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Method for producing siniperca chuatsi infectious spleen and kidney necrosis virus and siniperca chuatsi rhabdovirus through microcarrier suspension culturing CPB cells

A spleen and kidney necrosis virus and microcarrier culture technology, applied in the field of cell engineering, can solve the problems of high cost, high titer virus technical difficulties, long time, etc., and achieve the effects of low production cost, low pollution probability and small labor force.

Active Publication Date: 2017-11-07
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there are few research reports on the vaccine research of mandarin fish infectious spleen-kidney necrosis virus liquid and mandarin fish bullet-like virus liquid. The main difficulty lies in the technical difficulty, high cost and long time to obtain high-titer virus.

Method used

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  • Method for producing siniperca chuatsi infectious spleen and kidney necrosis virus and siniperca chuatsi rhabdovirus through microcarrier suspension culturing CPB cells
  • Method for producing siniperca chuatsi infectious spleen and kidney necrosis virus and siniperca chuatsi rhabdovirus through microcarrier suspension culturing CPB cells
  • Method for producing siniperca chuatsi infectious spleen and kidney necrosis virus and siniperca chuatsi rhabdovirus through microcarrier suspension culturing CPB cells

Examples

Experimental program
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Effect test

preparation example Construction

[0054] Preparation of seedling liquid for Siniperca infectious spleen-kidney necrosis virus: 71-73 hours after the second scale-up culture of CPB cells, let the microcarriers settle down, remove the supernatant, wash with an equal volume of incubation solution, and use the multiplicity of infection (MOI) after washing From 4.8 to 5.2, add mandarin fish infectious spleen-kidney necrosis virus to infect the cells, and add serum-free incubation solution to make the system volume half of the final volume, stir at 39-41r / min every 11-13min for 2.5-3.5min, and continue After 1.8 to 2.2 hours, add the remaining serum-containing incubation solution to the final volume, settle the microcarriers at 118 to 122 hours after inoculation, collect the supernatant, freeze and thaw, centrifuge to take the supernatant, and filter the filtrate to obtain virus seedlings liquid;

[0055] Preparation of mandarin rhizoma virus seedling preparation: 95-97 hours after the second scale-up culture of CPB...

Embodiment 1

[0078] Embodiment 1: A kind of method that utilizes stirred bottle and bioreactor microcarrier to cultivate CPB cell to produce mandarin fish infectious spleen-kidney necrosis virus and mandarin fish shell virus

[0079] Main experimental materials:

[0080] 1. Cells: CPB cells (see CN 104694483A);

[0081] 2. Virus species: Mandarin Infectious Spleen and Kidney Necrosis Virus ISKNV (QY) strain, Mandarin SCRV;

[0082] 3. Microcarrier: Cytodex1, product of GE Healthcare;

[0083] 4. Stirring bottle: double arm stirring bottle, Wheaton, USA;

[0084] 5. Bioreactor: BC type 3L bioreactor, Guangzhou Qizhi Bioengineering Equipment Co., Ltd.

[0085] 1. Handling of experimental equipment

[0086] 1. Treatment of the stirring bottle: Rinse the stirring bottle with tap water for more than 3 times, rinse with ultrapure water for more than 3 times, and sterilize with high-pressure steam at 121°C;

[0087] 2. Processing of 3L Bioreactor

[0088] (1) cleaning

[0089] a. Tanks and...

Embodiment 2

[0112] Embodiment 2: A kind of method that utilizes stirred bottle and bioreactor microcarrier to cultivate CPB cell to produce mandarin fish infectious spleen-kidney necrosis virus and mandarin fish shell virus

[0113] 1) Microcarrier pretreatment; put Cytodex 1 microcarrier powder into a common glass bottle siliconized with dichlorodimethylsilane, add Ca-free 2+ , Mg 2+ Hydrate in PBS at room temperature, then settle the microcarriers statically, remove PBS, wash with fresh PBS, add fresh PBS at last, cover the bottle and carry out high-pressure steam sterilization, after cooling to room temperature, use serum-containing L15 medium After washing, the serum-containing L15 medium was added, and transferred to a stirring bottle siliconized with dichlorodimethylsilane to equilibrate overnight.

[0114] 2) Initial culture of CPB cells on microcarriers in 125ml stirring flasks: microcarriers and CPB single cell suspensions are inoculated into cell culture stirring flasks contain...

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Abstract

The invention discloses a method for producing a siniperca chuatsi infectious spleen and kidney necrosis virus and a siniperca chuatsi rhabdovirus through microcarrier suspension culturing CPB cells. The method comprises the steps of selecting the CPB cells as a cell line for making vaccines; sequentially carrying out step-by-step magnifying microcarrier suspension culture on the CPB cells through stirring bottles and bioreactors, so that the CPB cells are used as hosts of virus infection; finally obtaining the siniperca chuatsi infectious spleen and kidney necrosis virus bulk and the siniperca chuatsi rhabdovirus bulk. The prepared ISKNV potency reaches up to 8.34LgTCID50 / mL, and the SCRV potency reaches up to 10.25LgTCID50 / mL. A microcarrier suspension culture process adopted by the invention is simple to operate, less in pollution probability, small in labor force compared with a roller bottle culture method, and lower in production cost.

Description

technical field [0001] The invention relates to a method for producing mandarin fish infectious spleen-kidney necrosis virus and mandarin fish fluke virus by culturing CPB cells with microcarriers, and belongs to the technical field of cell engineering. Background technique [0002] Infectious Spleen and Kidney Necrosis Virus (ISKNV) is a disease that mainly breaks out in mandarin fish. It is caused by Infectious Spleen and Kidney Necrosis Virus (ISKNV) in the Iridoviridae genus Spleen and Kidney Necrosis Virus (ISKNV). 90%. The spleen and kidney of the sick mandarin fish had clinical symptoms such as hemorrhage and swelling. The disease brings huge economic losses to the mandarin fish farming industry every year. [0003] Siniperca Chuatsi Rhabdovirus is caused by Siniperca Chuatsi Rhabdovirus (SCRV) in Rhabdoviridae, and the fatality rate exceeds 80%. The sick mandarin fish mainly showed clinical symptoms of skin hemorrhage. The disease brings huge economic losses to t...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N5/079C12N5/02C12R1/93
CPCC12N5/0618C12N7/00C12N2531/00C12N2710/00051C12N2760/20051
Inventor 李宁求付小哲胡松林强刘礼辉梁红茹黄志斌
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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