CN104774802B - Pond crucian carp fish dorsal fin cell line - Google Patents
Pond crucian carp fish dorsal fin cell line Download PDFInfo
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- CN104774802B CN104774802B CN201510206829.1A CN201510206829A CN104774802B CN 104774802 B CN104774802 B CN 104774802B CN 201510206829 A CN201510206829 A CN 201510206829A CN 104774802 B CN104774802 B CN 104774802B
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- crucian carp
- cell line
- dorsal fin
- carp fish
- pond crucian
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Abstract
The present invention relates to pond crucian carp fish dorsal fin cell line, the dorsal fin of described pond crucian carp fish dorsal fin cell line pond crucian carp fish obtains by original cuiture, Secondary Culture, and cell culture fluid used is that M199 culture mediums are made.The present invention also provides construction method and the application of the pond crucian carp fish dorsal fin cell line.Its advantage is shown：The pond crucian carp fish dorsal fin cell line that the present invention establishes can provide substantial amounts of pond crucian carp fish dorsal fin cell line with continuous passage.The cell line may be directly applied to the research that correlation function gene is immunized in pond crucian carp fish, and substantial amounts of manpower can have been saved with application cell system for the viral infection experiment of pond crucian carp fish, material resources and effect is preferable.Pond crucian carp fish dorsal fin cell line can be applied to the important research such as cell biology, virology, toxicology, molecular biology, science of heredity and immunology with multigelation.
Description
Technical field
The present invention relates to technical field of life science, is pond crucian carp fish dorsal fin cell line specifically.
Background technology
Pond crucian carp fish (C.auratus gibelio) is under the jurisdiction of carp type mesh (Percoiformes), Cyprinidae (Sparidae), crucian carp
Belong to (Carassius), C.auratus subspecies.Being distributed in the crucian in China includes the dyeing of black crucian carp, crucian carp and pond crucian carp, wherein crucian carp
Body number be 100, belong to diploid, pond crucian carp chromosome number be 150 ±, belong to triploid.Pond crucian carp fish individual has growth fast, and disease resistance is strong
It is the freshwater aquiculture kind of the main raising in China etc. good economic characters.
Cell culture technology is life science, cell engineering, genetic engineering and the main research of biomedical engineering
Means.What the Study on etiology for being configured to fishes virus disease of fish cell system and the vaccine research of virus provided the foundation
Biologic material has important researching value.It is world animal health group that sensitive Strain is identified and isolated from by cell line
The most effectual way of Office International Des Epizooties (OIE) recommendations is knitted, these cell lines are fish disease
The research of malicious, immunology, cytology etc. provides good experiment material.
Wolf and Quimby establishes first plant of fish cell system in the world, i.e. rainbow trout gonad in the 1960s
Cell line RTG-2.At present, it has been reported that fish cell system have more than 300 kind (Lakra W S, Swaminathan T R, Joy
K P.Development,characterization,conservation and storage of fish cell lines:
a review[J].Fish Physiology and Biochemistry,2011,37(1):1-20).Wherein it has been reported that
The cell line of crucian mainly has hybridized prussian carp cell line (Isogeneic ginbuna 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.), fancy carp fin ray cell line (Xiao Yi, Zeng Lingbing, creep, waits foundation and its biology of fancy carp isozyme cell lines
Learn characteristic [J] China cell biology journal, 2012,34 (8):767-774.), crucian heteroploid cell line (Chen Minrong, it is old
Hong Xi, the foundation of easy Chant orchids crucian heteroploid cell lines and biological characteristics [J] .1985.), crucian cheek epiphragma cell line
(HCC-87) (two plants of fish cell systems of Li Yanan, Zhang Yi, Mao Shujian-grass carp tail fin tissue lines (HGC-87) and the crucian cheek
The hard life sciences collection of thesis of foundation [J] hair trees of epiphragma cell line (HCC-87), 1992:112-119.).At present there is no on
Establish the relevant report of pond crucian carp fish dorsal fin cell line.
The content of the invention
The purpose of the present invention is to be directed to deficiency of the prior art, there is provided a kind of pond crucian carp fish dorsal fin cell line.
Another purpose of the present invention is to provide a kind of construction method of pond crucian carp fish dorsal fin cell line.
Another purpose of the present invention is to provide a kind of application of pond crucian carp fish dorsal fin cell line.
To achieve the above object, the present invention adopts the technical scheme that：A kind of pond crucian carp fish dorsal fin cell line, described pond crucian carp
The deposit number of fish dorsal fin cell line is CCTCC NO:C201525.
Described pond crucian carp fish dorsal fin cell line is that the dorsal fin of pond crucian carp fish obtains by original cuiture, Secondary Culture.
Cell culture fluid used in original cuiture and Secondary Culture is made for M199 culture mediums.
Cell culture fluid used in original cuiture and Secondary Culture is made by the following method：M199 culture mediums are taken, are added
Culture medium cumulative volume 10-20% hyclone is accounted for, final concentration of 20ng/ml Basic Fibroblast Growth Factor is final concentration of
1ng/ml EGF.
The method of original cuiture is：The dorsal fin of pond crucian carp fish is cut, then with 75% alcohol disinfecting, then with penicillin, strepto-
Element removes the mucus on pond crucian carp fish dorsal fin, is then rinsed with the D-PBS containing amphotericin B, after flushing, aseptically with cutting
Knife is immersed in trypsase and EDTA after dorsal fin is cut into small pieces；Then the dorsal fin of the pond crucian carp fish shredded is transferred to cell training
Support in bottle, and it is 25 DEG C to keep bottom temp, then adds serum, penicillin, streptomysin, anphotericin containing 10% tire ox
B, the M199 culture mediums of Basic Fibroblast Growth Factor and EGF.
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, use
Suction pipe removes old nutrient solution, with PBS, adds trypsin digestion cell, after microscope Microscopic observation cell rounding, adds
Fresh complete culture solution, blows and beats repeatedly, has hanged cell, with 1:2 are passed on, and are put into 27 DEG C of incubators and are carried out passage training
Support.
To realize above-mentioned second purpose, the present invention adopts the technical scheme that：A kind of structure of pond crucian carp fish dorsal fin cell line
Construction method, described construction method comprise the following steps：Original cuiture, Secondary Culture.
Described construction method comprises the following steps：
A) original cuiture
The dorsal fin of pond crucian carp fish is cut, then with 75% alcohol disinfecting, then is removed on pond crucian carp fish dorsal fin with penicillin, streptomysin
Mucus, then with containing amphotericin B D-PBS rinse, after flushing, after aseptically dorsal fin is cut into small pieces with scissors
It is immersed in trypsase and EDTA；Then the dorsal fin of the pond crucian carp fish shredded is transferred in Tissue Culture Flask, and keeps bottom
Temperature is 25 DEG C, then adds serum, penicillin, streptomysin, amphotericin B, basic fibroblast growth containing 10% tire ox
The M199 culture mediums of the factor and EGF；
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 culture is removed with suction pipe
Liquid, with PBS, trypsin digestion cell is added, after microscope Microscopic observation cell rounding, adds fresh complete culture
Liquid, blow and beat repeatedly, hanged cell, with 1:2 are passed on, and are put into 27 DEG C of incubators and are carried out Secondary Culture.
To realize above-mentioned 3rd purpose, the present invention adopts the technical scheme that：Described pond crucian carp fish dorsal fin cell line exists
Application in cell engineering, genetic engineering or biomedical engineering.
The invention has the advantages that：
1st, the pond crucian carp fish dorsal fin cell line that the present invention establishes can provide substantial amounts of pond crucian carp fish dorsal fin cell with continuous passage
System.
2nd, the cell line may be directly applied to the research that correlation function gene is immunized in pond crucian carp fish, for the virus of pond crucian carp fish
Infection experiment can save substantial amounts of manpower with application cell system, material resources and effect is preferable.
3rd, pond crucian carp fish dorsal fin cell line can be applied to cell biology, virology, toxicology, molecule with multigelation
The important research such as biology, science of heredity and immunology.
Brief description of the drawings
Accompanying drawing 1 is the cell (3 days or so) that the pond crucian carp fish dorsal fin tissue block under microscope nearby migrates out.
Accompanying drawing 2 is the pond crucian carp fish dorsal fin cell line after stable passage under microscope.
Accompanying drawing 3 is cell infection crucian herpesvirusⅡtype (CyHV-2) situation (A):Normal cell, scale bar.=
100 μm of (B and C) infection crucian herpesvirusⅡtypes (CyHV-2), 6 days after infection, 50 μm of arrows of scale bar.=100and
Head is signified to infect crucian herpesvirusⅡtype (CyHV-2), 8 days after infection, scale bar.=100. for sick cell (D)
Accompanying drawing 4 is that duplication viral in crucian herpesvirusⅡtype (CyHV-2) infection pond crucian carp fish dorsal fin cell line supernatant is bent
Line.
Embodiment
Embodiment provided by the invention is elaborated below in conjunction with the accompanying drawings.
The technical problem to be solved in the present invention is to provide it is a kind of can stablize passage establish pond crucian carp fish dorsal fin cell line
(SCC-DF cell line, Silver crucian carp dorsal fin cell line), the cell line is configured to
Study the immunology of pond crucian carp fish and screen unknown fishes virus and provide good biomaterial.
The construction method of the pond crucian carp fish dorsal fin cell line of embodiment 1
1st, cell culture fluid is prepared
GIBCO companies M199 culture mediums are taken, addition accounts for culture medium cumulative volume 10-20% (if percentage as described below is without spy
Do not mentionlet alone bright, be percent by volume) hyclone, final concentration of 20ng/ml Basic Fibroblast Growth Factor (bFGF), eventually
Concentration is 1ng/ml EGF (EGF), and 4 DEG C of storages are standby.
2nd, original cuiture
The dorsal fin of pond crucian carp fish is cut, then with 75% alcohol disinfecting, then with 1000IU mL-1Penicillin, 1000ug mL-1
Streptomysin removes the mucus on pond crucian carp fish dorsal fin, then uses mL containing 2.5ug-1Amphotericin B D-PBS rinse 3 times.Rinse
Afterwards, dorsal fin is aseptically cut into about 1mm with scissors3Fritter after be immersed in 0.25% trypsase and 0.02%EDTA
In.
Then it is 25cm the dorsal fin of the pond crucian carp fish shredded to be transferred into bottom2Tissue Culture Flask in, and keep bottom temperature
Spend for 25 DEG C.After 15 hours, the serum (FBS) that 2mL contains 10% tire ox, 200IU mL are added-1Penicillin, 200ug mL-1Chain
Mycin, 0.5ug mL-1Amphotericin B, FGF and EGF M199 culture mediums.After 24 hours, add 3mL and contain 10%FBS,
200IU mL-1Penicillin, 200ug mL-1Streptomysin, 0.5ug mL-1Amphotericin B, FGF and EGF culture medium.Change daily
50% fresh culture.
3rd, Secondary Culture
After the dorsal fin cell monolayer of the pond crucian carp fish of original cuiture is paved with Tissue Culture Flask, old culture is removed with suction pipe
Liquid, with twice of PBS, 0.25% pancreatin (containing EDTA) 1mL vitellophags are added, treat microscope Microscopic observation cell rounding
Afterwards, the fresh complete culture solutions of 10mL are added, blows and beats repeatedly, has hanged cell, with 1:2 are passed on, and add complete culture solution extremely
5mL/ bottles, it is put into 27 DEG C of incubators and carries out Secondary Culture.
The pond crucian carp fish dorsal fin cell line of embodiment 2
By cultured in vitro, present invention obtains the pond crucian carp fish dorsal fin cell line (SCC- that one plant of passage stable elements is homogeneous
DF cell line, Silver crucian carp dorsal fin cell line) passage is so far.
The pond crucian carp fish dorsal fin cell line that the present invention obtains has carried out preservation, pond crucian carp fish in China typical culture collection center
Dorsal fin cell line SCC-DF, its deposit number are CCTCC NO:C201525, Classification And Nomenclature are 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, the pond crucian carp fish dorsal fin cell line has following biological characteristics：
1) the primary cell being isolated near tissue block, in the Morphological Features of fibroblast-like cell；
2) ability of cell proliferation is general, and 7d or so can isolate a large amount of cells to each tissue block after treatment, have
The ability of continuous passage；
3) the cell line condition of culture is 25 DEG C of constant temperature cell culture incubators, without carbon dioxide.
This cell line cultivates the most frequently used culture medium M199 using fish cell system, and condition of culture is the same as common fish cell one
Cause.Pancreatin is needed to digest during passage, sub-bottle can cover with for 5 days.Cellular morphology is in typical threadiness.Therefore the cell line is a kind of
Stability of characteristics, the pond crucian carp fish dorsal fin cell line of uniform component, it is research aquatic livestock immunology and virological good biological material
Material.
3 viral infection experiment of embodiment
First, experimental method
1st, viral infection
Cultured grass goldfish abdomeinal fin cell line is reached in 6 orifice plates, adherent growth overnight.Blister sore containing crucian respectively
II, CyHV-2 of poison) and alkaline phosphate buffer, PBS (negative control group) takes 500 μ l infection cells, discarded after being incubated 1 hour
Supernatant, washed 3 times with PBS, the addition μ l of complete medium 500, which are placed in incubator, to be cultivated, and observes cell growth status daily.
2nd, the drafting of virus facsimile log in cell conditioned medium
Cell is reached in 24 orifice plates, by step virus infection in 1, takes supernatant within every 24 hours, high speed centrifugation discards precipitation,
Supernatant extracts DNA and carries out fluorescence quantitative PCR detection in the steps below.
3rd, the extraction of viral DNA
(1) isometric PBS is added, is well mixed, is centrifuged 5 minutes with 8000rpm, abandons supernatant；
(2) 20 μ L protease k. are added and is incubated 3 hours in 56 DEG C of water-baths；
(3) 8000rpm is centrifuged 5 minutes, is taken in 750 μ L of supernatant liquid in a new centrifuge tube, is added isometric phenol：Chlorine
It is imitative：Isoamyl alcohol (25:24:1)；Mixed 10 minutes with 50 revs/min of speed, then centrifuged 5 minutes with 8000 revs/min, take 650
μ L of supernatant liquid is in a new centrifuge tube；It is repeated 1 times；
(4) 600 μ L of supernatant liquid are taken to add isometric chloroform in a new centrifuge tube：Isoamyl alcohol (24:1).With 50 turns/
Minute speed mixes 10 minutes, and 8000 revs/min centrifuge 5 minutes；
(5) 480 μ L of supernatant liquid are taken in a new centrifuge tube, add 40 μ L 3mol/L sodium acetate and 960 μ L4 DEG C it is cold
But ethanol；Jog, it is seen that White Flocculus；
(6) subzero 20 DEG C place 20-30 minutes, after with 12000 revs/min centrifuge 10 minutes, abandoning supernatant；
(7) washed with 200ul 75% ethanol, 14000 revs/min centrifuge 5 minutes, abandon supernatant；Repetition is done once；
(8) 50ul milli-Q water is used after drying 10 minutes, in subzero 20 DEG C preservations.
4th, fluorescence quantitative PCR detection
Sense primer CyHV-2rtF2 (5 ' -3 ')：TTAGCGTCAGGTCCATAG
Anti-sense primer CyHV-2rtR1 (5 ' -3 ')：GGCGTGTAGAAATCAAAC
Reaction system：
Concussion mixes, trim centrifugation.
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 circulations；Melt
Solution curve：95 DEG C of 1min, 55 DEG C of 1min, 65 DEG C to 95 DEG C 0.1 DEG C/sec.
4th, experimental result
As shown in Figure 3A, negative infection group (uninfecting virus) cell growth is good；Fig. 3 B, C, virus infection 6 days or so
Contraction forms vacuole sample；Fig. 3 D, 8 days or so cells of infection form obvious virosis and become phenomenon (CPE).
Virus amplification curve result in cell conditioned medium is shown in Fig. 4, and virus was expanded at first 3 days, and platform is entered after 4 days
Phase.
Above testing result shows：The pond crucian carp fish dorsal fin cell line can be applied to crucian herpesviral II (CyHV-2)
Detection and corresponding virology, immunology related experiment.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of the inventive method is not departed from, can also make some improvement and supplement, and these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (2)
1. a kind of pond crucian carp fish dorsal fin cell line, it is characterised in that the deposit number of described pond crucian carp fish dorsal fin cell line is
CCTCC NO: C201525。
2. pond crucian carp fish dorsal fin cell line according to claim 1 is in cell engineering, genetic engineering or biomedical engineering
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 |
| 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 |
Citations (3)
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| CN1742083A (en) * | 2003-01-01 | 2006-03-01 | 耶路撒冷希伯来大学伊萨姆研发公司 | Immunizing fish against viral infection |
| 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 |
| CN103667176A (en) * | 2013-12-09 | 2014-03-26 | 中国水产科学研究院长江水产研究所 | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof |
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2015
- 2015-04-27 CN CN201510206829.1A patent/CN104774802B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1742083A (en) * | 2003-01-01 | 2006-03-01 | 耶路撒冷希伯来大学伊萨姆研发公司 | Immunizing fish against viral infection |
| 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 |
| CN103667176A (en) * | 2013-12-09 | 2014-03-26 | 中国水产科学研究院长江水产研究所 | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof |
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| Characterization of a novel cell line from the caudal fin of koi carp Cyprinus carpio;S.-L. Lin，et al;《Journal of Fish Biology》;20131231;第82卷;1888–1903 * |
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| 锦鲤鳍条组织细胞系的建立及其生物学特性;肖艺，等;《中国细胞生物学学报》;20121231;第34卷(第8期);767–774 * |
| 鲤疱疹病毒Ⅱ型对异育银鲫背鳍细胞的显微形态与免疫基因表达水平的影响;夏思瑶，等;《水产学报》;20161231;第12卷(第40期);1915－1922 * |
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