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Construction method of Epinephelus fuscoguttatus swim bladder cell line

A construction method and cell line technology, which can be applied to artificial cell constructs, animal cells, vertebrate cells, etc., can solve the problems such as the lack of establishment of the cell line of the bladder tissue of the brown-spotted grouper, and the lack of application research of the bladder cell line.

Inactive Publication Date: 2012-11-28
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the establishment of the swim bladder tissue cell line of the brown spotted grouper, and there is no successful report on the application of the swim bladder cell line in virus disease and environmental monitoring

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0009] 2. Preparation of the swim bladder tissue block of the brown-spotted grouper: the fresh-live brown-spotted grouper was kept temporarily in seawater with high double-antibody penicillin and streptomycin at a concentration of 1000 units / ml, and then sterilized in 75% alcohol for 1-24 hours. 2 minutes. The swim bladder tissue was aseptically dissected and removed in an ultra-clean workbench, rinsed twice with phosphate buffered saline, and cut into tissue pieces of about 1 cubic millimeter in DMEM / F12 culture medium containing 5% fetal bovine serum; Centrifuge at 800 rpm to collect the swim bladder tissue pieces; use DMEM / F12 culture medium (Gibco) containing 5% fetal bovine serum (Hyclone) and 0.05‰~0.15‰ carboxymethyl chitosan oligosaccharide with a pH value of 7.0~7.4 (Gibco) to fully Suspend and evenly inoculate in a 25 ml culture bottle (Corning) pretreated with 0.01% gelatin; put the culture bottle into an incubator at 22-24°C, and put it upright for 16-20 hours.

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Embodiment 1

[0014] Before the primary experiment on the swim bladder tissue of brown spotted grouper, 0.01 g of gelatin (Gibco) was dissolved in 100 ml of calcium-magnesium-free phosphate buffer, placed in a 50-degree water bath for 15 minutes to dissolve, and filtered through a 0.22-micron microporous filter. Sterilize by membrane filtration, draw 1 ml of gelatin solution and add it to a 25 ml culture bottle to coat the bottom of the culture bottle, place at room temperature for 1 hour, suck out the gelatin from the culture bottle, and store the culture bottle for later use. Put the fresh and live brown grouper in high double-antibody seawater with penicillin and streptomycin at a concentration of 1000 units / ml for 24 hours, then disinfect it in 75% alcohol for 2 minutes, wipe the surface of the fish with a cotton ball, and then dissect it Take out the swim bladder tissue, put it in a beaker filled with phosphate buffer, grab the whole swim bladder tissue with tweezers, rinse it twice to ...

Embodiment 2

[0017] Before the primary experiment on the swim bladder tissue of brown spotted grouper, dissolve 0.01 g of gelatin (Gibco) in 100 ml of calcium-magnesium-free phosphate buffer, place it in a 50-degree water bath for 20 minutes to dissolve it, and filter it through a 0.22-micron microporous filter. Sterilize by membrane filtration, draw 1 ml of gelatin solution and add it to a 25 ml culture bottle to coat the bottom of the culture bottle, place at room temperature for 1.5 hours, suck out the gelatin from the culture bottle, and set aside the culture bottle. Put the fresh and live brown grouper in high double-antibody seawater with penicillin and streptomycin at a concentration of 1000 units / ml for 24 hours, then disinfect it in 75% alcohol for 2 minutes, wipe the surface of the fish with a cotton ball, and then dissect it Take out the swim bladder tissue, put it in a beaker filled with phosphate buffer, grab the whole swim bladder tissue with tweezers, rinse it twice to wash o...

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Abstract

The invention relates to a method for constructing an air bladder cell system of blotchy rockcod, which comprises: taking an air bladder tissue of the blotchy rockcod as a material to start primary culture, and culturing the air bladder tissue in a DMEM / F12 liquid medium which contains fetal calf serum, carboxymethyl chito-oligosaccharide, basic fibroblast growth factors, I-type insulin-like growth factors, N-acetyl glucose hydrochloride and culture supernatant of heart cells of the blotchy rockcod in the log phase and has a pH value of between 7.0 and 7.4; and adopting a trypsin digestion method for subculturing. The air bladder cell system of the blotchy rockcod constructed by the method is subcultured for 62 generations currently. The technology is scientific and reasonable, is hopefulto be applied to ecotoxicological research, performs pollution monitoring and safety evaluation on various environmental pollutants in the sea, and can also be applied to separation, identification and in vitro breeding of fish viroids, and research in the aspects of interaction of viruses and host cells, and so on.

Description

technical field [0001] The invention relates to a method for establishing a swim bladder cell line by using the swim bladder tissue cells of the brown-spotted grouper—a method for constructing the swim-bladder cell line of the brown-spotted grouper. Background technique [0002] In recent years, with the large-scale outbreak of various marine fish viral diseases and the increasing pollution of the marine environment, the aquaculture industry has suffered huge losses. At the same time, various environmental pollutants in the ocean, including genotoxicants, mutagens, and carcinogens The existence of , environmental hormones and endocrine disruptors in water bodies and the enrichment in fish have brought great threats to human health. There is an urgent need for various fish virus vaccines and the establishment of a marine environmental monitoring system. Fish cell lines, especially various tissue cell lines of important marine economic fish, are just as an important in vitro r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06C12N5/071
Inventor 樊廷俊魏云波姜国建
Owner OCEAN UNIV OF CHINA
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