Kidney cell line of sparus latus and application of kidney cell line

Abstract

The invention discloses a sparus latus kidney cell line and application thereof. The preservation number of the YSK of the kidney cell of the sparus latus is GDMCC No: 65080, and the preservation number of the YSK of the kidney cell of the sparus latus is GDMCC No: 65080. The sparus latus kidney cell line YSK is constructed through primary culture and subculture, the cellular morphology of the sparus latus kidney cell line is mainly fibroblast-like, the cells grow rapidly, stable passage exceeds 60 generations, and the activity is good after ultralow temperature storage. The sparus latus kidney cell line YSK cell line can be applied to marine fish virus separation, virus infection pathogenic mechanism and exogenous gene function research, and meanwhile, a good in-vitro cell amplification system is provided for preparation of sparus latus dephosphorization virus SDDV vaccines.

Description

Technical Field

[0001] The invention belongs to the field of marine fish cell culture biotechnology, and specifically relates to a yellowfin sea bream kidney cell line and application thereof. Background Art

[0002] Yellowfin sea bream (Acanthopagrus latus), commonly known as yellow-legged fish, red-winged fish, and yellow-legged fish, is a fish belonging to the genus Acanthopagrus of the family Sparidae of the order Perciformes. It is one of the important economic fish species along the coast of South China. As a shallow warm-water bottom fish, yellowfin sea bream can adapt to changes in salinity and can grow in water bodies of different salinities. Yellowfin sea bream is rich in nutrition, has delicious meat, and has high economic value. It is a popular edible fish. With the development of yellowfin sea bream aquaculture, frequent outbreaks of diseases have brought challenges and obstacles to the development of the yellowfin sea bream aquaculture industry. Among them, viral diseases are important infectious diseases that endanger yellowfin sea bream aquaculture. Scaledrop syndrome virus (SDDV) is an important pathogen that causes ascites disease in yellowfin sea bream and belongs to the genus Cytomegalovirus of the family Iridoviridae. In addition, SDDV can infect young and adult Asian sea bass, resulting in a mortality rate of about 40-50%. At present, there is still a lack of effective treatment and prevention and control measures for the isolates of yellowfin sea bream dephosphorylation disease virus SDDV.

[0003] As an ideal in vitro culture system, fish cell lines have been widely used in basic and applied basic research such as virus isolation and identification, viral infection pathogenic mechanism, host gene function and large-scale preparation of viral vaccines due to their many advantages such as low cost, simple operation, good repeatability and controllable conditions. With the mutual penetration of interdisciplinary subjects, cell lines are also used in research fields such as immunology, toxicology, genetic developmental biology and oncology. Compared with other marine fish, there are fewer cell lines of yellowfin sea bream established so far, and yellowfin sea bream kidney cell lines have not been reported yet, which greatly limits the study of the pathogenic mechanism of yellowfin sea bream virus in vitro cell infection and the development of virus inactivated vaccines. Therefore, it is necessary to establish yellowfin sea bream cell lines that are sensitive to different viruses to provide good experimental materials for the study of viral infection mechanisms, viral gene functions and vaccine prevention and control.

[0004] This invention is supported by the project titled “Research on New Technologies for Immune Evaluation of Marine Aquaculture Animals” (2023YFC2812101). Summary of the invention

[0005] The first object of the present invention is to provide a yellowfin seabream kidney cell line YSK, whose deposit number is: GDMCC No:65080.

[0006] The yellowfin sea bream kidney cell line YSK grows rapidly, has high cell viability, has been stably passaged for more than 60 generations, and is sensitive to a variety of marine fish viruses.

[0007] The second object of the present invention is to provide the use of the yellowfin sea bream kidney cell line in expressing foreign genes.

[0008] The third object of the present invention is to provide the application of the yellowfin sea bream kidney cell line as a host cell for studying marine fish viruses, wherein the virus is yellowfin sea bream dephosphorylation virus, Singapore grouper iridovirus or neuronecrosis virus.

[0009] The fourth object of the present invention is to provide the use of the above-mentioned yellowfin sea bream kidney cell line in the cultivation of marine fish viruses and / or the detection of non-disease diagnosis purposes, wherein the virus is yellowfin sea bream dephosphorylation virus, Singapore grouper iridovirus or neuronecrosis virus.

[0010] The fifth object of the present invention is to provide the use of the above-mentioned yellowfin sea bream kidney cell line in the isolation of marine fish viruses, wherein the virus is yellowfin sea bream dephosphorylation virus, Singapore grouper iridovirus or neuronecrosis virus.

[0011] The sixth object of the present invention is to provide the use of the above-mentioned yellowfin sea bream kidney cell line as an in vitro virus infection model of aquatic animals, wherein the virus is yellowfin sea bream dephosphorylation virus, Singapore grouper iridovirus or neuronecrosis virus.

[0012] The seventh object of the present invention is to provide the use of the above-mentioned yellowfin sea bream kidney cell line in the development of an inactivated vaccine for yellowfin sea bream dephosphorylation disease virus.

[0013] Preferably, the yellowfin sea bream dephosphorylation disease virus inactivated vaccine uses virus liquid infected for 24h-96h as virus seed.

[0014] The eighth object of the present invention is to provide a method for preserving the above-mentioned yellowfin sea bream kidney cell line, which comprises the following steps: taking a cell suspension of the yellowfin sea bream kidney cell line in the logarithmic growth period, centrifuging, resuspending the cell pellet with a freezing solution pre-cooled at 4-5°C, transferring the cell suspension to a -70 to -90°C freezer overnight, and transferring to liquid nitrogen for long-term storage the next day; the freezing solution is an L-15 culture medium containing 20% ​​fetal bovine serum and 10% dimethyl sulfoxide in a volume ratio.

[0015] The ninth object of the present invention is to provide a method for culturing the above-mentioned yellowfin sea bream kidney cell line, which comprises the following steps: inoculating the yellowfin sea bream kidney cell line into a culture medium and culturing at 25-30°C; the culture medium is an L-15 culture medium containing 100-400 IU / mL penicillin, 100-400 μg / mL streptomycin and 10%-20% fetal bovine serum by volume.

[0016] Advantages of the present invention:

[0017] 1. The present invention obtains a yellowfin sea bream kidney cell line YSK, whose cell morphology is mainly fibroblast-like, the cells grow rapidly, have been stably propagated for more than 60 generations, and have good vitality after ultra-low temperature storage, laying a foundation for the preservation of yellowfin sea bream gene germplasm resources.

[0018] 2. The method for constructing the yellowfin sea bream kidney cell line provided by the present invention adopts a digestion culture method to culture primary cells. No other growth factors need to be added during the culture of primary cells and passage cells, and the culture conditions are not strict.

[0019] 3. The yellowfin sea bream kidney cell line provided by the present invention is sensitive to the yellowfin sea bream dephosphorylation disease virus SDDV, and the virus titer can reach 1.48×10 8 TCID 50 / mL.

[0020] 4. The yellowfin sea bream kidney cell line of the present invention can be directly applied to the study of virus-host cell interaction and exogenous gene function.

[0021] 5. The yellowfin sea bream kidney cell line of the present invention can be used for the preparation of SDDV inactivated vaccine.

[0022] The yellowfin seabream kidney cell line YSK was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on August 30, 2024, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, and the deposit number is: GDMCC No: 65080. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 These are morphological diagrams of YSK cells in Example 1 of the present invention, wherein Figure A and Figure B represent the cell morphology of the 20th and 60th generations of YSK cells, respectively.

[0024] Figure 2 It is a growth curve diagram of YSK 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.

[0025] Figure 3 These are fluorescence images of the YSK cell line transfected with pEGFP-N3 in Example 3 of the present invention; A represents the cell morphology under a phase contrast microscope; and B represents the fluorescence in the transfected cells under a fluorescence microscope.

[0026] Figure 4This is the sensitivity test of YSK cells to important fish viruses in Example 4 of the present invention; A represents the pathological observation of infected cells; B represents the expression of viral proteins in infected cells.

[0027] Figure 5 Figure 2 shows the replication of SDDV in YSK cells. (A) Electron microscopic observation of infected cells; (B) Virus titer in infected cells. DETAILED DESCRIPTION

[0028] In order to more fully understand and demonstrate the technical solutions, purposes and advantages of the present invention, the technical effects produced by the present invention are further described in detail and completely in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all. It should be pointed out that based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in the field without creative work are all within the scope of protection of the present invention.

[0029] Example 1: Construction of Yellowfin Sea Bream Kidney (YSK) Cell Line

[0030] (1) In the following examples, the fish selected were farmed yellowfin sea bream (Acanthopagrus latus) with a body length of 5 cm.

[0031] (2) The Leibovitz's-15 (L-15) basal medium, HEPES, double antibiotics (penicillin-streptomycin), 0.25% trypsin-EDTA digestion solution and fetal bovine serum in the following examples are products of GIBCO.

[0032] (3) Leibovitz's-15 (L-15) medium was prepared according to the company's product instructions, and 0.266% NaCl and 5 mM HEPES were added in a mass ratio, and the pH value was adjusted to 7.4. After filtering through a 0.22 μm filter membrane, it was divided into aliquots and stored at 4°C for later use. Different concentrations of fetal bovine serum or double antibiotics (penicillin-streptomycin) were added as needed for later use.

[0033] Rinsing medium: L-15 basal medium with penicillin and streptomycin added to a final concentration of 400 IU / mL and 400 μg / mL, and the pH was adjusted to 7.4.

[0034] Primary cell culture medium: Add 20% (volume ratio) fetal bovine serum to the rinsing medium.

[0035] Subculture cell culture medium: The culture medium used for cells of passages 1-10 is: primary cell culture medium; after cell passage 10, the serum concentration of the subculture cell culture medium is reduced to 10% by volume, and the dual antibody concentrations are: 100 IU / mL penicillin and 100 μg / mL streptomycin.

[0036] (4) Primary culture

[0037] Under sterile conditions, soak and rinse the fish body with 75% alcohol, then use sterile dissecting tools to open the fish abdominal cavity and take the kidney tissue of the fry. Rinse with rinsing solution three times, 5 minutes each time. Then use a blade to fully chop the kidney tissue blocks, add 1.5mL of 0.25% trypsin-EDTA digestion solution and digest at room temperature for 25 minutes, and add 1mL of primary cell culture medium to terminate the digestion reaction. The digested cell suspension is filtered with a 100-mesh sterile filter. The filtrate is transferred to a 1.5mL centrifuge tube and centrifuged at 1000rpm for 10 minutes. Discard the supernatant, resuspend the precipitate with primary cell culture medium, and after counting the cells, the cell suspension containing about 4×10 6 The cell suspension was transferred into a 25 cm 2 In a culture flask. Add 5 mL of primary cell culture medium to each culture flask. Place the culture flask in a 28°C incubator for culture. Replace the culture medium by half every 4 days. During this period, use a phase contrast microscope to observe and record the cell adhesion growth. On the second day of primary culture, primary cells began to grow adherently. In the early stage of culture, the cell morphology was diverse, with fibrous cells and epithelial cells coexisting. After 10 days, the primary cells covered about 85% of the bottom of the flask and began to be subcultured.

[0038] (5) Subculture

[0039] During the initial subculture, transfer the culture medium in the primary culture cell bottle to a sterile culture bottle. Digest the monolayer cells with 1 mL of 0.25% trypsin-EDTA digestion solution at room temperature, mix 5 mL of fresh subculture cell culture solution and 5 mL of primary cell culture solution, and add them to the culture bottle of the monolayer cells. Gently tap the bottle wall to make the monolayer cells fall off into the culture medium, and then gently blow with the culture medium to disperse the monolayer cells into single cells. Divide them evenly into two bottles at a volume ratio of 1:1 and continue to culture at 28°C. In the early stage of subculture, subculture once every 5 days according to the above method, and the culture medium is primary cell culture medium. When it is subcultured to the 10th generation, the fetal bovine serum in the subculture cell culture medium is reduced to 10%, and the antibiotic concentration is 100 IU / mL penicillin and 100 μg / mL streptomycin; subculture once every 3 days. The yellowfin sea bream kidney primary cells constructed in this example are fibroblast-like and epithelial-like in morphology. As the number of subcultures increases, the cell morphology becomes spindle-shaped, and fibroblasts are the main cells ( Figure 1 The cell morphology is currently stable and has been continuously passaged for more than 60 generations. It is named yellowfin seabream Kindey cell line (YSK).

[0040] (6) Cryopreservation and resuscitation ability of YSK cells

[0041] Take the cell suspension of the 40th generation in step (5) and centrifuge at 1000 rpm for 10 minutes. Resuspend the cell pellet in 1 mL of freezing solution (L-15 culture medium containing 20% ​​fetal bovine serum and 10% dimethyl sulfoxide by volume) precooled at 4°C, transfer the cell suspension into a cryotube and then into a low-temperature freezing box and place it in a -80°C ultra-low temperature refrigerator for freezing overnight, and transfer it into liquid nitrogen for long-term storage the next day.

[0042] After one month of cryopreservation, the above-mentioned frozen cells were revived. The cryopreserved tubes were taken out of liquid nitrogen and quickly placed in a 37°C water bath to thaw. The cell suspension was centrifuged at 1000rpm for 10 minutes to collect the cell precipitate, and the precipitate was resuspended in 1mL of cell culture medium. A small amount of cell suspension was taken and stained with 0.4% trypan blue for 5 minutes, and the number of dead and living cells was counted with a hemocytometer. The remaining cell suspension was mixed with 4mL of cell culture medium and transferred to a culture flask for culture at 28°C to observe the cell adhesion and growth. The cell survival rate of YSK cells after cryopreservation was 81%, and there was no significant difference in the morphology and proliferation ability of the surviving cells after adhesion compared with before cryopreservation.

[0043] The yellowfin seabream kidney cell line YSK was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on August 30, 2024, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, and the deposit number is: GDMCC No: 65080.

[0044] Example 2: Effects of different culture conditions on YSK cell growth

[0045] 1. Effects of different culture temperatures on the growth of YSK cells.

[0046] The 20th generation YSK cells of Example 1 were used to detect the growth of cells at different culture temperatures. The specific operation was as follows: 6×10 4 The cells were inoculated into a 12-well plate, and the culture medium (1 mL) was L-15 culture medium containing 10% fetal bovine serum by volume. The cells were cultured in incubators at 15°C, 28°C, and 37°C, respectively. Three wells of cells were taken from each experimental group on the 1st, 3rd, 5th, and 7th day of culture, and the cell growth curve was drawn after the cells were counted with a hemocytometer. Figure 2 As shown in A, the optimal growth temperature of YSK cells is 28°C, and the cells grow slowly at 15°C and 37°C.

[0047] 2. Effects of different concentrations of fetal bovine serum on the growth of YSK cells.

[0048] Using the 20th generation YSK cells of Example 1, 6×10 4Each well was inoculated with L-15 medium (1 mL) containing different concentrations of fetal bovine serum (FBS) at 5%, 10%, and 15% by volume, respectively, and cultured in a 28°C incubator. Three wells of cells were taken from each experimental group on the 1st, 3rd, 5th, and 7th day of culture, and the cell growth curve was drawn after counting the cells with a hemocytometer. Figure 2 As shown in B: The cell growth rate is proportional to the added serum concentration. On the third day of culture, the medium containing 15% and 10% serum significantly promoted cell proliferation, and 10% serum could maintain normal cell growth; when the serum concentration was 5%, cell proliferation was significantly slowed down, and the number of cells on the third day of culture was only one-third of that of the 10% serum concentration. From the perspective of cost saving, starting from the 10th generation, YSK cell culture preferably uses L-15 medium with a volume ratio of 10% serum concentration for culture.

[0049] Example 3: Successful transfection of exogenous genes in YSK cells

[0050] The 25th generation YSK cells in Example 1 were inoculated into a 24-well cell culture plate for overnight culture (the culture medium was the cell culture medium described in Example 1). When the cells were confluent with more than 85% of the monolayer, transfection was started. The transfection reagent was Lipofectamine 2000 (Ivitrogen), and pEGFP-N3 was transfected into the cells according to the operating instructions of the reagent. The expression of green fluorescent protein in the cells was observed under a fluorescence microscope 24 hours after transfection. Figure 3 As shown, a strong green fluorescence signal was observed in the transfected YSK cells, indicating that pEGFP-N3 was successfully transfected into the cells, suggesting that the CMV promoter can efficiently initiate the expression of foreign genes in YSK cells, and the YSK cell line can be used to study the functions of foreign genes.

[0051] Example 4: Virus infection experiment of YSK cell line

[0052] 1. Cytopathic effect (CPE) observation

[0053] The 35th generation YSK cells of Example 1 were inoculated into a 24-well culture plate and cultured overnight for virus infection (the cell culture medium was the cell culture medium described in Example 1). The yellowfin porgy dephosphorylation virus SDDV-ZH strain, the neuronecrosis virus RGNNV and the Singapore grouper iridovirus SGIV suspensions preserved in the laboratory were added to the culture medium, and the virus infection index (Multiplicity of infection, MOI) was 2. Uninfected cells (Mock) were used as negative controls. The CPE was observed and photographed at different times of infection using a phase contrast microscope. The results are shown in Figure 2. Figure 4A. The typical characteristics of cells infected with SGIV and SDDV-ZH strains are rounding of cells, increased refractive index, and individual rounded cells scattered on the cell monolayer. After 36 hours of infection, the rounded cells fell off and formed cavities in the cell monolayer. Vacuoles of different sizes appeared in the cytoplasm of RGNNV-infected cells. After 36 hours of infection, the cells necrotized and fell off, forming gaps in the monolayer of cells. On the contrary, when uninfected cells were observed at 24 hours, there was no obvious change in cell morphology. This shows that different marine fish virus infections can cause obvious cytopathic effects in YSK cells.

[0054] 2. Expression of viral proteins in infected cells

[0055] Take the 35th generation YSK cells of Example 1 and infect SDDV-ZH, RGNNV and SGIV respectively according to the method of step 1. At different time points after virus infection (SDDV: 24h, 48h and 96h; RGNNV: 12h, 24h and 36h; SGIV: 12h, 24h and 36h), harvest the cells and lyse them in RIPA buffer (ThermoFisher), and use western blotting to detect the expression of viral proteins in infected cells. The experimental procedure is as follows: the infected and uninfected cell samples are boiled and denatured, then separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore). The membrane containing the protein is stained with 5% skim milk (100mL PBS + 0.1% Tween 20 (PBST, v / v) + 5g skim milk powder (BD Biosciences, Inc.). Difco) was incubated at room temperature for 2 h for blocking, and then the corresponding specific antibodies were used as primary antibodies for incubation at 4°C overnight, including anti-β-tubulin antibody (1:3000, Abcam), anti-SDDV-MCP antibody (1:2000, homemade in our laboratory, patent: monoclonal antibody against yellowfin sea bream iridovirus SDDV isolate and its application, patent number: ZL202211202844.5), anti-SGIV-MCP antibody (1:3000, antigen SGIV-MCP prokaryotic Expression product, Wuhan Jinkairui Company was commissioned to prepare antibodies) or anti-RGNNV-CP antibody (1:3000, antigen RGNNV-CP prokaryotic expression product, Wuhan Jinkairui Company was commissioned to prepare antibodies). After washing the membrane three times with PBST, the membrane was incubated with the corresponding secondary antibody (horseradish peroxidase HRP-labeled goat anti-rabbit or goat anti-mouse-IgG antibody, 1:5000, Proteintech) at room temperature for 1 hour. Finally, the color was developed with ECL color development solution (Bioscience) in a Tanon 5200 automatic chemiluminescence imaging system.

[0056] The results are as follows Figure 4B, in the cell samples infected by SDDV-ZH, the expression of viral protein MCP increased significantly with time from 24h to 96h after infection. Similarly, in the cell samples infected by RGNNV and SGIV, the expression of viral capsid protein CP and major capsid protein MCP gradually increased from 12h to 36h of infection. No specific bands were detected in uninfected cells, indicating that viral proteins (SDDV MCP, RGNNV CP or SGIV MCP) were expressed in infected cells, further proving that the virus successfully proliferated in YHK cells.

[0057] Example 5: Replication of SDDV in YSK cells

[0058] 1. Electron microscopic observation of virus-infected cells

[0059] The 35th generation YSK cells of Example 1 were infected with SDDV-ZH according to step 1 of Example 4. The cells were collected 48 hours after virus infection, centrifuged at 2000 rpm for 10 minutes, and the cell pellet was fixed with 2.5% glutaraldehyde at 4°C for 1 hour. The fixative was discarded, and the cells were rinsed with PBS three times, each for 5 minutes; fixed with 1% (volume ratio) osmium acid at 4°C for 1 hour; dehydrated step by step with ethanol gradient (volume ratio 50%, 70%, 80%, 90%, 100%, 100%) for 10 minutes. The cells were embedded in epoxy resin Epon 812. The samples were then processed for ultrathin sections, and the sections were stained with 2% uranyl acetate and lead citrate for 1 hour, respectively, and then placed under a transmission electron microscope (Talos L120C, ThermoFisher Scientific Company) for recording and photography.

[0060] The results are as follows Figure 5 As shown in A, multiple single-layer membrane vacuolar structures were observed in the cytoplasm of SDDV-infected cells. The vacuolar structures contained hexagonal virus particles with a diameter of about 120-130nm. In addition, scattered hexagonal virus examples were also observed in the cytoplasm. This shows that SDDV can successfully proliferate in YSK cells. In summary, YSK cells can be used as an in vitro virus infection model to study the mechanism of viral infection and pathogenicity of marine fish and virus-host interaction.

[0061] 2. Determination of virus titer in infected cells

[0062] The 40th generation YSK cells of Example 1 were inoculated into a 24-well culture plate and cultured overnight. The SDDV infection was performed with an infection index of 2. The virus-infected cells were collected at 24 h, 48 h, and 96 h after infection, and the cells were repeatedly frozen and thawed three times at -20 °C. The virus was measured by the tissue infection dose (TCID 50) method to determine the virus titer. The specific method is as follows: ① Transfer the 42nd generation YSK cells into a 96-well cell culture plate and culture them in a constant temperature incubator at 28°C overnight. ② Make a 10-fold gradient dilution of the samples collected at different infection time points. ③ Inoculate 100μL of the diluted virus-infected cell sample to be tested in each well of cells, and then place it in a constant temperature incubator at 28°C for infection. ④ On the 6th day after infection, observe and record the number of cell wells with CPE, and calculate the TCID of the virus-infected sample according to the Reed-Muench method. 50 .

[0063] The results are as follows Figure 5 B, The virus titer in YSK-infected cells gradually increased with the extension of infection time. At 24h of infection, the virus titer in cells was not significantly different from that in cells infected at 0h. In cells infected at 48h, the virus titer increased from 7.76×10 5 TCID 50 / mL increased to 1.66×10 6 TCID 50 / mL, and as the infection time was extended to 96h, the virus in the infected cells increased to 1.48×10 8 TCID 50 The above results showed that SDDV could be amplified in YSK cells to obtain a higher virus titer, that is, YSK cells provided a good in vitro cell amplification system for the preparation of SDDV inactivated vaccine.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A yellowfin seabream kidney cell line YSK, characterized in that: The deposit number is: GDMCC No: 65080.

2. Use of the yellowfin sea bream kidney cell line according to claim 1 in expressing exogenous genes.

3. Use of the yellowfin sea bream kidney cell line described in claim 1 as a host cell for studying marine fish viruses, wherein the virus is yellowfin sea bream dephosphorylation virus, Singapore grouper iridovirus or neuronecrosis virus.

4. Use of the yellowfin sea bream kidney cell line according to claim 1 in the cultivation of marine fish viruses and / or the detection of non-disease diagnosis purposes, wherein the virus is yellowfin sea bream dephosphorylation virus, Singapore grouper iridovirus or neuronecrosis virus.

5. Use of the yellowfin sea bream kidney cell line according to claim 1 in the isolation of marine fish viruses, wherein the virus is yellowfin sea bream dephosphorylation virus, Singapore grouper iridovirus or neuronecrosis virus.

6. Use of the yellowfin sea bream kidney cell line according to claim 1 as an in vitro virus infection model for aquatic animals, wherein the virus is yellowfin sea bream dephosphorylation virus, Singapore grouper iridovirus or neuronecrosis virus.

7. Use of the yellowfin sea bream kidney cell line according to claim 1 in the development of an inactivated vaccine for yellowfin sea bream dephosphorylation disease virus.

8. The use according to claim 7, characterized in that: The yellowfin sea bream dephosphorylation disease virus inactivated vaccine uses virus liquid infected for 24h-96h as virus seed.

9. The method for preserving the yellowfin sea bream kidney cell line according to claim 1, characterized in that: The following steps are involved: A cell suspension of a yellowfin sea bream kidney cell line in the logarithmic growth period is taken and centrifuged, the cell pellet is resuspended in a freezing solution precooled at 4-5°C, the cell suspension is transferred to a -70 to -90°C freezer overnight, and the next day is transferred to liquid nitrogen for long-term storage; the freezing solution is an L-15 culture medium containing 20% ​​fetal bovine serum and 10% dimethyl sulfoxide in a volume ratio.

10. The method for culturing the yellowfin sea bream kidney cell line according to claim 1, characterized in that: The following steps are involved: The yellowfin sea bream kidney cell line is inoculated into a culture medium and cultured at 25-30 DEG C; the culture medium is an L-15 culture medium containing 100-400 IU / mL penicillin, 100-400 μg / mL streptomycin and 10%-20% fetal bovine serum in a volume ratio.

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