CN104974977B - A kind of epinephelus lanceolatus fish nephridial tissue cell line and its construction method - Google Patents
A kind of epinephelus lanceolatus fish nephridial tissue cell line and its construction method Download PDFInfo
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Abstract
The invention discloses a kind of epinephelus lanceolatus fish nephridial tissue cell line and its construction method, which specifically comprises the following steps: the processing of (1) nephridial tissue: taking epinephelus lanceolatus fish nephridial tissue and carries out disinfection, stripping and slicing, impregnates after processing stand-by；(2) originally culture: treated epinephelus lanceolatus fish nephridial tissue digests through pancreatin digestive juice, carries out originally culture after sterilizing distilled water processing；(3) secondary culture: primary cultured cell convergence degree carries out secondary culture when reaching 60-90%, and passage in every 5-7 days is primary.Nephridial tissue cell line constructed by the present invention is sensitive to grouper irido virus, and the cytopathy time is fast.Therefore, which can be advantageously applied to the separation, breeding and grouper irido virus vaccine research of grouper irido virus.Meanwhile also for other seawater fish virus be separately cultured and vaccine development etc. provides cell material.
Description
Technical field
The invention belongs to technical field of microbe cell line, and in particular to a kind of epinephelus lanceolatus fish nephridial tissue cell line and its
Construction method.
Background technique
By development in more than 20 years, China's sea-farming obtained great achievement, and cultivation scale, kind and yield occupy generation
Boundary forefront, sea-farming yield have risen to current 40% from past 10% in the specific gravity of marine fishery yield.Girth stone
Spot fish (Epinephelus Lanceolatus), also known as rough gentian grouper, are commonly called as rough gentian, are a kind of important sea-farmings in China
Economic fish, since its is Fresh & Tender in Texture, full of nutrition, its growth is fast in addition, adaptable, it has also become south China sea-farming
Important object.In recent years, while China's mariculture industry rapidly develops, also it is faced with basic research weakness, disease occurs
Frequently, the outstanding problems such as disease-resistant excellent variety shortage, this, which has become, restricts the health sustainable development of China's sea-farming industry
Key factor, especially face the Outbreak disease as caused by virus when, it is often helpless.Investigate thoroughly the sense of virus
Dye approach and Interaction between virus and cell mechanism are fundamentally to prevent and solve the pass of numerous marine economy Virus disease of fish
Key.Cell line is fish immunology, functional genomics and the strong tool of ecotoxicology research, and research biology
It evolves, the good material of development and heredity.Meanwhile cell line is also to carry out fish virology research, separation, identification fishes virus
And develop material necessary to viral vaccine.
The cell culture of fish originates in the early 1960s, so far, the fish cell system having built up is mostly
Freshwater fish and anadromous fish, are mainly used for virological investigation, and the cell line established by seawater fish is fewer.Due to lacking
The development progress of few sensitive cell line, the separation of seawater fish virus, culture and vaccine is slower, viral in marine fish culture mistake
Infection, the morbidity mechanism of action in journey can not also be furtherd investigate.Virus has certain cell tropism limitation in vivo and in vitro
Property.The presence of cell surface receptor often decides the host range of virus and the close preferendum to tissue and cell.In addition, sick
Each process of poison proliferation, such as adsorb and invasion, shell, the synthesis of virus composition and assembly and release all by cell because
The influence of element, moreover, itself is a extremely complex processes for virus multiplication." the animal of Yin Xia and Liu Jing Hua Qi chief editor
Virology " point out there is sensibility to the virus of which type about the cell of which type, without specific rule in a book
Property, it appears that experience can only be relied on, that is, finds and verifies by putting into practice.
The establishing techniques of fish primary cell line are currently a kind of more mature technology, but to be obtained quick for virus
The cell line of sense still deposits very big contingency and difficulty, so effective approach is with quantity to quality, that is, prepares various
The primary cell of the various tissues of fish is screened out from it sensitive cell line.The kidney of fish is the important of participation humoral immunity
Organ, while being also the important target organ of numerous fishes virus such as irido virus, grass carp reovirus, European eel virus.
Summary of the invention
The technical problem to be solved in the present invention is that in view of the above drawbacks of the prior art, the present invention is with epinephelus lanceolatus fish
Body nephridial tissue is object, has been successfully established epinephelus lanceolatus fish nephridial tissue cell line, to separate, identifying seawater fish virus and virus
Vaccine preparation provides important research platform and experimental material.Meanwhile the method that the present invention establishes also can be applied to from other fishes
Class, which is rich in haemocyte tissue, establishes cell line, such as liver, spleen tissue.
The technical solution adopted by the present invention to solve the technical problems is: providing a kind of epinephelus lanceolatus fish nephridial tissue cell
System, the biolvgical name of the epinephelus lanceolatus fish nephridial tissue cell line is known as GGKD, which, which lies in, is deposited on January 29th, 2015
State's Type Tissue Collection, preservation address are as follows: the Chinese Wuhan Wuhan University, postcode: 430072, preservation is registered on the books volume
Number be CCTCC NO:C201516, request the artificial ZhaoQing DaHuaNong Biological medicine Co., Ltd of preservation.
A kind of construction method of epinephelus lanceolatus fish nephridial tissue cell line of the present invention, specifically comprises the following steps:
(1) processing of nephridial tissue: taking epinephelus lanceolatus fish nephridial tissue and carry out disinfection, stripping and slicing, impregnates after processing stand-by；
(2) originally culture: it is treated take epinephelus lanceolatus fish nephridial tissue through pancreatin digestive juice digestion after carry out it is primary
Culture；
(3) secondary culture: primary cultured cell convergence degree carries out secondary culture, passage one in every 5-7 days when reaching 60-90%
It is secondary.
In the construction method of the present invention, specifically, the processing of nephridial tissue in step (1): by fresh and alive girth
Grouper is temporarily supported 24 hours in the dual anti-extra large Aeration in the water of the height containing penicillin and streptomysin, takes fresh and alive epinephelus lanceolatus fish, uses wine
Essence carries out disinfection to epinephelus lanceolatus fish, and solution takes nephridial tissue under aseptic condition, and stripping and slicing is soaked in rinsing liquid and is rinsed.
Preferably, the rinsing liquid is the M199 solution containing 400IU/ml penicillin, 400 μ g/ml streptomysins.
In the construction method of the present invention, specifically, originally culture in the step (2): step (1) is obtained
Nephridial tissue be added pancreatin digestive juice, digest 20-30min at room temperature, collect cell suspension after digestion；By cell suspension plus
It is (molten containing 400IU/ml penicillin, 400 μ g/ml streptomysins, the M199 of 5-10% fetal calf serum to enter 1-2ml serum-containing medium
Liquid) digestion is terminated, and filter, filtrate centrifugation is drawn, supernatant is abandoned, 1ml basic culture solution, 9ml aqua sterilisa is added in precipitating, overturns
15-30s is placed after mixing, cracks haemocyte；Then 1ml basic culture solution is added, is filtered on 100 mesh sterilizing strainer；Filtration
Liquid centrifugation is added increment culture solution and is resuspended, and after resuspension, is placed in 28 DEG C of constant incubators and starts originally culture, measure by half within every 2-3 days
It changes liquid mode and replaces culture solution.
Preferably, the basic culture solution is one of M199, MEM and L-15.
Preferably, the increment culture solution be containing 20% fetal calf serum, 0.046mM NaCl, 400IU/ml penicillin,
The M199 culture solution of 400 μ g/ml streptomysins, 20ng/ml epidermal growth factor and 20ng/ml fibroblast growth factor；Or
Contain 20% fetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml streptomysins, 20ng/ml table
The MEM culture solution of skin growth factor and 20ng/ml fibroblast growth factor；Or
Contain 20% fetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml streptomysins, 20ng/ml table
The L-15 culture solution of skin growth factor and 20ng/ml fibroblast growth factor.
In the construction method of the present invention, specifically, secondary culture in the step (3): primary cultured cell
When length to convergence degree reaches 60-90%, trypsin digestion is added, has hanged cell with secondary culture liquid, has then been inoculated in culture bottle
It is cultivated in inherent 28 DEG C of incubators, passage in every 5-7 days is primary.
Preferably, secondary culture liquid is to contain 5-20% fetal calf serum, 0.046mM NaCl, 100-400IU/ml mould
Element, 100-400 μ g/ml streptomysin, 0-20ng/ml epidermal growth factor and 0-20ng/ml fibroblast growth factor
M199 culture solution；Or
Contain 5-20% fetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml strepto-
The MEM culture solution of element, 0-20ng/ml epidermal growth factor and 0-20ng/ml fibroblast growth factor；Or
Contain 5-20% fetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml strepto-
The L-15 culture solution of element, 0-20ng/ml epidermal growth factor and 0-20ng/ml fibroblast growth factor.
Preferably, when reaching 2nd generation in step (3), by fibroblast growth factor, epidermal growth in culture solution
The concentration of the factor is reduced to 8ng/ml；Reach 5-10 for when, fetal calf serum concentration in culture solution is reduced to 15%, fibroblast
Growth factor, the concentration of epidermal growth factor are reduced to 4ng/ml, and penicillin concn is down to 100IU/ml, streptomysin concentration is down to
100μg/ml；Reach 15-20 for when, the concentration of fibroblast growth factor in culture solution, epidermal growth factor is reduced to
Zero, fetal calf serum content is down to 8-10%.
Implement epinephelus lanceolatus fish nephridial tissue cell line and its construction method of the invention, has the advantages that
(1) technical solution of the present invention is used, primary and secondary culture has been carried out to epinephelus lanceolatus fish nephridial tissue, has been achieved
Preferable culture effect can continuously reach for 65 generations to establish epinephelus lanceolatus fish nephridial tissue cell line.
(2) cell line can continuous passage, therefore a large amount of epinephelus lanceolatus fish nephridial tissue derived cell can be provided, and cell
Multiplying culture formula of liquid is simple, and without adding exogenous growth factors, passage cell can maintain good growth conditions, and can be to it
Carry out freezen protective.
(3) the nephridial tissue cell line constructed by the present invention is sensitive to grouper irido virus, and the cytopathy time is fast.Cause
This, which can be advantageously applied to the separation, breeding and grouper irido virus vaccine research of grouper irido virus.Together
When, also for other seawater fish virus be separately cultured and vaccine development etc. provides cell material.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples, in attached drawing:
Fig. 1 is the epinephelus lanceolatus fish nephridial tissue primary cultured cell figure of present pre-ferred embodiments 1；
Fig. 2 is the epinephelus lanceolatus fish nephridial tissue cultured cell line figure of present pre-ferred embodiments 1；
Fig. 3 is the chart for the influence that different culture solutions of the invention grow nephridial tissue cell；
Fig. 4 is the chart for the influence that different tire serum-concentrations of the invention grow nephridial tissue cell；
Fig. 5 is the 54th generation nephridial tissue cell metaphase chromosome division phasor of present pre-ferred embodiments 1；
Fig. 6 is the 54th generation nephridial tissue cell chromosome number analysis chart of present pre-ferred embodiments 1；
Fig. 7 is the CPE observation figure of the grouper irido virus infection nephridial tissue cell 0h of present pre-ferred embodiments 1；
Fig. 8 is the CPE observation figure of the grouper irido virus infection nephridial tissue cell of present pre-ferred embodiments 1 for 24 hours.
Fig. 9 is the CPE observation figure of the grouper irido virus infection nephridial tissue cell 60h of present pre-ferred embodiments 1.
Specific embodiment
In the following, being described further in conjunction with attached drawing and specific embodiment to the present invention:
The epinephelus lanceolatus fish that the present invention uses is purchased from Guangdong great Lin Yang Marine Bio Co., Ltd., not through molecular method verifying
Infect aquatic livestock virus, weight 160g.
Reagent: fetal calf serum (fetal bovine serum, FBS) is purchased from Gibco company；0.25% trypsase
(Trypsin) it is purchased from Gibco company；(penicillin penicillin, streptomysin streptomycin make mould PSN antibody mixed liquor
Rhzomorph nystatin) it is purchased from Gibco company；Recombination human basic fibroblast growth factor (recombinant human
Fibroblast growth factor-basic, FGF-basic), recombinant human epidermal growth factor (recombinant
Human epidermal growth factor, EGF) it is purchased from PeproTech company；Dimethyl sulfoxide (dimethyl
Sulfoxide, DMSO) it is purchased from Sigma company；MEM(Eagle's minimum essential medium),Leibovitz'
S L-15, M199 culture solution are purchased from Life Technologies company, filtration sterilization after culture solution is prepared by product description, and 4
It DEG C saves backup.
Kidney is the important blood forming organ of fish, when carrying out the culture of nephridial tissue cell primary using trypsin digestion, is digested
Haemocyte accounts for the overwhelming majority in the cell suspension of filter.When inoculating cell amount is high, culture surface is taken by haemocyte；Inoculating cell amount
When low, adherent tissue cell is few, dispersion and at single, and originally culture is difficult to success.Therefore, the original rich in haemocyte organ-tissue
It is commissioned to train feeding success, one of them important influence factor can be pasted in inoculating cell when how to effectively improve originally culture
The histiocytic amount of wall.
A kind of epinephelus lanceolatus fish nephridial tissue cell line of the invention, the biology of the epinephelus lanceolatus fish renal tissue cell line
Entitled GGKD, which, which lies in, is deposited in China typical culture collection center, preservation address on January 29th, 2015 are as follows: in
The Wuhan state Wuhan University, postcode: 430072, preservation number of registering on the books is CCTCC NO:C201516, and request preservation is artificial
ZhaoQing DaHuaNong Biological medicine Co., Ltd.
A kind of construction method of epinephelus lanceolatus fish nephridial tissue cell line of the invention, specifically comprises the following steps:
(1) processing of nephridial tissue: by fresh and alive epinephelus lanceolatus fish in the dual anti-seawater of height containing penicillin and streptomysin
Inflation is temporarily supported 24 hours in (wherein, the concentration of penicillin and streptomysin is 1000IU/ml), takes fresh and alive epinephelus lanceolatus fish, is used
Medicinal alcohol impregnates 1-5min and carries out overall disinfection to epinephelus lanceolatus fish, is then sterilized again with 75% alcohol, after disinfection, in nothing
Solution takes nephridial tissue in superclean bench under the conditions of bacterium, with blade by nephridial tissue stripping and slicing, is rinsed 4-6 times with rinsing liquid, removal
Haemocyte.
Wherein, rinsing liquid is the M199 solution containing 400IU/ml penicillin, 400 μ g/ml streptomysins.
(2) originally culture: pancreatin digestive juice is added in the nephridial tissue that step (1) is obtained, and digests 20-30min at room temperature,
Cell suspension is collected after digestion；1-2ml serum-containing medium is added, and (serum-containing medium is to contain 400IU/ml mould
The M199 solution of element, 400 μ g/ml streptomysins, 5-10% fetal calf serum) terminate digestion, and with 100 mesh sterilize strainer filtering, suction
Take filtrate into 1.5ml centrifuge tube, 1800 turns of centrifugation 8-10min abandon supernatant, and 1ml basic culture solution, 9ml sterilizing is added in precipitating
Water places 15-30s after being mixed by inversion, crack haemocyte；Then 1ml basic culture solution is added, the mistake on 100 mesh sterilizing strainer
Filter；Cell is collected by centrifugation in filtered solution, and increment culture solution is added and is resuspended, after resuspension, is placed in 28 DEG C of constant incubators and starts primary training
It supports, changes liquid mode by half amount within every 2-3 days and replace culture solution；The basic culture solution is one of M199, MEM and L-15.
Wherein, increment culture solution is to contain 20% fetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/
The M199 culture solution of ml streptomysin, 20ng/ml epidermal growth factor and 20ng/ml fibroblast growth factor；Or
Contain 20% fetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml streptomysins, 20ng/ml table
The MEM culture solution of skin growth factor and 20ng/ml fibroblast growth factor；Or
Contain 20% fetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml streptomysins, 20ng/ml table
The L-15 culture solution of skin growth factor and 20ng/ml fibroblast growth factor.
(3) secondary culture: primary cultured cell is long, and (convergence degree: cell is proliferated in bottle when reaching 60-90% to convergence degree
When, there is intercellular adhesion and ordered state, reach certain state is indicated with convergence degree %), 0.25% tryptose is added
Enzyme digests 2-3min at room temperature, has hanged cell with secondary culture liquid, has blown down attached cell, then by cell suspension inoculation in 2
25cm2Culture bottle in cultivated in 28 DEG C of incubators, every 5-7 days passage once.
Wherein, secondary culture liquid is to contain 5-20% fetal calf serum, 0.046mM (mmol/L) NaCl, 100-400IU/ml
Penicillin, 100-400 μ g/ml streptomysin, 0-20ng/ml epidermal growth factor and 0-20ng/ml fibroblast growth factor
M199 culture solution；Or
Contain 5-20% fetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml strepto-
The MEM culture solution of element, 0-20ng/ml epidermal growth factor and 0-20ng/ml fibroblast growth factor；Or
Contain 5-20% fetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml strepto-
The L-15 culture solution of element, 0-20ng/ml epidermal growth factor and 0-20ng/ml fibroblast growth factor.
The present invention carry out secondary culture during, inventor find when reaching 2nd generation, if by secondary culture liquid at
Fibroblast growth factor, epidermal growth factor concentration be reduced to 8ng/ml, high cell growth speed；And if reaching 5-10
Fetal calf serum concentration in secondary culture liquid is reduced to 15%, the concentration of fibroblast growth factor, epidermal growth factor by Dai Shi
It is reduced to 4ng/ml, penicillin concn, which is down to 100IU/ml, streptomysin concentration is down to 100 μ g/ml can not only accelerate the growth of cell；
But comprehensively consider the cost of culture solution and the survival ability of vitro growth rates and cell, 15-20 generation can reached
When, the concentration of fibroblast growth factor in secondary culture liquid, epidermal growth factor is reduced to zero, fetal calf serum content is down to
8-10%.Certainly, reach 15-20 for when, can be by fibroblast growth factor in secondary culture liquid, epidermal growth factor
Concentration is reduced to zero, and fetal calf serum content is down to 8-10%, reduces the cost of culture solution, while nor affecting on vitro growth rates
And the survival ability of cell.
Embodiment 1
The construction method of epinephelus lanceolatus fish nephridial tissue cell line, specifically comprises the following steps:
(1) processing of nephridial tissue: fresh and alive epinephelus lanceolatus fish is high in the penicillin and streptomysin that concentration is 1000IU/ml
It is dual anti-sea Aeration in the water temporarily support 24 hours, take fresh and alive epinephelus lanceolatus fish, with medicinal alcohol impregnate 1-5min to epinephelus lanceolatus fish into
Then row overall disinfection is sterilized again with 75% alcohol, after disinfection, solution takes kidney group in superclean bench aseptically
It knits, with blade by nephridial tissue stripping and slicing, is rushed with rinsing liquid (the M199 solution containing 400IU/ml penicillin, 400 μ g/ml streptomysins)
It washes 4-6 times, removes haemocyte.
(2) originally culture: pancreatin digestive juice is added in the nephridial tissue that step (1) is obtained, and digests 20-30min at room temperature,
Cell suspension is collected after digestion；1-2ml serum-containing medium is added and (contains 400IU/ml penicillin, 400 μ g/ml streptomysins, 5-
The M199 solution of 10% fetal calf serum) terminate digestion, and sterilized strainer filtering with 100 mesh, absorption filtrate is to 1.5ml centrifuge tube
In, 1800 turns of centrifugation 8-10min abandon supernatant, and precipitating is added 1ml M199 basic culture solution, 9ml aqua sterilisa, puts after being mixed by inversion
15-30s is set, haemocyte is cracked；Then 1ml M199 basic culture solution is added, is filtered on 100 mesh sterilizing strainer；Filtered solution from
The heart collects cell, and increment culture solution is added and is resuspended, after resuspension, is placed in 28 DEG C of constant incubators and starts originally culture, press within every 2-3 days
Half amount changes liquid mode and replaces culture solution；
Wherein, the increment culture solution is to contain 20% fetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400
The M199 culture solution of μ g/ml streptomysin, 20ng/ml epidermal growth factor and 20ng/ml fibroblast growth factor is (referred to as
M199 culture solution)；
(3) when primary cultured cell length to convergence degree is 60-90%, 0.25% trypsase room temperature secondary culture: is added
Lower digestion 2-3min has hanged cell with secondary culture liquid, has blown down attached cell, then by cell suspension inoculation in 2 25cm2's
It is cultivated in 28 DEG C of incubators in culture bottle, passage in every 5-7 days is primary；
Wherein, the secondary culture liquid be 5-20% fetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin,
The M199 of 100-400 μ g/ml streptomysin, 0-20ng/ml epidermal growth factor and 0-20ng/ml fibroblast growth factor is trained
Nutrient solution.
Embodiment 2
The construction method of epinephelus lanceolatus fish nephridial tissue cell line, the step of according to embodiment 1, the difference with embodiment 1 exists
In: in this embodiment,
The basic culture solution is MEM basic culture solution.
The increment culture solution is to contain 20% fetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml
MEM culture solution (the abbreviation MEM culture of streptomysin, 20ng/ml epidermal growth factor and 20ng/ml fibroblast growth factor
Liquid)；
The secondary culture liquid be containing 5-20% fetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin,
The MEM of 100-400 μ g/ml streptomysin, 0-20ng/ml epidermal growth factor and 0-20ng/ml fibroblast growth factor is trained
Nutrient solution.
Embodiment 3
The construction method of epinephelus lanceolatus fish nephridial tissue cell line, the step of according to embodiment 1, the difference with embodiment 1 exists
In: in this embodiment,
The basic culture solution is L-15 basic culture solution；
The increment culture solution is to contain 20% fetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml
L-15 culture solution (the abbreviation L-15 training of streptomysin, 20ng/ml epidermal growth factor and 20ng/ml fibroblast growth factor
Nutrient solution)；
The secondary culture liquid be containing 5-20% fetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin,
The L-15 of 100-400 μ g/ml streptomysin, 0-20ng/ml epidermal growth factor and 0-20ng/ml fibroblast growth factor is trained
Nutrient solution.
On the basis of above-described embodiment 1-3, further, when reaching 2nd generation, by FGF- in secondary culture liquid
Basic, EGF concentration are reduced to 8ng/ml；When reach 5-10 for when, fetal calf serum concentration in secondary culture liquid is reduced to 15%,
FGF-basic, EGF concentration are reduced to 4ng/ml, and penicillin concn is down to 100IU/ml, and streptomysin concentration is down to 100 μ g/ml；When
Reach 15-20 for when, FGF-basic, EGF concentration in secondary culture liquid are reduced to zero, fetal calf serum content is down to 8-10%.
1-3 of the embodiment of the present invention substantially increases inoculating cell using the most haemocytes of sterile water hypotonic lysis
In histiocytic content；Carry out the originally culture of nephridial tissue cell using the present invention, cell confluency degree reaches 50%- within 5-7 days
70%, significantly improve the success rate rich in haemocyte organ-tissue primitive cell culture.
Originally culture starts in tri- kinds of M199, MEM and L-15 different culture solutions respectively.Observation is the results show that M199 is trained
Attached cell is more in nutrient solution, and the speed of growth is fast；And attached cell is few in MEM, L-15 culture solution, ability of cell proliferation compared with
It is low.It can be seen that M199 culture solution is adapted for the best culture solution of epinephelus lanceolatus fish nephridial tissue cell primary culture.
Specifically, referring to cell growth state in embodiment 1, when the low generation of cell, Epithelial and fibroblast-like cells are same
When exist, as shown in Figure 1；With the progress of passage, epithelioid cell is gradually Main Morphology, as shown in Figure 2；Cell can be stablized
Proliferation, it is more than generation to have reached 65 for cell at present.
In the present invention, we have further investigated the influence of different culture solutions and the growth of FBS concentration versus cell.
1. influence of the different culture solutions to cell growth status.
Take the 22 × 10 of above-mentioned implementation 14A epinephelus lanceolatus fish nephridial tissue cell line (54-56 generation), cell is inoculated with respectively
In the M199 culture solution containing 10%FBS, the MEM culture solution containing 10%FBS and the L15 nutrient solution containing 10%FBS, trained at 28 DEG C
It supports.1 after culture, cell count is carried out with blood counting chamber within 3,5,7 days, it is bent to draw growth of the cell line in different culture solutions
Line.
As shown in figure 3, epinephelus lanceolatus fish nephridial tissue cell speed of growth in M199 culture solution is apparently higher than L15, MEM training
Nutrient solution.
2. the experiment of the form number of growing state of the cell under different FBS concentration, chromosome, virus infection.
According to the step of embodiment 1 respectively by 22 × 104A cell inoculation is in containing 2%, 5%, 10% or 15%FBS's
In M199 culture solution, cultivated at 28 DEG C.1 after culture, cell count is carried out with blood counting chamber within 3,5,7 days, draw cell line
In the growth curve of different FBS concentration.
The results show that as shown in figure 4, vitro growth rates to addition fetal calf serum concentration it is directly proportional, fetal calf serum concentration
When for 10-15%, cell growth is fast, but in view of the cost control reason of culture solution, in secondary culture, especially reaches the
After 15-20 generation, fetal calf serum concentration can suitably be reduced to 8-10%.
Result verification
1. recovery performance after the freezing of pair epinephelus lanceolatus fish nephridial tissue cell line cell obtained by means of the present invention
Power is verified, the specific steps are as follows:
1) Example 1 is in the cell of logarithmic growth phase, and single cell suspension, 160g centrifugation are obtained after pancreatin digests
10min discards supernatant.Appropriate configured cells frozen storing liquid is added into cell precipitation and (contains 20-30%FBS, 10%DMSO
M199 culture solution), be resuspended, be transferred in the sterile cryopreservation tube of 1.8ml；Cryopreservation tube is put into program temperature reduction box, -80 DEG C of refrigerators
Overnight, it is put into liquid nitrogen saves for a long time every other day；
2) it recovers to the above-mentioned cell frozen, cryopreservation tube is taken out from liquid nitrogen container, be put into 37 DEG C of water-baths fast
Speed is rocked to thawing, is added in the cell bottle of the culture solution containing 6-8ml, is cultivated in 28 DEG C of incubators, is changed liquid every other day.
Anabiosis rate is 82%-96% after different generation cell cryopreservations, and recovery cell can be adherent and grows division, and can be with
Normally passage, cellular morphology and proliferative capacity are same to freeze preceding no significant difference.
2. the chromosome analysis specific steps of pair epinephelus lanceolatus fish nephridial tissue cell line obtained by means of the present invention
It is as follows:
The epinephelus lanceolatus fish nephridial tissue cell line (54 generation) of above-mentioned implementation 1 is taken, it is respectively that the cell in 54 generations is adherent stable raw
Long 36-48h is added final concentration 0.6-1.0 μ g/ml colchicine and acts on 4-8h, and 160g centrifugation 10min recycling is thin after pancreatin digestion
Born of the same parents；37 DEG C of Hypotonic treatment 30min of 75mM KCl add 2ml fixer (methanol: glacial acetic acid 3:1) and pre-fix；160g centrifugation
10min abandons supernatant, is fixed the fixed 30min of liquid chamber temperature；It repeats fixing step 2-3 times；Appropriate fixation is stayed in last time fixation
Liquid is softly blown even；Fixed suspension is drawn, 15cm eminence drop rapidly firmly dispels drop on the glass slide of -20 DEG C of pre-coolings,
Room temperature is dried；Ji's nurse Sa dye liquor dyes 10min, the cell dyeing volume morphing of oily microscopic observation embodiment 1-3, enumerating chromosomes number
Mesh.
Chromosome number and caryogram are cytogenetic bases, are to identify that biological kind and gender etc. accurately refer to
Mark；During cell culture, chromosome is commonly used to the source of identification of cell, the reliability index whether converted.It is above-mentioned
Chromosome analysis is as the result is shown: the cell of embodiment 1 in 116 split coil methods of statistics, chromosome number from 32-58 not
Deng, but 53.4% split coil method chromosome number is 48, as seen in figs. 5-6, meets epinephelus lanceolatus fish chromosome number feature.
Although chromosome number is unevenly distributed, the diploid chromosome number mesh frequency of occurrences be it is highest, other aneuploids only account for
The ratio of very little.
3. the virus infection of pair epinephelus lanceolatus fish nephridial tissue cell line obtained by means of the present invention is tested, specific to walk
It is rapid as follows:
1) the epinephelus lanceolatus fish nephridial tissue cell line (62 generation) of above-described embodiment 1 is taken；
2) cytopathic effect (cytopathic effect, CPE) is observed: cell to be seeded is paved with 25cm2Cell bottle bottom
Afterwards, viral solution is added in Tissue Culture Flask, 28 DEG C are incubated for 1 hour, abandon viral solution, are changed to fetal calf serum containing 4-6%
Fresh M199 cell culture fluid, continue to cultivate, daily with inverted microscope sight look into cytopathy.
3) titer determination: after the complete lesion of cell, collecting viral suspension, by the virus liquid 10 of harvest be serially diluted again to
10-7, each dilution repeats 6 holes, is added and is vaccinated in 96 orifice plates of above-mentioned cell, cultivate in 28 DEG C of incubators, daily to observe
CPE is generated, and calculates virus titer according to Reed&Muench (Reed and Muench, 1938) method.
Cell rounding is a description of the om observation after virus infection, leads to cell rounding, death after virus infection.
As Figure 7-9, it the results show that grouper irido virus infection cell observes apparent CPE after 24 hours, infects 60 hours
80% or more cell rounding afterwards, the CPE feature of early stage are mainly cell circle contracting, and refractivity increases；After infection 60 hours, circle
Shrinking born of the same parents gradually become more, and entire cell monolayer forms a kind of netted connection；Last cell monolayer disintegrates completely.Grouper iris disease
Poison measures virus infection titer after infecting the above-mentioned complete CPE of cell, and titre reaches 107.6TCID50mL-1。
It will be apparent to those skilled in the art that can make various other according to the above description of the technical scheme and ideas
Corresponding change and deformation, and all these changes and deformation all should belong to the protection scope of the claims in the present invention
Within.
Claims (1)
1. the biolvgical name of a kind of epinephelus lanceolatus fish nephridial tissue cell line, the epinephelus lanceolatus fish nephridial tissue cell line is known as GGKD,
Deposit number is CCTCC NO:C201516.
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| CN112126619B (en) * | 2020-01-16 | 2021-09-07 | 中国水产科学研究院长江水产研究所 | Rhabdoviral sensitive finless eel kidney tissue cell line and application |
| CN111411073A (en) * | 2020-03-19 | 2020-07-14 | 华南农业大学 | Construction method of sea bass fry cell line |
| CN114774349B (en) * | 2022-04-07 | 2024-06-25 | 南京师范大学 | Takifugu obscurus primary kidney cell adherence culture method |
| CN117143806B (en) * | 2023-10-18 | 2024-02-23 | 华南农业大学 | Cell line of garrupa fries, construction method and application thereof |
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