Wubanli (hybrid of channa argus and channa maculata) brain cell line and construction method and application thereof

A construction method and technology for the snakehead snakehead, which are applied to the brain cell line of the snakehead snakehead and the field of construction thereof, can solve the problems of mechanical damage, difficult migration of cells, no cell line derived from the snakehead snakehead brain, and the like, and achieve stable characteristics, The effect of strong reproductive capacity

Active Publication Date: 2019-12-10
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

However, the disadvantages are that it is prone to mechanical damage and the cells are not easy to migrate out.
There is still no report on the cell line derived from the brain of Channa sinensis

Method used

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  • Wubanli (hybrid of channa argus and channa maculata) brain cell line and construction method and application thereof
  • Wubanli (hybrid of channa argus and channa maculata) brain cell line and construction method and application thereof
  • Wubanli (hybrid of channa argus and channa maculata) brain cell line and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Isolation and Primary Culture of Channa Brain Cells

[0049] Experimental Materials and Reagents

[0050] Experimental animals: 10g-30g of the black-spotted snakehead (verified by repeated molecular methods that this is a healthy black-spotted snakehead not infected with aquatic animal viruses).

[0051] Experimental equipment: scalpel, ophthalmic scissors, ophthalmic tweezers and gauze, etc.

[0052] Media and related reagents

[0053] The concentration of penicillin-streptomycin-amphotericin B third antibody solution is 10000u / ml;

[0054] Culture medium: M199 medium (gibco company) containing concentration of 100U / ml penicillin-streptomycin-amphotericin B third antibody and 20% fetal bovine serum (Gibco company);

[0055] Trypsin digestion solution: disodium hydrogen phosphate 2.3g, potassium dihydrogen phosphate 0.1g, sodium chloride 8.0g, potassium chloride 0.2g,

[0056] EDTA0.2g, trypsin 0.6g, water 1000ml; medical alcohol;

[0057] 0.01M PBS buffe...

Embodiment 2

[0068] Subculture and cryopreservation of the subculture and cryopreservation of the black spot snakehead brain cell line of embodiment 2

[0069] Reagent

[0070] Trypsin digestion solution: disodium hydrogen phosphate 2.3g, potassium dihydrogen phosphate 0.1g, sodium chloride 8.0g, potassium chloride 0.2g,

[0071] EDTA0.2g, trypsin 0.6g, water 1000ml.

[0072] Freezing solution: M199 medium containing 10% DMSO, 25% special grade fetal bovine serum.

[0073] experimental method

[0074] When the cells grow to 90%, discard the old medium, add 1ml of trypsin digestion solution for digestion; when the cells appear "white mist", add 2ml of black spot snakehead brain cell culture medium to stop the trypsin Shake gently to mix the cells and count. Centrifuge at 1000rpm for 5min, remove the culture medium, add 1.5ml cell freezing solution, and resuspend the cells.

[0075] Collect the cells into cryopreservation tubes, and mark the name of the cells, the number of cells and th...

Embodiment 3

[0077] Example 3 Determination of the Growth Characteristics of the Brain Cell Line of C. chinensis

[0078] Reagent

[0079] Trypsin digestion solution: disodium hydrogen phosphate 2.3g, potassium dihydrogen phosphate 0.1g, sodium chloride 8.0g, potassium chloride 0.2g, EDTA 0.2g, trypsin 0.6g, water 1000ml.

[0080] Medium: M199 medium with different concentrations of special grade fetal bovine serum. DMEM, MEM, L-15 medium

[0081] experimental method

[0082] 1 Determination of the best culture medium for the brain cells of Channa chinensis

[0083] Four cell culture media, DMEM, M199, MEM, and L-15, were selected, and FBS with a final concentration of 10% was added to prepare the cell culture medium. Adjust the cell density to 2 x 10 5 mL-1, each of the four mediums was inoculated in a 6-well plate at an amount of 2.5 mL / well, and cultured in an incubator at 27°C. Take 3 wells of cells from each experimental group every 1 day, collect and count the cells by Trypsin-...

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Abstract

The present invention discloses a wubanli (hybrid of channa argus and channa maculata) brain cell line and a construction method and an application thereof. Aiming at characteristics of wubanli braintissue cells, a collagenase I and pancreatin combined digestion method is used to separate the brain tissue cells for primary culture, then the wubanli brain cell line is successfully constructed andis continuously passed to 100 generations or more, a large number of wubanli brain-derived cells is provided, and besides, the cells can maintain a good growth state and can be freeze-stored. The wubanli brain cell line is sensitive to a variety of fish viruses, after virus inoculation, a cytopathic effect occurs, and besides, the wubanli brain cell line can also be used to study adhesion characteristics of channidae-derived aeromonas schubertii and can be directly applied in study of pathogenic characteristics and development of vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a brain cell line of the channa chinensis and its construction method and application. Background technique [0002] Snakeheads and snakeheads are relatively common commercial fish, and both belong to the order Perciformes, the suborder Channoidei, and the family Channidae. Commonly known as black fish, snakeheads are ferocious, aggressive, and have strong reproductive capacity. They are mostly distributed in the Yangtze River Basin and the water system to the north, while spotted snakeheads are mainly distributed in the south of the Yangtze River Basin, such as Guangdong, Hainan, Guangxi, Fujian, and Yunnan. Wait. Most of the male parents of black-spotted snakeheads come from black-spotted snakeheads in Hunan Province, Shandong Province and other regions, while their female parents are mostly local spotted snakeheads in Guangdong Province. The offspring produced by cros...

Claims

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Application Information

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IPC IPC(8): C12N5/079C12R1/91
CPCC12N5/0618
Inventor 王英英曾伟伟王庆李莹莹尹纪元石存斌常藕琴
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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