CN103849602B - A kind of bull testis clone and establishment method thereof and application - Google Patents
A kind of bull testis clone and establishment method thereof and application Download PDFInfo
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Abstract
The invention provides a kind of bull testis clone.Present invention also offers the establishment method of this clone, key step is: (1) original cuiture: aseptic acquisition is come into being bovine testicle, removes adhesion organization, adopts trypsin digestion to be separated the bull testis cell obtaining high vigor; By being separated the bull testis cell cultures of the high vigor obtained in DMEM substratum, be placed in 37 DEG C, 5%CO
2cultivate under condition, obtain primary bull testis cell; (2) transfection and screening: extraction purification contains the eukaryon expression plasmid pCI-neo-hTERT of hTERT gene, adopt lipofection, the bull testis cell of eukaryon expression plasmid pCI-neo-hTERT steps for importing (1) original cuiture of extraction purification is carried out transfection; (3) screening and enlarged culturing.Bull testis cell of the present invention is easy to cultivate, and the speed of growth is very fast, can stablize and go down to posterity, and Cell viability is high, purity is high and energy preservation very well; The inventive method is easy and simple to handle, easy to utilize.This clone can be applicable to produce swine Fever Vaccine, sheep pox, sore mouth virus vaccine etc.
Description
Technical field
The invention belongs to System in Animal Cell Biotechnology technical field, relate to a kind of new clone, be specifically related to a kind of bull testis clone and establishment method thereof and application.
Background technology
The main preventive measures of swine fever prevented and treated by vaccine.Can prevent kind of the virulence from returning strong and avoiding using isogenic animal to cause other diseases to propagate, as primary cell propagation CSFVC strain vaccines such as bull testis, Testis Caprae seu Ovis, sheep kidneys with heterologous animal cells produce vaccine.
Pestivirus suis (CSFV) can be bred in bull testis cell, does not but make cell produce pathology (CPE), and the propagation of virus can increase the passage number of primary bull testis cell and dimension treats the time.But for a long time, the hog cholera lapinised virus bull testis cell vaccine of China also exists many problems aborning: in swine fever bovine testicle cell seedling production process, because the new-born calve testis utilizing diary farm to eliminate is raw materials for production more, affect by the restriction of donor amount and often criticize output, if only apply the bovine testicle of collection on the same day, often because the few and inconvenient scheduling of production of quantity.For meeting Production requirement, usually adopt constant temperature to preserve in production, and the short period of time temperature too low (not constant temperature) usually caused due to the refrigeration mechanism of refrigerator affect the production of testicular cell cultivation and vaccine.
Along with the fast development of pig industry, swine fever bull testis cell vaccine demand is more and more large, relies on merely the primary cultivation of bovine testicle cell to be difficult to meet the needs of production, so it is extremely urgent to set up bull testis clone.Meanwhile, primary bull testis cell needs to gather bovine testicle tissue and is separated, and its source is very limited.Therefore, be badly in need of now improving its method, bull testis cell stablized and goes down to posterity down, reach high Cell viability, highly purified standard and good preservation can be obtained and continue.
Summary of the invention
The object of this invention is to provide a kind of high reactivity, highly purified bull testis clone.
The present invention establishes a kind of bull testis clone, reaches foregoing invention object.The preserving number of described clone is: CCTCCNO:C201399.
Above-mentioned bull testis clone can be used for producing swine Fever Vaccine, sheep pox vaccine and sore mouth virus vaccine, also can apply in Pestivirus suis, capripox virus, sheep of virus breed in testicular cell in research virus; Also can apply in isolation identification capripox virus and development capripox virus vaccine; Also can apply in external source gene transfection, apoptosis and cytogamy research.
The establishment method that above-mentioned clone is also provided of the present invention, the method carries out original cuiture by gathering nascent bovine testicle; Then the eukaryon expression plasmid pCI-neo-hTERT to primary cell importing coding hTERT and neo gene carries out transfection, and rear screening is containing the positive cell of this goal gene; Then carry out screening and enlarged culturing, with the bull testis clone of being immortalized, finally frozen and preservation is carried out to this cell.
The establishment method of above-mentioned bull testis clone, is characterized in that, comprise the steps:
(1) original cuiture: gather nascent bovine testicle, be cut into 0.5 ~ 2.0mm
3tissue block, be separated to obtain the bull testis cell of high vigor, cultivate, obtain primary bull testis cell,
(2) transfection and screening: extract, purifying contains the eukaryon expression plasmid pCI-neo-hTERT of hTERT gene, by extracting, purifying the primary bull testis cell of eukaryon expression plasmid pCI-neo-hTERT steps for importing (1) gained carry out transfection;
(3) screening and enlarged culturing: after cell transfecting eukaryon expression plasmid pCI-neo-hTERT when cell confluency to 80%, reach in another 24 well culture plate, screens liquid with G418 after 48h and carries out G418 screening;
After the complete cell death of control wells, the concentration of G418 being screened liquid reduces maintenance G418 screening, is inoculated in 96 well culture plates, then had digestive transfer culture to 24 well culture plate enlarged culturing by clone cell positive for G418 screening, proceed G418 screening, obtain the positive cell of anti-G418;
(4) positive cell of the anti-G418 of step (3) gained is carried out continuous passage culture in vitro in incubator, cultivate more than 60 generations, the bull testis clone of being immortalized; Described continuous passage culture in vitro adopts digestion method.
The establishment method of above-mentioned bull testis clone, is characterized in that, also comprises cell cryopreservation step, and this step is as follows in order:
Substratum in exchonge step (4) culturing bottle, continues to cultivate;
Use trysinization culturing cell, then add substratum termination reaction;
Counting;
Collect: centrifugal, remove supernatant liquor, add frozen storing liquid, mixing, makes cell resuspended, cultured cells is distributed into cryopreservation tube sealing;
Pre-freeze: 4 DEG C of pre-freeze cryopreservation tube 30 ~ 60min ,-20 DEG C of pre-freeze 1 ~ 2h ,-70 DEG C of pre-freeze 6 ~ 12h;
Frozen: the cryopreservation tube of-70 DEG C of pre-freezes to be dropped into rapidly in liquid nitrogen cabinet;
Shared by each composition of described frozen storing liquid, volume percent is: 10%DMSO, 60% foetal calf serum and 30%DMEM.
The establishment method of above-mentioned bull testis clone, is characterized in that, the cultivation described in step (1) refers to that the bull testis cell cultures of high vigor is in DMEM substratum completely;
The described substratum of DMEM is completely in liquid DMEM substratum, add the DMEM substratum that volume ratio is the foetal calf serum of 10%;
Described liquid DMEM substratum can use this liquid nutrient medium directly bought, and also can be to use the DMEM culture medium dry powder bought formulated.
The establishment method of above-mentioned bull testis clone, is characterized in that, described transfection comprises the steps:
(1) 24h before transfection, by bull testis cell with being digested to individual cells, is inoculated in 24 orifice plates, makes cell can reach the fusion of 80% ~ 90% same day in transfection;
(2) 4h before transfection, will be replaced with liquid DMEM substratum by DMEM substratum completely;
(3) with liquid DMEM substratum, the pCI-neo-hTERT plasmid of three part of 5 μ L is all diluted to 50 μ L, obtains the plasmid DNA of dilution;
(4) with liquid DMEM substratum dilute respectively 6 μ L, 9 μ L, 12 μ L Lipofectamine2000 to 50 μ L, obtain the Lipofectamine2000 of dilution;
(5) plasmid DNA of dilution of difference mixing step (3) and the Lipofectamine2000 of the dilution of step (4), obtain mixture;
(6) substratum in 24 orifice plates of sucking-off step (1), cleans with D-Hank ' s, adds the liquid DMEM substratum of 900 μ L;
(7) mixture of step (5) is added in different hole, mixing; Set up blank simultaneously;
(8) in volume fraction be 5%CO
2hatch in saturated humidity incubator, discard substratum, add DMEM substratum completely;
(9) observation of cell, and change liquid in time.
Bull testis clone of the present invention has following characteristics: high cell growth speed, healthy growth, and form is good, and apoptosis rate is low.Cell purity is more than 99.9%, and Cell viability is not less than 92.8%.
The biological characteristics of bull testis clone of the present invention detects indices and all reaches American type culture collection (AmericanTypeCultureCollection, ATCC) clone standard of perfection, and the expression rate of foreign gene in bull testis clone is more than 40%.
In the establishment method of bull testis clone of the present invention, the nascent bull testis tissue effect described in step (1) is best.
In the establishment method of bull testis clone of the present invention, the concrete steps of trysinization are: in culturing bottle, add 2.5g/L pancreatin 1.5 ~ 2.5mL, upset digestion 30 ~ 60S after after inversion culturing bottle is preheated to 37 DEG C in incubator.
In the establishment method of bull testis clone of the present invention, frozen cell can be recovered according to actual needs, concrete steps are: taken out from liquid nitrogen by cryopreservation tube and insert in 38 DEG C of water-baths, after rocking 1 point of kind, being moved into by cell to be added with in the culturing bottle of DMEM substratum completely blows and beats evenly, place containing 37 DEG C, 5%CO
2cO
2secondary Culture can be continued after continuing to cultivate 48h in incubator.
Advantage of the present invention and benefit: the present invention adjusts for the improvement of adherent culture method and medium component, and adopt the method for cell monoclonal, the bull testis cell of turning out can be made without contaminating cell such as epithelial cells, and cell purity compared with prior art has and significantly improves; Improvement for conditions of cryopreservation makes the cell of recovery compared with before frozen, cellular form and the speed of growth do not change, freeze-stored cell steady quality, and the Cell viability after cell cryopreservation can reach between 92.8% ~ 96.7%, and passage growth is stable, be significantly increased compared with existing cell culture technology cultured cells, be applicable to large scale culturing.For the adjustment of substratum and the improvement of cultural method, cost can also be made significantly to reduce.The inventive method compensate for the present situation that existing bull testis clone lacks, the lifting of bull testis clone survival rate and purity also makes swine fever and the biotechnological formulation development such as relative disease research and vaccine provide means, and provides science material and instrument for the development of the viral isolation identification such as sheep pox and vaccine; Also can be used for the use of foreign gene transfection, apoptosis and cytogamy research simultaneously.
Bull testis clone of the present invention can not only produce swine Fever Vaccine, can also study virus value-added fundamental characteristics and rule in testicular cell, in production of vaccine, the raising of Pestivirus suis output provides new thinking.Meanwhile, bull testis cell can also produce other virus vacciness: as sheep pox vaccine, sore mouth virus vaccine etc.
The inventive method is easy to cultivate, and the speed of growth is very fast, and working method is easy, and acquisition cell purity is high, survival rate is high and growth of going down to posterity is stable, easy to utilize.
Biomaterial preservation information
Classification And Nomenclature: bull testis clone WWX-BTC02
Deposit number: CCTCCNO:C201399
Preservation mechanism: China typical culture collection center (being called for short CCTCC)
Preservation organization address: China, Wuhan, Wuhan University's postcode: 430072
The preservation time: on June 24th, 2013
Accompanying drawing explanation
Fig. 1 is primary bull testis microcytoscope figure;
Fig. 2 is positive bull testis cell clone after transfection;
Fig. 3 is bull testis clone the 5th generation microcytoscope figure;
Fig. 4 is bull testis clone the 30th generation microcytoscope figure;
Fig. 5 is bull testis clone the 50th generation microcytoscope figure;
Fig. 6 is iBTCs karyogram;
Fig. 7 is bull testis clone soft agar assay result figure;
Fig. 8 is HELA cell soft agar positive control;
Fig. 9 is iBTCs growth curve chart.
Embodiment
Below in conjunction with accompanying drawing and preferred forms, the present invention will be further described, to make the public have overall to summary of the invention and understand fully, and not limiting the scope of the present invention.Preceding sections is own through fully disclosing the protection domain that the present invention can implement, and therefore all equivalent replacements any well known in the art carried out according to the disclosure of invention, all belong to infringement of the present invention.
G418 is a kind of aminoglycoside antibiotics, in molecular genetic test, is the most frequently used resistance screening reagent of stable transfection.
The value of the concentration of reagent of the present invention, temperature and its dependent variable just illustrates application of the present invention, and is not construed as limiting the invention.
The source of experiment material of the present invention:
Biomaterial:
It is Gibco company that foetal calf serum is available commercially from foetal calf serum, article No.: 10099-141.
Eukaryon expression plasmid pCI-neo-hTERT containing hTERT gene is presented by TanJin Agricultural College doctor Li Jixia.
Applicant states, above biomaterial all has preservation in applicant laboratory, can provide for proof test from the applying date in Two decades years to the public.
Source and the specification of reagent are as follows:
DMSO is available commercially from Sigma, Dimethylsulfoxide, cat.D8418, specification 100mL;
G418 is available commercially from Invitrogen, Catno.11811023, specification: 1g;
DMEM culture medium dry powder is purchased from Dulbecco ' sModifiedEagleMedium, Gibco company, specification: 10 × 1L; Substratum is Powdered powder, lowglucose low sugar; Article No.: Cat.no.31600-034, LotNo.843267;
Liquid DMEM substratum can use the liquid nutrient medium directly bought, and the present invention uses the DMEM culture medium dry powder bought formulated, and compound method is shown in embodiment 1;
DMEM substratum is add the Gibco foetal calf serum that volume ratio is 10% in the above-mentioned liquid DMEM substratum prepared voluntarily completely;
It is in the above-mentioned liquid DMEM substratum prepared voluntarily, add G418 obtain that G418 screens liquid;
D-Hank ' s, i.e. D-Hank'S liquid: NaCl8g, KCl0.4g, Na
2hPO
4h
2o0.06g, KH
2pO
40.06g, NaHCO
30.35g is dissolved in 1000m1 distilled water, and adjust ph is to 7.2-7.4, and autoclaving, saves backup at 4 DEG C.Reagent is analytical pure;
Pancreatin, is purchased from pancreatin Invitrogen, article No.: 27250-018,10g, 2.5g/L;
Lipofectamine2000Reagent, is available commercially from invitrogen company, and Chinese is liposome 2000, Cat.no.11668-027;
Other the chemical reagent do not listed is all conventional chemical reagent, analytical pure level, and the approach that is purchased obtains, and general chemical article company can buy.
Source and the specification of plant and instrument are as follows:
Endo-FreePlasmidMaxiKit, without intracellular toxin plasmid extraction kit, is purchased from Omega company, article No.: D6926-01; When equivalent is large, plasmid extraction can adopt plasmid to extract test kit in a large number, company: QIAGEN, article No.: 12163;
DNA/ plasmid purification reclaims test kit, is purchased from Omega company, article No.: D2500-01, GelExtractionKit;
The present invention's substratum used has two kinds: liquid DMEM substratum and complete DMEM substratum, wherein liquid DMEM substratum uses the DMEM culture medium dry powder be purchased (to be purchased from Dulbecco ' sModifiedEagleMedium, Gibco company) prepare voluntarily, and DMEM substratum is in liquid DMEM substratum, add the Gibco foetal calf serum that volume ratio is 10% completely;
Screening liquid used in the present invention adds G418 in liquid DMEM substratum, and 400 described μ g/mLG418 screen liquid and refer to that every mlG418 screens the DMEM substratum containing 400 μ gG418 in liquid; 200 described μ g/mLG418 screen liquid and refer to that every mlG418 screens the DMEM substratum containing 200 μ gG418 in liquid; G418 is a kind of microbiotic, adds G418 herein and namely screen liquid as G418 in liquid DMEM substratum;
Trysinization of the present invention pancreatin used is the pancreatin that mass volume ratio is 2.5g/L;
DMEM is a kind of substratum containing each seed amino acid and glucose, is develop on the basis of MEM substratum.Compare with MEM and add various Ingredient Amount, be divided into again high glycoform (lower than 4500mg/L) and low-sugar type (lower than 1000mg/L) simultaneously.High glycoform is conducive to cell and berths in a position growth, is suitable for growing comparatively fast, adheres to more difficult tumour cell etc.
DMEM is the abbreviation of dulbecco'smodifiedeaglemedium, so its feature mainly comprises following:
(1) aminoacids content is 2 times of Yi Geer substratum, and containing non-essential amino acid, as glycine etc.;
(2) vitamin contents is 4 times of Yi Geer substratum;
(3) containing the important substance-pyruvic acid in glycolytic pathway;
(4) iron ion containing trace.
BTC full name bovinetestiscells, i.e. bull testis cell, the clone that this patent is set up, called after bull testis clone WWX-BTC01, passes through to the bull testis histogen be separated exactly for the clone built up after cell transfecting telomerase gene.Source: by bull testis separate tissue, the healthy newborn calf of Niu Shi Accessories during Binzhou man of peasant household.
Plasmid DNA is the pCI-neo-hTERT plasmid DNA of purifying;
The preparation of the liquid DMEM substratum of embodiment 1.
Liquid DMEM substratum is for prepare voluntarily, and compound method is as follows:
(1) fresh tri-distilled water or millipore ultrapure water is prepared, the preparation process of described millipore ultrapure water is as follows: tap water is through first step metre filter, filter through second stage CTO activated charcoal filter again, after entering the filtration of third stage security personnel filter, by ultra-clean high-pressure hydraulic pump process, filtered by RO reverse osmosis membrane again, after eventually passing the process of multistage pure water post, obtain ultrapure water;
(2) bag DMEM culture medium dry powder (being purchased from DulbeccosModifiedEagleMedium, Gibco company) is added in the tri-distilled water of about whole half; Packing bag inner face need be washed with water 2 times, import in substratum, ensure that all dry powder is all dissolved into substratum.Magnetic agitation or hand mixing make it to dissolve completely;
(3) according to packing bag requiring the sodium bicarbonate adding 3.7g, the constant volume that adds water is to final volume 1L;
(4) degerming with aseptic 0.22 μm of membrane filtration, be sub-packed in aseptic serum bottle, 4 DEG C of Refrigerator stores.
DMEM substratum is in the DMEM substratum of the above-mentioned serum-free prepared voluntarily, add the Gibco foetal calf serum that volume ratio is 10% completely;
The preparation of embodiment 2. bull testis clone is cultivated
(1) original cuiture: aseptic collection is come into being bovine testicle, with PBS rinsing 3 ~ 5 times to remove adhesion organization, is cut into 0.5 ~ 2.0mm
3tissue block, adopt trypsin digestion digestion to be separated and obtain the primary bull testis cell of high vigor; Add DMEM substratum completely in the primary bull testis cell of high vigor separation obtained, be inoculated in (the liquid DMEM substratum containing 10% foetal calf serum) 24 orifice plates according to the concentration of 5000 cell/mL after counting, be placed in 37 DEG C, 5%CO
2cultivate under condition, obtain primary bull testis cell;
The described substratum of DMEM is completely in liquid DMEM substratum, add the DMEM substratum that volume ratio is the foetal calf serum of 10%.
(2) transfection and screening: the DH5 а bacterium containing pCI-neo-hTERT plasmid being kept at-80 DEG C is taken out, be seeded in the LB substratum containing ammonia benzyl, shaking culture 10h, get bacterium liquid and extract plasmid, carry out extracting that (plasmid extraction adopts plasmid to extract test kit in a large number with plasmid extraction kit, company: QIAGEN, article No.: 12163); Adopt DNA/ plasmid purification to reclaim test kit and purifying is carried out to plasmid, the eukaryon expression plasmid pCI-neo-hTERT of hTERT gene must be contained, adopt lipofection, the bull testis cell of eukaryon expression plasmid pCI-neo-hTERT steps for importing (1) original cuiture of coding hTERT and neo gene is carried out transfection, specifically comprising the steps: of transfection
24h before A, transfection, becomes individual cells, with 1 × 10 by bull testis cell 2.5g/L trysinization
5individual cells/well is inoculated in 24 orifice plates, makes cell can reach the fusion of 80% ~ 90% same day in transfection;
4h before B, transfection, will be replaced with liquid DMEM substratum by DMEM substratum completely;
C, to dilute 5 μ LpCI-neo-hTERT plasmid to volumes with liquid DMEM substratum be 50 μ L, and mix gently, plasmid concentration reaches 300ng/ μ L ~ 400ng/ μ L room temperature effect 5min, obtains the plasmid DNA of dilution;
D, dilute Lipofectamine2000 to 6 μ L, 9 μ L, 12 μ L respectively to 50 μ L with liquid DMEM substratum, mix gently, room temperature effect 5min, obtains the Lipofectamine2000 of dilution;
The plasmid DNA of dilution of E, mixed step C and the dilution of step D Lipofectamine2000, now cumulative volume is 100 μ L.Mix gently, room temperature effect 20min, obtains mixture;
F, by the old substratum sucking-off in 24 orifice plates, to clean with D-Hank ' s, add the liquid DMEM substratum of 900 μ L;
G, dropwise add the mixture of step e in different hole, before and after the edged of limit, waggle culture plate, mixes gently; Set up 2 hole untransfecteds as blank simultaneously;
H, 37 DEG C, Volume fraction is 5%CO
2hatch 4 ~ 6h in saturated humidity incubator, discard substratum, add DMEM substratum completely;
I, 24h ~ 48h observation of cell, and change liquid in time.；
(3) screening and enlarged culturing: until cell transfecting pCI-neo-hTERT eukaryon expression plasmid after 48 hours until cell confluency to 80% time, use 2.5g/L trysinization, reach in another 24 well culture plate with 1:1, add 400 μ g/mLG418 screening liquid (adding G418 in liquid DMEM substratum) after 48h and carry out G418 screening, within every three days, change once DMEM substratum completely, and the G418 screening liquid adding same concentration carries out G418 screening, when the 7th day, available pancreatin is in foramen primum peptic cell, discard Digestive system and add G418 and screen liquid, cultured continuously two weeks, commutation in every two days is with concentration screening liquid 1 time, observation of cell growth every day and death condition, after the complete cell death of control wells, the concentration of G418 being screened liquid is down to 200 μ g/mL maintenance G418 screenings, continue G418 and screen cell about month, within every two days, change 1 not good liquor, until positive colony cell is visible, clone cell positive for G418 screening to be digested and after counting, cell is carried out infinite dilution, be inoculated in 96 porocyte culture plates by every hole number≤2, mark the hole of each cell, until unicellular grow cloning cluster after, digest with 2.5g/L pancreatin, and cell is reached 24 well culture plates, in 37 DEG C, the CO of 5%
2carry out enlarged culturing under condition, continue to utilize G418 to screen, obtain the positive cell of anti-G418, herein, be equivalent to utilizing monoclonal method purifying cells in 96 well culture plates.
Under (4) 37 DEG C of conditions, adopt digestion method by step (3) gained positive cell in the CO of 5%
2carry out continuous passage culture in vitro in incubator, after sorting, cell presents the growth of long shuttle shape monolayer adherence, and iuntercellular exists contact inhibition, cultivates more than 60 generations, the bull testis clone of being immortalized.
(5) cell cryopreservation:
A, reject change liquid DMEM substratum 5 ~ 8mL in culturing bottle, continue to cultivate 24h;
B, use trysinization culturing cell, then add liquid DMEM substratum 5 ~ 8mL termination reaction;
C, use red blood cell count(RBC) plate calculate frozen front total cellular score;
Centrifugal 5 ~ the 8min of D, collection: 1000rpm, removes supernatant liquor, adds the frozen storing liquid 1mL of 4 DEG C of precoolings, blows and beats gently make cell resuspended after mixing with suction pipe; Shared by each composition of described frozen storing liquid, volume percent is: namely, in 100mL frozen storing liquid, DMSO accounts for 10% of cumulative volume to 10%DMSO ten 60% foetal calf serum+30%DMEM(, and foetal calf serum accounts for 60%, DMEM of cumulative volume and cultivates fiduciary point 30%);
E, cell culture is distributed into sterilizing cryopreservation tube in seal;
F, pre-freeze: 4 DEG C, by cryopreservation tube pre-freeze 30 ~ 60min, transfer to-20 DEG C of pre-freeze 1 ~ 2h, then proceed to-70 DEG C of pre-freeze 6 ~ 12h;
G, frozen: the cryopreservation tube of-70 DEG C of pre-freezes to be dropped into rapidly in liquid nitrogen cabinet, namely completes cell cryopreservation.
Above-mentioned obtained bull testis clone delivered to and be deposited in China typical culture collection center, it is referred to as CCTCC, and preservation enters volume and logins volume and be numbered CCTCCC201399, and depositary institution address is: Wuhan City, Hubei Province Wuhan University preservation center.
Postcode: 430072; Phone: 027-68754712
Preservation day: on June 24th, 2013, preserving number: CCTCCC201399, the Classification And Nomenclature of suggestion is bull testis clone.
The compliance test result of embodiment 3. embodiment 2 gained bull testis clone
To embodiment 2 cultivate bull testis clone carry out biological characteristics inspection, method and conclusion as follows.
One, morphological observation
Method: cell is in vitro in culturing process, and carry out routine examination to cell, whether observation of cell growth conditions, substratum colour-change situation and cell pollute.
Conclusion: as shown in Figures 1 to 4, when cell growth state is good, its form presents fusiformis or sealene triangle.The overall picture of cell colony is observed, institute's cultured cells all radially, flamboyancy or swirling tendency, prove that Growth of Cells is healthy, form is good.
Two, microorganism detection
1, bacterium, fungal detection
Method:
(1) get the freeze-stored cell of freeze-stored cell amount 0.5%, mix in 8mL nonreactive substratum, cell suspension with the centrifugal 20min of 1000rpm, and repeats twice, to eliminate antibiotic impact.Be resuspended in again in the substratum of 2mL antibiotic-free.
(2) be inoculated in Trypsin beans peptone and malt extract medium by cell with 0.8mL, the incubator being placed in 37 DEG C and 26 DEG C is respectively cultivated two weeks.
(3) set contrast as:
A. positive control: bacillus subtilis and Candida albicans bacterium are inoculated into respectively in the old and malt extract medium of Trypsin beans and cultivate, is placed in the incubator cultivation two weeks of 37 DEG C and 26 DEG C.
B. negative control: the old substratum of Trypsin beans and malt extract medium, the incubator being placed in 37 DEG C and 26 DEG C is respectively cultivated two weeks.
Conclusion: visual inspection inoculation has Soybean Trypsin arteries and veins substratum and the malt extract medium of bull testis cell, all presents limpid transparence, test tube is placed in basis of microscopic observation, the rounded transparence bright spot of cell is in the medium floating, without other foreign matter.Prove that in the whole process of and cell recovery frozen in cell cultures, cell is not by bacterium, fungal contamination.
Three, cell growth curve is drawn
Method:
(1) suspension preparation
Get the cell that growth conditions to be measured is good, increase to close to when converging, make cell suspension with ordinary method peptic cell, and count.
(2) inoculate
The cell of equal amts is inoculated respectively, in 37 DEG C of CO in 24 well culture plates
2continue in incubator to cultivate.
(3) count detection
Count from inoculation time, count the cell density in 3 holes every 24h, calculate total cellular score with blood counting chamber, calculate mean value, get the mean value in 3 holes, so terminate to the 8th group.
(4) draw
With incubation time (d) be X-coordinate, cell density for ordinate zou, result is drawn on graph paper, obtains the growth curve of culturing cell.
Conclusion:
By preparing cell suspension to cultivated bull testis cell ordinary method, counting, inoculate 24 well culture plates, every hole 1mL cell suspension, continuous 7d timing numeration is carried out to the cell being inoculated in 24 well culture plates, record each cell density, drafting forms culturing cell growth curve as shown in Figure 7, after inoculation, 2d cell count starts to increase, 3d enters logarithmic phase, 6d Growth of Cells is slow, phase of cell growth general trend is all S-type, the latent period of 24h is all had after cell inoculation, this phase is laundering period of cell, reparation period after the damage that to be cell cause due to trysinization during inoculation, after this cell is exponentially bred, i.e. exponential phase of growth, finally enter lag phase, Growth of Cells is slow, almost stop, visible have cells float.And cell division index (MI maximum (cultivating 2d) occurs before entering logarithmic phase, and is maintained at a higher level when cultivation 2nd ~ 4d, drops to initial level subsequently.The bull testis cell that the inventive method is cultivated
systempurity be 99.9%, be significantly increased compared with prior art culturing cell average purity 91%; And the cell population doublings time (PDT) increases significantly for PDT-35.9h with 48h that 24h and prior art collagenase digestion and existing stationary culture obtain compares tool, prove that cell purity is high, vitality is vigorous.
Four, Cell viability measures
Method:
The survival rate detecting freeze-stored cell can adopt trypan blue exclusion to test, and makes cell suspension, get after cell recovery to be checked 10 μ L cell suspensions and add 10 μ L1% trypan blue dye liquors, mixing.Blood counting chamber count, healthy viable cell cell space is complete, and cell is transparent, not painted, all painted in blue person be dead cell.Count 1000 cells, calculate cell survival rate.Add up two kinds of total cellular score, account for the per-cent reflection cell viability of total cellular score with viable cell.
Conclusion:
Before and after cell cryopreservation, Cell viability mensuration is carried out to bull testis cell.Result shows: frozen front Cell viability is between 96.8 ~ 98.7%, and frozen rear Cell viability is 92.8% ~ 96.7%, and living compared with in the of 78.6% with the frozen rear cell of existing cell culture technology increases significantly.And the cell of recovery is compared with before frozen, cellular form changes, but the speed of growth does not change, freeze-stored cell steady quality.
Five, the preparation of caryogram
Method:
1, add colchicine: the cell culture in vegetative period of taking the logarithm, add colchicine in substratum, final concentration is 0.1 ~ 0.4g/mL.
2, in 37 DEG C of incubators, continue cultivation 1 ~ 4h, make most cells be in metaphase.
3, gather split coil method cell: add 0.25% trypsin solution ordinary method and digest, then add substratum, stop trypsinase to the effect of cell.Proceed to 15mL centrifuge tube, with the centrifugal 8min of 1000rpm, collecting cell.
4, hypotonic: by sedimentation cell with being preheated to 37 DEG C, 0.075MKCl solution 2mL, resuspended, after piping and druming evenly, continue to add KC1 solution to l0mL, put into 37 DEG C of incubator incubation 15min.
5, pre-fix: add Fresh fixative (acetic acid: methyl alcohol==1:3) 1mL in suspension, beat gently.
6, fixing: by suspension with the centrifugal 8min of 1000rpm, remove supernatant, add Fresh fixative 8mL, beat, room temperature leaves standstill 20min.
7, heavily fixing: to repeat 2 times, after centrifugal, remove part supernatant, residue 1 ~ 1.5mL, mixing.
8, sheet is dripped: drip 2 ~ 3 cell suspensions in 45.Inclination slide glass, makes cell dispersal even, dry under room temperature.
9, dye: the Giemsa liquid dyeing l0min diluting 10 times with phosphate buffered saline buffer (pH6.8), tap water, dry air.
Claims (6)
1. a bull testis clone, its preserving number is: CCTCCNO:C201399.
2. the application of bull testis clone according to claim 1.
3. application according to claim 2, is characterized in that, described application refers to is producing the application in swine Fever Vaccine, sheep pox, sore mouth virus vaccine.
4. application according to claim 2, is characterized in that, described application refers to that described bull testis clone is in the application in testicular cell propagation of research Pestivirus suis, capripox virus and sheep of virus.
5. application according to claim 2, is characterized in that, described application refers to the application of described bull testis clone at development capripox virus vaccine.
6. application according to claim 2, is characterized in that, described application is the described application of bull testis clone in external source gene transfection, apoptosis and cytogamy research.
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| CN106222131B (en) * | 2016-08-16 | 2019-07-26 | 中国农业科学院兰州兽医研究所 | A natural passage cell line of lamb Sertoli cells and its use in isolation, culture and propagation of sheep pox virus |
| CN106754724A (en) * | 2016-12-09 | 2017-05-31 | 西北农林科技大学 | A kind of ox stem spermatogonium system of immortalization and its construction method |
| CN109837250B (en) * | 2019-01-15 | 2021-10-12 | 中国农业科学院兰州兽医研究所 | Immortalized cell line of lamb testicular supporting cell and establishment method and application thereof |
| CN109735499B (en) * | 2019-01-15 | 2021-10-15 | 中国农业科学院兰州兽医研究所 | Newborn bovine Sertoli cell immortalized cell line and its establishment method and application |
| CN111197028A (en) * | 2020-03-09 | 2020-05-26 | 崔立峰 | Human adipose-derived stem cell culture method |
| CN113355275B (en) * | 2021-06-11 | 2024-03-05 | 中国农业科学院兰州兽医研究所 | A kind of lamb testicular Sertoli cell, testicular Sertoli cell subclone and its isolation method and application |
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