CN103555657B - Tetraploid Loach, Misgurnus fin clone TEMF and method for building up thereof - Google Patents
Tetraploid Loach, Misgurnus fin clone TEMF and method for building up thereof Download PDFInfo
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- CN103555657B CN103555657B CN201310538211.6A CN201310538211A CN103555657B CN 103555657 B CN103555657 B CN 103555657B CN 201310538211 A CN201310538211 A CN 201310538211A CN 103555657 B CN103555657 B CN 103555657B
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Abstract
The invention provides a kind of Tetraploid Loach, Misgurnus fin clone TEMF and method for building up thereof. Described cell lies in and is deposited in Chinese Typical Representative culture collection center on August 19th, 2013, does is deposit number CCTCC? No:C2013111. Described Tetraploid Loach, Misgurnus fin clone TEMF supports the growth of SVCV and copies. The biological characteristics of described Tetraploid Loach, Misgurnus fin clone TEMF comprises: cell is that fibroblast sample, endochylema enrich adherent growth, tool contact inhibition growth characteristics; Cell doubling time is 41.45 hours; Characteristic chromosome number is 100.
Description
Technical field
The present invention relates to a kind of Tetraploid Loach, Misgurnus fin clone TEMF and method for building up thereof.
Background technology
Loach (Misgurnusanguillicaudatus) belongs to Cypriniformes in taxology(Cypriniformes), Cobitidae (Cobitidae), flower loach subfamily (Cobitinae),Loach belongs to (Misgurnus), is the distinctive wild Small Freshwater Fishes in Asia, is distributed widely inMost areas and Vietnam, Korea and Japan on the south China Liaohe River. The each freshwater of China,As all there is distribution in lake, pond, He Xi, ditch, rice field etc. Loach fine and tender taste, delicious,Nutritious, there is the title of " ginseng in water ", except higher edibility, it also has oneFixed nourishing medicinal efficacy is one of special aquatic products product salable on market. Important except havingOutside economic worth, the research material of the aspects such as loach or a kind of good cytology, science of heredity.
There is polyploid phenomenon in loach, natural tetraploid accounts for 2%. About grinding of Tetraploid Loach, MisgurnusStudy carefully to relate generally to diploid hybrid and produce triploid, up to now also not about in vitro cultureThe relevant report of Tetraploid Loach, Misgurnus fin cell.
A large amount of research reports confirm that the generation of tetraploid and tumour is relevant. Tetraploid Loach, Misgurnus cellThe foundation of system is not only for the Analysis on Mechanism of plasm resource protection, tetraploid characteristic, and with itThe research of carrying out tumour for model is also highly profitable.
Summary of the invention
In view of the above problems, the object of the present invention is to provide a kind of Tetraploid Loach, Misgurnus fin cloneTEMF and method for building up thereof.
Described Tetraploid Loach, Misgurnus fin clone TEMF is deposited in China on August 19th, 2013Typical case's culture collection center, deposit number is C2013111.
Described clone is supported the growth of SVCV and is copied.
The present invention also provides the method for building up of a kind of Tetraploid Loach, Misgurnus fin clone TEMF, comprisesFollowing steps:
I, prepare cell culture fluid
Used medium is commercially available DMEM/F12 culture medium, adds hyclone, people's alkalescenceFibroblast growth factor, I type IGF and chondroitin sulfate, be mixed withDescribed nutrient solution, hyclone accounts for 20% of nutrient solution cumulative volume, and human basic fibroblast is rawThe final concentration of the long factor is 10ng/ml, and the final concentration of I type IGF is20ng/ml, the final concentration of chondroitin sulfate is 10 μ g/ml, described nutrient solution leaves 4 DEG C in;
II, former culture
The fin tissue of clip Tetraploid Loach, Misgurnus under gnotobasis, first soaks with the Iodophor of 10wt%The described fin tissue of cutting 15 minutes, then with containing the penicillin that final concentration is 500IU/mLWith the mixed liquid dipping of 500 μ g/mL streptomysins of final concentration 30 minutes, then use aseptic PBSRinsing, is cut into 1mm by the fin tissue after rinsing3The fritter of left and right, is not evenly seeded in and cultivatesIn the Tissue Culture Flask of liquid, at the CO of 25 DEG C2In incubator, just putting dry doubling 24 hours, thenIn described blake bottle, add described nutrient solution, be placed on the CO of 25 DEG C2In incubator, continue trainingSupport;
III, the cultivation of going down to posterity
Grow up to after individual layer until the cell of Step II, remove the nutrient solution in described blake bottle, thenToward the trypsin solution that adds 0.25% in described blake bottle, described cell carry out digestion placeReason, sucked trypsin solution after 30 seconds, utilized and at the bottom of bottle, formed the continuation of one deck tryptose enzyme membraneDigest 1 minute, then add nutrient solution to blow and beat postdigestive cell, make cell suspension, willCell suspension proceeds in new blake bottle by predetermined ratio, and add nutrient solution, at 25 DEG CCO2In incubator, cultivate;
IV, go down to posterity according to the process of Step II I, after 30 generations, nutrient solution used is for containing 20vol%The DMEM/F12 culture medium of hyclone.
Tetraploid Loach, Misgurnus fin clone provided by the present invention can be used for carrying out virology, pathology,Toxicology, immunology, genetic breeding and germplasm are preserved scheduling theory research and viral vaccine developmentDeng application study.
Brief description of the drawings
Fig. 1 examines under a microscope, the aspect graph of Tetraploid Loach, Misgurnus fin clone of the present invention.
Fig. 2 is the chromosome analysis figure that the fin clone of Tetraploid Loach, Misgurnus shown in Fig. 1 is shown.
Fig. 3 is the result figure that the fin clone of Tetraploid Loach, Misgurnus shown in Fig. 1 is carried out to ssr analysis.
Fig. 4 is the figure that the fin clone of Tetraploid Loach, Misgurnus shown in Fig. 1 growth curve is shown.
Fig. 5 illustrates inoculation SVCV Tetraploid Loach, Misgurnus fin clone two days laterThe figure of form.
Detailed description of the invention
Below by detailed description of the invention to Tetraploid Loach, Misgurnus fin clone of the present invention and foundation thereofMethod describes.
1, the foundation of clone
Tetraploid Loach, Misgurnus fin clone TEMF sets up by following step.
(1) prepare cell culture fluid
Used medium is commercially available DMEM/F12 culture medium, add hyclone (FBS),Human alkaline fibroblast growth factor, I type IGF and chondroitin sulfate,Be mixed with cell culture fluid. Wherein, hyclone accounts for 20% of nutrient solution cumulative volume, people's alkalescenceThe final concentration of fibroblast growth factor is 10ng/ml, I type IGFFinal concentration is 20ng/ml, and the final concentration of chondroitin sulfate is 10 μ g/ml. The training preparingNutrient solution leaves 4 DEG C in.
(2) former culture
The fin tissue of clip Tetraploid Loach, Misgurnus under gnotobasis. Next, first use 10wt%'sThe fin tissue that Iodophor immersion is cut 15 minutes, then use penicillin and streptomysin mixed liquor (mouldElement final concentration is 500IU/mL, and streptomysin final concentration is 500 μ g/mL) soak 30 minutes.After immersion finishes, then use aseptic PBS (formula: 8gNaCl, 0.2gKCl, 3.63gNa2HPO4·12H2O、0.24gKH2PO4, be dissolved in distilled water and be settled to 1L) and rinsing twoTime. Then, the fin tissue after rinsing is cut into 1mm3The fritter of left and right, is evenly seeded in and does not haveThe 25cm of nutrient solution2In Tissue Culture Flask, at the CO of 25 DEG C2In incubator, just putting dry doubling 24Hour. Then in blake bottle, add cell culture fluid to 5mL/ bottle, at the CO of 25 DEG C2TrainingSupport in case and continue to cultivate.
(3) cultivation of going down to posterity
Grow up to after individual layer until the cell of former culture, the nutrient solution in blake bottle is transferred to totallyContainers for future use. Then,, toward the trypsin solution that adds 1mL0.25% in blake bottle, useIn cell is carried out to digestion process, after 30 seconds, suck trypsin solution. Now at the bottom of bottle, formOne deck tryptose enzyme membrane, continues digestion 1 minute, and blake bottle softly collides with. Then turn aforementionedThe nutrient solution that moves on to clean containers for future use is transferred in blake bottle again, blows and beats cell with suction pipe,Make cell suspension. Cell suspension is evenly assigned in two new blake bottles, and added cultivationLiquid is to 5mL, at the CO of 25 DEG C2In incubator, cultivate.
Again grow up to after individual layer until cell, go down to posterity according to the process of step (3). 30 generationsRear nutrient solution used is the DMEM/F12 culture medium containing 20vol%FBS.
2, the preservation of cell
First prepare cells frozen storing liquid, in this cells frozen storing liquid, contain FBS20%, DMEM/F1260% and dimethyl sulfoxide (DMSO) 20%. The cells frozen storing liquid preparing is placed in 4 DEG C of refrigerators and saves backup.
Get eugonic cell, with 0.25% trypsin solution digestion. After digestion finishes,Add nutrient solution re-suspended cell, the centrifugal 5min of 1500rpm, abandons supernatant, then thin with 1mLBorn of the same parents' cryopreserving liquid re-suspended cell. Adjust cell concentration to 1.0 × 106Individual/mL, proceeds to cell to freezeDeposit in pipe.
Above-mentioned cell suspension is lowered the temperature gradually, and concrete cooling process is as follows: first at 4 DEG C of refrigeratorsMiddle placement 30min, then proceed in-20 DEG C of refrigerators and place 2h, then proceed in-80 DEG C of refrigerators24h, finally puts into liquid nitrogen (196 DEG C) and preserves for a long time.
Detect the cell survival rate after frozen in liquid nitrogen. Get the cell that a pipe is preserved in liquid nitrogen,In 40 DEG C of water, thaw, after cell suspension melts, proceed to 3min in 25 DEG C of water-baths. Then willCell suspension proceeds to clean centrifuge tube, adds equivalent DMEM/F12 culture medium, 1500rpmCentrifugal 5min, abandons supernatant, with the DMEM/F12 culture medium re-suspended cell containing 20vol%FBS,Be transferred to 25cm2Tissue Culture Flask in, add nutrient solution to 5mL, at the CO of 25 DEG C2In incubator, cultivate 24h, then change fresh nutrient solution, continue to cultivate.
Meanwhile, resuspended cell liquid in the course of defrosting that takes a morsel, adds trypan blue (TrypanBlue) dyeing, and with blood counting chamber counting, calculate survival rate. Survival rate=(viable count/ TCS) × 100%.
The survival rate of freeze-stored cell is (81.57 ± 1.28) %.
3, chromosome analysis
Have to cultivation that in the blake bottle of Tetraploid Loach, Misgurnus fin clone, to add final concentration be 1 μ g/mLColchicine, blake bottle is placed in to the CO of 25 DEG C2In incubator, continue to cultivate 3～5h. SoDigest with 0.25% trypsin solution afterwards, postdigestive cell proceeded in centrifuge tube,The centrifugal 5min of 1500rpm, collecting cell. Hypotonic with 0.075mol/LKCl solution40min, Carnoy fixer is fixed 15min, drips sheet after fixing three times, dry, thenDye with Giemsa dye liquor.
Micro-Microscopic observation is taken pictures, counting chromosome number, and carry out karyotyping, according to dyingThe features such as the length of colour solid, position, kinetochore, brachium, divide into groups, match chromosome.
The karyotype of Tetraploid Loach, Misgurnus fin clone TEMF as shown in Figure 2.
4, ssr analysis
Identify set up by SSR (SimpleSequenceRepeats) technologyWhether Tetraploid Loach, Misgurnus fin clone TEMF has identical hereditary feature with loach.
First extract, respectively zebra fish musculature, loach musculature and Tetraploid Loach, Misgurnus finThe DNA of clone. Adopt conventional phenol-chloroform extraction method to extract base from loach musculatureBecause of group DNA. The extraction step of Tetraploid Loach, Misgurnus fin clone DNA is as follows: 1. take out-20DEG C freezing cell, puts into 1.5mL centrifuge tube, centrifugal rear removal supernatant at 4 DEG C after thawingLiquid; 2. add 250 μ L DNA cleavage liquid (contain 10mmol/LTris-Cl,50mmol/LKCl and 0.5%Tween-20, pH8.0), then add 5 μ L Proteinase Ks(concentration is 0.5mg/mL), in 55 DEG C of constant temperature water bath cracking 3h; 3. with reference to benzene phenol-chloroformExtraction process, uses isopyknic chloroform: isoamyl alcohol (24: 1) extracting once.
Next, carry out PCR (PolymeraseChainReaction) amplification.
According to the loach microsatellite markers of having reported, synthetic Mac9, Mac50 two are to drawingThing (concrete sequence is in table 1).
Table 1
PCR reaction system cumulative volume is 25 μ L, comprising:
PCR reaction condition is: 94 DEG C of denaturation 4min; Circular response 35 times, cycling conditionBe 94 DEG C of sex change 30s, annealing 30s (annealing temperature is in table 1), 72 DEG C are extended 30s; 72DEG C end wheel extends 10min.
After amplified reaction finishes, get 8 μ LPCR products and in 2% Ago-Gel, carry out electrophoresisDetect. Result as shown in Figure 3, shows that the continuous cell line TEMF that the present invention sets up hasThe hereditary feature identical with loach.
5, cell growth curve
Get cultivation the 50th generation Tetraploid Loach, Misgurnus fin cell, with 0.25% trypsin solutionVitellophag, then re-suspended cell, is 3.0 × 10 by inoculum density4～5.0×104Individual/mL,Every hole 500 μ L are inoculated in cell in 24 porocyte plates, are placed in the CO of 25 DEG C2In incubatorCultivate. Every 24h gets 3 porocytes blood counting chamber and counts, and carries out continuously 7d.
Taking incubation time (d) as abscissa, cell concentration (individual/mL) is ordinate, drawsThe growth curve of cell, as shown in Figure 4. Can according to formula T=t × Lg2/Lg (Nt/NO)Calculate the population doubling time of cell, wherein, NtFor the cell number after time t, N0InoculationCell number.
The population doubling time of the Tetraploid Loach, Misgurnus fin clone that the present invention sets up is41.45h。
6, cell and nucleus size
Size to cell is measured, and concrete grammar is as follows: get the tetraploid mud that goes down to posterity and cultivateLoach fin cell, with 0.25% trypsin solution digestion, re-suspended cell, then gets 200 μ LCell suspension, adds equal-volume trypan blue, joins in blood counting chamber, micro-after mixingUnder mirror, calculate the diameter of cell and add up.
Cell dia is 15.00 ± 0.04 μ m, and cell area is 176.52 ± 0.98 μ m2，Cell volume is 1764.64 ± 14.44 μ m3。
Nuclear size is measured, and concrete grammar is as follows: examine under a microscope adherentCell, chooses 100 round cell cores at random, calculates under the microscope their diameter and goes forward side by sideRow statistics.
Nucleus diameter is 12.52 ± 0.14 μ m, nuclear area is 122.98 ±2.88μm2, nucleus volume is 1026.36 ± 37.83 μ m3。
7, viral sensitiveness
Get the Tetraploid Loach, Misgurnus fin cell going down to posterity after 48 hours, suck nutrient solution, float with PBSWash after twice, add 0.1mL SVCV (SpingViremiaofCarpVirus, be called for short SVCV) virus liquid, rock gently blake bottle virus liquid uniform fold existedOn cell, be then placed in the CO of 25 DEG C2In incubator, continue to cultivate. After viruses adsorption 2h,Suck virus liquid, add 5mL to contain the DMEM/F12 culture medium of 20vol%FBS, 25℃、5％CO2Condition under continue cultivate.
Meanwhile, control group is set, replaces virus liquid with 0.1mLDMEM/F12 culture medium,Other steps are same as described above.
Day by day the variation of observation of cell form, and Taking Pictures recording cytopathic effect (CPE). ConnectAfter the viral 24h of kind, can be observed parts of fine born of the same parents and start to occur obvious shrinkage, become circle floating, go outNow obvious CPE. Along with the intensification of CPE, lysis death increases the weight of, a large amount of cells after 72hCome off or the death of disintegrating, attached cell is surplus 30% left and right only. Control group reacts without any pathology.
Prove thus, Tetraploid Loach, Misgurnus fin clone TEMF has SVCVNeurological susceptibility.
Comprehensively above-mentioned, the Tetraploid Loach, Misgurnus fin clone that the present invention sets up has following biological characteristicsProperty: cell is that fibroblast sample, endochylema enrich adherent growth, tool contact inhibition growth characteristics,Cell doubling time is 41.45 hours; Characteristic chromosome number is 100. This tetraploidLoach fin clone has the neurological susceptibility to SVCV.
Claims (1)
1. one kind is utilized Tetraploid Loach, Misgurnus fin clone TEMF growth and copies spring viremiaThe method of virus, described Tetraploid Loach, Misgurnus fin clone TEMF protected on August 19th, 2013Ensconce Chinese Typical Representative culture collection center, deposit number is C2013111,
Get the described Tetraploid Loach, Misgurnus fin clone TEMF going down to posterity after 48 hours, suck blake bottleIn nutrient solution, with after twice of PBS rinsing, add spring viremia disease described in 0.1mLThen the virus liquid of poison, rocks gently blake bottle and makes described virus liquid uniform fold on cell,Be placed in the CO of 25 DEG C2In incubator, continue to cultivate, after viruses adsorption 2h, suck virus liquid,Add 5mL to contain the DMEM/F12 culture medium of 20vol% hyclone, at 25 DEG C, 5%CO2Condition under continue cultivate.
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