Infectious hematopoietic necrosis vaccine and method for amplifying infectious hematopoietic necrosis virus on epithelioma papulosum cyprini
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A technology for hematopoietic organ necrosis and epithelial cells, applied in the biological field, can solve problems such as IHNV-free in vitro proliferation solutions
Pending Publication Date: 2021-07-23
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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However, there is currently no report on the systematic study of IHNV proliferation in vitro
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Embodiment 1
[0031] The preparation of embodiment 1, IHNV virus liquid
[0032] 1. Recovery and culture of EPC cells
[0033] Remove the frozen EPC from the liquid nitrogen tank (American Type Culture Collection, ATCC @ CRL-2872 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 (MEM medium+volume percentage content 10% fetal bovine serum+mass volume percentage content (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 single layer of EPC cells, digest the adhe...
Embodiment 2
[0063] Preparation and Application of Embodiment 2, IHNV Inactivated Vaccine
[0064] One, the preparation of IHNV inactivated vaccine
[0065] 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;
[0066] 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.
[0067] 2. Detection of relative immune pr...
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Abstract
The invention discloses an infectious hematopoietic necrosis vaccine and a method for amplifying infectious hematopoietic necrosis virus (IHNV) on epithelioma papulosum cyprini (EPC). The invention provides a method for preparing an infectious hematopoietic necrosis vaccine, and the method comprises the following steps: 1) inoculating IHNV to EPC according to an MOI value of 0.0001, carrying out culturing, and collecting supernatant, thereby obtaining proliferated IHNV; and 2) preparing the infectious hematopoietic necrosis vaccine by using the proliferated IHNV. Experiments in the invention prove that when the IHNV is inoculated to the EPC with the concentration of MOI = 0.0001, the required virus harvest time is short, and the virus titer is high and stable. The IHNV is amplified on a large scale based on the proliferation scheme, and an inactivated vaccine is prepared from BPL and formaldehyde. Results show that the vaccine has a good immune effect on Oncorhynchus mykiss, and the relative immune protection efficiency was up to 84%.
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 the epithelial cells of the crocodile. 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 (Infectious hematopoietic necrosis virus, hematopoietic necrosis, IHN). [0003] Previous studies have found that in the process of subculture of IHNV, when the inoculation dose is too large, there will be a large amount of IHNV defective virus packaged wrongly, and the defective virus does not have the ability to reproduce. With the increase of the number of passages, the defective virus will accumulate rapidly. Ultimately, IHNV cannot continue to be passed on in vitro; when the inoculation dose is too s...
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