CN111411073A - Construction method of sea bass fry cell line - Google Patents
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Abstract
The invention provides a construction method of a sea bass fry cell line, which comprises the following steps: (1) primary culture: carrying out pancreatin digestion on the tissues of the jewfish fry to obtain a cell suspension, and adding a primary cell culture solution to culture primary cells; (2) subculturing: when passage is carried out for 1-10 generations, the concentration of fetal calf serum in the passage cell culture solution is 20 percent; when passage is carried out for 11-20 generations, the fetal calf serum concentration in the passage cell culture solution is 15 percent; after passage for 20 generations, the fetal bovine serum concentration in the passage cell culture solution is 10 percent; (3) collecting passage cells to obtain the sea bass fry cell line. The sea bass fry cell line obtained by the construction method of the invention has good growth state, stable cell proliferation, continuous passage, ultralow temperature cryopreservation and resuscitation, and can be used for researches such as exogenous gene expression and virus infection separation.
Description
Technical Field
The invention belongs to the technical field of marine fish cell culture, and particularly relates to a method for constructing a cell line of a sea bass fry by using the sea bass fry.
Background
Lateolabrax japonicus belongs to the order Perciformes and genus Perciformes, and is carnivorous fish. Sea bass is a wide-salt and wide-warm fish, usually living in estuary areas, and also directly entering fresh water lakes. At present, the annual output of the sea bass in China is about 25 million tons, the sea bass is mainly concentrated in Guangzhou Zhu triangular paradise, and the sea bass becomes a supporting column industry for mariculture in the paradise of the Zhuhai city in Guangdong province. However, with the expansion of the culture scale of the sea bass, the increase of the intensification degree, the deterioration of the culture environment and other problems, diseases caused by pathogens such as bacteria, viruses or parasites frequently occur, and the disease problem gradually becomes a main bottleneck restricting the healthy development of the sea bass culture industry.
The iridovirus is one of the main pathogens of the virus diseases of the sea bass, the research on the pathogenic mechanism of virus infection and the interaction between the virus and host cells provides important theoretical information for clarifying the pathogenic characteristics, and the development of efficient vaccines is an effective way for fundamentally preventing and solving the virus diseases of the sea bass. The fish cell line is used as an in vitro culture system and is mainly applied to virus separation and identification, virus purification and preparation of virus vaccines. However, due to the advantages of good repeatability, controllable conditions and the like, with the development of biotechnology and interdiffusion of interdisciplines, the application of fish cell lines is becoming wide, and the method is gradually and widely applied to the research fields of fish virus molecular biology, immunology, toxicology, genetic development biology, oncology and the like. To date, although 180 fish cell lines have been established for virus isolation identification and virology studies, most cell lines are derived from freshwater fish and are not sensitive to marine fish viruses. Therefore, the establishment of a marine fish cell line sensitive to marine fish viruses is an urgent need for virology research and virus disease prevention and control.
To date, suitable cell lines for the isolated propagation of marine fish iridovirus, and particularly of cytostasis virus, have been very limited. It has been reported in the literature that cell lines derived from larval fish are suitable for isolation and purification of some viruses having tissue preference because they contain various cell types derived from tissues. For example, mandarin fish with plump iridovirus-Infectious Spleen and Kidney Necrosis Virus (ISKNV) is successfully separated from the cell line of the larval fish of the mandarin fish. In addition, the success probability of establishing the cell line is further improved because the cells of the larval fish have stronger differentiation capacity.
Disclosure of Invention
The first purpose of the invention is to provide a construction method of a sea bass fry cell line by utilizing sea bass fry.
In order to achieve the purpose, the invention adopts the technical scheme that:
a construction method of a sea bass fry cell line comprises the following steps:
(1) primary culture: carrying out pancreatin digestion on the tissues of the jewfish fry to obtain a cell suspension, and adding a primary cell culture solution to culture primary cells;
(2) subculturing: when passage is carried out for 1-10 generations, the concentration of fetal calf serum in the passage cell culture solution is 20 percent; when passage is carried out for 11-20 generations, the fetal calf serum concentration in the passage cell culture solution is 15 percent; after passage for 20 generations, the fetal bovine serum concentration in the passage cell culture solution is 10 percent;
(3) collecting passage cells to obtain the sea bass fry cell line.
Preferably, said sea bass fry is selected from sea bass fries hatched for 3-4 days.
Preferably, the basic culture medium adopted by the primary cell culture solution and the subculture cell culture solution is L-15 culture medium.
Preferably, the primary culture comprises the steps of: subjecting tissue of Lateolabrax japonicus larva to pancreatin to obtain cell suspension, centrifuging, collecting precipitate, adding primary cell culture solution, resuspending the precipitate, transferring into a culture bottle, culturing at 28 deg.C, replacing culture solution by half every 2-3 days, and performing subculture after 10 days.
Preferably, the formula of the culture solution of the subculture cells during passage 1-10 is that the basic culture medium L-15 contains 20% of fetal bovine serum, 0.266% of NaCl, 5mM of HEPES, 400IU/ml of penicillin, 400 mu g/ml of streptomycin and 400 mu g/ml of nystatin, and the pH value of the culture medium is 7.2-7.4.
Preferably, the formula of the culture solution of the subculture cells at the passage of 11-20 is that the basic culture medium L-15 contains 15% of fetal bovine serum, 0.266% of NaCl, 5mM of HEPES, 200IU/ml of penicillin, 200 mu g/ml of streptomycin and 200 mu g/ml of nystatin, and the pH value of the culture medium is 7.2-7.4.
Preferably, after 20 passages, the culture solution of the passaged cells is prepared by 10 percent of fetal bovine serum, 0.266 percent of NaCl, 5mM of HEPES, 100IU/ml of penicillin and 100 mu g/ml of streptomycin in a basic culture medium L-15, and the pH value of the culture medium is 7.2-7.4.
The invention also provides a sea bass fry cell line which is prepared by adopting the construction method.
The invention also provides application of the sea bass fry cell line obtained by the invention in expressing exogenous genes.
The invention also provides application of the jewfish larva cell line obtained by the invention in virus infection.
Compared with the prior art, the invention has the following advantages:
1. the cell line of the sea bass larval obtained by the construction method has good growth state, stable cell proliferation and the cell form is more than a skin sample as a main form, not only can be continuously passaged (the cells are passed to more than 80 generations at present), but also can be frozen at ultralow temperature and revived, and the establishment of the cell line lays a foundation for the related research of sea bass germ plasm resource preservation.
2. The construction method of the sea bass fry cell line provided by the invention adopts the whole fish tissue cutting and pancreatin digestion methods to culture primary cells, does not need to add other growth factors in a basic culture medium in the cell primary culture and passage processes, properly adjusts the formula of the culture medium in the passage culture process, determines the optimal culture adjustment suitable for the cell growth, and simultaneously reduces the culture cost.
3. The improved sea bass fry cell line is sensitive to various sea fish iridovirus, so that an important cell platform is provided for separating and identifying sea fish viruses and preparing virus vaccines.
4. The improved jewfish larva cell line can be directly applied to the research on virus infection pathogenesis, virus-host cell interaction and exogenous gene function.
Drawings
FIG. 1 is a phase contrast microscope observation of morphology of sea bass fry cells, A-B shows the morphology of primary culture cells of sea bass fry cells; c represents the 60 th cell morphology of the sea bass fry cells.
FIG. 2 is a graph of growth curves of a larval cell line of sea bass under different culture conditions; a represents the influence of different culture media on the growth of the larval cells of the sea bass; b shows the effect of different fetal calf serum concentrations on the growth of the larval cells of the sea bass.
FIG. 3 is a diagram of the karyotype analysis of a larval cell line of sea bass; A-B represents the metaphase chromosome split phase and chromosome number distribution of 35 th generation sea bass cell; C-D shows metaphase chromosome split phase and chromosome number distribution of 55 th generation jewfish fry cells.
FIG. 4 is a fluorescent picture of the cell line of Lateolabrax japonicus larva transfected with pEGFP-N3; a represents 25 generations of cells; b represents 45 generation cells.
FIG. 5 is a schematic diagram showing the observation of iridovirus cytopathic effect of a sea bass fry cell line infected with sea fish; a represents the cytopathic observation result of SGIV or SPIV infected sea bass fry cells; b represents ultrastructural observations of cells infected with SGIV or SPIV.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.
Example 1 construction method of Lateolabrax japonicus fry cell line
(1) Selecting the fry: selecting a sea bass fry which is hatched for 3-4 days, wherein the length of an experimental fish body is 5-7 mm.
(2) Preparing a rinsing solution and a cell culture solution:
the basic culture medium comprises L eibovitz's-15 (L15) culture medium, M199 culture medium and Eagle's Minimal Essential Medium (MEM) culture medium which are all products of Gibco company, wherein each culture medium is prepared according to the company product specification, 0.266% NaCl and 5mM HEPES are additionally added, the mixture is filtered by a 0.22 mu M filter membrane and subpackaged for use at 4 ℃ and then 10-20% fetal calf serum and 1-4 times PSN antibody mixed solution (penicillin, streptomycin and nystatin) are added when the culture medium is used.
The rinse solution is composed of basic culture medium L-15 containing 400IU/m L penicillin, 400 μ g/m L streptomycin and 100 μ g/m L nystatin, and has pH of 7.2-7.4.
The primary cell culture solution is composed of a basic culture medium L-15 containing 20% fetal calf serum, 0.266% NaCl, 5mM HEPES, 400IU/ml penicillin, 400 ug/ml streptomycin and 400 ug/ml nystatin, and the pH of the culture medium is 7.2-7.4.
The reagents and sources used in the invention are respectively: fetal Bovine Serum (FBS), 0.25% Trypsin (Trypsin), PSN antibody cocktail were purchased from Gibco; dimethyl sulfoxide mock (dimethyl sulfoxide, DMS0) was purchased from Sigma company; colchicine and giemsa dyes were purchased from Sigma.
(3) Primary culture
Anesthetizing Lateolabrax japonicus fry incubated for 3-4 days under aseptic condition, soaking and rinsing in sterile water and rinsing solution respectively, and cutting the fry into pieces of 1-3mm with blade3Then 0.25% trypsin is added to the tissue blocks for digestion at room temperature for 30min, and the cell suspension after digestion is filtered by a 100-mesh sterile filter screen. The filtrate was aspirated into a 1.5ml centrifuge tube and centrifuged at 1000rpm for 10 min. Discard the supernatant, resuspend the pellet with primary cell culture medium, and transfer to 25cm2In a culture flask. 5ml of primary cell culture solution was added to each flask. The culture flask was placed in an incubator at 28 ℃. The culture solution is replaced by half the amount of the culture solution every 2-3 days. During the period, the adherent growth of the cells is observed by a phase contrast microscope. The next day of primary culture the cells began adherent growth, a portion of the cells migrated from the cell pellet or tissue block (FIG. 1A), and after 10 days the primary cells were confluent at the bottom of the flask to form a monolayer, and subculture was started. In the initial stage of primary culture, the cell morphology is various, and the fiber-like cells and the epithelial-like cells coexist.
(4) Subculturing
At the first subculture, the medium in the primary culture cell flask was transferred to a sterile flask. And (3) soaking and washing a primary cell monolayer by using 0.25% pancreatin, then adding new pancreatin to digest the monolayer cells for 1-2min at room temperature, and observing the cell rounding condition under a microscope. Gently tapping the cell vial wall to detach and disperse the cells into individual cells. 5ml of fresh subculture cell broth and 5ml of primary cell broth were added, the cell suspension was gently pipetted, followed by mixing at volume 1: 1, flask-divided subculture.
The cell culture fluid is passaged once every 3 to 5 days, and when the cell culture fluid reaches the 10 th generation, the serum content in the cell culture fluid is reduced to 15 percent, and the concentration of the antibiotic is 2 times of the normal use concentration. By passage 20, serum levels in the cell culture broth were reduced to 10% and antibiotic concentrations were at normal use concentrations (100IU/ml penicilin, 100. mu.g/ml streptomycin, 100. mu.g/ml nystatin). After passage 20, nystatin may not be added to the cell culture.
The subculture medium takes L-15 medium as a basic medium, and culture solutions of different subculture cells are optimized on the basis of the embodiment, specifically as follows:
the subculture cell culture medium (1-10 generations) is composed of basic medium L-15 containing 20% fetal calf serum, 0.266% NaCl, 5mM HEPES, 400IU/ml penicillin, 400. mu.g/ml streptomycin and 400. mu.g/ml nystatin, and has pH of 7.2-7.4.
The subculture cell culture medium (11-20 generations) is composed of basic medium L-15 containing 15% fetal calf serum, 0.266% NaCl, 5mM HEPES, 200IU/ml penicillin, 200. mu.g/ml streptomycin and 200. mu.g/ml nystatin, and has pH of 7.2-7.4.
The subculture cell culture solution (after 20 generations) is composed of a basic culture medium L-15 containing 10% fetal bovine serum, 0.266% NaCl, 5mM HEPES, 100IU/ml penicillin and 100 μ g/ml streptomycin, and has a pH value of 7.2-7.4.
The larval sea bass cell line constructed in this example was passaged from primary cells to 80 generations, had a good cell growth state, and could be continuously and stably propagated and passaged, and was named as a larval sea bass cell line (L ateolarax japonica fin, L JF). the cell morphology was mainly dominated by the epithelioid cells (fig. 1C).
Example 2 examination of the Effect of different culture solutions and FBS concentrations on the growth of Lateolabrax japonicus fry cells
1. The effect of different culture solutions on the growth of cells.
The 40 th generation of the larval cells of the sea bass in the embodiment 1 is used for detecting the growth curves of the cells in different culture media, and the specific operation is as follows: the medium in the flask was aspirated off, the flask was rinsed 1 time with pancreatin, 2ml of 0.25% pancreatin was added to digest the cells until they completely fell off, and the cell suspension was gently blown up by adding medium to make the cells single. Cell density was counted using a hemocytometer.
Respectively 0.4 × 105The cells are inoculated in a 24-well plate, the culture mediums are L-15, MEM and M199 culture mediums of 10% fetal bovine serum respectively, and the cells are cultured in a constant temperature incubator at 28 ℃, and the cells are counted by using a blood counting chamber 1, 3, 5 and 7 days after the culture, and the growth curves of the sea bass fry cells under different culture mediums are drawn.
The effect of different media on cell growth as shown in FIG. 2A, L JF cells grew rapidly in L-15 medium with a doubling time of about 2.5 days cells also maintained good growth in MEM medium but at a rate slightly less than that of L-15 medium unlike L-15 and MEM, cells only remained essentially viable in M199 medium.
2. Effect of different fetal bovine serum concentrations on cell growth.
After counting the cells, 0.4 × 10 cells were added5Inoculating the cells into a 24-well plate, culturing in a constant-temperature incubator at 28 ℃ with serum concentrations of 5%, 10%, 15% or 20% in L-15 culture medium, counting the cells by using a blood counting plate respectively after culturing for 1, 3, 5 and 7 days, and drawing growth curves of the sea bass fry cells in culture media with different serum concentrations.
The effect of different serum concentrations on cell growth is shown in fig. 2B: the growth speed of the cells is in direct proportion to the concentration of the added serum, and the normal growth of the cells can be maintained by 10 percent of the serum; at a serum concentration of 5%, cell proliferation was significantly slowed to only half of the 10% serum concentration. The culture medium containing 15% serum obviously stimulates cell proliferation; increasing serum concentrations to 20% had no significant promoting effect on cell growth compared to 15% serum.
In summary, cells of the L JF cell line are suitably grown in L-15 medium containing 10% -15% serum at 28 ℃ under conditions such that the cells multiply for about 2.5 days, and for cost savings, serum is used at a concentration of 10% from passage 20.
Example 3 verification of the cryopreservation and recovery abilities of cells of the Lateolabrax japonicus fry cell line of the present invention
1. Cell cryopreservation
Taking 35 generations of monolayer adherent cells in example 1, obtaining a single cell suspension after trypsinization, centrifuging at 1000rpm for 10min, discarding supernatant, resuspending cell precipitates by using a freezing medium (L-15 culture medium containing 20% fetal calf serum and 10% DMSO) precooled at 4 ℃, transferring 1ml of each tube into a 2ml freezing tube, carrying out freezing procedures of 4 ℃, 30min, minus 20 ℃, 2h, minus 80 ℃ overnight, and transferring into liquid nitrogen for long-term storage every other day.
2. Recovery of cryopreserved cells
And (4) recovering the frozen cells after freezing for 15 days. The cells were removed from the liquid nitrogen and quickly thawed in a 37 ℃ water bath. The thawed cell suspension was centrifuged at 1000rpm to collect the cell pellet, and the pellet was resuspended in subculture medium. A small amount of cell suspension was stained with 0.4% trypan blue for 5min, and the number of dead and live cells was counted using a hemocytometer. And transferring the rest cell suspension into a culture bottle for culture at 28 ℃, and observing the adherence and growth conditions of the cells.
The recovery rate of the cells after being frozen for 15 days is 81%, the survival cells can grow and divide adherently, and the cell morphology and the proliferation capacity have no obvious difference compared with those before being frozen.
Example 4 analysis of chromosomal karyotype of cells of the Lateolabrax japonicus fry cell line obtained by the present invention
The larval cell lines of the sea bass (35 generations and 55 generations) in the example 1 are taken, the cells of different generations are grown for 48h in an adherent way, colchicine with the final concentration of 0.8 mug/ml is added for 6h, and the cells are recovered by centrifugation at 1000rpm for 10min after trypsinization. Hypotonic treatment with 75mM KCl in 37 deg.C water bath for 30min, and pre-fixing by adding 3ml of fixing solution (methanol: glacial acetic acid ═ 3:1) to the cell treatment solution. Centrifuging at 1000rpm for 10min to recover cell precipitate, adding 8ml of fixative, fixing at room temperature for 30min, and repeating the fixing step for 2-3 times. About 100. mu.l of the fixing solution was left for the last fixing and gently and uniformly blown. Sucking the fixative-cell suspension, dropping onto a glass slide pre-cooled at-20 deg.C at a height of 15cm, quickly blowing off the drop with force, and air drying at room temperature. And (3) soaking the dried glass slide in a giemsa staining solution for staining for 10min, washing the staining solution with purified water, and drying at room temperature. The cell chromosome morphology was observed under an oil microscope, photographed and the number of chromosomes counted.
Chromosome number and karyotype are the basis of cytogenetics and are often used to identify the source of a cell, a reliable indicator of whether transformation has occurred. 150 and 200 dividing phases were counted for the 35 and 55 cell generations of example 1, respectively. As a result, as shown in fig. 3A to 3B, the number of chromosomes in the division phase of 35 generations of cells was distributed mostly between 32 and 70, the characteristic chromosome number was 56, the division phase having the diploid chromosome number (2 n: 56) accounted for 24% of all the statistical cells, the cell having 48 chromosomes accounted for 16.7% of all the statistical cells, and the chromosome morphology was mostly telomeric. As shown in fig. 3C to 3D, the number of chromosomes in the division phase of 55 generation cells was distributed mostly between 30 and 72, the characteristic chromosome number was 48, the division phase having diploid chromosome number (2 n: 82) accounted for 32.5% of all the statistical cells, and most of the chromosomes had telomeric chromosomes. Although the number of chromosomes is unevenly distributed, the frequency of diploid chromosome number is the highest, and other aneuploidies account for a small proportion.
Example 5 verification of transfection Effect of exogenous Gene of Lateolabrax japonicus fry cell line
The larval cells of sea bass (25 th and 45 th generations) in example 1 were seeded in 24-well cell culture plates for overnight culture, and the cells were confluent in a monolayer of 70-80% to start transfection, then pEGFP-N3 with endotoxin removed was transfected into the cells according to the instruction manual of Ivitrogen L ipofectamine 2000, and after 36h of transfection, the expression of green fluorescent protein in the cells was observed under a fluorescent microscope.
The observation result of the fluorescence microscope is shown in figure 4, strong green fluorescence signals are observed in transfected sea bass cells of different generations, and the green fluorescence is enhanced along with the increase of the number of passages, which indicates that pEGFP-N3 is transfected into the cells, and the M L V promoter can efficiently start the expression of exogenous genes in L JF cells, and indicates that the sea bass fry cell line can be used for in vitro research on the functions of the exogenous genes.
Example 6 Virus infection test of the cell line of Lateolabrax japonicus fry obtained in the present invention
1. Cytopathic effect (CPE) observation
The larval cells of the sea bass (30 generations) in example 1 are inoculated to a 24-hole culture plate for 18h of virus infection, suspensions of 2 kinds of iridovirus (neogarland grouper iridovirus SGIV and sea bass iridovirus SPIV) infected cells stored in a laboratory are respectively added into a culture medium, the inoculation indexes are respectively 1 MOI and 2 MOI, and the culture is continued after uniform mixing. The cytopathic effect was observed daily with a phase contrast microscope. The cytopathic process was recorded by photography.
The microscopic observation results are shown in fig. 5A: SGIV infected cells are typically characterized by cell shrinkage, rounding, aggregation, and significant void formation in the cell monolayer. Approximately 60% of the cells round after 36h of infection, and the entire cell monolayer forms a network. SPIV infected cells are typically characterized by cell rounding, and individual rounded cells are dispersed on a monolayer of cells. Approximately 80% of the cells become rounded 5 days after infection, and voids, i.e., CPE, form in the cell monolayer after the rounded cells slough off.
2. Electron microscopy of infected cells
The juvenile sea bass cells (passage 30) of example 1 were infected with SGIV and SPIV respectively, the cells were scraped at 36h of SGIV infection and 5 days of SPIV infection, centrifuged at 2000rpm for 10min, the cell pellet was fixed with 2.5% glutaraldehyde at 4 ℃ for 1h, the fixative was discarded, rinsed three times with PBS, 5min each, 1% osmic acid for 1h, then dehydrated stepwise with ethanol gradient (50%, 70%, 80%, 90%, 100%), 10min stepwise, epoxy resin Epon812 was soaked and embedded, the microtome slices were then stained with uranyl acetate and lead citrate for 1h each, and finally observed under a transmission electron microscope (Talos L120C, Thermo Fisher scientific) at 120KV and photographed by CCD recording.
As shown in FIG. 5B, in both SGIV and SPIV infected cells, a large number of virus particles are observed, which indicates that two iridovirus can be replicated and assembled in L JF cells in large quantities.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for constructing a larval cell line of sea bass, which is characterized by comprising the following steps:
(1) primary culture: carrying out pancreatin digestion on the tissues of the jewfish fry to obtain a cell suspension, and adding a primary cell culture solution to culture primary cells;
(2) subculturing: when passage is carried out for 1-10 generations, the concentration of fetal calf serum in the passage cell culture solution is 20 percent; when passage is carried out for 11-20 generations, the fetal calf serum concentration in the passage cell culture solution is 15 percent; after passage for 20 generations, the fetal bovine serum concentration in the passage cell culture solution is 10 percent;
(3) collecting passage cells to obtain the sea bass fry cell line.
2. The method of claim 1, wherein said sea bass fry is selected from the group consisting of 3-4 day-hatched sea bass fries.
3. The method of claim 1, wherein the primary cell culture fluid and the subculture cell culture fluid are both L-15 medium as basal medium.
4. The method of construction according to claim 1, wherein the primary culture comprises the steps of: subjecting tissue of Lateolabrax japonicus larva to pancreatin to obtain cell suspension, centrifuging, collecting precipitate, adding primary cell culture solution, resuspending the precipitate, transferring into a culture bottle, culturing at 28 deg.C, replacing culture solution by half every 2-3 days, and performing subculture after 10 days.
5. The method of claim 1, wherein the culture medium for the passage cells at passage 1-10 is prepared from basic culture medium L-15 containing 20% fetal calf serum, 0.266% NaCl, 5mM HEPES, 400IU/ml penicillin, 400 μ g/ml streptomycin and 400 μ g/ml nystatin, and has pH of 7.2-7.4.
6. The method of claim 1, wherein the culture medium for the passage cells at passage 11-20 is prepared from a basic culture medium L-15 containing 15% fetal calf serum, 0.266% NaCl, 5mM HEPES, 200IU/ml penicillin, 200 μ g/ml streptomycin and 200 μ g/ml nystatin, and has a pH of 7.2-7.4.
7. The method of claim 1, wherein the culture medium for passaging after 20 passages is prepared from a basal medium L-15 containing 10% fetal bovine serum, 0.266% NaCl, 5mM HEPES, 100IU/ml penicillin and 100 μ g/ml streptomycin, and has a pH of 7.2-7.4.
8. A larval cell line of sea bass prepared by the method of any one of claims 1 to 7.
9. Use of a lateolabrax japonicus fry cell line according to claim 8 for expressing an exogenous gene.
10. Use of a sea bass fry cell line according to claim 8 in a viral infection.
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| CN112920987A (en) * | 2021-02-04 | 2021-06-08 | 中国水产科学研究院南海水产研究所 | Separation and primary culture method of vascular sac cells of lateolabrax japonicus |
| CN113249308A (en) * | 2021-05-14 | 2021-08-13 | 中国水产科学研究院南海水产研究所 | Perch arterial ball cell line and application and culture method thereof |
| CN117904032A (en) * | 2024-03-12 | 2024-04-19 | 华南农业大学 | Sea bass fin line cell line and application thereof |
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| CN112920987A (en) * | 2021-02-04 | 2021-06-08 | 中国水产科学研究院南海水产研究所 | Separation and primary culture method of vascular sac cells of lateolabrax japonicus |
| CN113249308A (en) * | 2021-05-14 | 2021-08-13 | 中国水产科学研究院南海水产研究所 | Perch arterial ball cell line and application and culture method thereof |
| CN113249308B (en) * | 2021-05-14 | 2022-02-15 | 中国水产科学研究院南海水产研究所 | Perch arterial ball cell line and application and culture method thereof |
| CN117904032A (en) * | 2024-03-12 | 2024-04-19 | 华南农业大学 | Sea bass fin line cell line and application thereof |
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