3 times, use 2-5mL D-PBS solution each time; then take 0.2mL to a concentration of 0.25 % Trypsin digests the adherent cells for 2 minutes, then removes the trypsin; add 4 mL of DMEM medium containing 9% (volume fraction) FBS to the cell fluid, mix well, transfer the cell fluid to T 75 In the culture flask, place...

preparation Embodiment 3

[0073] The preparation method of IHNV virus inactivated vaccine is as follows:

[0074] S1. Recovery of FHM cells: Take 1.5 mL of FHM cells frozen in liquid nitrogen, place the cell liquid in a constant temperature water bath at 38°C for 1 min, then centrifuge at 800 r / min for 6 min, remove the supernatant, and obtain recovered FHM cells;

[0075] S2. Basic culture of FHM cells: Add 6 mL of DMEM medium containing 10% (volume fraction) FBS to the FHM cells obtained in step S1, and then transfer the cell liquid to T 25 Culture in a culture flask;

[0076] S3. FHM cell expansion culture: After the cells are 90%-100% full in step S2, remove the culture medium, rinse with D-PBS 3 times, use 2-5mL D-PBS solution each time; then take 1mL to a concentration of 0.3% Digest the adherent cells with the trypsin for 2 minutes, then remove the trypsin; add 6 mL of DMEM medium containing 10% (volume fraction) FBS to the cell fluid, mix well, and transfer the cell fluid to T 75 In the culture flask,...

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Abstract

The invention discloses a preparation method of a rainbow trout infectious haematopoietic necrosis virus (IHNV) inactivated vaccine. The method comprises the following steps of S1, recovering and purifying an FHM cell; S2, basically culturing the FHM cell; S3, amplifying and culturing the FHM cell; S4, culturing an IHNV virus; S5, purifying the IHNV virus; S6, inactivating the IHNV virus. The IHNVinactivated vaccine prepared through the method is applied to preventing infectious haematopotietic necrosis; the IHNV inactivated vaccine successfully prepared through the method is good in inactivation effect, high in safety and high in relative immune protective rate; meanwhile, the preparation method is simple in process, beneficial to reducing a large-scale production cycle and cost of the virus, and capable of being used for industrially preparing the IHNV inactivated vaccine for preventing infectious haematopotietic necrosis.

Description

Technical field [0001] The invention relates to a preparation method and application of a rainbow trout infectious hematopoietic necrosis virus (IHNV) inactivated vaccine. Background technique [0002] Rainbow trout is a kind of precious cold-water fish. Its meat is fresh and tender, delicious, high in protein and fat, cholesterol content is almost zero, EPA content is several times higher than other fish, it has the reputation of "water ginseng", so it is widely accepted People love. Rainbow trout has been farmed in my country since 1959. The main species include hardhead trout, Nelson’s super rainbow trout, Jalo strain rainbow trout and golden trout (yellow rainbow trout). At present, there are more than 400 rainbow trout farms in my country, which are distributed in 100 counties in 23 provinces, cities, and autonomous regions. The breeding methods are mostly high-density flowing water aquaculture. However, in recent years, due to the gradual expansion of rainbow trout farmin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/205A61P31/14C12N7/00C12N7/02C12N7/06
CPCA61K39/12A61K2039/5252A61K2039/552C12N7/00C12N2760/20034C12N2760/20051C12N2760/20063
Inventor 魏文燕汪开毓李良玉杨马杨倩刘韬唐洪张小丽刘家星朱玲杨壮志何洁陈霞何琦瑶陈健何晟毓
Owner CHENGDU ACAD OF AGRI & FORESTRY SCI
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