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Construction method of Epinephelus fuscoguttatus fin cell line

A construction method and technology of fin cells, applied in the field of construction of brown grouper fin cell lines, can solve problems such as the establishment of brown grouper tissue cell lines, successful production of virus vaccines, etc.

Inactive Publication Date: 2009-02-25
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no report on the establishment of tissue cell lines of brown spotted grouper, and there is no successful report on the large-scale production of virus vaccines through corresponding cell lines

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0009] 1. Preparation of fin tissue blocks of brown-spotted grouper: fresh and 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 24 hours. ~2 minutes. Aseptically remove the fin tissue in an ultra-clean workbench, rinse again with 75% alcohol for 0.5-1 minute, rinse twice with phosphate buffer, and cut the fin tissue in DMEM / F12 culture medium containing 5% fetal bovine serum. into about 1mm 3 Tissue pieces; collect fin tissue pieces by centrifugation at 800 rpm, add 0.5% hyaluronidase and 0.2% type II collagenase for joint digestion for 1 to 2 hours; centrifuge at 1000 rpm to collect tissue pieces; use 5% fetal bovine Suspend serum and 0.05‰~0.15‰ carboxymethyl chitosan oligosaccharides in DMEM / F12 culture medium with a pH value of 7.0~7.4, fully suspend them, and evenly inoculate them in a 25 ml culture bottle; put the culture bottle in...

Embodiment 1

[0015] Put the fresh and live brown grouper into the high double-antibody seawater with a concentration of 1000 units / ml of penicillin and streptomycin for 24 hours, then disinfect it in 75% alcohol for 2 minutes for the first disinfection; wipe the fish with a cotton ball Remove the fin tissue from the body surface, place it in a glass petri dish filled with phosphate buffer, use tweezers to pick up a cotton ball soaked in phosphate buffer and wipe the fin tissue along the direction of the fin rays to remove the mucus on the surface of the fin tissue; Rinse in 75% alcohol for 1 minute, carry out secondary disinfection; rinse the fin tissue twice with phosphate buffer, fully wash away the alcohol, transfer it to a penicillin vial containing 5% fetal bovine serum DMEM / F12 culture medium, and cut into Tissue pieces of 1 cubic millimeter; centrifuge at 800 rpm to collect fin tissue pieces, add 0.5% hyaluronidase and 0.2% type II collagenase for combined digestion for 2 hours; cent...

Embodiment 2

[0018] Put the fresh and live brown grouper into the high double-antibody seawater of penicillin and streptomycin with a concentration of 1000 units / ml for temporary cultivation for 24 hours, then disinfect it in 75% alcohol for 1.5 minutes for the first disinfection; then transfer the fish to into the ultra-clean workbench; wipe the surface of the fish with a cotton ball, remove the fin tissue, place it in a glass petri dish filled with phosphate buffer solution, and use tweezers to pick up the cotton ball soaked in phosphate buffer solution along the direction of the fin rays. Wipe the fin tissue to remove the mucus on the surface of the fin tissue; then rinse in 75% alcohol for 0.5 minutes for secondary disinfection; rinse the fin tissue twice with phosphate buffer solution, fully wash off the alcohol, and then transfer to the solution containing 5% fetal bovine serum DMEM / F12 culture medium in penicillin vials cut into 1 mm3 tissue pieces; centrifuge at 800 rpm to collect f...

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Abstract

The invention relates to a method for constructing a brown spotted rockfish fin cell line. The method is as follows: brown spotted rockfish fin tissue are taken as a material, the hyaluronidase with the concentration of 0.5% and collagenase II with the concentration of 0.2% combining digestion method is used for initiating primary culture, and the material is cultured in a DMEM / F12 culture medium which has the PH value of 7.0-7.4 and contains fetal calf serum, alkaline fibroblast growth factor, insulin-like growth factor I and carboxymethyl chito-oligosacaride, and the trypsin digestion method is adopted for subculturing. The brown spotted rockfish fin cell line constructed by the method is passed to the sixty-first generation at present. The method has scientific and reasonable process, and is expected to be applied to separating, identifying and reproducing fish viruses, preparing viral vaccines, studying the interaction between viruses and host cells, etc.

Description

technical field [0001] The invention relates to a method for establishing a fin cell line by using the fin tissue cells of the brown-spotted grouper - a method for constructing the fin cell line of the brown-spotted grouper. Background technique [0002] Brown-spotted grouper (Epinephelus fuscoguttatus) is a rare marine fish with high economic value widely distributed in the tropical and subtropical waters of the Indian Ocean and the Pacific Ocean. In recent years, the scale of grouper farming in my country has become larger and larger, but infectious viral diseases have also begun to spread widely, causing its mortality rate to increase year by year and causing major economic losses. Finding out the infection route of fish viruses and the interaction mechanism between viruses and cells is the key to fundamentally preventing and solving many marine economic fish virus diseases such as grouper, and fish cell lines are the key to studying virus infection routes, infection It ...

Claims

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Application Information

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