CN100335617C - Process for structuring large brill fin clone - Google Patents
Process for structuring large brill fin clone Download PDFInfo
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- CN100335617C CN100335617C CNB2005100451859A CN200510045185A CN100335617C CN 100335617 C CN100335617 C CN 100335617C CN B2005100451859 A CNB2005100451859 A CN B2005100451859A CN 200510045185 A CN200510045185 A CN 200510045185A CN 100335617 C CN100335617 C CN 100335617C
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Abstract
The present invention relates to a method for structuring a turbot fin cell system. The fin texture and the pelvic fin texture of a turbot are used as materials; first, the fin texture is sheared, and free fin cells and loosen texture blocks are obtained by a step digestion method through trypsinase, hyaluronidase and II-type collagenase; next, the free fin cells and the loosen texture blocks are cultured in L-15 culture solution containing 20% of fetal bovine serum, carboxymethyl-chitosan, N-acetyl glucosamine hydrochloride, D-glucosamine hydrochloride, human epidermal growth factors, human basic fibroblast growth factors and culture solution for flounder gill cells; finally, the subculture is carried out by a trypsinase digestion method. The method is scientific and reasonable, and generation continuing cells obtained by the method are transmitted to the 62nd generation at present. The turbot fin cells are not transfected by any virus or oncogene, so that the turbot fin cells can be directly applied to relevant theoretical research and the development of viral vaccines.
Description
Technical field
The present invention relates to a kind of method of utilizing the large brill fin tissue to set up fin clone, i.e. a kind of construction process of large brill fin clone.
Background technology
In recent years, it is increasing that the turbot of China is cultured scale, but the spreading venereal diseases viral disease also begins wide-scale distribution thereupon, causes the mortality ratio of turbot to rise year by year, caused great financial loss for the turbot aquaculture.How to solve the prevention and the treatment problem of turbot virus disease, become the major issue of being badly in need of solution in the world today in the turbot aquaculture.In prevention and treatment research to the marine cultured animal virus disease, scholars are consistent to think to have only the marine cultured animal of foundation continuity clone, could fundamentally investigate thoroughly the route of infection and infection mechanism of virus, just can obtain the last success of its virus disease treatment the marine cultured animal cell; In addition, utilize this clone to prepare its specificity virus vaccine, could successfully carry out the epidemic prevention of its virus disease effectively.
In marine organisms, especially in turbot, so far also not by setting up the successful report that corresponding clone is studied the breeding of virus and infected mechanism and scale operation virus vaccines.Therefore, set up turbot clone, be to investigate thoroughly that fundamentally some Causative virus to the route of infection of turbot cell and the precondition of infection mechanism thereof, also are the places of the key of development of turbot virus vaccines and production, will already bring huge economic benefit for turbot.
Summary of the invention
Purpose of the present invention is utilized the large brill fin tissue exactly, and a kind of technology of setting up large brill fin clone---a kind of construction process of large brill fin clone is provided.
The turbot of living is in the sterilization seawater of the penicillin of 1000 units per ml and Streptomycin sulphate and supported 24 hours temporarily having added concentration, after the taking-up turbot is put and is soaked 1～2 minute in 70% alcohol, aseptic dorsal fin and the abdomeinal fin cut in Bechtop, through 70% after alcohol-pickled 30～60 seconds, with putting into the sterilization small beaker after the cleaning of L-15 nutrient solution, add 5 milliliters of L-15 nutrient solutions that contain 5% foetal calf serum, with eye scissors the fin tissue being cut into fragment packs into and sterilizes in the centrifuge tube, centrifugal collecting precipitate, suspend evenly with 2 milliliters of L-15 nutrient solutions, add 2 milliliter of 0.5% trypsinase, enzymolysis is 20～30 minutes behind the mixing.The trypsin digestion thing is packed in the disinfectant centrifuge tube, centrifugal collecting precipitate, suspend evenly with 2 milliliters of L-15 nutrient solutions, add 2 milliliter of 1.5% Unidasa and 2 milliliters of 0.6%II Collagen Type VI enzymes, the mixing enzymolysis is packed into after 1～2 hour in the disinfectant centrifuge tube, free fin cell of centrifugal collection and open texture piece throw out, with the special-purpose multiplication culture liquid of 2 milliliters of large brill fin cells, divide and be filled in 2 25 milliliters of culturing bottles, just putting dry doubling after 6 hours, add the special-purpose multiplication culture liquid of 4 milliliters of fin cells for every bottle, put in 20～22 ℃ of biochemical incubators and cultivate, change the special-purpose multiplication culture liquid of large brill fin cell once every 3～5 days half amounts.
The prescription of the special-purpose multiplication culture liquid of used large brill fin cell is: the L-15 nutrient solution, 20% foetal calf serum, 0.05 ‰～0.15 ‰ carboxymethyl chitosan, 0.05 ‰～0.15 ‰ N-acetyl glucosamine hydrochloride, 0.05 ‰～0.15 ‰ glucosamine hydrochloride, 0.01 ‰～0.02 ‰ human epidermal growth factor, 0.01 ‰～0.02 ‰ rh-bFGF and 20% paralichthys olivaceus gilled cell nutrient solution.
Can pass more than 60 generations through the large brill fin cell that goes down to posterity that this method obtained, the constructed large brill fin clone of the present invention now reached for the 62nd generation.It is that the principal feature of technology is that this cell is built: clone can continuous passage, can provide a large amount of large brill fin cells; Cell line cell can directly apply to theoretical investigation, the breeding of virus and the development of virus vaccines; Be applied to the production of turbot virus vaccines and culture the turbot immunity high efficiency, have a extensive future.
Embodiment
As embodiment, the present invention provides concrete preparation and construction process in detail.
1, the processing of large brill fin tissue: the turbot of will living was supported 12～24 hours in having added the sterilization seawater that contains penicillin 1000 units per ml and Streptomycin sulphate 1000 units per ml temporarily.After the taking-up turbot is put and is soaked 1～2 minute in 70% alcohol, aseptic dorsal fin and the abdomeinal fin cut in Bechtop, through 70% after alcohol-pickled 30～60 seconds, in the sterilization culture dish, clean 2 times with commercially available L-15 nutrient solution, put into 10 milliliters of sterilization small beakers, add 5 milliliters of L-15 nutrient solutions that contain 5% foetal calf serum, the fin tissue is cut into the fragment of organizing of 1 cubic millimeter of size with eye scissors.
2, the acquisition of the enzymolysis of large brill fin tissue and free cell: the fin tissue is shredded thing pack in 10 milliliters of glass centrifuge tubes of disinfectant, 1200 rev/mins centrifugal 15 minutes, the collecting precipitation thing suspends evenly with 2 milliliters of L-15 nutrient solutions, add 2 milliliter of 0.5% commercially available trypsinase, enzymolysis is 20～30 minutes behind the mixing.The trypsin digestion thing is packed in 10 milliliters of glass centrifuge tubes of disinfectant, 1200 rev/mins centrifugal 15 minutes, the collecting precipitation thing suspends evenly with 2 milliliters of L-15 nutrient solutions, adds 2 milliliter of 1.5% commercially available Unidasa and 2 milliliter of 0.6% commercially available II Collagen Type VI enzyme, and enzymolysis is 1～2 hour behind the mixing.
3, the former foster startup of being commissioned to train of large brill fin cell: Unidasa and II Collagen Type VI enzyme zymolyte are packed in 10 milliliters of glass centrifuge tubes of disinfectant, 1200 rev/mins centrifugal 20 minutes, collect free fin cell and open texture piece throw out, after suspending evenly with the special-purpose multiplication culture liquid of 2 milliliters of large brill fin cells, divide to be filled in 2 25 milliliters of plastic culture bottles, just putting dry doubling 6 hours; Add the special-purpose multiplication culture liquid of 4 milliliters of large brill fin cells for every bottle, put in 20～22 ℃ of biochemical incubators and cultivate, observe and record cultivation situation; Change liquid once every 3～5 days half amounts, 2.5 milliliters of old nutrient solutions of promptly aseptic taking-up add 2.5 milliliters of special-purpose multiplication culture liquid of new large brill fin cell again.
4, the succeeding transfer culture of large brill fin cell: after treating that the large brill fin cell grows up to individual layer, the original fluid in the sucking-off culturing bottle, adding 0.5 ml concn is 0.25～0.3% trypsin solution in each culturing bottle, leaves standstill enzymolysis 2～3 minutes; Trypsin solution is removed in suction, adds the special-purpose multiplication culture liquid of 5 milliliters of large brill fin cells in every culturing bottle, with making the large brill fin cell suspension at the bottom of the dropper piping and druming culturing bottle in 3～5 minutes; Take out 2.5 milliliters of large brill fin cell suspensions respectively from each culturing bottle, join respectively in the new culturing bottle, each culturing bottle is added 2.5 milliliters of above-mentioned nutrient solutions, makes it final volume to 5 milliliter; After treating that the large brill fin cell grows up to individual layer once more, still with the cultivation of going down to posterity of above-mentioned same procedure.
Embodiment 1
1, the processing of large brill fin tissue: the turbot of will living was supported 12 hours in having added the sterilization seawater that contains penicillin 1000 units per ml and Streptomycin sulphate 1000 units per ml temporarily.The taking-up turbot is put and is soaked after 1 minute in 70% alcohol, aseptic dorsal fin and the abdomeinal fin cut in Bechtop, through 70% after alcohol-pickled 30 seconds, in the sterilization culture dish, clean 2 times with commercially available L-15 nutrient solution, put into 10 milliliters of sterilization small beakers, add 5 milliliters of L-15 nutrient solutions that contain 5% foetal calf serum, the fin tissue is cut into the fragment of organizing of 1 cubic millimeter of size with eye scissors.
2, the acquisition of the enzymolysis of large brill fin tissue and free cell: the fin tissue is shredded thing pack in 10 milliliters of glass centrifuge tubes of disinfectant, 1200 rev/mins centrifugal 15 minutes, the collecting precipitation thing suspends evenly with 2 milliliters of L-15 nutrient solutions, add 2 milliliter of 0.5% commercially available trypsinase, enzymolysis is 20 minutes behind the mixing.The trypsin digestion thing is packed in 10 milliliters of glass centrifuge tubes of disinfectant, 1200 rev/mins centrifugal 15 minutes, the collecting precipitation thing suspends evenly with 2 milliliters of L-15 nutrient solutions, adds 2 milliliter of 1.5% commercially available Unidasa and 2 milliliter of 0.6% commercially available II Collagen Type VI enzyme, and enzymolysis is 2 hours behind the mixing.
3, the former foster startup of being commissioned to train of large brill fin cell: Unidasa and II Collagen Type VI enzyme zymolyte are packed in 10 milliliters of glass centrifuge tubes of disinfectant, 1200 rev/mins centrifugal 20 minutes, collect free fin cell and open texture piece throw out, after suspending evenly with the special-purpose multiplication culture liquid of 2 milliliters of large brill fin cells, divide to be filled in 2 25 milliliters of plastic culture bottles, just putting dry doubling 6 hours; Add the special-purpose multiplication culture liquid of 4 milliliters of large brill fin cells for every bottle, put in 20 ℃ of biochemical incubators and cultivate, observe and record cultivation situation; Change liquid once every 3 days half amounts, 2.5 milliliters of old nutrient solutions of promptly aseptic taking-up add 2.5 milliliters of special-purpose multiplication culture liquid of new large brill fin cell again.
4, the succeeding transfer culture of large brill fin cell: after treating that the large brill fin cell grows up to individual layer, the original fluid in the sucking-off culturing bottle, adding 0.5 ml concn is 0.25% trypsin solution in each culturing bottle, leaves standstill enzymolysis 2～3 minutes; Trypsin solution is removed in suction, adds the special-purpose multiplication culture liquid of 5 milliliters of large brill fin cells in every culturing bottle, with making the large brill fin cell suspension at the bottom of the dropper piping and druming culturing bottle in 3～5 minutes; Take out 2.5 milliliters of large brill fin cell suspensions respectively from each culturing bottle, join respectively in the new culturing bottle, each culturing bottle is added 2.5 milliliters of above-mentioned nutrient solutions, makes it final volume to 5 milliliter; After treating that the large brill fin cell grows up to individual layer once more, still with the cultivation of going down to posterity of above-mentioned same procedure.
Embodiment 2
1, the processing of large brill fin tissue: the turbot of will living was supported 24 hours in having added the sterilization seawater that contains penicillin 1000 units per ml and Streptomycin sulphate 1000 units per ml temporarily.The taking-up turbot is put and is soaked after 2 minutes in 70% alcohol, aseptic dorsal fin and the abdomeinal fin cut in Bechtop, through 70% after alcohol-pickled 60 seconds, in the sterilization culture dish, clean 2 times with commercially available L-15 nutrient solution, put into 10 milliliters of sterilization small beakers, add 5 milliliters of L-15 nutrient solutions that contain 5% foetal calf serum, the fin tissue is cut into the fragment of organizing of 1 cubic millimeter of size with eye scissors.
2, the acquisition of the enzymolysis of large brill fin tissue and free cell: the fin tissue is shredded thing pack in 10 milliliters of glass centrifuge tubes of disinfectant, 1200 rev/mins centrifugal 15 minutes, the collecting precipitation thing suspends evenly with 2 milliliters of L-15 nutrient solutions, add 2 milliliter of 0.5% commercially available trypsinase, enzymolysis is 30 minutes behind the mixing.The trypsin digestion thing is packed in 10 milliliters of glass centrifuge tubes of disinfectant, 1200 rev/mins centrifugal 15 minutes, the collecting precipitation thing suspends evenly with 2 milliliters of L-15 nutrient solutions, adds 2 milliliter of 1.5% commercially available Unidasa and 2 milliliter of 0.6% commercially available II Collagen Type VI enzyme, and enzymolysis is 1 hour behind the mixing.
3, the former foster startup of being commissioned to train of large brill fin cell: Unidasa and II Collagen Type VI enzyme zymolyte are packed in 10 milliliters of glass centrifuge tubes of disinfectant, 1200 rev/mins centrifugal 20 minutes, collect free fin cell and open texture piece throw out, after suspending evenly with the special-purpose multiplication culture liquid of 2 milliliters of large brill fin cells, divide to be filled in 2 25 milliliters of plastic culture bottles, just putting dry doubling 6 hours; Add the special-purpose multiplication culture liquid of 4 milliliters of large brill fin cells for every bottle, put in 22 ℃ of biochemical incubators and cultivate, observe and record cultivation situation; Change liquid once every 5 days half amounts, 2.5 milliliters of old nutrient solutions of promptly aseptic taking-up add 2.5 milliliters of special-purpose multiplication culture liquid of new large brill fin cell again.
4, the succeeding transfer culture of large brill fin cell: after treating that the large brill fin cell grows up to individual layer, the original fluid in the sucking-off culturing bottle, adding 0.5 ml concn is 0.3% trypsin solution in each culturing bottle, leaves standstill enzymolysis 2～3 minutes; Trypsin solution is removed in suction, adds the special-purpose multiplication culture liquid of 5 milliliters of large brill fin cells in every culturing bottle, with making the large brill fin cell suspension at the bottom of the dropper piping and druming culturing bottle in 3～5 minutes; Take out 2.5 milliliters of large brill fin cell suspensions respectively from each culturing bottle, join respectively in the new culturing bottle, each culturing bottle is added 2.5 milliliters of above-mentioned nutrient solutions, makes it final volume to 5 milliliter; After treating that the large brill fin cell grows up to individual layer once more, still with the cultivation of going down to posterity of above-mentioned same procedure.
Claims (1)
1, a kind of construction process of large brill fin clone, the turbot of at first will living is in the sterilization seawater of the penicillin of 1000 units per ml and Streptomycin sulphate and supported 24 hours temporarily having added concentration, after the taking-up turbot is put and is soaked 1～2 minute in 70% alcohol, aseptic dorsal fin and the abdomeinal fin cut in Bechtop, through 70% after alcohol-pickled 30～60 seconds, with putting into the sterilization small beaker after the cleaning of L-15 nutrient solution, add 5 milliliters of L-15 nutrient solutions that contain 5% foetal calf serum, with eye scissors the fin tissue being cut into fragment packs into and sterilizes in the centrifuge tube, centrifugal collecting precipitate, suspend evenly with 2 milliliters of L-15 nutrient solutions, add 2 milliliter of 0.5% trypsinase, enzymolysis is 20～30 minutes behind the mixing.The trypsin digestion thing is packed in the disinfectant centrifuge tube, centrifugal collecting precipitate, suspend evenly with 2 milliliters of L-15 nutrient solutions, add 2 milliliter of 1.5% Unidasa and 2 milliliters of 0.6%II Collagen Type VI enzymes, the mixing enzymolysis is packed into after 1～2 hour in the disinfectant centrifuge tube, free fin cell of centrifugal collection and open texture piece throw out, with the special-purpose multiplication culture liquid of 2 milliliters of large brill fin cells, divide and be filled in 2 25 milliliters of culturing bottles, just putting dry doubling after 6 hours, add the special-purpose multiplication culture liquid of 4 milliliters of fin cells for every bottle, put in 20～22 ℃ of biochemical incubators and cultivate, change large brill fin cell proliferation nutrient solution once every 3～5 days half amounts, described special-purpose multiplication culture liquid formula is the L-15 nutrient solution, and 20% foetal calf serum, 0.1 ‰ carboxymethyl chitosans, 0.1 ‰ N-acetyl glucosamine hydrochloride, 0.1 ‰ glucosamine hydrochloride, 0.015 ‰ human epidermal growth factor, 0.015 the culture supernatant of ‰ rh-bFGF and 20% paralichthys olivaceus gilled cell.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101372682B (en) * | 2008-10-15 | 2010-08-11 | 中国海洋大学 | Construction method of Epinephelus fuscoguttatus fin cell line |
| CN102399743A (en) * | 2011-12-16 | 2012-04-04 | 中国水产科学研究院长江水产研究所 | Cell line of pterygiophore tissue of cryprinus carpiod and construction method |
| CN102757932A (en) * | 2012-08-07 | 2012-10-31 | 中国科学院昆明动物研究所 | Construction method of sinocyclocheilus grahami grahami fin cell line |
| CN102757934B (en) * | 2012-08-07 | 2013-05-29 | 中国科学院昆明动物研究所 | Construction method of fin cell line of anabarilius grahami |
| CN102925407B (en) * | 2012-11-22 | 2015-02-18 | 大连海洋大学 | In-vitro culture method of triploid loach fin cells |
| CN105200005A (en) * | 2014-08-13 | 2015-12-30 | 中国科学院海洋研究所 | Paralichthys olivaceus muscle satellite cell line establishing method, specific primer for identifying paralichthys olivaceus muscle satellite cell marker gene and application of specific primer |
| CN104762252B (en) * | 2015-04-27 | 2017-06-16 | 上海海洋大学 | A kind of construction method of grass goldfish abdomeinal fin cell line |
| CN104830760B (en) * | 2015-06-01 | 2017-12-22 | 中国海洋大学 | A kind of method for building up of turbot muscle cell system |
| CN113943693A (en) * | 2021-09-27 | 2022-01-18 | 广东海洋大学 | Construction method of tilapia ventral fin cell line |
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| 壳多糖及其衍生物在动物细胞培养中应用研究进展 王绪敏，中国海洋药物，第5期 2000 * |
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