CN104774802A - Silver crucian carp back fin cell line - Google Patents
Silver crucian carp back fin cell line Download PDFInfo
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- CN104774802A CN104774802A CN201510206829.1A CN201510206829A CN104774802A CN 104774802 A CN104774802 A CN 104774802A CN 201510206829 A CN201510206829 A CN 201510206829A CN 104774802 A CN104774802 A CN 104774802A
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Abstract
The invention relates to a silver crucian carp back fin cell line. The silver crucian carp back fin cell line is obtained through primary culture and subculture conducted on the back fin of a silver crucian carp, and a used cell culture fluid is prepared by using an M199 culture medium. The invention further provides an establishing method and application of the silver crucian carp back fin cell line. The silver crucian carp back fin cell line has the advantages that the established silver crucian carp back fin cell line can be subjected to continuous passage, and a large amount of silver crucian carp back fin cell lines can be provided. The silver crucian carp back fin cell line can be directly applied to research on silver crucian carp immunity related function genes and can be applied to a silver crucian carp virus infection experiment, a large amount of manpower and material resources are saved, and effect is better. The silver crucian carp back fin cell line can be subjected to multigelation and can be applied to important research in cell biology, virology, molecular biology, genetics, immunology and the like.
Description
Technical field
The present invention relates to technical field of life science, specifically, is pond crucian carp fish dorsal fin clone.
Background technology
Pond crucian carp fish (C.auratus gibelio) is under the jurisdiction of carp type order (Percoiformes), Cyprinidae (Sparidae), crucian carp genus (Carassius), the subspecies of C.auratus.The crucian being distributed in China comprises black crucian carp, crucian carp and pond crucian carp, and wherein the chromosome number of crucian carp is 100, belongs to diploid, and pond crucian carp chromosome number is 150 ±, belong to triploid.It is fast that pond crucian carp fish individuality has growth, the economic characters that disease resistance is by force etc. good, is the freshwater aquiculture kind of the main raising of China.
Cell culture technology is life science, the research means that cell engineering, genetically engineered and biomedical engineering are main.The biologic material that the Study on etiology being configured to fishes virus disease of fish cell system and the vaccine research of virus provide the foundation has important researching value.Identify that with being separated responsive virus strain be the most effectual way that OIE Office International Des Epizooties (OIE) is recommended by cell strain, the research of the aspects such as these cells are fish virology, immunology, cytology provides good experiment material.
Wolf and Quimby establishes the first strain fish cell system, i.e. rainbow trout gonadal cell system RTG-2 in the world in the sixties in 20th century.At present, the fish cell system reported has more than 300 to plant (Lakra WS, Swaminathan T R, Joy K P.Development, characterization, conservation andstorage of fish cell lines:a review [J] .Fish Physiology and Biochemistry, 2011,37 (1): 1-20).The cell strain of the crucian wherein reported mainly contains hybridized prussian carp clone (Isogeneicginbuna crucian carp, Lake Suwa) (Hasegawa S, Nakayasu C, Okamoto N, et al.Fin cell line from isogeneic ginbuna crucian carp [J] .In Vitro Cellular & Developmental Biology-Animal, 1997, 33 (4): 232-233.), brocade carp fin ray clone (Xiao Yi, Zeng Lingbing, Xu Jin, Deng. the foundation of bright and beautiful carp isozyme clone and biological characteristics [J] thereof. Chinese cytobiology journal, 2012, 34 (8): 767-774.), crucian heteroploid cell line (Chen Minrong, Chen Hongxi, easy Chant is blue. the foundation of crucian heteroploid cell line and biological characteristics [J] .1985.), crucian cheek epiphragma clone (HCC-87) (Li Yanan, Zhang Yi, Mao Shujian. the foundation [J] of two strain fish cell systems-grass carp tail fin tissue lines (HGC-87) and crucian cheek epiphragma clone (HCC-87). Mao Shujian. life science collection of thesis, 1992:112-119.).There is no the relevant report about setting up pond crucian carp fish dorsal fin clone at present.
Summary of the invention
The object of the invention is for deficiency of the prior art, a kind of pond crucian carp fish dorsal fin clone is provided.
Of the present invention again one object be that a kind of construction process of pond crucian carp fish dorsal fin clone is provided.
Another object of the present invention provides a kind of application of pond crucian carp fish dorsal fin clone.
For achieving the above object, the technical scheme that the present invention takes is: a kind of pond crucian carp fish dorsal fin clone, and the deposit number of described pond crucian carp fish dorsal fin clone is CCTCC NO:C201525.
Described pond crucian carp fish dorsal fin clone is that the dorsal fin of pond crucian carp fish obtains through original cuiture, Secondary Culture.
Original cuiture and Secondary Culture cell culture fluid used are that M199 substratum obtains.
Original cuiture and Secondary Culture cell culture fluid used obtain by the following method: get M199 substratum, add the foetal calf serum accounting for substratum cumulative volume 10-20%, final concentration is the Basic Fibroblast Growth Factor of 20ng/ml, and final concentration is the Urogastron of 1ng/ml.
The method of original cuiture is: the dorsal fin cutting pond crucian carp fish, then 75% alcohol disinfecting is used, again with the mucus on penicillin, Streptomycin sulphate removing pond crucian carp fish dorsal fin, then rinse with the D-PBS containing amphotericin B, after flushing, be immersed in after aseptically dorsal fin being cut into small pieces with scissors in trypsinase and EDTA; Then the dorsal fin of the pond crucian carp fish shredded is transferred in Tissue Culture Flask, and keep bottom temp to be 25 DEG C, then add the M199 substratum of the serum containing 10% tire ox, penicillin, Streptomycin sulphate, amphotericin B, Basic Fibroblast Growth Factor and Urogastron.
The method of Secondary Culture is: after the dorsal fin cell monolayer of the pond crucian carp fish of original cuiture is paved with Tissue Culture Flask, old nutrient solution is removed with suction pipe, clean with PBS, add trypsin digestion cell, after microscope Microscopic observation cell rounding, add fresh complete culture solution, repeatedly blow and beat, hang cell, gone down to posterity with 1:2, put into 27 DEG C of incubators and carry out Secondary Culture.
For realizing above-mentioned second object, the technical scheme that the present invention takes is: a kind of construction process of pond crucian carp fish dorsal fin clone, described construction process comprises the following steps: original cuiture, Secondary Culture.
Described construction process comprises the following steps:
A) original cuiture
Cut the dorsal fin of pond crucian carp fish, then use 75% alcohol disinfecting, then with the mucus on penicillin, Streptomycin sulphate removing pond crucian carp fish dorsal fin, then rinse with the D-PBS containing amphotericin B, after flushing, be immersed in after aseptically dorsal fin being cut into small pieces with scissors in trypsinase and EDTA; Then the dorsal fin of the pond crucian carp fish shredded is transferred in Tissue Culture Flask, and keep bottom temp to be 25 DEG C, then add the M199 substratum of the serum containing 10% tire ox, penicillin, Streptomycin sulphate, amphotericin B, Basic Fibroblast Growth Factor and Urogastron;
B) Secondary Culture
After the dorsal fin cell monolayer of the pond crucian carp fish of original cuiture is paved with Tissue Culture Flask, old nutrient solution is removed with suction pipe, clean with PBS, add trypsin digestion cell, after microscope Microscopic observation cell rounding, add fresh complete culture solution, repeatedly blow and beat, hang cell, gone down to posterity with 1:2, put into 27 DEG C of incubators and carry out Secondary Culture.
For realizing above-mentioned 3rd object, the technical scheme that the present invention takes is: the described application of pond crucian carp fish dorsal fin clone in cell engineering, genetically engineered or biomedical engineering.
The invention has the advantages that:
1, the pond crucian carp fish dorsal fin clone that the present invention sets up can continuous passage, can provide a large amount of pond crucian carp fish dorsal fin clone.
2, this clone can directly apply to the research of pond crucian carp fish immunity correlation function gene, and the virus infection experiment for pond crucian carp fish can application cell system, has saved a large amount of manpowers, material resources and effect is better.
3, pond crucian carp fish dorsal fin clone can multigelation, can be applicable to the important research such as cytobiology, virusology, toxicology, molecular biology, genetics and immunology.
Accompanying drawing explanation
Accompanying drawing 1 is the cell (about 3 days) moved out near the pond crucian carp fish dorsal fin tissue block under microscope.
The pond crucian carp fish dorsal fin clone after going down to posterity stablized under microscope by accompanying drawing 2.
Accompanying drawing 3 is cell infection crucian herpesvirusⅡtype (CyHV-2) situations. (A): normal cell, scalebar.=100 μm. (B and C) infects crucian herpesvirusⅡtype (CyHV-2), infect latter 6 days, scalebar.=100and 50 μm. arrow indication is sick cell. (D) infects crucian herpesvirusⅡtype (CyHV-2), infect latter 8 days, scale bar.=100.
Accompanying drawing 4 is facsimile logs that crucian herpesvirusⅡtype (CyHV-2) infects virus in pond crucian carp fish dorsal fin cell line supernatant.
Embodiment
Below in conjunction with accompanying drawing, embodiment provided by the invention is elaborated.
The technical problem to be solved in the present invention be to provide a kind of can stablize go down to posterity set up pond crucian carp fish dorsal fin clone (SCC-DF cell line, Silver crucian carp dorsal fin cell line), the immunology being configured to research pond crucian carp fish of this clone and the unknown fishes virus of screening provide good biomaterial.
The construction process of embodiment 1 pond crucian carp fish dorsal fin clone
1, cell culture fluid is prepared
Get GIBCO company M199 substratum, add and account for substratum cumulative volume 10-20% (the following stated per-cent unless otherwise noted, be volume percent) foetal calf serum, final concentration is the Basic Fibroblast Growth Factor (bFGF) of 20ng/ml, final concentration is the Urogastron (EGF) of 1ng/ml, deposit for 4 DEG C, for subsequent use.
2, original cuiture
Cut the dorsal fin of pond crucian carp fish, then use 75% alcohol disinfecting, then use 1000IU mL
-1penicillin, 1000ug mL
-1mucus on Streptomycin sulphate removing pond crucian carp fish dorsal fin, then with containing 2.5ug mL
-1the D-PBS of amphotericin B rinse 3 times.After flushing, aseptically with scissors, dorsal fin is cut into about 1mm
3fritter after be immersed in 0.25% trypsinase and 0.02%EDTA.
Then the dorsal fin of the pond crucian carp fish shredded is transferred to bottom for 25cm
2tissue Culture Flask in, and keep bottom temp be 25 DEG C.After 15 hours, add the serum (FBS) that 2mL contains 10% tire ox, 200IUmL
-1penicillin, 200ug mL
-1streptomycin sulphate, 0.5ug mL
-1amphotericin B, the M199 substratum of FGF and EGF.After 24 hours, add 3mL and contain 10%FBS, 200IU mL
-1penicillin, 200ugmL
-1streptomycin sulphate, 0.5ug mL
-1amphotericin B, the substratum of FGF and EGF.Change the fresh culture of 50% every day.
3, Secondary Culture
After the dorsal fin cell monolayer of the pond crucian carp fish of original cuiture is paved with Tissue Culture Flask, old nutrient solution is removed with suction pipe, with PBS cleaning twice, add 0.25% pancreatin (containing EDTA) 1mL peptic cell, after microscope Microscopic observation cell rounding, add the complete culture solution that 10mL is fresh, repeatedly blow and beat, hanged cell, go down to posterity with 1:2, add complete culture solution to 5mL/ bottle, put into 27 DEG C of incubators and carry out Secondary Culture.
Embodiment 2 pond crucian carp fish dorsal fin clone
Through isolated culture, present invention obtains a strain homogeneous pond crucian carp fish dorsal fin clone (SCC-DF cell line, Silver crucian carp dorsal fin cell line) of stabilizing component that goes down to posterity and go down to posterity so far.
The pond crucian carp fish dorsal fin clone that the present invention obtains has carried out preservation in China typical culture collection center, pond crucian carp fish dorsal fin clone SCC-DF, its deposit number is CCTCC NO:C201525, Classification And Nomenclature is pond crucian carp fish (Carassius C.auratus), the address of China typical culture collection center is Wuhan University of Wuhan City of Hubei China province, and preservation date is on March 27th, 2015.
Further, described pond crucian carp fish dorsal fin clone has following biological characteristics:
1) cell near primary separation self-organization block, the Morphological Features in fibroblast-like cell;
2) ability of cell proliferation is general, and each tissue block after treatment about 7d all can isolate a large amount of cell, has the ability of continuous passage;
3) this clone culture condition is 25 DEG C of constant temperature cell culture incubators, without the need to carbonic acid gas.
This clone adopts fish cell system to cultivate the most frequently used substratum M199, and culture condition is consistent with common fish cell.Need trysinization when going down to posterity, sub-bottle can cover with for 5 days.Cellular form is in typical threadiness.Therefore this clone is the pond crucian carp fish dorsal fin clone of a kind of stability of characteristics, uniform component, is research aquatic animal immunology and virological good biological material.
Embodiment 3 virus infection is tested
One, experimental technique
1, the infection of virus
Cultured grass goldfish abdomeinal fin clone is reached in 6 orifice plates, adherent growth overnight.Respectively containing crucian simplexvirus II, and alkaline phosphate buffer CyHV-2), PBS (negative control group) gets 500 μ l cells infecteds, hatch supernatant discarded after 1 hour, 3 times are washed with PBS, add perfect medium 500 μ l to be placed in incubator and to cultivate, every day observation of cell growing state.
2, the drafting of virus facsimile log in cell conditioned medium
Reached by cell in 24 orifice plates, infect virus by step in 1, within every 24 hours, get supernatant, high speed centrifugation discards precipitation, and supernatant extracts DNA in the steps below and carries out fluorescence quantitative PCR detection.
3, the extraction of viral DNA
(1) add isopyknic PBS damping fluid, mix, with 8000rpm centrifugal 5 minutes, abandon supernatant;
(2) add 20 μ L proteolytic enzyme k. and be incubated 3 hours in 56 DEG C of water-baths;
(3) centrifugal 5 minutes of 8000rpm, to get in 750 μ L supernatant liquors in a new centrifuge tube, adds isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1); 10 minutes are mixed with the speed of 50 revs/min, more centrifugal 5 minutes with 8000 revs/min, get 650 μ L supernatant liquors in a new centrifuge tube; Repeat 1 time;
(4) get 600 μ L supernatant liquors in a new centrifuge tube, add isopyknic chloroform: primary isoamyl alcohol (24:1).Mix 10 minutes with 50 revs/min of speed, 8000 revs/min centrifugal 5 minutes;
(5) get 480 μ L supernatant liquors in a new centrifuge tube, add the sodium-acetate of 40 μ L 3mol/L and the cooling ethanol of 960 μ L4 DEG C; Jog, visible white floss;
(6) subzero 20 DEG C place 20-30 minute, after centrifugal 10 minutes with 12000 revs/min, abandoning supernatant;
(7) by the washing with alcohol of 200ul 75%, 14000 revs/min centrifugal 5 minutes, abandons supernatant; Repeat to do once;
(8) milli-Q water with 50ul after 10 minutes is dried, in subzero 20 DEG C of preservations.
4, fluorescence quantitative PCR detection
Upstream primer CyHV-2rtF2 (5 '-3 '): TTAGCGTCAGGTCCATAG
Downstream primer CyHV-2rtR1 (5 '-3 '): GGCGTGTAGAAATCAAAC
Reaction system:
Concussion mixing, trim is centrifugal.
Real-time PCR reaction conditions: 95 DEG C of 10min, (95 DEG C of 10s, 59 DEG C of 15s, 72 DEG C of 20s) 39 circulation; Melt curve analysis: 95 DEG C of 1min, 55 DEG C of 1min, 65 DEG C to 95 DEG C 0.1 DEG C/sec.
4, experimental result
As shown in Figure 3A, negative infection group (uninfecting virus) Growth of Cells is good; Fig. 3 B, C, infect virus about 6 days and shrink formation cavity sample; Fig. 3 D, infect about 8 days cells formed obvious virus disease cash resemble (CPE).
Virus amplification curve in cell conditioned medium the results are shown in Figure 4, and virus increased at first 3 days, entered plateau after 4 days.
Above detected result shows: this pond crucian carp fish dorsal fin clone can be applied to the detection of crucian simplexvirus II (CyHV-2) and corresponding virusology, immunology related experiment.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (9)
1. a pond crucian carp fish dorsal fin clone, is characterized in that, the deposit number of described pond crucian carp fish dorsal fin clone is CCTCC NO:C201525.
2. a pond crucian carp fish dorsal fin clone, is characterized in that, described pond crucian carp fish dorsal fin clone is that the dorsal fin of pond crucian carp fish obtains through original cuiture, Secondary Culture.
3. pond crucian carp fish dorsal fin clone according to claim 2, is characterized in that, original cuiture and Secondary Culture cell culture fluid used are that M199 substratum obtains.
4. pond crucian carp fish dorsal fin clone according to claim 3, it is characterized in that, original cuiture and Secondary Culture cell culture fluid used obtain by the following method: get M199 substratum, add the foetal calf serum accounting for substratum cumulative volume 10-20%, final concentration is the Basic Fibroblast Growth Factor of 20ng/ml, and final concentration is the Urogastron of 1ng/ml.
5. pond crucian carp fish dorsal fin clone according to claim 3, it is characterized in that, the method of original cuiture is: the dorsal fin cutting pond crucian carp fish, then 75% alcohol disinfecting is used, again with the mucus on penicillin, Streptomycin sulphate removing pond crucian carp fish dorsal fin, then rinse with the D-PBS containing amphotericin B, after flushing, be immersed in after aseptically dorsal fin being cut into small pieces with scissors in trypsinase and EDTA; Then the dorsal fin of the pond crucian carp fish shredded is transferred in Tissue Culture Flask, and keep bottom temp to be 25 DEG C, then add the M199 substratum of the serum containing 10% tire ox, penicillin, Streptomycin sulphate, amphotericin B, Basic Fibroblast Growth Factor and Urogastron.
6. pond crucian carp fish dorsal fin clone according to claim 3, it is characterized in that, the method for Secondary Culture is: after the dorsal fin cell monolayer of the pond crucian carp fish of original cuiture is paved with Tissue Culture Flask, remove old nutrient solution with suction pipe, clean with PBS, add trypsin digestion cell, after microscope Microscopic observation cell rounding, add fresh complete culture solution, repeatedly blow and beat, hang cell, gone down to posterity with 1:2, put into 27 DEG C of incubators and carry out Secondary Culture.
7., according to the construction process of the arbitrary described pond crucian carp fish dorsal fin clone of claim 1-6, it is characterized in that, described construction process comprises the following steps: original cuiture, Secondary Culture.
8. the construction process of pond crucian carp fish dorsal fin clone according to claim 7, it is characterized in that, described construction process comprises the following steps:
A) original cuiture
Cut the dorsal fin of pond crucian carp fish, then use 75% alcohol disinfecting, then with the mucus on penicillin, Streptomycin sulphate removing pond crucian carp fish dorsal fin, then rinse with the D-PBS containing amphotericin B, after flushing, be immersed in after aseptically dorsal fin being cut into small pieces with scissors in trypsinase and EDTA; Then the dorsal fin of the pond crucian carp fish shredded is transferred in Tissue Culture Flask, and keep bottom temp to be 25 DEG C, then add the M199 substratum of the serum containing 10% tire ox, penicillin, Streptomycin sulphate, amphotericin B, Basic Fibroblast Growth Factor and Urogastron;
B) Secondary Culture
After the dorsal fin cell monolayer of the pond crucian carp fish of original cuiture is paved with Tissue Culture Flask, old nutrient solution is removed with suction pipe, clean with PBS, add trypsin digestion cell, after microscope Microscopic observation cell rounding, add fresh complete culture solution, repeatedly blow and beat, hang cell, gone down to posterity with 1:2, put into 27 DEG C of incubators and carry out Secondary Culture.
9. according to the arbitrary described application of pond crucian carp fish dorsal fin clone in cell engineering, genetically engineered or biomedical engineering of claim 1-6.
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Cited By (3)
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| CN107629996A (en) * | 2017-08-29 | 2018-01-26 | 中国水产科学研究院珠江水产研究所 | The construction method of one plant of grass carp pectoral fin cell line |
| CN107641612A (en) * | 2017-09-30 | 2018-01-30 | 湖南师范大学 | Triploid cruciancarp caudal fin cell system 3nFC and its construction method and application |
| CN108546672A (en) * | 2018-04-20 | 2018-09-18 | 上海海洋大学 | A kind of construction method of hybridized prussian carp caudal fin cell system and its application |
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| CN107629996A (en) * | 2017-08-29 | 2018-01-26 | 中国水产科学研究院珠江水产研究所 | The construction method of one plant of grass carp pectoral fin cell line |
| CN107641612A (en) * | 2017-09-30 | 2018-01-30 | 湖南师范大学 | Triploid cruciancarp caudal fin cell system 3nFC and its construction method and application |
| CN108546672A (en) * | 2018-04-20 | 2018-09-18 | 上海海洋大学 | A kind of construction method of hybridized prussian carp caudal fin cell system and its application |
| CN108546672B (en) * | 2018-04-20 | 2021-06-25 | 上海海洋大学 | Construction method and application of carassius auratus gibelio fin cell line |
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