CN117904032A

CN117904032A - Sea bass fin line cell line and application thereof

Info

Publication number: CN117904032A
Application number: CN202410275029.4A
Authority: CN
Inventors: 黄友华; 刘梦珂; 黄晓红; 秦启伟; 林菲菲; 刘绮婷
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2024-03-12
Filing date: 2024-03-12
Publication date: 2024-04-19

Abstract

The invention discloses a sea bass fin line and application thereof. The sea bass fin line (Lateolabrax japonicus FIN CELL LINE) LJFin is obtained by primary culture and subculture construction and is preserved in the Guangdong province microorganism strain collection (GDMCC) at 1-12 days 2024, and the preservation number is: GDMCC No:64278.LJFin cell line can be applied to seawater fish or freshwater fish virus separation, virus infection pathogenic mechanism and exogenous gene function research, and simultaneously, a good in vitro cell expansion system is improved for vaccine preparation.

Description

Sea bass fin line cell line and application thereof

Technical Field

The invention belongs to the technical field of marine fish cell culture, and particularly relates to a sea bass fin line and application thereof.

Background

The sea bass belongs to the order of the sea bass and the genus of the sea bass, is a meat-eating fish with wide salt and wide temperature, and usually living in estuary areas or salty and fresh water lakes. However, with the expansion of the cultivation scale, the degradation of the cultivated species, the degradation of the cultivation environment, etc., the frequent outbreak of diseases has gradually become one of the main bottlenecks restricting the healthy development of the sea bass cultivation industry. Among them, viral diseases are important infectious diseases that jeopardize sea bass culture. Iridovirus and nerve necrosis virus are the two most prominent viral pathogens of sea bass, and currently there is still a lack of measures to effectively prevent and control both viral diseases.

As an ideal in vitro culture system, the fish cell line has the advantages of low cost, simple operation, good repeatability, controllable conditions and the like, and has been widely applied to basic and application basic researches such as virus separation and identification, virus infection pathogenic mechanism, host gene function, virus vaccine preparation and the like. Along with the development of biotechnology and interdisciplinary interpenetration, cell lines are also used in the fields of research such as immunology, toxicology, metabonomics, genetic developmental biology and oncology. To date, suitable cell lines for the isolation and proliferation of the sea bass iridovirus are very limited, which greatly limits the research on the pathogenic mechanism of sea bass iridovirus infection and the development of virus inactivated vaccines. Therefore, the establishment of a sea bass cell line sensitive to different viruses is urgently needed for researching, preventing and controlling the infection mechanism of the viral diseases.

Disclosure of Invention

The first object of the present invention is to provide a sea bass fin cell line (Lateolabrax japonicus FIN CELL LINE) LJFin, which has the accession number: GDMCC No:64278.

The second object of the invention is to provide the application of the sea bass fin line cell line in expressing exogenous genes.

A third object of the present invention is to provide an application of the above-mentioned jewfish fin line as a host cell for studying aquatic animal viruses, wherein the viruses are jewfish iridovirus, nerve necrosis virus or megalopsis iridovirus.

A fourth object of the present invention is to provide an application of the above-mentioned jewfish fin line in culturing of aquatic animal viruses, and/or diagnosis and detection of non-disease, and therapeutic purposes, wherein the viruses are jewfish iridovirus, nerve necrosis virus or largemouth jewfish iridovirus.

The fifth object of the present invention is to provide the application of the above-mentioned sea bass fin line cell line in the isolation of aquatic animal viruses, wherein the viruses are sea bass iridovirus, nerve necrosis virus or largemouth black bass iridovirus.

The sixth object of the present invention is to provide the application of the above-mentioned sea bass fin cell line as an in vitro virus infection model of aquatic animals, wherein the virus is sea bass iridovirus, nerve necrosis virus or largemouth black bass iridovirus.

The seventh object of the invention is to provide the application of the sea bass fin line cell line in animal genetic breeding research or animal nutrition research.

The eighth object of the invention is to provide the application of the sea bass fin line cell line in the development of sea bass iridovirus SPIV inactivated vaccine.

Preferably, the sea bass iridovirus SPIV inactivated vaccine uses virus liquid within 5 passages as virus seeds.

The ninth object of the invention is to provide a sea bass iridovirus SPIV inactivated vaccine prepared by using the sea bass fin line cell line.

The invention has the advantages that:

1. The sea bass fin line LJFin is obtained, the cell morphology is mainly a fibroblast, the cell growth state is good, the stable passage is over 80 generations, the activity is good after ultralow-temperature preservation, and a foundation is laid for preservation of sea bass gene germplasm resources.

2. The construction method of the sea bass fin line provided by the invention adopts a tissue blocking method to perform primary cell culture. Other growth factors are not required to be added in the primary cell and the subculture cell, and the culture conditions are not harsh.

3. The sea bass fin cell line provided by the invention is sensitive to sea bass iridovirus, and the virus titer can reach 7.9X10 ⁷TCID₅₀/mL.

4. The sea bass fin cell line can be directly applied to the research of virus-host cell interaction and exogenous gene functions.

5. The jewfish fin cell line can be applied to preparation of the SPIV inactivated vaccine.

The sea bass fin line (Lateolabrax japonicus FIN CELL LINE) LJFin was deposited at the cantonese microbiological bacterial deposit center (GDMCC), 1.12, 2024, address: building 5, road 100, college 59, guangzhou city martyrs, post code: 510070, accession number: GDMCC No:64278.

Drawings

FIG. 1 is a morphology of LJFin cells in example 1 of the present invention, and A and B are shown as the morphology of the LJFin cells, the 7 th day primary cells and the 40 th generation cells, respectively.

FIG. 2 is a graph showing the growth of LJFin cells under different culture conditions in example 2 of the present invention; a represents the effect of different temperatures on cell growth; b represents the effect of different fetal bovine serum concentrations on cell growth.

FIG. 3 is a chart showing chromosome karyotyping of LJFin cell lines in example 3 of the present invention; a represents the metaphase chromosome number distribution of the 55 th generation LJFin cells. B represents the metaphase chromosome division phase of the 55 th generation LJFin cells.

FIG. 4 is a fluorescent image of LJFin cell line transfected pEGFP-N3 in example 4 of the present invention; a represents cell morphology under a phase contrast microscope; b represents fluorescence in transfected cells under a fluorescence microscope.

FIG. 5 is a graph showing the detection of the sensitivity of LJFin cells to fish important viruses in example 5 of the present invention; a represents lesion observation of infected cells; b represents the expression of viral proteins in the infected cells; c represents ultrastructural observation of infected cells.

FIG. 6 shows the infectious change of SPIV in LJFin cells after serial passage in example 6 of the present invention. The figures show the viral titers in SPIV infected cells at passages 5, 10 and 15.

Detailed Description

The technical effects produced by the present invention will be described in further detail and fully in connection with the specific embodiments for a more complete understanding of the technical aspects, objects, and advantages of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. It should be noted that other embodiments obtained without departing from the inventive concept are within the scope of protection of the present invention for a person skilled in the art. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Example 1: construction of a sea bass Fin (LJFin) cell line

(1) In the following examples, the fish body was selected as a cultured Japanese sea bass (Lateolabrax japonicus) having a body length of 3 cm.

(2) Leibovitz's-15 (L-15) basal medium, HEPES, antibiotics, 0.25% pancreatin digest and foetal calf serum in the following examples were manufactured by GIBCO company.

(3) Leibovitz's-15 (L-15) culture medium is prepared according to the specification of the company product, naCl and 5mM HEPES with the mass ratio of 0.266% are additionally added, the pH value is regulated to 7.4, and the mixture is filtered by a 0.22 mu m filter membrane and then split-packed for standby at 4 ℃; at the later stage, fetal bovine serum or antibiotics (penicillin, streptomycin and nystatin) with different concentrations are added according to the requirement.

Rinsing the medium: the final concentration of penicillin, streptomycin and nystatin were added at 400 IU/mL, 400 μg/mL and 400 μg/mL to the basal medium, and the pH was adjusted to 7.4.

Primary cell culture fluid: to the rinse medium was added 20% fetal bovine serum by volume.

Passaging cell culture fluid: the culture medium used for the cells of passage 1-10 is: adding 20% fetal bovine serum and diabody (100 IU/mL penicillin and 100 μg/mL streptomycin) in volume ratio into the L-15 basal medium, and adjusting pH to 7.4; after passage of the cells 10, the serum concentration of the passage cell culture medium was reduced to 10% by volume, and the double antibody concentrations were 100 IU/mL penicillin and 100. Mu.g/mL streptomycin, respectively.

(4) Primary culture

The sea bass with the body length of about 3 cm is taken and rinsed 3 h in a rinsing culture medium under the aseptic condition. The fin was cut off and transferred to a sterile petri dish, and the rinsing medium was repeatedly rinsed 3 times. The fin was minced with a sterile razor blade and the rinsing medium was repeatedly rinsed 3 times. The minced fin tissue blocks are attached to the bottom of a cell culture flask coated with fetal bovine serum, 10-15 blocks per flask. The flask was placed upside down in an incubator at 28℃2 h. Slowly adding 1.5 mL primary cell culture solution, placing the culture flask in an incubator, and continuously culturing at 28 ℃. On day 5 of primary culture, cells began to migrate from the edges of the tissue mass. On day 15 of culture, the tissue mass-migrating cells had been confluent with 80% of monolayer, and subculturing was started.

(5) Subculture

At the time of the first passage, the medium in the primary culture flask was transferred to a sterile flask. The monolayer cells were digested with 1mL of 0.25% pancreatin digestate at room temperature, and 5mL fresh passaged cell culture broth and 5mL primary cell culture broth were added, and the monolayer cells were dispersed into single cells by gentle blowing. Evenly dividing the culture medium into two bottles according to the volume ratio of 1:1, and continuously culturing at 28 ℃. The passage was continued once every 5 days as described above, and when passage was made to passage 10, the fetal bovine serum in the culture broth was reduced to a volume ratio of 10% and the antibiotic concentration was the normal use concentration (100 IU/mL penicillin and 100. Mu.g/mL streptomycin). After 10 passages, passage was made every 3 days.

The primary cell morphology of the sea bass fin tissue migration constructed in the embodiment is a fibroblast-like and an epithelial-like. As the passage times increased, the cells were mostly fibroblast-like (FIG. 1). Currently, cells are morphologically stable, and have been serially passaged over 80 passages, designated as the sea bass fin cell line (Lateolabrax japonicus FIN CELL LINE, LJFin).

(6) LJFin cryopreservation and resuscitation Capacity of cells

Taking 30 generations of cell suspension in the step (5), and centrifuging the cell suspension by 1000 rpm to obtain 10 min. The cell sediment is resuspended by 1mL frozen stock solution (L-15 culture medium contains 20% fetal bovine serum and 10% dimethyl sulfoxide) precooled at 4 ℃, the cell suspension is put into a frozen stock tube and then put into a low-temperature frozen stock box to be frozen overnight in an ultralow-temperature refrigerator at-80 ℃, and the cell suspension is put into liquid nitrogen for long-term storage at intervals.

And recovering the frozen cells after 30 days of freezing. The frozen tube was removed from the liquid nitrogen and quickly thawed in a 37 ℃ water bath. The cell suspension was centrifuged at 1000 rpm for 10: 10min to collect the cell pellet, which was resuspended in 1 mL passage cell culture medium. A small amount of the cell suspension was stained with 0.4% trypan blue 5min and the number of dead living cells counted with a hemocytometer. And uniformly mixing the rest cell suspension with 4 mL passage cell culture solution, transferring into a culture flask for culturing at 28 ℃, and observing the adherence and growth condition of cells. The survival rate of LJFin cells after cryopreservation is 78.3%, and the morphology and proliferation capacity of the surviving cells after adherence are not obviously different from those of the cells before cryopreservation.

Example 2: effect of different culture conditions on LJFin cell growth

1. Effect of different culture temperatures on LJFin cell growth.

Using the 35 th generation LJFin cells of this example 1, the growth of the cells at different culture temperatures was examined as follows: 1X 10 ⁵ cells were inoculated into 12-well plates, respectively, and medium (1 mL) was L-15 medium containing 15% fetal bovine serum by volume, and the cells were placed in 16 ℃,22 ℃,28 ℃ and 37 ℃ incubator, respectively, for culturing. 3-well cells were removed from each experimental group every alternate day and the blood cell counting plate counted the cells. Cells were plotted after a continuous count of 7 d. As shown in FIG. 2A, LJFin cells were grown at an optimal temperature of 28℃and at a cell growth rate slightly below 28℃at 22 ℃. But the cells grew slowly at 16℃and 37 ℃.

2. Effect of different concentrations of fetal bovine serum on LJFin cell growth.

Using the 35 th generation LJFin cells of this example 1, 1X 10 ⁵ cells were inoculated into 12 well plates, respectively, and L-15 medium (1 mL) of different concentrations of fetal bovine serum was added to each well, and the fetal bovine serum concentrations were set to 5%, 10%, 15% and 20% by volume, respectively, and cultured in a constant temperature incubator at 28 ℃. 3-well cells were removed from each experimental group every alternate day and the blood cell counting plate counted the cells. After a continuous count of 7 d, the growth curve of the cells was plotted. As shown in fig. 2B: the cell growth speed is in direct proportion to the concentration of added serum, and 10% of serum can maintain the normal growth of cells; when the serum concentration is 5%, the cell proliferation is obviously slowed down, which is only half of the serum concentration of 10%. The medium containing 15% and 20% serum significantly promoted cell proliferation. From the standpoint of overall and cost saving, LJFin cells were suitably grown at 28℃and serum was used at a concentration of 10% from passage 10 onwards.

Example 3: chromosome karyotyping of LJFin cells

The 55 th generation LJFin cells in example 1 were cultured in a passaging cell culture medium at 28℃for 48 hours, 1. Mu.g/mL of colchicine-treated cells were treated for 6 hours, and after digestion with 0.25% pancreatin digest, the cells were recovered by centrifugation at 1000: 1000 rpm and 10: 10 min. Cell pellets were hypotonic treated with 75 mM KCl in a 37 ℃ water bath for 30min, centrifuged at 1000 rpm for 10min, and the cell pellets were resuspended in 8 mL fixative (volume ratio methanol: glacial acetic acid=3:1) for 30min at room temperature and fixation was repeated 1 time. Sucking the fixing liquid-cell suspension liquid drop on the precooled glass slide, quickly blowing off the liquid drop, and naturally airing at room temperature. The slide was stained 10min with 1-fold of giemsa staining solution (Saccharum biosciences (Zhenjiang) Inc.: bioseth, cat# RS 3080), rinsed and dried at room temperature. Chromosome morphology was observed under an oil microscope, and 200 split phases were photographed and the number of chromosomes was counted, respectively.

As a result, as shown in FIG. 3, the number of chromosomes in the 55 th generation cell division phase was mostly distributed between 22 and 72, the number of characteristic chromosomes was 48, the division phase with diploid chromosome number (2n=48) accounted for 21% of all the statistical cells (FIG. 3A), and the chromosome morphology was mostly telomere chromosomes (FIG. 3B).

Example 4: transfection efficiency of exogenous Gene in LJFin cells

The LJFin cells of passage 50 of example 1 were seeded into 24-well cell culture plates and cultured overnight (the cell culture medium was the passaged cell culture medium described in example 1) and transfection was initiated when the cells were confluent with a monolayer of more than 90%. The transfection reagent was Lipofectamine 2000 (Ivitro Inc.), and pEGFP-N3 was transfected into cells according to the instructions of the reagent. The expression of the green fluorescent protein in the cells was then observed under a fluorescent microscope. As shown in FIG. 4, a strong green fluorescent signal was observed in LJFin cells transfected with 24h, and cells showing the green fluorescent signal account for about 40% of total cells, indicating that pEGFP-N3 was successfully transfected into cells, and that the CMV promoter was able to efficiently initiate exogenous gene expression in LJFin cells, suggesting that LJFin cell line could be used to study exogenous gene function.

Example 5: virus infection experiments of LJFin cell lines

1. Cytopathic Effect (cytopathic effect, CPE) observations

The virus infection was performed after inoculating the LJFin th generation cells of example 1 to 24-well culture plates overnight (the cell culture medium was the passage cell culture medium described in example 1), and the virus suspensions of laboratory preserved sea bass iridovirus SPIV, nerve necrosis virus RGNNV and larch iridovirus LMBV were added to the culture medium, respectively, with an infection index (Multiplicity of infection, MOI) of 2, while the uninfected cells were used as controls. Cytopathic effects were recorded by observing and photographing at different times of infection with a phase contrast microscope, respectively. As a result, FIG. 5A, SPIV infected cells are typically characterized by cell rounding, increased refractive index, and individual rounded cells dispersed throughout a monolayer of cells. About 80% of cells become rounded after 96 h infection, and voids form in the cell monolayer after the rounded cells fall off. A typical lesion of RGNNV infected cells is characterized by the presence of variable size, variable number of vacuoles within the cytoplasm of the cell. As the infection time increases, the infected cells necrotize and shed, forming voids in the monolayer of cells. The pathological features of LMBV infected cells are shown as: the cells become round and the volume is reduced. As the infection time progresses, the rounded cells shed and the cell monolayer forms a void.

2. Expression of viral proteins in infected cells

The LJFin th generation cells of example 1 were used to infect SPIV, RGNNV and LMBV respectively according to the procedure of step 1, cells were harvested in RIPA buffer (ThermoFisher Co.) after viral infection at 12 h, 24 h, 48 h or 96 h, and expression of viral proteins in the infected cells was examined by Western blotting. Infected and uninfected cell samples were boiled denatured and separated using 10% SDS-PAGE and transferred to PVDF membrane (Millipore Co.). Subsequently, the membranes were blocked by incubating 4 h with 5% skim milk (100 mL PBS +0.1% Tween 20 (PBST, v/v) +5 g skim milk powder (BD Difco Co.) at room temperature and incubated overnight with the corresponding specific antibodies, including anti- β -tubulin antibodies (1:3000, abcam Co.), anti-SPIV-MCP antibodies (1:2000, antigen SPIV-MCP prokaryotic expression products, antibody prepared by Wuhan Jin Kairui Co.), anti-LMBV-MCP antibodies (1:3000, self-made by the present laboratory, patent number: ZL 202210174632.4) or anti-RGNNV-CP antibodies (1:3000, antigen RGNNV-CP prokaryotic expression products, antibody prepared by Wuhan Jin Kairui Co.) after washing the membrane three times with PBST, the membrane was incubated with the corresponding secondary antibodies (1:5000, proteintech Co.) at room temperature for 1 hour, finally, automated incubation with ECL color solution (Biol color development system prepared by Walsh, 5230 s) was imaged by a chemiluminescent system of 5242 seconds.

As a result, as shown in fig. 5B, in the SPIV-infected cell sample, the expression amount of viral protein MCP was significantly increased from 24 to h to 96 h after infection with the increase in time; likewise, the expression level of viral protein CP increased significantly with time from 12 h to 48 h after infection in RGNNV-infected cell samples, and the expression level of viral protein MCP increased significantly with time from 24 h to 48 h after infection in LMBV-infected cell samples. Whereas no specific bands were seen in uninfected cells, indicating that viral proteins (SPIV MCP, RGNNV CP or LMBV MCP) were expressed in infected cells, further indicating that detection of successful proliferation of the virus in LJFin cells.

3. Electron microscope observation of virus infected cells

The LJFin th generation of cells from example 1 were used to infect SPIV, RGNNV and LMBV respectively according to step 1, the cells were harvested at 48 h or 96 h for viral infection respectively, centrifuged at 2000 rpm for 10 min, and the cell pellet was fixed at 4℃with 2.5% glutaraldehyde for 1 h. The fixative was discarded and the PBS was rinsed three times, 5min times each; 1h of osmium acid with the volume ratio of 1% is fixed at 4 ℃; ethanol gradient (volume ratio 50%, 70%, 80%, 90%, 100%) was dehydrated stepwise 10 min. Epoxy Epon812 impregnates the embedding. Then the sample is subjected to ultrathin section treatment, and after the section is respectively dyed by 2% uranyl acetate and lead citrate to be 1h, the section is placed under a transmission electron microscope (Talos L120C, thermo FISHER SCIENTIFIC company) for observation, recording and photographing.

As shown in FIG. 5C, a large number of hexagonal virus particles were observed in the cytoplasm of both SPIV and LMBV-infected cells. Whereas a large number of scattered spherical virions were observed in the vacuoles of RGNNV infected cells, the virions were approximately 30nm in diameter. The electron microscope observations further indicate that all three viruses can successfully proliferate in LJFin cells. The LJFin cells are used as an in vitro virus infection model, and can be used for researching the mechanism of virus infection pathogenicity of aquatic animals and researching virus-host interaction.

Example 6: infectivity of serial passage of SPIV in LJFin cells

When the LJFin th generation cell of example 1 was infected with SPIV and cytopathy reached 90% or more, virus solution was harvested, freeze-thawed once at-20℃and then re-infected with LJFin th generation cell of example 50, and the second virus infection passage was performed. The cells infected with the virus were harvested by infecting the cells sequentially at 5 th, 10 th and15 th passages, and the titer of the virus was measured by the method of virus half tissue infection (TCID ₅₀). The specific method comprises the following steps: the cells infected with the SPIV amplified by different times are frozen and thawed 1 time in a refrigerator at the temperature of minus 20 ℃, and then the sample of the cells infected with the virus to be detected is diluted by a gradient of 10 times. The LJFin th generation cells were transferred to 96-well cell culture plates (2000 cells per well) and cultured overnight in a constant temperature incubator at 28 ℃. And inoculating 100 mu L of diluted virus infected cell samples to be tested into each hole of cells. On day 7 post infection, the number of cell wells where CPE occurred was observed and recorded, and TCID ₅₀ of virus infected samples was calculated according to the Reed-Muench method.

As a result, as shown in FIG. 6, infection of LJFin th generation of virus with a virus produced highly infectious progeny virus, the virus titer was 7.9X10 ⁷TCID₅₀/mL, the virus titer at 10 th generation was slightly decreased to 3.4X10 ⁷TCID₅₀/mL, and the virus titer at 15 th generation was significantly decreased compared to 5 th generation, about 1.8X10 ⁷TCID₅₀/mL. Indicating that infection by the progeny virus produced by SPIV decreases with increasing passage times. It was suggested that within 5 passages of the passage number, virus fluid was required as virus seed in the preparation of SPIV vaccine. The results show that LJFin is the preparation of the SPIV inactivated vaccine, and a good in-vitro cell amplification system is improved.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. A jewfish fin cell line (Lateolabrax japonicus FIN CELL LINE) LJFin, characterized by a deposit number of: GDMCC No:64278.

2. Use of the jewfish fin line of claim 1 for expressing a foreign gene.

3. Use of the jewfish fin line of claim 1 as a host cell for studying aquatic animal viruses, the viruses being jewfish iridovirus, nerve necrosis virus or largemouth jewfish iridovirus.

4. Use of the jewfish fin line of claim 1 for culturing of aquatic animal viruses, said viruses being jewfish iridovirus, nerve necrosis virus or largemouth jewfish iridovirus and detection of diagnostic and therapeutic purposes of non-disease.

5. Use of the jewfish fin line of claim 1 as an in vitro model for viral infection in aquatic animals, said virus being jewfish iridovirus, neurotravovirus or jewfish iridovirus.

6. The use of the jewfish fin line of claim 1 in the development of a jewfish iridovirus SPIV inactivated vaccine.

7. The use according to claim 6, wherein the inactivated vaccine for sea bass iridovirus SPIV uses as viral seed a viral fluid within 5 passages.