CN110295137A - A kind of crow snakehead kidney cell line and its construction method and application - Google Patents
A kind of crow snakehead kidney cell line and its construction method and application Download PDFInfo
- Publication number
- CN110295137A CN110295137A CN201910626036.3A CN201910626036A CN110295137A CN 110295137 A CN110295137 A CN 110295137A CN 201910626036 A CN201910626036 A CN 201910626036A CN 110295137 A CN110295137 A CN 110295137A
- Authority
- CN
- China
- Prior art keywords
- snakehead
- black
- cell line
- kidney cell
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of black snakehead kidney cell lines, its classification naming is black snakehead kidney cell line CAMK, China typical culture collection center is stored on May 8th, 2019, preservation accession designation number is CCTCC NO:C201994, the construction method of above-mentioned crow snakehead kidney cell line are as follows: take kidney after taking black snakehead to sterilize, it is cut into small pieces after being impregnated in the culture medium of the dual anti-solution containing Pen .- Strep, the digestion of pancreatin digestive juice, complete medium, which is resuspended, to be mixed, it obtains resuspension mixing liquid constant temperature incubation to cell in Tissue Culture Flask and is paved with plank, after the digestion of pancreas enzyme -EDTA digestive juice, complete medium is added to continue to cultivate, after passage 3 times, it freezes up to black snakehead kidney cell line.Succeed for the first time in the present invention and has cultivated black snakehead kidney cell line in vitro, it is used to be the most common culture medium of fish cell culture and carry out primary cell screening and culturing in simple condition, guarantee that the cell finally obtained can be proliferated without special reagent and condition, provides better space for its application from now on.
Description
Technical field
The present invention relates to a kind of cell line and its construction method and applications, and in particular to it is a kind of crow snakehead kidney cell line and its
Construction method and application, belong to medical biotechnology field.
Background technique
About the research of fish cell system, very big progress is achieved both at home and abroad.1962 by Wolf and Quimby
The rainbow trout gonadal cell system (RGT-2) of foundation starts, big head muscle cell system, Xiphophorus helleri embryo cell line SWT, Atlantic Ocean trout
Viscera tissue cell line AS and Paraguay Scad juvenile fish cell line etc. a variety of include that sea water and fresh water fish cell system also sets up successively.
The establishing techniques of fish primary cell line are currently a kind of more mature technology, but to obtain the cell sensitive for virus
System still deposits very big contingency and difficulty, so establishing one plant to need the effective approach of fish cell system is confronted with quantity
Amount, that is, prepare large batch of primary cell strain, to filter out sensitive cell line.
Snakehead (Channa argus) and snakehead (Channa maculata) they are the important breed variety of China fish, because
Its adaptability is especially strong, and by the fish that is commonly called as making a living, they belong to Perciformes (Perciformes), Anabantoidei
(Anabantoidei), murrel section (Channidae), Ophiocephalus (Channa).Black snakehead is that snakehead and snakehead are miscellaneous by the remote edge of fish
The Interspecific Hybrids of friendship the advantages of both combining, and embody during actual breeding production hybrid itself
Advantage has the advantages that the speed of growth is fast, resistance is strong, high survival rate, resistance to transport, easily tames and dociles and eat expanded pellet feed etc..
It is relatively fewer to the research in terms of the disease and its prevention and treatment of murrel section fish in China since murrel section fish disease resistance is stronger.With
The intensive degree of Zhe Li section fish culture is continuously improved, cultivates the gradually expansion of scale and cultivation density, causes murrel section fish
There is a situation where infectious diseases to increase increasingly, and various diseases, especially Disease frequently occurs, and constrains the fish culture of murrel section
The development of industry and faster popularization.
It is to separate disease by cell that " gold standard " of detection aquatic livestock virus is recommended by the World Health Organization (OIE)
Poison.In order to more preferably efficiently separate the virus of aquatic livestock, early detect virus, prevent the outburst of disease, perhaps
Scholar mostly both domestic and external is still in the research for carrying out fish cell system.It is there is no at present about the correlation for establishing black snakehead kidney cell line
Report.
Summary of the invention
In view of this, the present invention provides a kind of black snakehead kidney cell line and its construction method and application, it is specific using such as
Lower technical solution:
A kind of crow snakehead kidney cell line, classification naming are black snakehead kidney cell line CAMK, are saved on May 8th, 2019
It in China typical culture collection center, and proves to survive, preservation accession designation number is CCTCC NO:C 201994, preservation address
For Wuhan, China, Wuhan University, China typical culture collection center.
Black snakehead kidney cell line of the invention has following biological characteristics:
(1) stability of characteristics, form are uniform, and typical epithelial cell form is presented in cell, and 70 generation of continuous passage culture still protects
Hold the Morphological Features；
(2) cell has stronger proliferative capacity, is passed on by the ratio of 1:4, can be paved with bottom of bottle within 24 hours.Continuous training
In feeding 70 generations, still keep the proliferation and growth characteristics.
The present invention also provides the construction methods of above-mentioned black snakehead kidney cell line, comprising the following steps:
(1) black snakehead is taken to impregnate 1-2min with 75% alcohol, the kidney of clip crow snakehead, is placed in dense under aseptic condition
Degree is that 3-5min is spare in the culture medium of the dual anti-solution of Pen .- Strep containing 1000u/ml；
(2) kidney of black snakehead is cut into small pieces, the digestion of pancreatin digestive juice, complete medium, which is resuspended, to be mixed, and obtains being resuspended mixed
Even liquid；
(3) mixing liquid will be resuspended to be added in Tissue Culture Flask, culture is paved with plank to cell in 27 DEG C of incubators, uses pancreas
After the digestion of enzyme-EDTA digestive juice, complete medium is added and continues to cultivate；
(4) it after passing on 3 times, freezes up to black snakehead kidney cell line.
Succeed for the first time in the present invention and cultivated black snakehead kidney cell line in vitro, it is used be fish cell culture most
Common culture medium and primary cell screening and culturing is carried out in simple condition, guarantees the cell finally obtained without special examination
Agent and condition can be proliferated, and provide better space for its application from now on.The cell is when cultivating 3 days, hence it is evident that extends
Growth；It is paved with plank after 7 days, after being digested, there is stronger fertility, typical epithelial cell form is presented.Cell
Continuous passage culture reached for 70 generations for 13 months, still maintained above-mentioned Morphological Features, growth and multiplication characteristic.Therefore, the cell
It is the uniform black snakehead kidney cell line of a kind of stability of characteristics, form, is the good material for studying aquatic livestock virus.
It further, further include the operation for supplementing complete medium, specifically: mixing liquid will be resuspended in step (2) in culture
When culture is taped against plank 1/3 to cell in case, complete medium is supplemented.
Further, it is the dual anti-solution of 100u/ml Pen .- Strep and 20% superfine tire that complete medium, which is containing concentration,
The M199 culture medium of cow's serum.
The present invention also provides application of the above-mentioned black snakehead kidney cell line in aquatic livestock virus purification.
Further, above-mentioned black snakehead kidney cell line is in aquatic livestock virus purification as research aquatic livestock virus place
Chief cell uses.
Detailed description of the invention
Fig. 1 is the black snakehead kidney cell line experimental result of the embodiment of the present invention 1；
Fig. 2 is the optimum condition inquiry experiment result of the black snakehead kidney cell line of the embodiment of the present invention 3；
Fig. 3 is the chromosome analysis result of the black snakehead nephrocyte of the embodiment of the present invention 4；
Fig. 4 is sensitivity experiments result of the black snakehead kidney cell line of the embodiment of the present invention 5 to different Aquatic animals virus；
Fig. 5 is sensitivity experiments result of the 6CAMK of the embodiment of the present invention to Luohu viral (TiLV)；
Fig. 6 is the TCID50 measurement result that 6TiLV of the embodiment of the present invention is inoculated with E-11 and CAMK cell；
Fig. 7 is 6 indirect immunofluorescene assay result of the embodiment of the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
The separation and originally culture of black snakehead nephrocyte
1. experimental material and reagent
Experimental animal: the black snakehead of 150g-300g (is uninfected by the strong of aquatic livestock virus by the verifying of molecular method repeatedly
Health crow snakehead).
Experimental apparatus: scalpel, eye scissors, ophthalmology tweezers, penicillin bottle, alcohol swab and gauze etc..
Culture medium and related reagent:
The dual anti-solution concentration of Pen .- Strep is 10000u/ml；
Culture medium: being the dual anti-solution of 100U/ml Pen .- Strep and 20% fetal calf serum (Gibco) containing concentration
M199 culture medium (gibco company)；Pancreatin digestive juice (gibco company)；0.01M PBS buffer solution (NaCl 80g,
Na2HPO412H2O 29g, KH2PO4 2g, KCl 25 2g, water 1000ml).
2. experimental method
The black snakehead of health after testing, temporarily supports 15 days in laboratory, is detected no disease, start to test, specific to walk
It is rapid as follows:
It is fresh and alive crow snakehead 1~2min is impregnated in 75% alcohol, be placed in it is sterile in super-clean bench remove brain tissue, floated with PBS
It after washing 2 times, is placed in the sterile penicillin bottle of 5ml (the M199 culture solution containing 5% fetal calf serum), is cut into operating scissors
About 1mm3Fragment is added 5ml PBS and collects to 15ml centrifuge tube, with 600 turns/min centrifugation 1 minute, abandons supernatant, this step is repeatedly
Operation 3 times, can be such that the protein ingredients such as cell fragment sufficiently remove, in order to avoid influencing the digestion of digestive juice, tissue block about 5 is added
The Type I collagen enzyme of times volume digests 15-25 minutes at 37 DEG C, until the digestive juice muddiness of the fritter containing tissue can terminate digestion.It goes
Fall digestive juice, PBS is washed twice, adds 0.25% trypsase, and room temperature digests 3-5min, and complete culture solution termination is added and disappears
Change, filtered with 200 mesh nylon gauzes into new batch cultur ware, 5ml cell culture fluid flushing gauze is added and refilters one
It is secondary, filtered solution is collected into 15ml centrifuge tube, low-speed centrifugal removes supernatant, collects cell.Tissue block on gauze can weigh again
Multiple digestion, filtering.With the abundant suspension cell of M199 culture solution containing 20%FBS, it is uniformly inoculated in 25cm2Culture bottle in, training
The condition of supporting is 27 DEG C.Plank (about 7 days) are paved with to cell, after carrying out protopepsia with pancreatin digestive juice, new culture medium is added
It is cultivated；
It after passage 3 times, freezes, can continue to stablize proliferation growth after recovery, which is named as CAMK；Every 3 days
It passes on 1 time, experimental result such as Fig. 1, in figure, Figure 1A indicates that primary cell, Figure 1B indicate that 5 generation crow snakehead nephrocytes, Fig. 1 C indicate
15 generation crow snakehead nephrocytes, Fig. 1 D indicate 50 generation crow snakehead nephrocytes.
Embodiment 2
The secondary culture of black snakehead kidney cell line and freeze conservation
When cell grows to 90 ﹪, old culture medium is discarded, 1ml pancreatin digestive juice is added and is digested；It is in cell
When existing " white haze " shape, the frozen stock solution of 5ml is added, to terminate the effect of pancreatin, gently heavy curtain is dynamic, mixes cell, counts.From
After the heart, frozen stock solution is added.Cell is collected into cryopreservation tube, in the title of cell dated on cryopreservation tube, cell number and freezes the date.
After one month, according to the method that regular growth is recovered, cell recovery is carried out, ability of cell proliferation is still very strong, shape
State is good.
The black snakehead kidney cell line (CAMK) of acquisition is stored in China typical culture collection on May 8th, 2019
The heart (CCTCC), and prove to survive, preservation accession designation number is CCTCC NO:C C201994.
Embodiment 3
The optimum condition of black snakehead kidney cell line determines
1. the determination of black snakehead nephrocyte optimal medium
Tetra- kinds of cell culture mediums of DMEM, M199, MEM, L-15 are selected, and adds final concentration of 10% FBS and prepares cell
Culture solution.Adjusting cell density is 2 × 105mL-1, four kinds of culture mediums are respectively inoculated in 6 orifice plates by the amount in the hole 2.5mL/, in 27 DEG C
It is cultivated in incubator.Every 1d takes out 3 hole cells in each experimental group, collects cell with Trypsin-EDTA digestion method and counts
Number co-cultures 7d, continuous counter 7 times, draws its growth curve.Determine that its optimum medium is M199 or L-15 culture medium (figure
2A)。
2. the determination of the most suitable serum-concentration of black snakehead nephrocyte
The culture solution that FBS concentration is 5%, 10%, 15%, 20% is prepared respectively, and adjustment cell density is 2 × 105mL-1,
Four kinds of serum-concentration culture mediums are respectively inoculated in 6 orifice plates by the amount in the hole 2.5mL/, cultivate in 27 DEG C of incubator.Every 1d is from each
3 hole cells are taken out in experimental group, is collected and cell and is counted with Trypsin-EDTA digestion method, co-culture 7d, continuous counter 7 times,
Its growth curve is drawn, discovery FBS concentration is 10%~20% culture (Fig. 2 B) for being suitable for black snakehead nephrocyte.
3. the determination of the most suitable cultivation temperature of black snakehead nephrocyte
15 DEG C of selection, 22 DEG C, 27 DEG C, 32 DEG C of four different cultivation temperatures, using the DMEM culture solution of addition 10%FBS,
Adjusting cell density is 2 × 105mL-1, cell suspension is inoculated in 6 orifice plates by the amount in the hole 2.5mL/ and is placed in four different cultures
In temperature incubation chamber.Every 1d takes out 3 hole cells in each experimental group, collects cell with Trypsin-EDTA digestion method and counts
Number co-cultures 7d, continuous counter 7 times, draws its growth curve, 22 DEG C, 27 DEG C of discovery is suitable for the culture of black snakehead nephrocyte
(Fig. 2 C).It collects cell and counts, co-culture 7d, continuous counter 7 times, draw its growth curve.
As shown in Figure 2, in the M199 culture medium of superfine fetal calf serum 20%, 27 DEG C of growths increase black snakehead kidney cell line
It grows best.
Embodiment 4
The chromosome analysis of black snakehead nephrocyte
The colchicine of final concentration of 10 μ g/mL, 27 DEG C of processing are added in logarithmic growth phase for 60th generation crow snakehead nephrocyte
Cell is collected after 12h, and with the KCl Hypotonic treatment 20min of 0.075mol/L, the Kano fixer of 1mL pre-cooling, 1000r/ is added
After min centrifugation 5min removes supernatant, fixed 3 times with the Kano fixer of pre-cooling, each 15min.Dropping through a cold drop method, after dry,
25min is dyed with 5% Giemsa.Microscopy selects 100 split coil methods to carry out karyotyping and statistics respectively.(figure as the result is shown
3, in figure, A indicates that black snakehead nephrocyte chromosome, B indicate crow snakehead nephrocyte P60 for chromosome), the black snakehead kidney in the 60th generation
The chromosome of cell 43% is 2n=45, consistent with the chromosome number of black snakehead body cell.
Embodiment 5
Black snakehead kidney cell line to the sensitivity experiments of different Aquatic animals virus (connect TiLV, FV3, LMBV, GCRV,
SVCV)
By the different virus of certain concentration (Tilapia mossambica lake virus TiLV, Basidiobolus spp FV3, Micropterus salmoides virus LMBV,
Grass carp reovirus GCRV, spring viremia of carp virus SVCV) it is inoculated in black snakehead kidney cell line of the invention, cell is observed after inoculation to be produced
Raw CPE situation, and qualitative detection is carried out with PCR, as a result (A indicates blank control group, and B indicates TiLV group, and C is indicated as shown in Figure 4
FV3 group, D indicate GCRV group, and E indicates SVCV group, and F indicates LMBV group), occur apparent cytopathy (CPE), explanation into the cell
Black snakehead kidney cell line can virus of proliferation TiLV, FV3, LMBV, GCRV, SVCV, provided effectively for the research of aquatic animal
Material.
Embodiment 6
CAMK tests Tilapia mossambica lake viral susceptibility
Sensibility of the 1.CAMK to Luohu viral (TiLV)
By cell inoculation in 25cm2When cell monolayer reaches 80%-90% in culture bottle, cell 2 times additions are cleaned with PBS
Luohu virus (TiLV) suspension 1mL, standing adsorption 1h, the culture medium for inhaling abandoning viral suspension serum-free clean cell surface 2 times,
The culture medium containing 3%FBS is added, cellular change is observed under inverted microscope and photographs to record.When CPE (cytopathic effect)
Occur being used as viral susceptibility index, shows cell to virus sensitivity.
The result shows that: CAMK confluent monolayer cell, 3d after infection are infected with Tilapia mossambica lake viral (TiLV), cell occurs
Contraction is rounded, diopter increases, cell monolayer shrinkage phenomenon.5d after infection, cell fragment increase, cell detachment, and single layer is in
Broken fishing net shaped, occurs typical cells pathological effect (CPE) (Fig. 5 .B), and control group is normal (Fig. 5 .A).It can be observed under Electronic Speculum
The virion (Fig. 5 .C, D) of TiLV.
The TCID of 2.TiLV inoculation CAMK cell50Measurement
The sensitive cell line of generally acknowledged TiLV is the E-11 cell from line moon murrel in the world at present, in order to compare CAMK and
E-11 measures the principle of virus titer according to Reed-Muench method, takes well-grown CAMK and E-11 to the sensibility of TiLV
Cell samples counting after digesting, with M199 by cell according to 5 × 104The concentration of a/mL is diluted, with the first of 100 holes μ L/
Beginning amount is inoculated into 96 orifice plate cultures makes cell sufficiently adherent for 24 hours.Next day takes out cell plates and abandons growth-promoting media to the greatest extent, and 100 μ L are added in every hole
Then M199 maintaining liquid containing 3%FBS is distinguished viral sample to be checked by the sequence of extension rate from low to high with 100 holes μ L/
It is added in 1~10 each hole of column of 96 orifice plates, 11~12 column L-15 maintaining liquid of the 100 μ L of each addition containing 3%FBS is as negative right
According to the experiment sets 3 repetitions.By cell plates in 37 DEG C, 5%CO2Cytopathy is observed after cultivating 96h in incubator
(cytopathic effect, CPE) and terminal is recorded, the potency of virus is calculated by Reed-Muench method, is arranged two in parallel
Experiment.Formula are as follows:
The viral highest dilution of virus titer=10lgCPE > 50%+(percentage -50% of CPE > 50%)/(CPE >
The percentage of 50% percentage-< 50%) TCID50/100μL
As the result is shown: the virus titer range that TiLV infects CAMK cell line is 100.125TCID50/ ml (2d after infection)-
107.3TCID50/ ml (9d after infection) is higher than E-11 cell 106.7TCID50/ ml (9d after infection).As a result such as Fig. 6.
3. indirect immunofluorescence experiment
CAMK cell is inoculated with into 96 orifice plates, when long full to 90%, 30 μ L vial supernatants are added in every hole, train at 22 DEG C
It supports and is incubated in case, until there is typical case CPE；Culture solution is removed, (- 20 DEG C) methanol that pre-cooling is added fixes 10min at room temperature,
It is washed 3 times with PBS；0.5%Triton is added to carry out being permeabilized 10min, is washed 3 times with PBS；Add 37 DEG C of 5%BSA closings
30min is washed 3 times with PBS；The anti-CAMK primary antibody of mouse is added, 37 DEG C of incubation 1h are washed 3 times with PBS；Fluorescein isothiocynate is added
(FITC) the sheep anti mouse secondary antibody marked, 37 DEG C of incubation 1h are washed 3 times with PBS；Finally observed under inverted fluorescence microscope.
Indirect immunofluorescence experiment the result shows that, using for TiLV antibody as primary antibody incubated cell, marked with FITC
The secondary antibody of note can detecte a large amount of green fluorescence, and control group does not find that specific green fluorescence, TiLV can infect
CAMK cell, and arrived by TiLV detection of specific antibody, as a result as (A indicates that control group, B indicate that the CAMK of infection TiLV is thin to Fig. 7
Born of the same parents).
After the black snakehead nephridial tissue cell for infecting research foundation with TiLV, it can lead to cell and typical cells lesion effect occur
(CPE) is answered, the TiLV cultivated is infected through continuous passage, can generate typical case CPE.It is observed through Electronic Speculum, it is seen that a large amount of disease
Virion.Pathogenesis, detection method, vaccine preparation and the host cell that the cell can be applied to research TiLV interact
And the research such as control and prevention of disease.
Claims (7)
1. a kind of crow snakehead kidney cell line, which is characterized in that the crow snakehead kidney cell line is black snakehead kidney cell line CAMK, in
It is stored in China typical culture collection center on May 8th, 2019, preservation accession designation number is CCTCC NO:C 201994.
2. a kind of construction method of black snakehead kidney cell line described in claim 1, which comprises the following steps:
(1) black snakehead is taken to carry out disinfection, the kidney of clip crow snakehead, is placed in dual anti-containing Pen .- Strep under aseptic condition
It is spare in the culture medium of solution；
(2) kidney of black snakehead is cut into small pieces, the digestion of pancreatin digestive juice, complete medium, which is resuspended, to be mixed, and obtains that mixing liquid is resuspended；
(3) mixing liquid will be resuspended to be added in Tissue Culture Flask, culture to cell is paved with plank in 27 DEG C of incubators, with pancreatin-
After the digestion of EDTA digestive juice, complete medium is added and continues to cultivate；
(4) it after passing on 3 times, freezes up to black snakehead kidney cell line.
3. a kind of construction method of black snakehead kidney cell line according to claim 2, which is characterized in that further include having supplemented
The operation of full culture medium, the operation specifically: resuspension mixing liquid is cultivated in incubator to cell in step (2) and is taped against plate
When son 1/3, complete medium is supplemented.
4. a kind of construction method of black snakehead kidney cell line according to claim 2 or 3, which is characterized in that in step (1)
The operation of the disinfection is to impregnate 1-2min with 75% alcohol.
5. a kind of construction method of black snakehead kidney cell line according to claim 2 or 3, which is characterized in that in step (1)
Described to be placed in the culture medium of the dual anti-solution containing Pen .- Strep, operating to be placed in concentration is the blueness containing 1000u/ml
3-5min in the culture medium of the dual anti-solution of mycin-streptomysin.
6. a kind of construction method of black snakehead kidney cell line according to claim 2 or 3, which is characterized in that described complete
Culture medium is containing concentration be the dual anti-solution of 100u/ml Pen .- Strep and 20% superfine fetal calf serum M199 culture medium.
7. a kind of application of black snakehead kidney cell line described in claim 1 in aquatic livestock virus purification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910626036.3A CN110295137B (en) | 2019-07-11 | 2019-07-11 | Channa argus kidney cell line and construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910626036.3A CN110295137B (en) | 2019-07-11 | 2019-07-11 | Channa argus kidney cell line and construction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110295137A true CN110295137A (en) | 2019-10-01 |
| CN110295137B CN110295137B (en) | 2020-10-30 |
Family
ID=68030942
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910626036.3A Active CN110295137B (en) | 2019-07-11 | 2019-07-11 | Channa argus kidney cell line and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110295137B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112410290A (en) * | 2020-11-05 | 2021-02-26 | 广东富伦德生物科技有限公司 | Micropterus salmoides cardiac fibroblast line and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591638A (en) * | 2009-06-23 | 2009-12-02 | 中国水产科学研究院黄海水产研究所 | The construction process of kidney cell line of scophthalmus maximus |
| CN103695366A (en) * | 2013-12-20 | 2014-04-02 | 中国检验检疫科学研究院 | Goldfish kidney cell line and application thereof |
| CN108865974A (en) * | 2018-05-11 | 2018-11-23 | 中国水产科学研究院珠江水产研究所 | Tilapia mossambica brain cell line and its application |
| CN109207422A (en) * | 2018-09-26 | 2019-01-15 | 福建省农业科学院生物技术研究所 | A kind of European eel kidney cell system EK and its application |
-
2019
- 2019-07-11 CN CN201910626036.3A patent/CN110295137B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591638A (en) * | 2009-06-23 | 2009-12-02 | 中国水产科学研究院黄海水产研究所 | The construction process of kidney cell line of scophthalmus maximus |
| CN103695366A (en) * | 2013-12-20 | 2014-04-02 | 中国检验检疫科学研究院 | Goldfish kidney cell line and application thereof |
| CN108865974A (en) * | 2018-05-11 | 2018-11-23 | 中国水产科学研究院珠江水产研究所 | Tilapia mossambica brain cell line and its application |
| CN109207422A (en) * | 2018-09-26 | 2019-01-15 | 福建省农业科学院生物技术研究所 | A kind of European eel kidney cell system EK and its application |
Non-Patent Citations (1)
| Title |
|---|
| S ABDUL MAJEED: "Develpment characterization and application fo a new fibroblastic-like cell line from kidney of a freshwater air breathing fish Channa striatus", 《ACTA TROPICA》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112410290A (en) * | 2020-11-05 | 2021-02-26 | 广东富伦德生物科技有限公司 | Micropterus salmoides cardiac fibroblast line and application thereof |
| CN112410290B (en) * | 2020-11-05 | 2023-07-18 | 广东富伦德生物科技有限公司 | Lateolabrax japonicus myocardial fibroblast line and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110295137B (en) | 2020-10-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103667176B (en) | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof | |
| CN108865974B (en) | Tilapia mossambica brain cell line and its application | |
| CN112410290B (en) | Lateolabrax japonicus myocardial fibroblast line and application thereof | |
| CN109609460B (en) | A kind of human glioma cell line and its method for building up and application | |
| CN110551689B (en) | Channa argus brain cell line and construction method and application thereof | |
| CN108384762B (en) | Porcine alpha-enterocoronavirus and culture method and application thereof | |
| CN113025574B (en) | Micropterus salmoides brain cell line and application thereof | |
| CN104974977B (en) | A kind of epinephelus lanceolatus fish nephridial tissue cell line and its construction method | |
| CN109294974A (en) | A kind of hybridized prussian carp myeloid tissue cell line and its construction method are applied with it | |
| CN100535108C (en) | Immunizing fish against viral infection | |
| CN104152403B (en) | A kind of method that establishing goose embryonic epithelium cell line and the goose embryonic epithelium cell line of foundation | |
| CN106222131B (en) | A kind of lamb sustentacular cell of testis nature continuous cell line and its purposes in being separately cultured and be proliferated in capripox virus | |
| CN110295137A (en) | A kind of crow snakehead kidney cell line and its construction method and application | |
| CN110527661B (en) | Carassius xyphoides larva cell line and construction method and application thereof | |
| CN114196616B (en) | Fancy carp fin tissue cell line and application thereof | |
| CN107227296A (en) | A kind of method of myelomonocyte separation, identification and induction differentiation | |
| CN108531442B (en) | Insect cell line cultured in serum-free suspension manner and application thereof | |
| CN114058566B (en) | Mandarin skin cell line and application thereof | |
| CN113234660B (en) | Grass carp ureter tissue cell line and application thereof | |
| CN105903011A (en) | Swine pseudorabies live vaccine and preparation method thereof | |
| CN115044556A (en) | Carp brain cell line and application thereof | |
| CN109207422A (en) | A kind of European eel kidney cell system EK and its application | |
| CN109517804A (en) | It is a kind of with the vibrio mimicus bacteriophage OY-1 across kind of crack characteristic, preparation method and applications | |
| CN114874974A (en) | Epinephelus coioides intestinal cell line ECGI-21 and application thereof | |
| CN110283793B (en) | Method for separating and culturing porcine pseudorabies virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |