CN103710298A - Goldfish snout cell line and application thereof - Google Patents
Goldfish snout cell line and application thereof Download PDFInfo
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- CN103710298A CN103710298A CN201310711835.3A CN201310711835A CN103710298A CN 103710298 A CN103710298 A CN 103710298A CN 201310711835 A CN201310711835 A CN 201310711835A CN 103710298 A CN103710298 A CN 103710298A
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Abstract
The invention discloses a goldfish snout cell line and application thereof. The goldfish snout cell line is stored in a general microbiology center of the China Committee for Culture Collection of Microorganisms (CGMCC) in December 4, 2013, and proved to be alive, and the registration number for preservation is CGMCC No.8558. The goldfish snout cell line disclosed by the invention is stable in characteristic, and uniform in ingredient, and is a good material for researching aquatic animal viruses.
Description
Technical field
The present invention relates to a kind of goldfish kiss terminal cell system and application thereof, belong to medical biotechnology field.
Background technology
Research about fish cell system, has obtained very large progress both at home and abroad.The rainbow trout gonadal cell system (RGT-2) being set up by Wolf and Quimby for 1962 starts, and bighead muscle cell system, Xiphophorus helleri embryo cell line SWT, Atlantic Ocean trout viscera tissue clone ASHe Paraguay Scad juvenile fish clone etc. are multiple comprises that sea water and fresh water fish cell system also sets up successively.The technology of setting up of fish primary cell line is a kind of comparatively proven technique at present, but obtain for the responsive clone of virus and still depositing very large randomness and difficulty, so setting up a strain, to need fish cell be that effective approach is to quality with quantity, prepare large batch of primary cell strain, thereby filter out responsive clone.
" golden standard " of detection aquatic animal virus recommended by the World Health Organization (OIE) is to carry out isolated viral by cell.In order to be better effectively separated to the virus of aquatic animal, early detect virus, prophylactic outburst, many scholars both domestic and external are still in the research of carrying out fish cell system.There is no at present about setting up the relevant report of goldfish kiss terminal cell system.
Summary of the invention
First technical problem that the present invention will solve is to provide the goldfish kiss terminal cell system (GS) of a kind of stability of characteristics, uniform component, is the sensitive cell line of some aquatic animal viruses, is the good material of further studying unknown virus.
Second technical problem that the present invention will solve is to provide the preparation method that a kind of goldfish kiss terminal cell is.
The 3rd technical problem that the present invention will solve is to provide described goldfish kiss terminal cell and ties up to the Preliminary Applications to the separated aspect of aquatic animal virus.
For solving the problems of the technologies described above, the present invention adopts following technical proposals:
Through screening, the present invention has obtained the goldfish kiss terminal cell system of stability of characteristics, uniform component, this goldfish kiss terminal cell system (GS) has been kept at China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 4th, 2013, and proof survival, its preservation accession designation number is CGMCC No.8558, and preserving address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101, its Classification And Nomenclature is goldfish kiss terminal cell system.
Further, described clone has following biological characteristics:
(1) cell presents typical epithelial cell form, and continuous passage was cultivated for 50 generations and still kept this Morphological Features;
(2) cell has stronger multiplication capacity, and 50 generations of cultured continuously still keep this propagation and growth characteristics.
The preparation method that the present invention also provides a kind of above-mentioned goldfish kiss terminal cell to be, the method comprises the following steps:
(1) get one of goldfish, with medical alcohol, soak 1-5min, the kiss end of clip goldfish under aseptic condition, being put in concentration is in the dual anti-solution of penicillin-Streptomycin sulphate of 1000u/ml, places 10-15min;
(2) trysinization liquid digestion 10min, is then cut into small pieces, resuspended, centrifugal with substratum, discards supernatant liquor, then with substratum, carries out resuspendedly, centrifugal, discards supernatant, finally adds substratum, resuspended mixing;
(3) the resuspended mixing liquid of being prepared by step (2) joins in Tissue Culture Flask, in 25 ℃ of incubators, cultivates;
(4), when treating cell bed board to 1/3, change fresh substratum once;
(5) treat that cell is paved with plank, after tentatively digesting with trysinization liquid, add new substratum to cultivate;
(6) go down to posterity after 3 times, frozen, after recovery, can continue to stablize proliferate, this clone called after GS.
Further, described substratum is to contain the M199 substratum that concentration is the dual anti-solution of 100u/ml penicillin-Streptomycin sulphate and the superfine foetal calf serum of 20 ﹪.
Further, the preparation of described trysinization liquid: Sodium phosphate dibasic 2.3g, potassium primary phosphate 0.1g, sodium-chlor 8.0g, Repone K 0.2g, EDTA0.2g, pancreatin 0.6g, water 1000ml.
Further, goldfish kiss terminal cell of the present invention is that GS can be used as the use of research aquatic animal virus host cell, for the separation of aquatic animal virus.
Beneficial effect of the present invention is as follows:
That in experiment, uses is the most frequently used substratum cultivated of fish cell and carries out primary cell screening and culturing in simple condition, guarantees that the final cell obtaining just can breed without special reagent and condition, for its application from now on provides better space.This goldfish kiss terminal cell system is that success is cultivated in vitro first.When cultivating 7 days, obviously there is elongation growth in this cell; After 15 days, be paved with plank, after digesting, there is stronger fecundity, present typical epithelial cell form.Cell continuous passage is cultivated and within 16 months, was reached for 50 generations, still keeps above-mentioned Morphological Features, growth and multiplication characteristic.Therefore, this cell is the goldfish kiss terminal cell system of a kind of stability of characteristics, uniform component, is the good material of research aquatic animal virus.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail;
The form of the goldfish kiss terminal cell system under Fig. 1 phase microscope;
Fig. 2 goldfish kiss terminal cell is suspension characteristic analysis
Determining of the best serum-concentration of Fig. 3 goldfish kiss terminal cell system;
Determining of the optimum culturing temperature of Fig. 4 goldfish kiss terminal cell system;
The multiplication tracing analysis of Fig. 5 goldfish kiss terminal cell system;
The Preliminary Applications of the virus of proliferation of Fig. 6 goldfish kiss terminal cell system.
Embodiment
For understanding better the present invention, will further illustrate the solution of the present invention by specific embodiment below, protection scope of the present invention should comprise the full content of claim, but is not limited to this.
Embodiment 1 goldfish kisses teloblastic separation and former culture
Experiment material and reagent
The goldfish of laboratory animal: 10g-30g (the healthy goldfish of aquatic animal virus is not infected in checking through molecular method repeatedly).
Experimental apparatus: scalper, eye scissors, ophthalmology tweezers and gauze etc.
Substratum and related reagent:
The dual anti-strength of solution of penicillin-Streptomycin sulphate is 10000u/ml;
Substratum: containing concentration is the M199 substratum (gibco company) of the dual anti-solution of 100U/ml penicillin-Streptomycin sulphate and the superfine foetal calf serums of 20 ﹪ (Hangzhou Sijiqing Biological Engineering Material Co., Ltd.);
Trysinization liquid: Sodium phosphate dibasic 2.3g, potassium primary phosphate 0.1g, sodium-chlor 8.0g, Repone K 0.2g, EDTA0.2g, pancreatin 0.6g, water 1000ml;
Medical alcohol;
PBS damping fluid (NaCl80g, the Na of 0.01M
2hPO
412H
2o29g, KH
2pO
42g, KCl2g, water 1000ml).
Experimental technique
Through the healthy goldfish detecting, in laboratory, support 15 days temporarily, without other diseases, start experiment.Concrete grammar:
(1) get a goldfish and in medical alcohol, soak 5min, under aseptic condition, the kiss end of clip goldfish, being put in concentration is in the dual anti-solution of penicillin-Streptomycin sulphate of 1000u/ml, places 15min;
(2) trysinization liquid digestion 10min, cuts into fritter (about 1mm) with scalper.Add substratum, resuspended.Centrifugal 1000rmp, 10min.Discard supernatant liquor, again add substratum, carry out resuspendedly, centrifugal, discard supernatant liquor, finally add substratum, resuspended mixing.
(3) paving culturing bottle, puts in 25 ℃ of incubators and cultivates.
(4) the adherent situation of observation of cell after cultivating 2 days, suitably adds substratum (guaranteeing the pH of substratum).When treating cell bed board to 1/3, (approximately 7 days), change fresh substratum once.
(5) treat that cell is paved with plank (approximately 15 days), after tentatively digesting, adds new substratum to cultivate with trysinization liquid;
(6) go down to posterity after 3 times, frozen, after recovery, can continue to stablize proliferate, this clone called after GS;
(7) every 7 days, go down to posterity 1 time.
Experimental result:
Fig. 1 is the form of the goldfish kiss terminal cell system under phase microscope; Fig. 2 is that goldfish kiss terminal cell is suspension characteristic analysis, and result shows that cell homogeneity is relatively good, concentrates on diameter 12-18 micron.
Reagent
Trysinization liquid: Sodium phosphate dibasic 2.3g, potassium primary phosphate 0.1g, sodium-chlor 8.0g, Repone K 0.2g, EDTA0.2g, pancreatin 0.6g, water 1000ml.
Frozen storing liquid: the M199 substratum that contains the superfine foetal calf serum of 10 ﹪ DMSO, 25 ﹪.
Experimental technique
When Growth of Cells to 90 ﹪, discard old substratum, add 2.5ml trysinization liquid to digest;
When treating that cell presents " white mist " shape, add the frozen storing liquid of 2ml, to stop the effect of pancreatin, heavy curtain is moving gently, and cell is mixed, counting.
Collecting cell, in cryopreservation tube, indicates title, cell count and the frozen date of cell on cryopreservation tube.
After one month, according to the method for conventional cell recovery, carry out cell recovery, ability of cell proliferation is still very strong, in good condition.
The goldfish kiss terminal cell system (GS) obtaining has been kept at China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 4th, 2013, and proof survival, and its preservation accession designation number is CGMCC No.8558.
The optimum condition of embodiment 3 goldfish kiss terminal cell systems is determined
One, reagent
Trysinization liquid: Sodium phosphate dibasic 2.3g, potassium primary phosphate 0.1g, sodium-chlor 8.0g, Repone K 0.2g, EDTA0.2g, pancreatin 0.6g, water 1000ml.
Substratum: the M199 substratum of the superfine foetal calf serum of different concns.
Two, experimental technique
1, determining of the suitableeest serum
Prepare respectively superfine foetal calf serum concentration and be 10%, 15%, 20% nutrient solution, adjusting cell density is 1.8 * 10
5mL
-1, three kinds of serum-concentration substratum are respectively inoculated in 24 orifice plates by the amount in 1mL/ hole, in 25 ℃, in incubator, cultivate.Every 2d takes out 3 porocytes from each experimental group, and digestion collecting cell counting, cultivate 12d altogether, and continuous counter 6 times, draws its growth curve.
2, optimum temperuture determines
Select 15 ℃, 20 ℃, 25 ℃, 30 ℃ four different culture temperature, use and add the too M199 nutrient solution of bovine serum of 20% superfine, adjusting cell density is 1.8 * 10
5mL
-1, cell suspension is inoculated in 24 orifice plates by the amount in 1mL/ hole and is placed in four different culture temperature incubators.Every 2d takes out 3 porocytes from each experimental group, and collecting cell counting, cultivate 12d altogether, and continuous counter 6 times, draws its growth curve.
Three, experimental result
The determining of the suitableeest serum (Fig. 3), goldfish kiss terminal cell ties up in the M199 substratum of superfine foetal calf serum 20%, increases breeding best.
Definite (Fig. 4) of optimum temperuture, goldfish kiss terminal cell ties up to 25 ℃ of growths, breeds best.
The growth curve of embodiment 4 goldfish kiss terminal cell systems
One, reagent
Trysinization liquid: Sodium phosphate dibasic 2.3g, potassium primary phosphate 0.1g, sodium-chlor 8.0g, Repone K 0.2g, EDTA0.2g, pancreatin 0.6g, water 1000ml.
Substratum: the M199 substratum of 20% superfine foetal calf serum (FBS).
Two, experimental technique
The cell of taking the logarithm vegetative period, collects counting cells, uses the M199 containing 20%FBS to cultivate, and adjusting cell concn is 8.38 * 10
4mL
-1, by the amount in 1mL/ hole, be seeded in 24 orifice plates, put 25 ℃ of cultivations in incubator.Get 3 porocytes every day, cell suspension is made in trysinization digestion, mixes with cell counter, to count afterwards, experiment repeats 3 times, carries out continuously 9d, and the incubation time (d) of take is X-coordinate, cell concn (cell/mL) is ordinate zou, draws growth curve; According to formula T=t * lg2/lg (N
t/ N
0) calculate the population doubling time of cell, wherein, N
0for inoculating cell number, N
tfor the cell count after time t.
Three, experimental result
It is lag phase that goldfish kiss terminal cell ties up in the 24h that goes down to posterity, and logarithmic phase, at 24-120h, enters into the stage of stable development (Fig. 5) when 120h
The Preliminary Applications of embodiment 5 goldfish kiss terminal cell systems
One, reagent
Trysinization liquid: Sodium phosphate dibasic 2.3g, potassium primary phosphate 0.1g, sodium-chlor 8.0g, Repone K 0.2g, EDTA0.2g, pancreatin 0.6g, water 1000ml.
Substratum: the M199 substratum of 20% superfine foetal calf serum (FBS).
The Koi herpesvirus of being bought by ATCC (KHV) Reference Strains (ATCCVR1592).
Two, experimental technique
To goldfish kiss terminal cell, system digests with trysinization liquid, and the M199 substratum 1:1 that contains 20% superfine foetal calf serum (FBS) goes down to posterity, and in 25 ℃ of incubators, cultivates.Cell proliferation accesses KHV virus in 24h, is placed in 20 ℃ of incubators, observes every day and changes.
Three, experimental result
Shown in Fig. 6, there is obvious cytopathy (CPE) in cell, illustrate that goldfish kiss terminal cell is can virus of proliferation KHV, for the research of KHV provides strong material.
Obviously; the above embodiment of the present invention is only for example of the present invention is clearly described; and be not the restriction to embodiments of the present invention; for those of ordinary skill in the field; can also make other changes in different forms on the basis of the above description; here cannot give all embodiments exhaustive, every still row in protection scope of the present invention of apparent variation that technical scheme of the present invention extends out or change that belong to.
Claims (7)
1. goldfish kiss terminal cell is a GS, and its preserving number is CGMCC No.8558.
2. goldfish kiss terminal cell according to claim 1 is GS, it is characterized in that, described clone has following biological characteristics:
(1) cell presents typical epithelial cell form, and continuous passage was cultivated for 50 generations and still kept this Morphological Features;
(2) cell has stronger multiplication capacity, and 50 generations of cultured continuously still keep this propagation and growth characteristics.
3. goldfish kiss terminal cell claimed in claim 1 is a preparation method of GS, it is characterized in that, the method comprises the following steps:
(1) get one of goldfish, with medical alcohol, soak 1-2min, the kiss end of clip goldfish under aseptic condition, being put in concentration is in the dual anti-solution of penicillin-Streptomycin sulphate of 1000u/ml;
(2) trysinization liquid digestion 10min, is then cut into small pieces, resuspended, centrifugal with substratum, discards supernatant liquor, then with substratum, carries out resuspendedly, centrifugal, discards supernatant, finally adds substratum, resuspended mixing;
(3) the resuspended mixing liquid of being prepared by step (2) joins in Tissue Culture Flask, in 25 ℃ of incubators, cultivates;
(4), when treating cell bed board to 1/3, change fresh substratum once;
(5) treat that cell is paved with plank, after tentatively digesting with trysinization liquid, add new substratum to cultivate;
(6) go down to posterity after 3 times, frozen, after recovery, can continue to stablize proliferate, this clone called after GS.
4. preparation method according to claim 3, is characterized in that, described substratum is to contain the M199 substratum that concentration is the dual anti-solution of 100u/ml penicillin-Streptomycin sulphate and the superfine foetal calf serum of 20 ﹪.
5. preparation method according to claim 3, is characterized in that, the preparation of described trysinization liquid: Sodium phosphate dibasic 2.3g, potassium primary phosphate 0.1g, sodium-chlor 8.0g, Repone K 0.2g, EDTA0.2g, pancreatin 0.6g, water 1000ml.
6. goldfish kiss terminal cell claimed in claim 1 is that GS is as the application of research aquatic animal virus host cell.
7. goldfish kiss terminal cell claimed in claim 1 is the application of GS in the separation of aquatic animal virus.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310711835.3A CN103710298B (en) | 2013-12-20 | 2013-12-20 | Goldfish snout cell line and application thereof |
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|---|---|---|---|
| CN201310711835.3A CN103710298B (en) | 2013-12-20 | 2013-12-20 | Goldfish snout cell line and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110527661A (en) * | 2019-09-06 | 2019-12-03 | 中国水产科学研究院珠江水产研究所 | A kind of Xiphophorus helleri prelarva cell line and its construction method and application |
| CN116836912A (en) * | 2023-06-26 | 2023-10-03 | 中国水产科学研究院珠江水产研究所 | Lateolabrax japonicus kistrodon cell line and construction method and application thereof |
-
2013
- 2013-12-20 CN CN201310711835.3A patent/CN103710298B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| 孔祥婷: "苏州大学硕士学位论文：黄颡鱼细胞培养研究", 《中国优秀硕士学位论文全文数据库(基础科学辑)》, no. 9, 15 September 2009 (2009-09-15), pages 17 - 18 * |
| 孟凡华等: "鲤鱼吻端和尾鳍细胞的生长形态及增殖能力比较研究", 《内蒙古师范大学学报（自然科学汉文版）》, vol. 42, no. 1, 31 January 2013 (2013-01-31), pages 68 - 70 * |
| 郭莹等: "大泷六线鱼鳍、吻端和肾脏组织原代培养的研究", 《大连海洋大学学报》, vol. 27, no. 5, 30 October 2012 (2012-10-30), pages 387 - 391 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110527661A (en) * | 2019-09-06 | 2019-12-03 | 中国水产科学研究院珠江水产研究所 | A kind of Xiphophorus helleri prelarva cell line and its construction method and application |
| CN110527661B (en) * | 2019-09-06 | 2021-03-23 | 中国水产科学研究院珠江水产研究所 | Carassius xyphoides larva cell line and construction method and application thereof |
| CN116836912A (en) * | 2023-06-26 | 2023-10-03 | 中国水产科学研究院珠江水产研究所 | Lateolabrax japonicus kistrodon cell line and construction method and application thereof |
| CN116836912B (en) * | 2023-06-26 | 2024-03-01 | 中国水产科学研究院珠江水产研究所 | Lateolabrax japonicus kistrodon cell line and construction method and application thereof |
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