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Proliferation method of siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV) based on epithelioma papulosum cyprinid (EPC)

A technology of spleen and kidney necrosis virus and tumor cells, which is applied in the field of proliferation of mandarin fish infectious spleen and kidney necrosis virus ISKNV to achieve the effects of improving affinity, easy cultivation and easy acquisition

Inactive Publication Date: 2016-09-21
WUHAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Currently, there is no targeted fish medicine, and vaccine immunization is the only effective way to control the disease. Therefore, the large-scale amplification technology and purity requirements of ISKNV virus are particularly important for the production of inactivated vaccines.

Method used

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  • Proliferation method of siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV) based on epithelioma papulosum cyprinid (EPC)
  • Proliferation method of siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV) based on epithelioma papulosum cyprinid (EPC)

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Embodiment 1

[0035] A method for the proliferation of mandarin fish infectious spleen and kidney necrosis virus ISKNV based on carp epithelial tumor cell EPC, comprising the steps:

[0036] (1) Cultivation of EPC cells: inoculate EPC cells in M199 medium containing 10% (mass concentration) fetal bovine serum, and culture at 25°C for 2 days until the cells grow into a monolayer and the number of cells is >10 6 / mL;

[0037] (2) Preparation of ISKNV virus seeds: get EPC cells, add therein the infection solution containing ISKNV virus (infection solution is the M199 medium containing 2wt% fetal bovine serum, ISKNV virus), after constant temperature cultivation, collect the supernatant of the culture solution ;

[0038] (3) Rejuvenation of ISKNV virus species: take another EPC cell, add the collected culture supernatant to it, infect the EPC cell with the ISKNV virus in the culture supernatant, culture at a constant temperature of 25°C for 10 days, and collect the culture supernatant again ;...

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Abstract

The invention belongs to the field of biotechnology and in particular relates to a proliferation method of siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV) based on epithelioma papulosum cyprinid (EPC). The proliferation method comprises the following steps: culturing an EPC cell line; inflecting EPC cells by using ISKNV, culturing at constant temperature, then collecting culture solution supernatant; inflecting the EPC cells again by using ISKNV in the culture solution supernatant, repeatedly operating for 2-3 times, rejuvenating ISKNV seeds, taking culture solution supernatant nitrogen and saving the ISKNV seeds as ISKNV amplification seeds; inflecting the EPC cells by using the rejuvenated ISKNV seeds, culturing at constant temperature till CPE is greater than 80%, and collecting supernatant to obtain ISKNV particles. The EPC cell line is used as sensitive cells infected by ISKNV; the EPC cell line is high in degree of commodity, easy to get, easy to culture and distant in genetic relationship with siniperca chuatsi; the purification difficulty of ISKNV for inactivating vaccines can be greatly reduced; the adverse effects are reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for multiplying mandarin fish infectious spleen and kidney necrosis virus ISKNV based on carp epithelial tumor cell EPC. Background technique [0002] Infectious spleen and kidney necrosis virus (ISKNV) was isolated from mandarin fish in 1998. The virus replicates and assembles in the cytoplasm, with a diameter of 150nm, and is a cytoplasmic double-stranded linear DNA virus. ISKNV has a very high lethality rate to a variety of sea and freshwater aquaculture fish such as mandarin fish, grouper, and large yellow croaker, and is one of the most serious viral diseases harmful to aquaculture fish in my country. ISKNV can infect mandarin fish spleen, kidney, liver, gills, brain, gonads, heart and digestive tract and other tissues and organs, and spleen and kidney are the main infected organs. Infection experiments showed that ISKNV can infect hosts in four ways: (1) ...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12R1/93
CPCC12N7/00C12N2710/00051
Inventor 周伟东邓平喻运珍喻婷张立强周勇徐洪亮艾桃山林蠡周裕和
Owner WUHAN ACADEMY OF AGRI SCI
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