CN104974977A - Epinephelus lanceolatus kidney tissue cell line and construction method thereof - Google Patents
Epinephelus lanceolatus kidney tissue cell line and construction method thereof Download PDFInfo
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Abstract
The present invention discloses an Epinephelus lanceolatus kidney tissue cell line and a construction method thereof. The construction method specifically comprises: (1) kidney tissue treatment: taking Epinephelus lanceolatus kidney tissue, carrying out disinfection and block cutting, treating, and soaking for spare; (2) primary culture: digesting the treated Epinephelus lanceolatus kidney tissue with a trypsin digestion liquid, carrying out sterilized double-distilled water treatment, and then carrying out primary culture; and (3) subculturing: carrying out subculturing when the primary culture cell confluency achieves 60-90%, wherein the passage is performed every 5-7 days. According to the present invention, the constructed kidneytissue cell line is sensitive to grouper iridovirus, and the cell pathological change is rapid, such that the cell line can be well used for grouper iridovirus separation and propagation and grouper iridovirus vaccine research, and further provides the cell material for separation culture, vaccine research and the like of other marine fish viruses.
Description
Technical field
The invention belongs to technical field of microbe cell line, be specifically related to a kind of epinephelus lanceolatus fish nephridial tissue clone and construction process thereof.
Background technology
Through the development of more than 20 years, China's sea farming obtained great achievement, and cultivation scale, kind and output all occupy prostatitis, the world, and sea farming output had risen to current 40% at the proportion of marine fishery yield from 10% of the past.Epinephelus lanceolatus fish (Epinephelus Lanceolatus), also known as rough gentian cabrilla, be commonly called as rough gentian, it is the important sea farming economic fish of China one class, because it is Fresh & Tender in Texture, nutritious, in addition its growth is fast, and strong adaptability, has become the important object of south China sea farming.In recent years, while China's mariculture industry develops rapidly, also be faced with fundamental research weakness, the outstanding problems such as frequent, disease-resistant improved seeds shortage occur disease, this has become the key factor of the healthy sustainable development of restriction China sea farming industry, especially, when the Outbreak in the face of causing due to virus is sick, be usually at a loss what to do.Investigate thoroughly that viral route of infection and Interaction between virus and cell mechanism fundamentally prevent and solve the key of numerous marine economy Virus disease of fish.Clone is the strong instrument of fish immunology, functional genomics and environmental toxicology research, is also the good material of research organic evolution, growth and heredity.Meanwhile, clone is also carry out fish virology research, is separated, identifies fishes virus and the necessary material of development virus vaccines.
The cell cultures of fish originates in early 1960s, and up to now, the fish cell system set up mostly is freshwater fish and anadromous fish, is mainly used in virological investigation, and the clone set up by seawater fish is fewer.Owing to lacking sensitive cell line, the development progress of the separation of seawater fish virus, cultivation and vaccine is comparatively slow, and the infection of virus in marine fish culture process, the morbidity mechanism of action also cannot be furtherd investigate.Virus has certain cell tropism restricted in vivo and in vitro.The existence of cell surface receptor, often decide virus host range and to tissue and the close preferendum of cell.In addition, each process of virus multiplication, as absorption with invade, shell, the synthesis of virus composition and assemble and the impact being all subject to cellular factor such as to discharge, and, the process of a virus multiplication inherently very complexity." animal virology " one that Yin Xia and Liu Jing Hua Qi edits is pointed out in book, and the virus of cell to which kind about which type has susceptibility, does not have clear and definite regularity, seems to rely on experience, namely finds by putting into practice and verifies.
The establishing techniques of fish primary cell line is one comparatively proven technique at present, but the clone obtained for virus is responsive still deposits very large randomness and difficulty, so effective approach is to quality with quantity, namely prepare the primary cell of the various tissue of various fish, therefrom filter out responsive clone.The kidney of fish is the vitals participating in humoral immunization, is also the important target organ of numerous fishes virus such as irido virus, GCRV, European eel virus simultaneously.
Summary of the invention
The technical problem to be solved in the present invention is, for the above-mentioned defect of prior art, the present invention with epinephelus lanceolatus fish body nephridial tissue for object, successfully establish epinephelus lanceolatus fish nephridial tissue clone, for separation, qualification seawater fish virus and virus vaccines preparation provide important research platform and experiment material.Meanwhile, the method that the present invention sets up also can be applied to be rich in hemocyte tissue from other fish sets up clone, as the tissue such as liver, spleen.
The technical solution adopted for the present invention to solve the technical problems is: provide a kind of epinephelus lanceolatus fish nephridial tissue clone, the biolvgical name of this epinephelus lanceolatus fish nephridial tissue clone is called GGKD, this cell lies in and is deposited in China typical culture collection center on January 29th, 2015, preservation address is: China. Wuhan. and Wuhan University, postcode: 430072, preservation is registered on the books and is numbered CCTCC NO:C201516, and request preservation people is ZhaoQing DaHuaNong Biological medicine Co., Ltd.
The construction process of a kind of epinephelus lanceolatus fish nephridial tissue clone of the present invention, specifically comprises the steps:
(1) process of nephridial tissue: get epinephelus lanceolatus fish nephridial tissue and carry out disinfection, stripping and slicing, process after soak stand-by;
(2) original cuiture: the epinephelus lanceolatus fish nephridial tissue of getting after above-mentioned process carries out original cuiture after the digestion of trysinization liquid;
(3) Secondary Culture: carry out Secondary Culture when primary cultured cell degree of converging reaches 60-90%, every 5-7 days goes down to posterity once.
In described construction process of the present invention, particularly, the process of nephridial tissue in step (1): fresh and alive epinephelus lanceolatus fish is supported 24 hours temporarily in the dual anti-extra large Aeration in the water of the height containing penicillin and Streptomycin sulphate, get fresh and alive epinephelus lanceolatus fish, with alcohol, epinephelus lanceolatus fish is carried out disinfection, under aseptic condition, solution takes nephridial tissue, stripping and slicing, is soaked in rinsing liquid and carries out rinsing.
Preferably, described rinsing liquid is the M199 solution containing 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates.
In described construction process of the present invention, particularly, original cuiture in described step (2): the nephridial tissue that step (1) obtains is added trysinization liquid, at room temperature digests 20-30min, collecting cell suspension after digestion; Cell suspension is added 1-2ml and stop digestion containing serum free culture system liquid (the M199 solution containing 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 5-10% foetal calf serum), and filter, draw filtrate centrifugal, abandon supernatant, 1ml basic culture solution, 9ml aqua sterilisa is added in precipitation, 15-30s is placed, cracking hemocyte after putting upside down mixing; Then add 1ml basic culture solution, 100 order sterilizing filter screens filter; Filtered solution is centrifugal, and it is resuspended to add increment nutrient solution, after resuspended, be placed in 28 DEG C of constant incubators and start original cuiture, within every 2-3 days, change liquid mode by half amount and change nutrient solution.
Preferably, described basic culture solution is the one in M199, MEM and L-15.
Preferably, described increment nutrient solution is the M199 nutrient solution containing 20% foetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 20ng/ml Urogastron and 20ng/ml fibroblast growth factor; Or
MEM nutrient solution containing 20% foetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 20ng/ml Urogastron and 20ng/ml fibroblast growth factor; Or
L-15 nutrient solution containing 20% foetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 20ng/ml Urogastron and 20ng/ml fibroblast growth factor.
In described construction process of the present invention, particularly, Secondary Culture in described step (3): primary cultured cell grows to degree of converging when reaching 60-90%, add tryptic digestion, cell has been hanged with Secondary Culture liquid, then be inoculated in the inherent 28 DEG C of incubators of culturing bottle and cultivate, every 5-7 days goes down to posterity once.
Preferably, Secondary Culture liquid is the M199 nutrient solution containing 5-20% foetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml Streptomycin sulphate, 0-20ng/ml Urogastron and 0-20ng/ml fibroblast growth factor; Or
MEM nutrient solution containing 5-20% foetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml Streptomycin sulphate, 0-20ng/ml Urogastron and 0-20ng/ml fibroblast growth factor; Or
L-15 nutrient solution containing 5-20% foetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml Streptomycin sulphate, 0-20ng/ml Urogastron and 0-20ng/ml fibroblast growth factor.
Preferably, go down to posterity when reaching 2nd generation in step (3), the concentration of fibroblast growth factor, Urogastron in nutrient solution is reduced to 8ng/ml; Reach 5-10 for time, foetal calf serum concentration in nutrient solution is reduced to 15%, and the concentration of fibroblast growth factor, Urogastron reduces to 4ng/ml, and penicillin concn is down to 100IU/ml, Streptomycin sulphate concentration is down to 100 μ g/ml; Reach 15-20 for time, the concentration of fibroblast growth factor, Urogastron in nutrient solution is reduced to zero, and foetal calf serum content is down to 8-10%.
Implement epinephelus lanceolatus fish nephridial tissue clone of the present invention and construction process thereof, there is following beneficial effect:
(1) adopt technical scheme of the present invention, primary and Secondary Culture has been carried out to epinephelus lanceolatus fish nephridial tissue, has achieved good culture effect, thus establish epinephelus lanceolatus fish nephridial tissue clone, can reach for 65 generations continuously.
(2) clone can continuous passage, therefore can provide a large amount of epinephelus lanceolatus fish nephridial tissue derived cells, and cell proliferation nutrient solution formula is simple, without the need to adding exogenous growth factors, passage cell can maintain good growth conditions, and can carry out freezen protective to it.
(3) the nephridial tissue clone constructed by the present invention is responsive to grouper irido virus, and the cytopathy time is fast.Therefore, this clone can be advantageously applied to the separation of grouper irido virus, breeding and grouper irido virus vaccine research.Meanwhile, also for the separation and Culture of other seawater fish virus and vaccine development etc. provide cell material.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying 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 that the different nutrient solution of the present invention is on the chart of the impact of nephridial tissue Growth of Cells;
Fig. 4 is that the different tire serum-concentration of the present invention is on the chart of the impact of nephridial tissue Growth of Cells;
Fig. 5 is the 54th generation nephridial tissue cell Metaphase Chromosome division phasor of present pre-ferred embodiments 1;
Fig. 6 be present pre-ferred embodiments 1 the 54th generation nephridial tissue cell chromosome number analysis chart;
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 24h of present pre-ferred embodiments 1.
Fig. 9 is the CPE observation figure of the grouper irido virus infection nephridial tissue cell 60h of present pre-ferred embodiments 1.
Embodiment
Below, by reference to the accompanying drawings and embodiment, the present invention is described further:
The epinephelus lanceolatus fish that the present invention adopts, purchased from Guangdong great Lin Yang Marine Bio Co., Ltd., does not infect aquatic animal virus, body weight 160g through molecular method checking.
Reagent: foetal calf serum (fetal bovine serum, FBS) is purchased from Gibco company; 0.25% trypsin Trypsin) purchased from Gibco company; PSN antibody mixed solution (penicillin penicillin, Streptomycin sulphate streptomycin, nystatin nystatin) 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) is all purchased from PeproTech company; Dimethyl sulfoxide (DMSO) (dimethyl sulfoxide, DMSO) available from Sigma; MEM (Eagle's minimum essential medium), Leibovitz ' s L-15, M199 nutrient solution are purchased from Life Technologies company, and nutrient solution presses the rear filtration sterilization of product description preparation, and 4 DEG C save backup.
Kidney is the important hemocytopoietic organ of fish, and adopt trypsin digestion to carry out nephridial tissue cell primary when cultivating, in the cell suspension that digestion is filtered, hemocyte accounts for the overwhelming majority.During inoculating cell amount height, culture surface is taken by hemocyte; When inoculating cell amount is low, adherent tissue cell is few, and disperse and become single, original cuiture is difficult to successfully.Therefore, be rich in the original cuiture success of hemocyte organ-tissue, one of them important influence factor is the amount of energy adherent tissue cell in inoculating cell when how effectively to improve original cuiture.
A kind of epinephelus lanceolatus fish nephridial tissue clone of the present invention, the biolvgical name of this epinephelus lanceolatus fish renal tissue clone is called GGKD, this cell lies in and is deposited in China typical culture collection center on January 29th, 2015, preservation address is: China. Wuhan. and Wuhan University, postcode: 430072, preservation is registered on the books and is numbered CCTCC NO:C201516, and request preservation people is ZhaoQing DaHuaNong Biological medicine Co., Ltd.
The construction process of a kind of epinephelus lanceolatus fish nephridial tissue clone of the present invention, specifically comprises the steps:
(1) process of nephridial tissue: by fresh and alive epinephelus lanceolatus fish in the dual anti-seawater of height containing penicillin and Streptomycin sulphate (wherein, the concentration of penicillin and Streptomycin sulphate is 1000IU/ml) in inflation support 24 hours temporarily, get fresh and alive epinephelus lanceolatus fish, soak 1-5min with medical alcohol and overall disinfection is carried out to epinephelus lanceolatus fish, then again sterilize with 75% alcohol, after sterilization, in Bechtop aseptically, solution takes nephridial tissue, with blade by nephridial tissue stripping and slicing, rinse 4-6 time with rinsing liquid, remove hemocyte.
Wherein, rinsing liquid is the M199 solution containing 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates.
(2) original cuiture: the nephridial tissue that step (1) obtains is added trysinization liquid, at room temperature digests 20-30min, collecting cell suspension after digestion; Add 1-2ml to stop digesting containing serum free culture system liquid (described be M199 solution containing 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 5-10% foetal calf serum containing serum free culture system liquid), and with 100 order sterilizing strainer filterings, draw filtrate in 1.5ml centrifuge tube, 1800 leave heart 8-10min, abandon supernatant, precipitation adds 1ml basic culture solution, 9ml aqua sterilisa, places 15-30s, cracking hemocyte after putting upside down mixing; Then add 1ml basic culture solution, 100 order sterilizing filter screens filter; Filtered solution centrifugal collecting cell, it is resuspended to add increment nutrient solution, after resuspended, be placed in 28 DEG C of constant incubators and start original cuiture, within every 2-3 days, change liquid mode by half amount and change nutrient solution; Described basic culture solution is the one in M199, MEM and L-15.
Wherein, the nutrient solution that rises in value is the M199 nutrient solution containing 20% foetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 20ng/ml Urogastron and 20ng/ml fibroblast growth factor; Or
MEM nutrient solution containing 20% foetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 20ng/ml Urogastron and 20ng/ml fibroblast growth factor; Or
L-15 nutrient solution containing 20% foetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 20ng/ml Urogastron and 20ng/ml fibroblast growth factor.
(3) Secondary Culture: primary cultured cell grows to (degree of converging: when cell is bred in bottle when degree of converging reaches 60-90%, appear at intercellular adhesion and ordered state, reach certain state right % that uses foreign currency to represent), 2-3min is digested under adding 0.25% trypsinase room temperature, cell has been hanged with Secondary Culture liquid, blow down attached cell, then by cell suspension inoculation in 2 25cm
2the inherent 28 DEG C of incubators of culturing bottle in cultivate, every 5-7 days goes down to posterity once.
Wherein, Secondary Culture liquid is the M199 nutrient solution containing 5-20% foetal calf serum, 0.046mM (mmol/L) NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml Streptomycin sulphate, 0-20ng/ml Urogastron and 0-20ng/ml fibroblast growth factor; Or
MEM nutrient solution containing 5-20% foetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml Streptomycin sulphate, 0-20ng/ml Urogastron and 0-20ng/ml fibroblast growth factor; Or
L-15 nutrient solution containing 5-20% foetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml Streptomycin sulphate, 0-20ng/ml Urogastron and 0-20ng/ml fibroblast growth factor.
The present invention carries out in the process of Secondary Culture, and contriver finds when reaching 2nd generation, if the concentration of fibroblast growth factor, Urogastron in Secondary Culture liquid is reduced to 8ng/ml, high cell growth speed; And if reach 5-10 for time, foetal calf serum concentration in Secondary Culture liquid is reduced to 15%, the concentration of fibroblast growth factor, Urogastron reduces to 4ng/ml, and penicillin concn is down to 100IU/ml, Streptomycin sulphate concentration is down to the growth that 100 μ g/ml can not only accelerate cell; But, consider the cost of nutrient solution and the survival ability of vitro growth rates and cell, can reach 15-20 for time, the concentration of fibroblast growth factor, Urogastron in Secondary Culture liquid is reduced to zero, and foetal calf serum content is down to 8-10%.Certainly, reach 15-20 for time, the concentration of fibroblast growth factor, Urogastron in Secondary Culture liquid can be reduced to zero, foetal calf serum content is down to 8-10%, reduce the cost of nutrient solution, also do not affect the survival ability of vitro growth rates and cell simultaneously.
embodiment 1
The construction process of epinephelus lanceolatus fish nephridial tissue clone, specifically comprises the steps:
(1) process of nephridial tissue: be that the high dual anti-extra large Aeration in the water of the penicillin of 1000IU/ml and Streptomycin sulphate is supported 24 hours temporarily in concentration by fresh and alive epinephelus lanceolatus fish, get fresh and alive epinephelus lanceolatus fish, soak 1-5min with medical alcohol and overall disinfection is carried out to epinephelus lanceolatus fish, then again sterilize with 75% alcohol, after sterilization, in Bechtop aseptically, solution takes nephridial tissue, with blade by nephridial tissue stripping and slicing, rinse 4-6 time with rinsing liquid (the M199 solution containing 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates), remove hemocyte.
(2) original cuiture: the nephridial tissue that step (1) obtains is added trysinization liquid, at room temperature digests 20-30min, collecting cell suspension after digestion; Add 1-2ml and stop digestion containing serum free culture system liquid (the M199 solution containing 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 5-10% foetal calf serum), and with 100 order sterilizing strainer filterings, draw filtrate in 1.5ml centrifuge tube, 1800 leave heart 8-10min, abandon supernatant, precipitation adds 1ml M199 basic culture solution, 9ml aqua sterilisa, places 15-30s, cracking hemocyte after putting upside down mixing; Then add 1ml M199 basic culture solution, 100 order sterilizing filter screens filter; Filtered solution centrifugal collecting cell, it is resuspended to add increment nutrient solution, after resuspended, be placed in 28 DEG C of constant incubators and start original cuiture, within every 2-3 days, change liquid mode by half amount and change nutrient solution;
Wherein, described increment nutrient solution is the M199 nutrient solution (being called for short M199 nutrient solution) containing 20% foetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 20ng/ml Urogastron and 20ng/ml fibroblast growth factor;
(3) Secondary Culture: primary cultured cell grows to degree of converging when being 60-90%, digests 2-3min, hanged cell, blow down attached cell with Secondary Culture liquid under adding 0.25% trypsinase room temperature, then by cell suspension inoculation in 2 25cm
2the inherent 28 DEG C of incubators of culturing bottle in cultivate, every 5-7 days goes down to posterity once;
Wherein, described Secondary Culture liquid is the M199 nutrient solution of 5-20% foetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml Streptomycin sulphate, 0-20ng/ml Urogastron and 0-20ng/ml fibroblast growth factor.
Embodiment 2
The construction process of epinephelus lanceolatus fish nephridial tissue clone, according to the step of embodiment 1, is with the difference of embodiment 1: in this embodiment,
Described basic culture solution is MEM basic culture solution.
Described increment nutrient solution is the MEM nutrient solution (being called for short MEM nutrient solution) containing 20% foetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 20ng/ml Urogastron and 20ng/ml fibroblast growth factor;
Described Secondary Culture liquid is the MEM nutrient solution containing 5-20% foetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml Streptomycin sulphate, 0-20ng/ml Urogastron and 0-20ng/ml fibroblast growth factor.
Embodiment 3
The construction process of epinephelus lanceolatus fish nephridial tissue clone, according to the step of embodiment 1, is with the difference of embodiment 1: in this embodiment,
Described basic culture solution is L-15 basic culture solution;
Described increment nutrient solution is the L-15 nutrient solution (being called for short L-15 nutrient solution) containing 20% foetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 20ng/ml Urogastron and 20ng/ml fibroblast growth factor;
Described Secondary Culture liquid is the L-15 nutrient solution containing 5-20% foetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml Streptomycin sulphate, 0-20ng/ml Urogastron and 0-20ng/ml fibroblast growth factor.
On the basis of above-described embodiment 1-3, further, when reaching 2nd generation, FGF-basic, EGF concentration in Secondary Culture liquid is reduced to 8ng/ml; When reach 5-10 for time, foetal calf serum concentration in Secondary Culture liquid is reduced to 15%, FGF-basic, EGF concentration reduces to 4ng/ml, penicillin concn is down to 100IU/ml, and Streptomycin sulphate concentration is down to 100 μ g/ml; When reach 15-20 for time, FGF-basic, EGF concentration in Secondary Culture liquid is reduced to zero, and foetal calf serum content is down to 8-10%.
Embodiment of the present invention 1-3 adopts the most hemocyte of sterilized water hypotonic lysis, substantially increases histiocytic content in inoculating cell; Adopt the present invention to carry out the original cuiture of nephridial tissue cell, within 5-7 days, namely cell confluency degree reaches 50%-70%, improves the success ratio being rich in hemocyte organ-tissue primary cell culture significantly.
Original cuiture starts respectively in M199, MEM and L-15 tri-kinds of different nutrient solutions.Observations shows, and in M199 nutrient solution, attached cell is many, and fast growth; And attached cell is few in MEM, L-15 nutrient solution, ability of cell proliferation is lower.Visible M199 nutrient solution is the best nutrient solution being suitable for the cultivation of epinephelus lanceolatus fish nephridial tissue cell primary.
Particularly, see cell growth state in embodiment 1, during the low generation of cell, Epithelial and fibroblast-like cells exist simultaneously, as shown in Figure 1; Along with the carrying out of going down to posterity, epithelioid cell is Main Morphology gradually, as shown in Figure 2; Cell can stablize propagation, and current cell has reached 65 generations more than.
In the present invention, we have investigated the impact of different nutrient solution and the growth of FBS concentration versus cell further.
1. the impact of different nutrient solution cell growth situation.
Get 22 × 10 of above-mentioned enforcement 1
4individual epinephelus lanceolatus fish nephridial tissue clone (54-56 generation), is inoculated in the M199 nutrient solution containing 10%FBS, the MEM nutrient solution containing 10%FBS respectively and contains in the L15 nutritive medium of 10%FBS, cultivating at 28 DEG C by cell.After cultivation, within 1,3,5,7 day, carry out cell counting with blood counting chamber, draw the growth curve of clone in different nutrient solution.
As shown in Figure 3, epinephelus lanceolatus fish nephridial tissue cell in M199 nutrient solution the speed of growth apparently higher than L15, MEM nutrient solution.
2. the growing state of cell under different FBS concentration, chromosomal form number, virus infection experiment.
According to the step of embodiment 1 respectively by 22 × 10
4individual cell be inoculated in containing 2%, 5%, 10% or 15%FBS M199 nutrient solution in, at 28 DEG C cultivate.After cultivation, within 1,3,5,7 day, carry out cell counting with blood counting chamber, draw the growth curve of clone in different FBS concentration.
Result shows, as shown in Figure 4, vitro growth rates is directly proportional to interpolation foetal calf serum concentration, when foetal calf serum concentration is 10-15%, Growth of Cells is fast, but in view of the cost control reason of nutrient solution, when Secondary Culture, after particularly reaching 15-20 generation, foetal calf serum concentration suitably can be reduced to 8-10%.
Result verification
1. the frozen rear recovery sexuality of the epinephelus lanceolatus fish nephridial tissue cell line cell pair obtained by method of the present invention is verified, concrete steps are as follows:
1) Example 1 is in the cell of logarithmic phase, after trysinization, obtain single cell suspension, and the centrifugal 10min of 160g, discards supernatant liquor.The cells frozen storing liquid (the M199 nutrient solution containing 20-30%FBS, 10%DMSO) configured in right amount is added in cell precipitation, resuspended, be transferred in the aseptic cryopreservation tube of 1.8ml; Cryopreservation tube is put into program temperature reduction box ,-80 DEG C of refrigerator overnight, put into the medium-term and long-term preservation of liquid nitrogen every other day;
2) above-mentioned frozen cell is recovered, cryopreservation tube is taken out from liquid nitrogen container, put into 37 DEG C of water-baths and rock fast to thawing, add in the cell bottle containing 6-8ml nutrient solution, cultivate in 28 DEG C of incubators, change liquid every other day.
After different generation cell cryopreservation, anabiosis rate is 82%-96%, and recovery cell can adherent and growth division, and can normally go down to posterity, and cellular form and multiplication capacity are with frozen front no significant difference.
2. the chromosome analysis concrete steps of the epinephelus lanceolatus fish nephridial tissue clone pair obtained by method of the present invention are as follows:
Get the epinephelus lanceolatus fish nephridial tissue clone (54 generation) of above-mentioned enforcement 1, respectively by the cell attachment stable growth 36-48h in 54 generations, add final concentration 0.6-1.0 μ g/ml colchicine effect 4-8h, after trysinization, the centrifugal 10min of 160g reclaims cell; 75mM KCl 37 DEG C of Hypotonic treatment 30min, then add 2ml stationary liquid (methyl alcohol: Glacial acetic acid 3:1) and pre-fix; The centrifugal 10min of 160g, abandons supernatant, is fixed the fixing 30min of liquid chamber temperature; Repeat fixing step 2-3 time; Fixedly stay appropriate stationary liquid for the last time, softly blow even; Draw fixing suspension, 15cm eminence is dripped on the slide glass of-20 DEG C of precoolings, and firmly dispelled by drop rapidly, room temperature is dried; Ji's nurse Sa dye liquor dyeing 10min, the cell dyeing volume morphing of oily Microscopic observation embodiment 1-3, enumerating chromosomes number.
Chromosome number and caryogram are cytogenetic bases, are the indexs comparatively accurately such as identification of organism kind and sex; In the process of cell cultures, karyomit(e) is commonly used to the source of identification of cell, whether the reliability index of conversion occurs.The result display of above-mentioned chromosome analysis: the cell of embodiment 1 is in 116 split coil method of statistics, and from 32-58 not etc., but the split coil method chromosome number of 53.4% is 48 to chromosome number, as seen in figs. 5-6, meets epinephelus lanceolatus fish chromosome number feature.Although chromosome number skewness, the diploid chromosome number order frequency of occurrences is the highest, and other aneuploids only account for very little ratio.
3. the virus infection experiment of the epinephelus lanceolatus fish nephridial tissue clone pair obtained by method of the present invention, concrete steps are as follows:
1) the epinephelus lanceolatus fish nephridial tissue clone (62 generation) of above-described embodiment 1 is got;
2) cytopathic effect (cytopathic effect, CPE) is observed: cell to be seeded is paved with 25cm
2after at the bottom of cell bottle, added by viral solution in Tissue Culture Flask, hatch 1 hour, abandon viral solution for 28 DEG C, be replaced by the fresh M199 cell culture fluid containing 4-6% foetal calf serum, continue to cultivate, every day is seen with inverted microscope and looks into cytopathy.
3) titer determination: after the complete pathology of cell, collect viral suspension, by virus liquid 10 times of serial dilutions to 10 of results
-7, each extent of dilution repeats 6 holes, adds and is vaccinated with in 96 orifice plates of above-mentioned cell, cultivates in 28 DEG C of incubators, observes CPE every day and produces, calculate virus titer according to Reed & Muench (Reed and Muench, 1938) method.
Cell rounding is a description of the om observation after virus infection, causes cell rounding, death after virus infection.As Figure 7-9, result shows, and grouper irido virus cells infected observed obvious CPE after 24 hours, infected the cell rounding of after 60 hours more than 80%, and early stage CPE feature is mainly the contracting of cell circle, and refractivity increases; After infecting 60 hours, circle shrinking born of the same parents become many gradually, and whole cell monolayer forms a kind of netted connection; Last cell monolayer disintegrates completely.Grouper irido virus measures virus infection titer after infecting the complete CPE of above-mentioned cell, and titre reaches 10
7.6tCID
50mL
-1.
To one skilled in the art, according to technical scheme described above and design, other various corresponding change and deformation can be made, and all these change and deformation all should belong within the protection domain of the claims in the present invention.
Claims (10)
1. an epinephelus lanceolatus fish nephridial tissue clone, the biolvgical name of this epinephelus lanceolatus fish nephridial tissue clone is called GGKD, and preserving number is CCTCC NO:C201516.
2. a construction process for epinephelus lanceolatus fish nephridial tissue clone, is characterized in that, specifically comprise the steps:
(1) process of nephridial tissue: get epinephelus lanceolatus fish nephridial tissue and carry out disinfection, stripping and slicing, process after soak stand-by;
(2) original cuiture: the epinephelus lanceolatus fish nephridial tissue after above-mentioned process carries out original cuiture after the digestion of trysinization liquid;
(3) Secondary Culture: carry out Secondary Culture when primary cultured cell degree of converging reaches 60-90%, every 5-7 days goes down to posterity once.
3. the construction process of epinephelus lanceolatus fish nephridial tissue clone according to claim 2, it is characterized in that, the process of nephridial tissue in described step (1): fresh and alive epinephelus lanceolatus fish is supported 24 hours temporarily in the dual anti-extra large Aeration in the water of the height containing penicillin and Streptomycin sulphate, get fresh and alive epinephelus lanceolatus fish, with alcohol, epinephelus lanceolatus fish is carried out disinfection, under aseptic condition, solution takes nephridial tissue, stripping and slicing, is soaked in rinsing liquid and carries out rinsing.
4. the construction process of epinephelus lanceolatus fish nephridial tissue clone according to claim 3, is characterized in that, the rinsing liquid described in described step (1) is the M199 solution containing 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates.
5. the construction process of epinephelus lanceolatus fish nephridial tissue clone according to claim 2, it is characterized in that, original cuiture in described step (2): the nephridial tissue that step (1) obtains is added trysinization liquid, at room temperature digest 20-30min, collecting cell suspension after digestion; Cell suspension is added 1-2ml and stop digestion containing serum free culture system liquid, and filter, absorption filtrate is centrifugal, abandons supernatant, adds 1ml basic culture solution, 9ml aqua sterilisa, place 15-30s after putting upside down mixing, cracking hemocyte in precipitation; Then add 1ml basic culture solution, 100 order sterilizing filter screens filter; Filtered solution is centrifugal, and it is resuspended to add increment nutrient solution, after resuspended, be placed in 28 DEG C of constant incubators and start original cuiture, within every 2-3 days, change liquid mode by half amount and change nutrient solution.
6. the construction process of epinephelus lanceolatus fish nephridial tissue clone according to claim 5, it is characterized in that, described basic culture solution is the one in M199, MEM and L-15.
7. the construction process of epinephelus lanceolatus fish nephridial tissue clone according to claim 5, it is characterized in that, described increment nutrient solution is the M199 nutrient solution containing 20% foetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 20ng/ml Urogastron and 20ng/ml fibroblast growth factor; Or
MEM nutrient solution containing 20% foetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 20ng/ml Urogastron and 20ng/ml fibroblast growth factor; Or
L-15 nutrient solution containing 20% foetal calf serum, 0.046mM NaCl, 400IU/ml penicillin, 400 μ g/ml Streptomycin sulphates, 20ng/ml Urogastron and 20ng/ml fibroblast growth factor.
8. the construction process of epinephelus lanceolatus fish nephridial tissue clone according to claim 2, it is characterized in that, Secondary Culture in described step (3): primary cultured cell grows to degree of converging when reaching 60-90%, add tryptic digestion, cell has been hanged with Secondary Culture liquid, then be inoculated in the inherent 28 DEG C of incubators of culturing bottle and cultivate, every 5-7 days goes down to posterity once.
9. the construction process of epinephelus lanceolatus fish nephridial tissue clone according to claim 8, it is characterized in that, the Secondary Culture liquid in described step (3) is the M199 nutrient solution containing 5-20% foetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml Streptomycin sulphate, 0-20ng/ml Urogastron and 0-20ng/ml fibroblast growth factor; Or
MEM nutrient solution containing 5-20% foetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml Streptomycin sulphate, 0-20ng/ml Urogastron and 0-20ng/ml fibroblast growth factor; Or
L-15 nutrient solution containing 5-20% foetal calf serum, 0.046mM NaCl, 100-400IU/ml penicillin, 100-400 μ g/ml Streptomycin sulphate, 0-20ng/ml Urogastron and 0-20ng/ml fibroblast growth factor.
10. the construction process of epinephelus lanceolatus fish nephridial tissue clone according to claim 8, it is characterized in that, go down to posterity when reaching 2nd generation in step (3), the concentration of fibroblast growth factor, Urogastron in nutrient solution is reduced to 8ng/ml; Reach 5-10 for time, foetal calf serum concentration in nutrient solution is reduced to 15%, and the concentration of fibroblast growth factor, Urogastron reduces to 4ng/ml, and penicillin concn is down to 100IU/ml, Streptomycin sulphate concentration is down to 100 μ g/ml; Reach 15-20 for time, the concentration of fibroblast growth factor, Urogastron in nutrient solution is reduced to zero, and foetal calf serum content is down to 8-10%.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106508891A (en) * | 2016-10-27 | 2017-03-22 | 中国科学院昆明动物研究所 | Construction method of kidney cell line of Schizothorax taliensis |
| CN111411073A (en) * | 2020-03-19 | 2020-07-14 | 华南农业大学 | Construction method of sea bass fry cell line |
| CN112126619A (en) * | 2020-01-16 | 2020-12-25 | 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 |
| CN114774349A (en) * | 2022-04-07 | 2022-07-22 | 南京师范大学 | Fugu obscurus primary kidney cell adherent culture method |
| CN117143806A (en) * | 2023-10-18 | 2023-12-01 | 华南农业大学 | Cell line of garrupa fries, construction method 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 |
| CN102634481A (en) * | 2012-04-23 | 2012-08-15 | 上海海洋大学 | In vitro culture method for scatophagus argus kidney cells |
| CN103060259A (en) * | 2012-12-06 | 2013-04-24 | 沙珍霞 | Building method of fish head and kidney tissue derived cell lines |
| CN103484425A (en) * | 2013-10-12 | 2014-01-01 | 国家海洋局第三海洋研究所 | Pseudosciaena crocea head kidney cell line and construction method thereof |
-
2015
- 2015-04-30 CN CN201510219115.4A patent/CN104974977B/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 |
| CN102634481A (en) * | 2012-04-23 | 2012-08-15 | 上海海洋大学 | In vitro culture method for scatophagus argus kidney cells |
| CN103060259A (en) * | 2012-12-06 | 2013-04-24 | 沙珍霞 | Building method of fish head and kidney tissue derived cell lines |
| CN103484425A (en) * | 2013-10-12 | 2014-01-01 | 国家海洋局第三海洋研究所 | Pseudosciaena crocea head kidney cell line and construction method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Y-S LAI ET AL.: "Two iridovirus-susceptible cell lines established from kidney and liver of grouper,Epiephelus awoara(Temminck＆Schlegel),and partial characterization of grouper iridovirus", 《JOURNAL OF FISH DISEASES》 * |
| 张博等: "近10年鱼类细胞培养研究进展及应用展望", 《海洋科学》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106508891A (en) * | 2016-10-27 | 2017-03-22 | 中国科学院昆明动物研究所 | Construction method of kidney cell line of Schizothorax taliensis |
| CN106508891B (en) * | 2016-10-27 | 2019-09-20 | 中国科学院昆明动物研究所 | A kind of construction method of Dali schizothoracin kidney cell system |
| CN112126619A (en) * | 2020-01-16 | 2020-12-25 | 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 |
| 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 |
| CN114774349A (en) * | 2022-04-07 | 2022-07-22 | 南京师范大学 | Fugu obscurus primary kidney cell adherent culture method |
| CN117143806A (en) * | 2023-10-18 | 2023-12-01 | 华南农业大学 | Cell line of garrupa fries, construction method and application thereof |
| 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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