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Infectious hematopoietic organ necrosis vaccine and method for amplifying virus of infectious hematopoietic organ necrosis vaccine on muscle cells of Pimephales promelas

A technology for necrosis of hematopoietic organs and muscle cells, applied in the biological field, can solve the problems of prolonging the production cycle, increasing the probability of pollution, wrong packaging, etc., and achieve the effect of stable virus titer, high virus titer and good immune effect

Pending Publication Date: 2021-07-16
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Previous studies have found that in the process of subculture of IHNV, when the inoculation dose is too large, a large number of IHNV defective viruses will be mispackaged to form a large number of IHNV defective viruses, and the defective viruses do not have the ability to reproduce. As the number of passages increases, the defective viruses will accumulate rapidly. Ultimately, IHNV cannot continue to be passed on in vitro; when the inoculation dose is too small, it will take a long time for the virus to proliferate, prolong the production cycle, increase the probability of contamination, and increase the production cost of the vaccine

Method used

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  • Infectious hematopoietic organ necrosis vaccine and method for amplifying virus of infectious hematopoietic organ necrosis vaccine on muscle cells of Pimephales promelas
  • Infectious hematopoietic organ necrosis vaccine and method for amplifying virus of infectious hematopoietic organ necrosis vaccine on muscle cells of Pimephales promelas
  • Infectious hematopoietic organ necrosis vaccine and method for amplifying virus of infectious hematopoietic organ necrosis vaccine on muscle cells of Pimephales promelas

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Experimental program
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Effect test

Embodiment 1

[0031] The preparation of embodiment 1, IHNV virus liquid

[0032] 1. Recovery and culture of FHM cells

[0033] Take out the FHM (American Type Culture Collection, American Type Culture Collection, No.: CCL-42 TM ), quickly put it into a 30°C constant temperature water bath, and shake it from time to time to completely melt it. Move it into a 15ml centrifuge tube under aseptic conditions, centrifuge at 1000g for 2min, discard the supernatant, add 1ml of cell culture medium (M199 medium + volume percentage 10% fetal bovine serum + mass volume percentage (g: ml) 1% double antibody) Gently blow evenly. Transfer the blown cell solution into a T-25 cell culture bottle, add 4ml of cell culture solution, blow it evenly, put it in a 25°C carbon dioxide constant temperature cell incubator for static culture, and observe the cell state under an inverted microscope every day. When the T-25 cell culture flask is covered with a monolayer of FHM cells, digest the adherent cells with 1...

Embodiment 2

[0065] Preparation and Application of Embodiment 2, IHNV Inactivated Vaccine

[0066] One, the preparation of IHNV inactivated vaccine

[0067] IHN-BPL inactivated vaccine: add β-propiolactone (BPL) to the amplified IHNV virus liquid obtained in 3 of Example 1 of 80ml, so that the final concentration of BPL is 3mM, mix and place on a shaker at 24°C , 100r / min carried out inactivation for 24h, adding sodium thiosulfate solution (final concentration is 20mM) to terminate the inactivation, to obtain IHN-BPL inactivated seedlings;

[0068] IHN-formaldehyde inactivated vaccine: add formaldehyde to 80ml of the amplified IHNV virus liquid obtained in 3 of Example 1, so that the final concentration of formaldehyde is 5mM, mix well, place on a shaker at 24°C, and inactivate at 100r / min After 24 hours, sodium bisulfite solution (final concentration: 1 mM) was added to terminate the inactivation to obtain IHN-formaldehyde inactivated vaccine.

[0069] 2. Detection of relative immune pr...

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Abstract

The invention discloses an infectious hematopoietic organ necrosis vaccine and a method for amplifying a virus of the infectious hematopoietic organ necrosis vaccine on muscle cells of Pimephales promelas. The invention provides the method for proliferating infectious hematopoietic organ necrosis virus in vitro. The method comprises the following steps of: inoculating the infectious hematopoietic organ necrosis virus to muscle cells of Pimephales promelas according to the MOI value of 0.001, culturing, and collecting supernate, so as to proliferate the infectious hematopoietic organ necrosis virus on the muscle cells of Pimephales promelas. According to the present invention, the IHNV is inoculated to the FHM cell at the concentration of MOI = 0.001, the required virus collection time is short, and the virus titer is high and stable. According to the proliferation scheme, viruses are amplified on a large scale, the inactivated vaccine is prepared from BPL and formaldehyde, the result shows that the immune effect of the vaccine on rainbow trout is good, and the relative immune protection efficiency can reach 80% or above.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a vaccine for infectious hematopoietic organ necrosis and a method for amplifying the virus on fat-headed catfish muscle cells. Background technique [0002] Infectious hematopoietic necrosis virus (IHNV) belongs to Rhabdoviridae (Rhabdoviridae), Nora rhabdovirus (Novirhabdovirus), is a single-stranded negative-sense RNA virus, is infectious hematopoietic necrosis disease (Infectious hematopoietic necrosis virus (IHNV) hematopoietic necrosis, IHN) pathogen. IHN first broke out in the United States in the 1950s, was introduced to China in the 1980s, and is now widely distributed in many salmon farming countries around the world. The disease is a viral disease that can lead to 100% mortality of salmonid juveniles. It is an animal disease that must be declared by the World Organization for Animal Health (Office International Des Epizooties, OIE), and it is a second-class di...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/205A61P31/14C12R1/93
CPCC12N7/00A61K39/12A61P31/14C12N2760/20051C12N2760/20034A61K2039/5252A61K2039/552
Inventor 徐黎明陈桂花卢彤岩赵景壮任广明邵轶智
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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